Continuing Education Activity

Amegakaryocytic thrombocytopenia is a severe form of thrombocytopenia with reduced or absent megakaryocytes in the bone marrow. It can be congenital or acquired. There is an eventual progression to impairment in the production of all three cell lines. This activity reviews the evaluation and management of amegakaryocytic thrombocytopenia and explains the interprofessional team's role in evaluating and treating patients with this condition.

Objectives:

• Describe the etiology of amegakaryocytic thrombocytopenia.
• Summarize the epidemiology of amegakaryocytic thrombocytopenia.
• Review pathophysiology of amegakaryocytic thrombocytopenia.
• Outline some interprofessional team strategies that the healthcare team can use to improve outcomes in patients with lipoid proteinosis.

Introduction

Amegakaryocytic thrombocytopenia is a severe form of thrombocytopenia with reduced or absent megakaryocytes in the bone marrow. It can be congenital or acquired.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare, severe form of thrombocytopenia with reduced or absent megakaryocytes in the bone marrow since birth, especially seen in the neonatal period. There is an eventual progression to impaired production of all three cell lines and should be considered inherited pancytopenia.[1][2][3][4]

Acquired amegakaryocytic thrombocytopenia (AAMT) is seen in later years of life and is again characterized by diminished or absent megakaryopoiesis with otherwise normal bone marrow.[5][6][7]

Etiology

CAMT

CAMT is an autosomal recessive disease with two main types described by Ballmaier et al. in 2001.[4]

Type 1 results from a stop codon or frameshift mutation, causing a loss of the intracellular domain of the MPL receptor with complete loss of function of the receptor. This results in early progression to bone marrow failure, with the mean age being one year and 11 months.[3][8][9]

Type 2 results from a splicing defect or amino acid substitution, which can affect the MPL receptor's glycosylation and result in an inability to react with thrombopoietin (THPO). These mutations can also cause a loss of hydrogen bonds within the MPL receptor, making it unstable. However, some residual receptor function exists. Bone marrow failure usually occurs at age 3 to 6 years, with the mean age being five years.[9] In this variant, the platelet count can transiently get better in the first year of life before getting worse with the development of pancytopenia.[4]

However, alternate etiologies have been proposed, including an X-linked variety of CAMT, a homozygous mutation in thrombopoietin, an anti-HLA A2 antibody, and some rare mutations in genes besides c-Mpl which could interfere with MPL receptor signaling and lead to CAMT.[8][10][11][10]

AAMT

AAMT can be considered as a heterogeneous group of disorders leading to a common downstream clinical manifestation. Three mechanisms have been proposed:[12][13][12]

1. Suppression of maturation of megakaryocytes by an exogenous agent: AAMT is seen in association with Ebstein Barr virus (EBV), parvovirus B19, hepatitis C virus, interferon therapy, cytomegalovirus, benzene exposure, alcohol abuse, vitamin B12 deficiency, and radioiodine therapy.[14][15]
2. Suppression of megakaryocyte maturation by endogenous stimuli due to antibody-mediated or T-cell autoimmunity: AAMT has been seen in association with thymoma with a more aggressive disease course, adult-onset Still disease, eosinophilic fasciitis, systemic lupus erythematosus, systemic sclerosis, Graves' disease, and hyperestrogenic states.[12][16][17][16]
3. An early manifestation of a stem cell abnormality: AAMT has been described as a precursor to acute myeloid leukemia, myelodysplastic syndrome, aplastic anemia, and non-Hodgkin's lymphoma.[18][6] A case report has shown an association with large granular lymphocyte leukemia.[19] Some other reported cytogenetic abnormalities include Philadelphia chromosome and 5q deletion.[18]

Epidemiology

CAMT

CAMT has been seen in consanguineous families. A slight female preponderance exists.[4] Less than 100 cases have been reported.[8] However, the incidence of CAMT is thought to be underestimated because isolated CAMT can be misdiagnosed as neonatal alloimmune thrombocytopenia, and the late pancytopenic phase of the disease cannot be distinguished from primary aplastic anemia.[20]

AAMT

Similarly, the incidence rate of AAMT is higher than reported because many cases are misdiagnosed as immune thrombocytopenic purpura.[18] Most affected females are diagnosed in the age group of 40 to 60 years, whereas most affected males lie at both ends of the age distribution with a peak in the 60s.[21]

Pathophysiology

Megakaryopoeisis begins with the hematopoietic stem cell (HSC) in the bone marrow, which matures into a multipotent progenitor cell, a committed megakaryocyte progenitor cell, an immature megakaryocyte, and finally into a mature megakaryocyte, which gives rise to blood platelets.[22] It is important to note that THPO is a cytokine needed at every step of megakaryopoiesis to increase the number, size, and ploidy of megakaryocytes and promote the expression of platelet-specific markers.[3] THPO also enhances the expression of VEGF, HoxB4, HoxA9, promoting HSC growth and survival.[8] THPO is mainly produced in the liver but can also be produced in the kidney's proximal tubular cells and the bone marrow stromal cells.[23] 

The THPO receptor also called the MPL receptor, is found in the bone marrow, liver, spleen, and CD34+cells.[3] THPO binding initiates a cascade of signaling events within the target cell via the JAK-Stat kinase family of proteins along with MAP and PI3 kinase pathways.[23] MPL signaling is vital for the differentiation of multipotent progenitor cells to megakaryocyte and erythrocyte progenitor cells. Problems in the signaling can lead to impaired megakaryocyte and erythrocyte production, as seen in later stages of CAMT.[24]

CAMT

The c-Mpl gene is located in the 1p34 locus and consists of 12 exons.[25] CAMT can occur due to inheritance of homozygous mutations of the c-Mpl gene, which is seen in families with consanguinity, or due to two different inherited mutations of c-Mpl resulting in a compound heterozygous state.[9] Close to 41 mutations have been defined in the literature. The most frequent location for mutations is within the 1st and 5th exons of the c-Mpl gene (75% of all mutations seen in CAMT), with 60% in exon 2 and 3 alone.[4][8][25] Some examples of mutations include C268T, G304C, G305C, G578A, F104S, P635L, R102P, R257C, R257L, W154R, 1,499delT, and Q186X mutations.[8][26]

It is interesting to note that a homozygous mutation p.R119C of the THPO gene impairs the secretion and function of THPO, producing the same clinical picture of CAMT.[11][27]

Thompson and Nguyen described a rare variant of CAMT associated with radioulnar synostosis due to mutation in HOXA11, which regulates megakaryocyte differentiation.[2][28] Mutations in the MECOM gene have also been associated with CAMT.[28]

AAMT

AAMT can be caused by antibody or T-cell-mediated autoimmunity.[15] Proposed pathophysiology includes anti-THPO antibodies, antibodies against antigens on megakaryocyte progenitor cells, antibodies against granulocyte monocyte colony-stimulating factor or antibodies against megakaryocyte colony-forming unit, failure of terminal megakaryocyte differentiation, suppression of megakaryocyte colony-forming unit by T cells and adherent monocytes, monoclonal T-cell population destroying megakaryocyte lineage,[5] or a defect in cytokine (IL 7, stem cell factor, TGF-beta 1) mediated regulation of megakaryopoiesis.[5][16][7][29][7][16][18][30][18]

Anti-MPL antibodies have been seen with systemic lupus erythematosus and systemic sclerosis.[15][31] Hepatitis C infection causes the generation of anti-MPL antibodies, which are first absorbed onto MPL receptors on platelets resulting in their destruction in the spleen and creating an immune thrombocytopenic purpura-like picture. Later, these antibodies bind to the MPL receptor on megakaryocytes and block the function of THPO, resulting in AAMT.[32] Interestingly, interferon therapy, which is used to treat patients with hepatitis C can result in the generation of anti-MPL antibodies by itself.[32]

Histopathology

On peripheral smear, platelets appear normal in size and morphology.[2][9] Bone marrow evaluation in patients with amegakaryocytic thrombocytopenia typically demonstrates normal overall cellularity with a reduction or absence of megakaryocytes, without evidence of dysplasia.[3][9] Sometimes the megakaryocytes that are present may look immature or small.[8] Immunohistochemical staining for CD-61 megakaryocyte antigen shows diminished megakaryocytes in core biopsy.[6] 

For comparison, the number of megakaryocytes usually seen on bone marrow biopsy is 5 to 10 per low power field. In patients with CAMT, bone marrow studies early in the disease course can have minimal findings that can be misleading, and serial bone marrow biopsies may be required to elucidate the diagnosis.[9] Later, once pancytopenia develops, patients have hypocellular marrow with decreased progenitors in all lineages, making it challenging to distinguish CAMT from other causes of aplastic anemia.[8]

History and Physical

CAMT

CAMT generally presents with severe thrombocytopenia within the first month of life or even earlier in the fetal period, which can manifest as petechiae, intracranial bleeds, recurrent rectal bleeding, or pulmonary hemorrhage.[3][8] It is interesting to note that decreased P selectin expression on neonatal platelets can reduce platelet activation and predispose the high bleeding tendency pre or perinatally in patients with CAMT.[33] 

A family history of thrombocytopenia may be present.[3] There are no characteristic congenital phenotypic abnormalities associated with CAMT. Some studies have noted neurological defects like strabismus, cerebellar agenesis, hypoplasia of the corpus callosum and brainstem, facial malformations, and cortical dysplasia. There are two postulated mechanisms regarding the cause of neurological defects in these patients. First, MPL is detected in neurons of the brain, and the absence or deficiency of MPL as seen in CAMT can lead to developmental delay. The second hypothesis revolves around early intracranial bleeding causing long-term neurological sequelae.[2][4][8][9][26]

AAMT

AAMT is a diagnosis of exclusion, and patients usually present with bleeding complications not responding to standard treatment for immune thrombocytopenia like steroids or intravenous immunoglobulin therapy.[6] Patients with AAMT can present with petechiae, purpura, ecchymosis, easy bruising, epistaxis, or fatigue. There is an absence of splenomegaly.[7] One case report described an AAMT patient who presented with massive hemoperitoneum due to hemorrhagic corpus luteum.[34]

Evaluation

Bone marrow biopsy is the mainstay of the diagnosis of CAMT or AAMT (see the section on histopathology).

CAMT

Approximately 1% to 5% of newborns are found to have thrombocytopenia, of which 5% to 10% have platelet counts below 50,000.[4] The mean platelet count at diagnosis of CAMT is approximately 20,000.[8] Bone marrow biopsy should be routinely done for all children with severe thrombocytopenia since birth, and c-Mpl gene testing should be performed when a reduced number of megakaryocytes are seen on biopsy.[20] 

The c-Mpl gene analysis is done with bidirectional sequencing of all 12 exons, including coding regions, splice sites, and intron-exon boundaries. This testing is conducted at GeneDx in Maryland (with 95% to 97% sensitivity to detect CAMT mutations) and Prevention Genetics in Wisconsin.[3][8] Other genetic testing includes chromosome analysis and FISH studies. The samples that can be used for this purpose include whole blood, bone marrow, skin fibroblasts, buccal brushings, amniotic fluid, or CD34+ cells.[3][35] 

The findings of homozygous or compound heterozygous mutations in c-Mpl are considered confirmatory for the diagnosis of CAMT.[4] However, patients must also be screened for THPO mutations because CAMT caused by these mutations can easily be reversed with an MPL agonist like romiplostim.[11] THPO levels can be measured to support the diagnosis of CAMT.  In CAMT, due to MPL mutations, THPO levels rise tenfold because THPO internalization and destruction by MPL receptors do not occur. However, in patients with THPO mutations, the levels of THPO are low because of secretion defect.[9][11]

AAMT

Since the etiology of AAMT is so varied, no standard algorithm can be laid out to come to the diagnosis. A strong clinical suspicion for AAMT and a knowledge of the disease are needed to request a bone marrow biopsy and confirm the diagnosis.

Treatment / Management

CAMT

1. Allogeneic hematopoietic stem cell transplant (HSCT) is the only curative option for patients with CAMT with c-Mpl mutations (the majority of cases).[8] HSCT should be considered as early as possible. Some studies suggest that HLA typing of patients and their siblings should be done at the time of diagnosis itself. The average age at which CAMT patients undergo HSCT is 38 months, ranging from 7 to 89 months.[8] Ideally, HSCT should be done before the development of the pancytopenic phase because patients might require multiple transfusions and risk alloimmunization and infections, which compromise transplant outcomes.[8][20] Conditioning regimens for HSCT include the use of busulfan, cyclophosphamide, and total body irradiation.[4] In patients with THPO mutation, this approach has no benefit because THPO is produced by the liver.[27]

2. Supportive treatment with irradiated, leukocyte-reduced platelet transfusions, antifibrinolytics like tranexamic acid, avoidance of non-steroidal anti-inflammatory drugs, and aspirin is important. Once pancytopenia develops, packed red blood cells and antibiotics may be needed.

3. Romiplostim-a THPO peptide mimetic and Eltrombopag-a small molecule agonist of the MPL receptor, which induces conformational changes in the receptor, can be used for CAMT cases with THPO mutations.[11][23] There is no benefit of using these medications for patients with c-Mpl mutations.

4. Experimental therapies: Gene therapy with lentiviral vectors have been used to repair the mutant c-Mpl gene. However, concerns about the leukemogenicity of this approach remain.[8] CRISPR-Cas9 gene editing to repair the mutation has been tried.[36] LGD-4665, minibodies, and diabodies are experimental drugs that stimulate some forms of mutant MPL receptors to increase megakaryocyte proliferation.[23][37]

AAMT

The goal of therapy must be to treat the underlying etiology of AAMT, e.g., resection of the thymus in thymoma-associated AAMT.[38] Hoffman suggested using plasmapheresis, cyclophosphamide, cyclosporine, and prednisone for antibody-mediated AAMT and cyclosporine, cytokine therapy, and anti-thymocyte globulin for T-cell mediated AAMT.[39] If a specific thrombopoiesis inhibition site cannot be identified, empiric therapy with different immunosuppressive or immunomodulatory agents is warranted.[29] Some experts suggest that it is advisable to start with the least toxic and most cost-effective medications with periodic treatment response monitoring.[34] 

The following medications and therapies have been used for the treatment of AMMT: steroids which suppress immune B and T cell-mediated autoimmunity, high dose intravenous immunoglobulin therapy, which binds antibodies against THPO and megakaryocytes, rituximab which suppresses the production of autoantibodies by B cells, cyclosporine which is a calcineurin inhibitor, anti-thymocyte globulin which suppresses T cell-mediated autoimmunity and stimulates hematopoiesis directly, bone marrow transplant, cyclophosphamide, azathioprine especially in patients with systemic lupus erythematosus, lithium carbonate, vincristine, mycophenolate mofetil, danazol, eltrombopag, recombinant IL-11. A case report described successful treatment of adult-onset Still’s disease with tocilizumab and cyclosporine.[15] Some refractory cases of AAMT have been treated with combination therapy of anti-thymocyte globulin and cyclosporine.[18][30][40][30]

Differential Diagnosis

CAMT

Differential diagnosis includes thrombocytopenia due to birth asphyxia or placental insufficiency, TORCH (toxoplasmosis, rubella, CMV, herpes) infections. sepsis, congenital syphilis, varicella, parvovirus B19, Wiskcott Aldrich syndrome, Fanconi anemia, and dyskeratosis congenita.[4][33] Neonatal autoimmune thrombocytopenia can be distinguished from CAMT by normal to increased megakaryocytes in the bone marrow.[8][9] In thrombocytopenia with absent radius, patients have skeletal or cardiac defects, and a spontaneous increase in platelet counts is observed after the first year of life.[3]

AAMT

Etiology for thrombocytopenia in adults is extensive. Each cause must be painstakingly ruled out before one can come to the diagnosis of AAMT, which is ultimately confirmed on bone marrow biopsy. Thrombocytopenia can be caused by:

• Infections like Epstein–Barr virus, varicella, leptospirosis, anaplasmosis, dengue, babesiosis, and tick-borne diseases
• Medications like daptomycin, valproic acid, linezolid, penicillins
• Beverages like alcohol, tonic water containing quinine, and herbal supplements
• Vitamin deficiency like B12 and folic acid deficiency
• Decreased THPO production due to liver dysfunction
• Blood cancers or solid organ cancers with metastasis to the bone
• Chemotherapy and radiation therapy used to treat blood cancers or metastatic cancer
• Rheumatologic conditions like systemic lupus erythematosus with or without the anti-phospholipid syndrome
• Peripheral destruction of platelets by shearing stress as seen in the use of intra-aortic balloon pumps and aortic aneurysms
• Splenic sequestration of platelets
• Consumptive coagulopathies like disseminated intravascular coagulation, heparin-induced thrombocytopenia with thrombosis, or hemolytic uremic syndrome

Prognosis

CAMT

Once pancytopenia develops, the prognosis of CAMT patients is poor. A study has shown 30% of CAMT patients die from bleeding complications, and another 20% die from complications related to HSCT.[3]

AAMT

In AAMT, patients can achieve remission immediately after targeted therapy or develop a long relapsing and remitting course. However, more commonly, patients progress to aplastic anemia or require HSCT to achieve remission.[6] Once AAMT progresses to aplastic anemia, patients have a poor prognosis.[7]

Complications

CAMT

Progression to bone marrow failure and aplastic anemia is seen at a mean age of 13 months for patients with CAMT type 1 and 5 years for patients with CAMT type 2.[8] The risk of developing pancytopenia in CAMT is higher than reported numbers and occurs in probably >90% of cases.[4] 

CAMT patients are at an increased risk of development of myelodysplastic syndrome and acute myeloid leukemia. The pathogenesis of the above conditions can be explained by karyotype instability, mutator effect, i.e., an increased accumulation of chromosomal aberrations over time, or an expansion of abnormal cell clones in the bone marrow.[1][4][8] Some studies have shown increased amounts of myelosuppressive cytokines like TNF-alpha and IFN-gamma, contributing to bone marrow failure in CAMT patients.[20]

AAMT

AAMT can progress to aplastic anemia from an average of 1 month to 2 years after diagnosis.[7] AAMT can progress to myelodysplastic syndrome.[14] A single case report describes mononeuritis multiplex as a complication of AAMT.[14]

Deterrence and Patient Education

Amegakaryocytic thrombocytopenia is a rare disease. It can either be congenital due to genetic defects or acquired due to various diseases. In CAMT cases, parents must be educated about the importance of early diagnosis with invasive procedures like a bone marrow biopsy and the need for HSCT once the diagnosis is confirmed. In AAMT cases, patients must be educated about avoiding activities like skiing or gymnastics due to the high risk of intracranial bleeds. The treatment of AAMT is sometimes frustrating due to the failure of multiple immunosuppressive and immunomodulatory agents, and patients may ultimately land up needing supportive platelet transfusions more than once a week.

Enhancing Healthcare Team Outcomes

Amegakaryocytic thrombocytopenia is a rare disease requiring a high degree of suspicion for diagnosis before serious complications like fatal hemorrhage or pancytopenia occur. It requires collaboration between primary care physicians and specialists, including hematologists, to avoid any delay in care. Communication between team members is crucial for managing these cases, including the nursing staff, to pick up any slight bleeding or new symptoms that can reflect serious complications. Shared decision-making about treatment options and their pros and cons must take place with patients and their families.

Enrollment of patients in national and international studies is essential because of the rarity of the disease. At the moment, there are no large multicenter randomized control trials; hence hematologists have to depend on case reports describing success with various therapies for CAMT and AAMT.[4][21]