Ali Amid3 0000-0001-6926-7502 (ali.amid@cw.bc.ca)
  1. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada
  2. Department of Pathology and Laboratory Medicine, Division of Hematopathology, BC Children’s Hospital, Vancouver, BC, Canada
  3. Division of Hematology & Oncology, BC Children’s Hospital, Vancouver, BC, Canada
Correspondence to: Audi Setiadi, BC Children’s Hospital, Room 2K29 - 4480 Oak Street, Vancouver BC V6H 3N1. Tel: (604) 875-2345 x2269. Fax: (604) 875-2815. audi.setiadi@cw.bc.ca
Conflict of interest disclosure: No conflict of interest
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Running title: Atypical Megakaryocytes in Diamond Blackfan Anemia
Figures: 1 (3 panels)
Key words: Diamond Blackfan Anemia; RPL5 mutation; Diamond Blackfan Anemia type 6, megakaryocytes; dysplastic megakaryocytes; myelodysplastic syndrome
Diamond-Blackfan anemia (DBA) is congenital erythroid aplasia associated with defects in ribosomal biogenesis, classically presenting with macrocytic anemia in infancy and congenital anomalies¹. Ribosomal protein S19 (RPS19) is the most commonly mutated gene, while Ribosomal protein L5 (RPL5) mutation accounts for the minority (7%) of cases and is associated with more severe phenotype thanRPS19 mutation2,3. Approximately 80% of patients with DBA respond to corticosteroid therapy, whereas 20% require chronic red blood cell (RBC) transfusion4. However, they are also at risk of developing myeloid malignancies later in life, including myelodysplastic syndrome (MDS)4,5,6. There is an overlap in presentation between DBA and MDS, therefore careful bone marrow assessment with ancillary testing are crucial to detect disease transformation. Megakaryocytic dysplasia at disease presentation in DBA is a highly atypical finding that has not been well described in the literature. We report two cases of DBA with RPL5 mutation (DBA type 6) showing peculiar megakaryocytic morphology with monolobated nuclei at baseline, mimicking MDS.
Two unrelated infants presented with reticulocytopenic anemia, mild dysmorphic features and cardiac anomalies at birth. Patient 1 was a term male infant with hemoglobin (Hb) of 81 g/L, reticulocyte count of 10 x 10⁹/L, and mean corpuscular volume (MCV) of 99.2 fL. Patient 2 was a female infant born at 36 weeks gestation with Hb of 67 g/L, reticulocyte count of 56 x 10⁹/L, and MCV of 110 fL. Both otherwise had normal blood cell counts and smears. Bone marrow biopsies were first performed at three months of age for patient 1 and one year of age for patient 2. Bone marrow in both patients showed normal to mildly increased cellularity with paucity of erythroid precursors and no dysplasia in erythroid or granulocytic lineages. Blasts were not increased (<1%). Unexpectedly, however, both marrows showed increased number of monolobated megakaryocytes, observed in approximately half of the lineage (Figs. 1A-C). Exome sequencing revealed heterozygous mutation in RPL5 gene c.[67C>T];[=], p.[Arg23*];[=] for patient 1 and c.67C>T, p.[Arg23Ter] for patient 2, leading to the diagnosis of DBA type 6. Cytogenetic analysis were normal and next generation sequencing of the bone marrow samples were negative for mutations in myeloid-malignancy related genes.
Both patients were managed with red blood cell transfusions (RBC) as needed and steroids. Patient 1 had follow-up bone marrow biopsies at age five (Fig. 1B) and eight years, which revealed similar findings as previous with persistence of monolobated megakaryocytes and no clonal evolution. At nine years of age, he had been weaned off steroids and only required one RBC transfusion in the past 2 years. Patient 2 was started on steroids at one year of age and was able to discontinue transfusion therapy.
Both DBA and MDS can present with macrocytosis and reticulocytopenic anemia. However, bone marrow in DBA is classically characterized by the absence or <5% of erythroid progenitors and absence of dysplasia at baseline. Megakaryocytic monolobation is considered a dysplastic feature in MDS, particularly the 5q deletion subtype5. This observation raised a concern of transformation to MDS. However, megakaryocytic atypia was present as early as three months of age for Patient 1, there was no dysplasia in other cell lineages, genetic testing was negative for MDS-related mutations, and both patients were eventually weaned off steroids and became independent of transfusions years later, following the expected clinical course of DBA. Although megakaryocytes in infants may show less nuclear segmentation or ploidy compared to older children, the number of non-lobated megakaryocytes in these cases was out of proportion to normal age-related changes, and persisted beyond five years of age in Patient 1. To our knowledge, baseline megakaryocytic dysplasia has never been published in DBA literature, apart from one poster abstract that also described an increased number of non-lobulated megakaryocytes in patients with RPL5 and RPL11 mutations7.
These findings shed light into common pathogenesis between the two disorders. Deletion of 5q in MDS results in haploinsufficiency ofRPS14 , a ribosomal gene closely related to those implicated in DBA. RPS14 is involved in the MDM2/p53 axis, which is essential in erythroid and megakaryocytic differentiation8,9. The inability to assemble ribosomes leads to free ribosomal proteins which bind Murine Double Minute-2 (MDM2) and in turn suppress ubiquitination of p53. This allows for accumulation of p53, resulting in erythroid hypoplasia and dysmegakaryopoiesis8,9. Interestingly, both RPL5 and RPL11 proteins have also been shown to be implicated in p53 activation through interaction with the MDM2 protein10,11,12. Through this shared MDM2/p53 pathway, it is plausible that RPL5 and RPL11 haploinsufficiency leads to dysmegakaryopoiesis similar to that in MDS with 5q deletion.
Childhood MDS is often considered a higher risk disease, with 5-year overall survival of 63% and usually requires stem cell transplant5,6. It is imperative to recognize that the “dysplastic” megakaryocytes may represent a baseline finding in DBA with RPL5 mutations instead of progression to MDS, since the long-term prognosis and management for the two conditions are different. Further studies are needed to investigate whether DBA with RPL5mutations represent a distinct subcategory that shares similar pathophysiology with MDS with 5q deletion and explore common therapeutic strategies for both conditions.