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Congenital thrombocytopenias are rare conditions that show a spectrum of clinical findings and varying degrees of heritability with the unifying feature of thrombocytopenia. Recent developments, especially in identification of molecular defects, have improved the ability to identify and distinguish cases of congenital thrombocytopenia.1, 2, 3 Notwithstanding considerable progress, approximately half of patients with congenital thrombocytopenia do not have a specific diagnosis because the underlying causes have not (yet) been identified.4, 5

A subset of congenital thrombocytopenias have an increased risk of developing myeloid neoplasms. Leukemias have been reported with varying frequency in association with inherited thrombocytopenias, including Wiskott–Aldrich syndrome,6, 7, 8, 9 ankyrin repeat domain 26 (ANKRD26)-related thrombocytopenia,6, 10 shortened telomere syndrome,11, 12, 13, 14 familial platelet disorder with predisposition to acute myeloid leukemia caused by germline heterozygous mutations in RUNX1,15, 16, 17 ETV6-related thrombocytopenia,1, 2, 3 and myosin heavy chain 9 (MYH9)-related disorder18, 19 (Table 1). Although these diseases have known molecular defects and expected clinical course, the distinction between the baseline condition and disease progression may be difficult, even with relevant clinical history. Data are limited on bone marrow biopsy findings for congenital thrombocytopenias. Most studies report the common finding of varying degrees of immature or dysplastic megakaryocytes,8, 9, 10, 20, 21, 22, 23, 24 but there is little information comparing the histopathologic findings between the entities, and there are no studies following bone marrow biopsies over time.

Table 1 Summary of congenital thrombocytopenias

This study analyzes bone marrow alterations in cases of congenital thrombocytopenia with long-term follow-up. This is the first comprehensive integrated evaluation of these rare cases.

Materials and methods

Samples

A computer-assisted search of the pathology files of the Weill Cornell Medicine New York-Presbyterian Hospital from the last 20 years was performed for congenital thrombocytopenia patients with bone marrow biopsy and follow up at our institution. Both bone marrow biopsies and aspirates were performed from posterior superior iliac crest. The clinical information, complete blood counts, and relevant radiographic images were also obtained following institutional review board approval. Complete blood count data (hemoglobin, mean corpuscular volume, white blood cell count, absolute neutrophil count, absolute lymphocyte count, and platelet count) of all the patients from the time of diagnosis was concurrently reviewed and recorded for statistical analysis. The degree of anemia was determined based on the World Health Organization toxicity grading system, as follows: grade 1 (mild) 9.5–10.9 g/dl, grade 2 (moderate) 8–9.4 g/dl, grade 3 (severe) 6.5–7.9 g/dl, and grade 4 (life-threatening) <6.5 g/dl. Thrombocytopenia was graded according to the National Cancer Institute criteria into grade 1 (75–150 × 109/L), grade 2 (50–75 × 109/L), grade 3 (10–50 × 109/L), and grade 4 (<10 × 109/L).

Morphology

A detailed bone marrow biopsy and aspirate analysis included quantification of the marrow cellularity, the myeloid to erythroid ratio, evidence of reticulin and collagen fibrosis, presence of dysplasia, aspirate and core biopsy blast count, megakaryocyte morphology and overall number, aberrant megakaryocyte antigen expression, and presence of megakaryoblasts. Dysplasia was defined according to the World Health Organization criteria.

Special Stains, Immunohistochemistry, and Flow Cytometry

Reticulin stain was performed on every case and was graded according to a modified Bauermeister/Manoharan scale. Trichrome staining was performed. The blast count was evaluated by manual count of the bone marrow aspirate and with immunohistochemical staining for CD34 (BioGenex MU236-UC, Fremont, Ca with dilution of 1:50) on the bone marrow biopsy. Immunohistochemical stain for CD42b (Leica NCL-CD42b, Newcastle-upon-Tyne, UK with dilution of 1:50) was performed to simplify the quantification of the megakaryocytes and to identify the presence of megakaryoblasts. Four-color flow cytometric analysis was performed on eight bone marrow aspirates to assess for B-cell and T-cell clonality, increased myeloid blasts, and evidence of immunophenotypic abnormalities in myeloid lineage cells.

Cytogenetic Analysis

Cytogenetic studies were performed on overnight unstimulated bone marrow cultures. The findings were described according to the 2009 and 2013 International System for Human Cytogenetic Nomenclature. Fluorescence in situ hybridization was carried out on five cases using the LSI D5S23/D5S721/EGR1 probes (Abbott Molecular, Des Plaines, IL, USA). Diepoxybutane clastogen assay for Fanconi anemia (Quest Diagnostics, Madison, NJ, USA) was performed in one case.

Molecular Analysis

Molecular studies were performed in six cases. Myelodysplastic syndrome molecular profile, including ASXL1, RUNX1, EZH2, ETV6, and TP53, testing was performed via polymerase chain reaction (Genoptix Medical Laboratory, Carlsbad, CA, USA) on bone marrow aspirates in two cases. ANKRD26 polymerase chain reaction (University of Pavia, Pavia, Italy) was performed in two cases. MPL mutation, WAS, ADAMTS13, MYH9, and RUNX1 polymerase chain reaction (Blood Center of Wisconsin, Milwaukee, WI, USA) was performed in one case. MYH9 polymerase chain reaction (Children’s Hospital of Philadelphia, Philadelphia, PA, USA) was performed in one case. TERT mutation testing was performed in one case. Telomere length by quantitative polymerase chain reaction was performed in one case. Human leukogen antigen testing (LabCorp, Burlington, NC, USA) was performed in one case. JAK2V617F polymerase chain reaction (Weill Cornell, New York, NY, USA) was performed in one case on peripheral blood. RUNX1-RUNX1T1 (AML1-ETO) t(8;21) by real-time polymerase chain reaction was performed in one case. Osteopetrosis testing, including TCIRG1, TCIRG1 deletion, CLCN7 deletion, OSTM1, OSTM1 deletion, and PLEKHM1 (Connective Tissue Gene Tests, Allentown, PA, USA), was performed on one case. GATA1 mutation (Prevention Genetics, Marshfield, WI, USA) was performed in one case. SBDS gene mutation, and dyskeratosis congenita gene testing DKC1, TINF2 Exon 6, TERC, NHP2 exon 4, NOP10 exon 2, and TERT (Ambry genetics, Aliso Viejo, CA, USA) was performed in one case.

Results

Clinical Presentation

A total of eight cases of inherited thrombocytopenia were identified. Six were male and two female patients with age range at first bone marrow biopsy from 1.3 to 47 years (Table 2). Underlying diseases included thrombocytopenia-absent radius syndrome, ANKRD26-related thrombocytopenia, congenital thrombocytopenia with radial–ulnar synostosis, shortened telomere syndrome, MYH9-related disorder, Wiskott–Aldrich syndrome, and familial platelet disorder with predisposition to acute myeloid leukemia. One disorder was not further characterized.

Table 2 Summary of patient and bone marrow biopsy characteristics

Thrombocytopenia-absent radius syndrome

This male patient initially presented at 1 month of age with a characteristic congenital absence of both radii and both ulnas with further deformities of both humerii (Figure 1) and both tibias requiring knee replacement twice for his right knee. Up until the age of 20, his platelet counts were usually 25–45 × 103/μl and would fall with intercurrent infections. By the age of 27 years, his platelet counts were between 5–15 × 103/μl. He was found to have hepatomegaly and massive splenomegaly. Significant extramedullary hematopoiesis was seen on magnetic resonance imaging and in his spleen post-splenectomy at age 34.

Figure 1
figure 1

Lateral (top) and posterior–anterior (bottom) radiograph of right forearm of Case 1 (thrombocytopenia-absent radius syndrome) showing absent radius and associated ulnar, carpal, and clavicular deformities.

During subsequent follow-up, the patient’s white blood cell count was initially over 100 × 103/μl but gradually decreased and was found to be persistently elevated (22–38 × 103/μl) with neutrophilia and myeloid left-shift. He has been treated with steroids, intravenous immunoglobulin, aminocaproic acid, and required frequent platelet transfusions. A course of eltrombopag was given with initial substantial platelet increase followed by transient appearance of blasts resulting in cessation of this treatment. The patient was diagnosed with acute myeloid leukemia at age 47 and was treated with chemotherapy followed by allogeneic stem cell transplant. The patient currently has stable disease with moderate thrombocytopenia and neutrophilia, but no increase in blasts.

ANKRD26-related thrombocytopenia

This male patient with a long-standing history of thrombocytopenia (on average <10 × 103/μl, lowest 2 × 103/μl) was virtually asymptomatic until the age of 47 years, then presented with bruising, frequent epistaxis, and months of swelling and petechiae in the lower extremities. Multiple family members (grandfather, father, aunt, uncle, four cousins, and son) were reported to have thrombocytopenia. Notably, his father’s sister also developed chronic myelogenous leukemia and his father expired due to acute myeloid leukemia. Treatments included steroids, romiplostim, rituxumab, cyclophosphamide, and thalidomide. Despite therapy, the patient developed progressive anemia with a decrease in hemoglobin from 13 g/dl in 2011 to 4–6 g/dl in 2014. At that time, he was started on azacitidine, followed by matched unrelated stem cell transplant. Nine months following transplant, the patient has stable grade 3 thrombocytopenia and no evidence of anemia.

Shortened telomere syndrome

This male patient initially presented at the age of 41 years. He had frequent nose bleeds and was pancytopenic with platelet count of 10 × 103/μl, hemoglobin between 7.5 and 9.0 g/dl, and white blood cell count of 2.5 × 103/μl. The patient had a strong history of multiple family members with idiopathic pulmonary fibrosis and thrombocytopenia. He was treated with danazol. Because of the concern for disease progression, he received a matched unrelated stem cell transplant with a relatively uneventful post-transplant course and normal complete blood counts 2.5 years post-transplant.

MYH9-related disorder

This female patient presented at the age of 38 years old with a platelet count of 78 × 103/μl and occasional petechiae. She sought workup because her son was born with a low platelet count and tested positive for MYH9 mutation. The patient has not required treatment except at delivery of her next child.

Familial platelet disorder with predisposition to acute myeloid leukemia

This female patient presented at the age of 36 years old with a history of hepatitis C and asymptomatic thrombocytopenia (60–120 × 103/μl). Five other immediate family members had thrombocytopenia and abnormal platelet function studies. One brother with thrombocytopenia subsequently developed acute myeloid leukemia. Patient’s son was studied for delayed development and mild thrombocytopenia. Platelet function assay of the patient and her brother showed decreased platelet aggregation with arachidonic acid, collagen, adenosine diphosphate 20 μM, and epinephrine, and normal aggregation with ristocetin. The patient was treated with trials of intravenous immunoglobulin, romiplostim, and etrombopag with partial response to the latter two agents. She is clinically stable.

Congenital thrombocytopenia with radial–ulnar synostosis

This male patient presented at several months of age with platelet counts between 30–40 × 103/μl with easy bruising and petechiae. Several months later he was discovered to have inability to supinate his arms bilaterally, and pulmonary hypertension. He was followed up to 16 years of age, maintaining stable platelet counts fluctuating between 30–50 × 103/μl. During this time, he developed one episode of a severe left knee hemarthrosis. The most recent platelet counts on no treatment were 60–70 × 103/μl.

Unspecified congenital thrombocytopenia with giant platelets

This male patient presented at birth with decreased platelets, abnormal bruising, and a perinatal intracranial bleed. The patient’s cousin had osteopetrosis. The patient was treated with prednisone, intravenous immunoglobulin, and romiplostim. After clinical follow-up of 2 years, he requires platelet transfusions regularly with baseline platelets fluctuating between 3–60 × 103/μl, but is otherwise clinically stable.

Wiskott–Aldrich syndrome

This male patient was initially diagnosed around 12 months of age at our institution and presented again at 25 years old with severe eczema, psoriasis, bleeding diathesis, and thrombocytopenia (30–40 × 103/μl). He had multiple other male family members affected with similar symptoms. He was managed with daily ethrombopag and had been clinically stable. He chose to undergo stem cell transplant primarily to address severely symptomatic psoriasis and eczema, and unfortunately passed away from peri-transplant complications.

Morphologic and Immunophenotypic Features

Thrombocytopenia-absent radius syndrome

Multiple bone marrow biopsies were obtained during follow-up of 28 years starting after the age of 20. All biopsies showed markedly hypercellular (virtually 100%) bone marrows (Figure 2a) with markedly elevated myeloid to erythroid ratio (>10:1) and a decreased megakaryopoesis with a predominance of micromegakaryocytes (Figure 3), best highlighted by CD42b immunostain (Figure 4a). Bone marrow eosinophilia and basophilia were also noted. This was consistent with several magnetic resonance imaging studies which all revealed the absence of fat in the medullary space. When the patient was 34 years old, a splenectomy was performed which confirmed extramedullary hematopoesis. Subsequent bone marrow biopsies showed similar findings until, at the age of 39 years, the number of myeloid blasts slowly began to increase up to 23% 8 years later. The increase in blasts correlated with development of myeloid and erythroid dysplasia and marrow fibrosis. He developed acute myeloid leukemia and underwent successful stem cell transplant complicated by chronic graft-versus-host disease.

Figure 2
figure 2

Bone marrow cellularity. Hematoxylin and eosin of bone marrow biopsies demonstrating hypercellularity in (a) Case 1: thrombocytopenia-absent radius syndrome, (b) Case 2: ANKRD26-related thrombocytopenia, (c) Case 3: shortened telomere syndrome, (d) Case 4: MYH9-related disorder. Hypocellular marrow was observed in (e) Case 5: familial platelet disorder with predisposition to acute myeloid leukemia and (f) Case 6: congenital thrombocytopenia with radial–ulnar synostosis. Normocellular marrow was observed in (g) Case 7: unspecified congenital thrombocytopenia with giant platelets and (h) Case 8: Wiskott–Aldrich syndrome.

Figure 3
figure 3

Micromegakaryocyte morphology as seen in bone marrow aspirate from Case 1: thrombocytopenia-absent radius syndrome.

Figure 4
figure 4

Presence of micromegakaryocytes was a notable feature in six patients. CD42b immunohistochemical stain highlights megakaryocytes (a) Case 1: thrombocytopenia-absent radius syndrome, (b) Case 2: ANKRD26-related thrombocytopenia, (c) Case 3: shortened telomere syndrome, (d) Case 4: MYH9-related disorder, (e) Case 5: familial platelet disorder with predisposition to acute myeloid leukemia, (f) Case 6: congenital thrombocytopenia with radial–ulnar synostosis, (g) Case 7: unspecified congenital thrombocytopenia with giant platelets, and (h) Case 8: Wiskott–Aldrich syndrome.

ANKRD26-related thrombocytopenia

The initial bone marrow biopsy showed markedly increased cellularity (90%) (Figure 2b) with an increased myeloid to erythroid ratio (3.6:1) and increased number of micromegakaryocytes highlighted by CD42b staining (Figure 4b). There was increased fibrosis (MF-2) (Figure 5a) and no increase in blasts. Subsequent biopsies correlating with worsening anemia showed increase in cellularity (95–100%) over time, increasing myeloid to erythroid ratio (up to 5:1), new evidence of erythroid and myeloid dysplasia and up to 7% blasts. Splenectomy showed evidence of extramedullary hematopoiesis with no increase in blasts.

Figure 5
figure 5

Additional characteristics of each case included: (a) Moderate reticulin fibrosis (MF-2) with reticulin staining in Case 2: ANKRD26-related thrombocytopenia and (b) Case 3: shortened telomere syndrome. Abnormally large and giant platelets were observed in the peripheral blood smears of (c) Case 4: MYH9-related disorder and (d) Case 7: unspecified congenital thrombocytopenia with giant platelets.

Shortened telomere syndrome

Several bone marrow biopsies were evaluated. The initial biopsy showed increased cellularity (80%) (Figure 2c) for the patient’s age, increased myeloid to erythroid ratio (3.4:1), 5% myeloblasts by immunohistology, megaloblastoid features in the erythroid lineage, increased fibrosis (MF-2) (Figure 5b) and overt dysplasia in the myeloid and megakaryocyte lineages. Megakaryocytes were composed predominantly of small, hypolobated dysplastic-appearing forms (Figure 4c). Post-transplant bone marrow biopsy showed normal cellularity (50%) for age, normal myeloid to erythroid ratio, and adequate megakaryocytes.

MYH9-related disorder

A bone marrow biopsy showed increased cellularity (85%) (Figure 2d), increased myeloid to erythroid ratio, myeloid left-shift, and a markedly increased number of megakaryocytes varying from atypically small to normal in size (Figure 4d). There was increased (MF-2) marrow fibrosis. Rare enlarged platelets were observed on review of the peripheral blood smear (Figure 5c).

Familial platelet disorder with predisposition to acute myeloid leukemia

A bone marrow biopsy showed a mildly hypocellular bone marrow for age (~40%) (Figure 2e), a normal myeloid to erythroid ratio, an increased number of micromegakaryocytes and scattered bare megakaryocyte nuclei (Figure 4e).

Congenital thrombocytopenia with radial–ulnar synostosis

The patient was followed with bone marrow biopsy from 15 months to 12 years of age. The earliest biopsy showed bone marrow with normal cellularity (Figure 2f) and myeloid to erythroid ratio, but with decreased number of megakaryocytes. The megakaryocytes present tended to be immature small forms (Figure 4f). Over time, there was progressive bone marrow failure with 25% cellularity at 12 years old and virtually absent megakaryocytes. There was no evidence of myeloid/erythroid dysplasia or marrow fibrosis.

Unspecified congenital thrombocytopenia with giant platelets

The patient had two bone marrow biopsies at the age of 1 and 2 years old. The biopsies showed appropriate cellularity (>90%) for age (Figure 2g), normal myeloid to erythroid ratio, and increased number of megakaryocytes showing increased pleomorphism (Figure 4g). Peripheral blood smears showed abundant giant platelets (Figure 5d).

Wiskott–Aldrich syndrome

A bone marrow biopsy showed normal cellularity (70%) for age (Figure 2h), normal myeloid to erythroid ratio, and a markedly increased number of megakaryocytes varying from small to normal in size (Figure 4h).

Molecular Studies and Cytogenetics

Thrombocytopenia-absent radius syndrome

The patient had multiple bone marrow biopsies with normal male karyotype until 20 years later when a bone marrow biopsy 1 year before the diagnosis of acute myeloid leukemia was found to have new cytogenetic abnormalities with partial loss of 3p and 12q. At the same time, molecular analysis demonstrated evidence of two missense frameshift mutations in the CALR gene (c.1153_1154insCTTGT and c.1154A>C; K385T). Neither mutation has been previously described.

ANKRD26-related thrombocytopenia

There was a heterozygous single nucleotide substitution (c.-134G>A) in 5′-untranslated region of ANKRD26 detected with targeted polymerase chain reaction. Extensive additional molecular genetic workup was performed including ASXL1, RUNX1, EZH2, ETV6, TP53, and JAK2V617F polymerase chain reaction which were all negative. Subsequently, a 21-gene myeloid molecular sequencing panel was performed when disease progression was suspected which demonstrated mutation in CBL gene (c.111T>G; pY371D).

Shortened telomere syndrome

Molecular testing showed TERT mutation with telomere length by quantitative polymerase chain reaction reported to be in the first percentile. Cytogenetics showed an extra chromosome 1, which had isochromosome 1q, suggesting four copies of 1q. A myelodysplastic syndrome molecular profile showed RUNX1 and ETV6 mutations.

MYH9-related disorder

Cytogenetic analysis showed a constitutive pericentric inversion of one of the homologs of chromosome 9; 46,XX,inv9(p12q13)c[20]. Molecular testing showed heterozygosity in the MYH9 gene for a missense variant mutation defined as c.4198 C>T and predicted to result in the amino acid substitution p.Arg1400Trp.

Familial platelet disorder with predisposition to acute myeloid leukemia

Cytogenetics showed a normal female karyotype. RUNX1-RUNX1T1 t(8;21) by real-time polymerase chain reaction was not detected. The patient and her brother had normal results with conventional polymerase chain reaction testing for RUNX1, ANKRD26, MPL, WAS, ADAMTS13, and MYH9 gene mutations. Next-generation sequencing performed on the sample from the patient’s son showed a large RUNX1 deletion.

Congenital thrombocytopenia with radial–ulnar synostosis

Hox11a, c-mpl, Wiskott–Aldrich syndrome protein, and Fanconi anemia molecular testing were normal. Cytogenetics showed a normal male karyotype.

Unspecified congenital thrombocytopenia with giant platelets

An extensive genetic workup was normal (ANKRD26, MPL, ADAMTS13, WAS, MYH9, RUNX1, TCIRG1, TCIRG1 deletion, CLCN7 deletion, OSTM1, OSTM1 deletion, PLEKHM1, diepoxybutane clastogen assay for Fanconi anemia, GATA1 mutation, SBDS gene mutation, DKC1, TINF2 Exon 6, TERC, NHP2 exon 4, NOP10 exon 2, and TERT). Cytogenetics showed a normal male karyotype.

Wiskott–Aldrich syndrome

Cytogenetics showed a normal male karyotype. Genetic testing showed a point mutation in exon 2 of the WAS gene.

Discussion

More than 20 individual inherited thrombocytopenia entities—all with a very low incidence in the general population—have been identified thus far. They remain under-diagnosed by hematologists and oncologists and are often classified as idiopathic thrombocytopenic purpura, or even myelodysplastic syndrome or myeloproliferative neoplasm. Information in the literature regarding their histopathologic findings is very limited. Patients with congenital thrombocytopenia can present with bone marrow features mimicking myelodysplastic syndrome and myeloproliferative neoplasm. A high incidence of hematologic malignancy has been previously established in certain categories of familial thrombocytopenia.5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 18, 19 The distinction between the baseline condition and disease progression to malignancy can be challenging, especially in the absence of relevant clinical history.

The distinctive histologic findings, clinical features, and familial inheritance patterns of common types of congenital thrombocytopenia are summarized in Table 1.5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 18, 19, 25, 26, 27, 28, 29 As congenital thrombocytopenia cases are heterogeneous in etiology and clinical features, it was expected that there could be distinctive microscopic findings differentiating each disease (Figure 4). Persistent eosinophilia in thrombocytopenia with absent radii, giant platelets in MYH9-related disease, and progressive bone marrow aplasia in congenital thrombocytopenia with radial–ulnar synostosis have been previously described.21, 28 Genetic mutations in ANKRD26, MYH9, TERT, and RUNX1 genes were discovered in patients and their family members and were crucial in unequivocal assignation of ANKRD26-related thrombocytopenia, MYH9-related disorder, shortened telomere syndrome, and familial platelet disorder with predisposition to acute myeloid leukemia, respectively.5

Bone marrow core biopsy is an important tool to provide baseline characteristics of the congenital thrombocytopenia and to evaluate for disease progression. In the current era of molecular diagnosis based on peripheral blood samples, bone marrow examination is often bypassed. In this series of eight cases, three distinct morphologic patterns were observed. Four out of the eight patients (thrombocytopenia-absent radius syndrome, ANKRD26-related thrombocytopenia, shortened telomere syndrome, and MYH9-related disorder) had an initial bone marrow morphology that was reminiscent of myelodysplastic/myeloproliferative neoplasm, including marked bone marrow hypercellularity (4/4), increased myeloid to erythroid ratio (4/4), mild increase in blasts (1/4), fibrosis (2/4), and numerous micromegakaryocytes (4/4). Two out of eight patients (congenital thrombocytopenia with radial–ulnar synostosis and familial platelet disorder with predisposition to acute myeloid leukemia) had morphologic features of bone marrow failure, including hypocellularity and decreased number of megakaryocytes. Megakaryocytes in these two cases had atypically small morphology. Finally, the remaining two cases (Wiskott–Aldrich syndrome and unspecified congenital thrombocytopenia with giant platelets) had normal bone marrow morphology with an appropriate increase in unremarkable-appearing megakaryocytes.

Presence of micromegakaryocytes was a notable feature in six out of eight patients. Micromegakaryocytes were morphologically undistinguishable from small hypolobated megakaryocytes observed in myelodysplastic syndrome. They were best appreciated with CD42b immunohistochemical staining that highlights cells with the glycoprotein Ib receptor for von Willebrand factor. Micromegakaryocytes were not readily apparent on hematoxylin- and eosin-stained slides, therefore, immunohistochemical staining was found to be an important ancillary test for optimal characterization of suspected congenital thrombocytopenia.

Moderate reticulin fibrosis (MF-2) was present in the initial bone marrow biopsies of patients with ANKRD26-related thrombocytopenia and shortened telomere syndrome. The patient with thrombocytopenia-absent radius syndrome developed marrow fibrosis in the course of disease progression. Bone marrow fibrosis may be seen in a variety of benign conditions and malignant disorders, and is well known to be associated with myeloid neoplasm.30, 31, 32 Although idiopathic pulmonary fibrosis and cirrhosis are well recognized in shortened telomere syndrome,11, 12, 13, 33 bone marrow fibrosis has not been specifically reported as a feature in congenital thrombocytopenia patients. Thus, reticulin fibrosis may be more likely associated with disease progression.

In this study, three out of eight (38%) patients had evidence of morphologic disease progression as noted on multiple consecutive bone marrow biopsies (Table 3). All patients were in the myelodysplastic/myeloproliferative neoplasm-like morphologic group. The patient with thrombocytopenia-absent radius syndrome developed evidence of erythroid and myeloid dysplasia, slowly progressive elevated bone marrow blasts, bone marrow fibrosis, evidence of new cytogenetic abnormalities, and presence of CALR gene mutations. The patient with ANKRD26-related thrombocytopenia had a worsening anemia and multiple bone marrow biopsies, which showed progressive increase in cellularity and the myeloid to erythroid ratio, new erythroid and myeloid dysplasia and an increase in blasts, as well as mutation in CBL gene. The patient with shortened telomere syndrome diagnosis presented at the age of 41 with pancytopenia, overt trilineage dysplasia, a borderline increase in blasts and presence of RUNX1 and ETV6 mutations.

Table 3 Bone marrow changes in patients with disease progression

Molecular abnormalities are a salient feature in both congenital thrombocytopenia and in myeloid neoplasms. In this study, additional acquired genetic and cytogenetic defects over the course of disease in congenital thrombocytopenia were strongly associated with disease progression. At baseline, four of eight congenital thrombocytopenia patients were identified to have characteristic disease-specific mutations (TERT gene mutation defining shortened telomere syndrome, ANKRD26, MYH9, and WAS genes). Over time, all three patients with clinical disease progression concerning for myeloid neoplasm developed new cytogenetic and/or molecular abnormalities. The patient with thrombocytopenia-absent radius syndrome had partial loss of 3p and 12q, correlated to an increase in blasts and clinical deterioration. The molecular analysis demonstrated evidence of two missense frameshift mutations in the CALR gene. Neither mutation has been previously described. Mutations in exon 9 of the CALR gene has been described in a large subset of patients with patients with JAK2 wild-type essential thrombocythemia and primary myelofibrosis.34, 35 Calreticulin is a highly conserved protein with pleiotropic roles related to distribution in the endosplasmic reticulum, cytosol, and the cell surface. In vitro transfection of mutant calreticulin leads to hyperactivation of the pathway.35 Calreticulin immunostaining highlights the megakaryocytes in bone marrows specimens from patients with myeloid neoplasms.36 Thus, CALR-mutated myeloproliferative neoplasms appear to correspond to stem cell-derived neoplasms with aberrant and preferential expansion of the megakaryocyte lineage.34 A study of 154 patients with familial myeloproliferative neoplasm demonstrated that all CALR mutations were somatically acquired.37 In this study, it is unclear when the patient acquired this mutation and whether there was a possibility of germline CALR mutation associated with thrombocytopenia-absent radius syndrome pathogenesis. To the best of our knowledge, CALR mutation has not been previously described in patients with congenital thrombocytopenia or in patients with de novo acute myeloid leukemia.

Familial thrombocytopenia with ANKRD26 mutation is a recently described entity that is one of the more common forms of congenital thrombocytopenia.10, 38 Approximately 45 families have been identified with an autosomal dominant transmission. It is characterized by normal platelet size, moderate thrombocytopenia, and absent or mild bleeding tendency. Predisposition to myeloid neoplasms, especially acute myeloid leukemia and myelodysplastic syndrome has been reported in the affected family members. Results of bone marrow examination have been previously described in four patients. Similar to the patient in this study, all showed an increased number of very small hypolobated megakaryocytes (‘dysmegakaryopoiesis’).10, 38 In this study, the patient with ANKRD26-related thrombocytopenia demonstrated a mutation in the CBL gene at the time of morphologic disease progression. CBL is a tumor-suppressor gene that encodes a multivalent adaptor protein with E3 ubiquitin ligase activity.39 Somatically acquired, mostly homozygous CBL mutations have been found to occur with variable prevalence in myeloproliferative disorders, including juvenile myelomonocytic leukemia and acute myeloid leukemia.40, 41 Heterozygous germline mutations in CBL gene have been found in patients with Noonan syndrome.42 The significance of CBL mutation is the setting of disease progression in ANKRD26-related thrombocytopenia is uncertain. Finally, the patient with shortened telomere syndrome had evidence of morphologic disease progression and concurrent detection of RUNX1 and ETV6 gene mutations. Both mutations have been described in germline setting of familial thrombocytopenia15, 43 and in somatic setting of myeloid neoplasms including acute myeloid leukemia.44, 45

The patient with familial platelet disorder with predisposition to acute myeloid leukemia is presumed to have RUNX1 abnormality due to strong family history of thrombocytopenia with autosomal dominant transmission and presence of large RUNX1 gene deletion in her son, identified by sequencing but not with conventional polymerase chain reaction techniques. Familial platelet disorder with predisposition to acute myeloid leukemia is an autosomal dominant disorder characterized by mild to moderate thrombocytopenia and abnormalities of platelet function. More than a third of these patients develop myelodysplastic syndrome and/or acute myeloid leukemia.16 Bone marrow morphology has not been systematically evaluated in familial platelet disorder with predisposition to acute myeloid leukemia, but when reported, dysmegakaryopoiesis has been documented.46 Dysmegakaryopoiesis may reflect the abnormal megakaryopoiesis secondary to the germline RUNX1 mutation, since RUNX1 is a transcription factor that plays a key role in megakaryocyte maturation, differentiation, ploidization, and proplatelet formation.47 RUNX1 mutations are distributed throughout the gene and comprise complete deletions of RUNX1, splice-site mutations, missense, nonsense, and frameshift mutations. Germline testing on individuals with suspected RUNX1 mutations should include tests sensitive to deletions, duplications, and rearrangements, which may go undetected by standard sequencing techniques, as was the case in the family described in this study.48

The only effective treatment to increase the platelet count in patients with congenital thrombocytopenia—other than short-term effects of platelet transfusion and infrequent responses to intravenous immunoglobulin and steroids—are thrombopoietin receptor agonists. Their use has been specifically described in Wiskott–Aldrich syndrome49 and MYH9-related disorder.50 These agents have been well-described by our group and others to result in bone marrow findings reminiscent of myeloproliferative neoplasm.51 Therefore, use of these agents may further complicate morphologic evaluation of bone marrow biopsies in these cases and could even result in induction of true myeloproliferative neoplasm or even leukemia as had been seen in cases of myelodysplastic syndrome and of severe aplastic anemia.52

In summary, congenital thrombocytopenias are a heterogeneous group of diseases with variable clinical characteristics and morphologic features. We identified three distinct types based on bone marrow morphology: a hyperplastic myelodysplastic/myeloproliferative neoplasm-like group, a hypoplastic bone marrow failure-like group, and a group with normal marrow morphology. Three of four patients in the myelodysplastic/myeloproliferative neoplasm-like group had evidence of disease progression, compared with none in the other groups. Features of disease progression included development of non-megakaryocytic dysplasia, increase in blasts, and new cytogenetic/molecular abnormalities. Analysis is complicated by the fact that patients in the myelodysplastic/myeloproliferative neoplasm-like group had evidence of persistently elevated bone marrow cellularity (3/3), high myeloid to erythroid ratio (3/3), megakaryocyte atypia (3/3), and marrow fibrosis (2/3) for years before definitive diagnosis of disease progression. In addition, although the cytogenetic abnormalities in patient with thrombocytopenia-absent radius syndrome were definitely a new finding, the significance of CALR, CBL, RUNX1, and ETV6 gene mutations is less clear since these mutations have not been evaluated at the disease onset. Myeloid neoplasm was diagnosed at ages 40, 47, and 52 in these patients with lifelong congenital thrombocytopenia. Thus, myelodysplastic/myeloproliferative neoplasm-like congenital thrombocytopenias appear to have a long and indolent clinical course with very slow progression to leukemia in a subset of cases.

On the basis of these findings, we suggest that bone marrow biopsy is an important part in the diagnostic workup and subsequent monitoring of patients with congenital thrombocytopenia. Adequate and complete clinical history is paramount, since there is a significant morphologic overlap with myeloid neoplasms. Conservative approach is warranted in congenital thrombocytopenia patients with myelodysplastic/myeloproliferative neoplasm-like morphologic findings. Further studies on the evaluation of both bone marrow morphology and molecular genetic characteristics and their contribution to thrombocytopenia and disease progression are warranted to further elucidate the pathogenesis of not only these rare hematologic disorders but their progression to malignancy.