Amegakaryocytic Thrombocytopenia Research Articles

Reduced-intensity conditioning is effective for allogeneic hematopoietic stem cell transplantation in infants with MECOM-associated syndrome

Mutations in the MECOM encoding EVI1 are observed in infants who have radioulnar synostosis with amegakaryocytic thrombocytopenia. MECOM-associated syndrome was proposed based on clinical heterogeneity. Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for progressive bone marrow failure. However, data regarding allogeneic HSCT for this rare disease are limited. We retrospectively assessed overall survival, conditioning regimen, regimen-related toxicities and long-term sequelae in six patients treated with allogeneic HSCT. All patients received a reduced-intensity conditioning (RIC) regimen consisting of fludarabine, cyclophosphamide or melphalan, and rabbit anti-thymocyte globulin and/or low-dose total body/thoracic-abdominal/total lymphoid irradiation, followed by allogeneic bone marrow or cord blood transplantation from unrelated donors between 4 and 18 months of age. All patients survived and achieved stable engraftment and complete chimerization with the donor type. Moreover, no patient experienced severe regimen-related toxicities, and only lower grades of acute graft-versus-host disease were observed. Three patients treated with low-dose irradiation had relatively short stature compared to three patients not treated with irradiation. Therefore, allogeneic HSCT with RIC is an effective and feasible treatment for infants with MECOM-associated syndrome. Future studies are needed to evaluate the use of low-dose irradiation to avoid risks of other long-term sequelae.

Role of Platelet Indices and Thrombopoietin (TPO) Serum Levels in the Differential Diagnosis of Thrombocytopenia

Background: The differential diagnosis of an isolated thrombocytopenia may be challenging, especially when it comes to differentiate immune thrombocytopenia (ITP) and an isolated thrombocytopenia in the context of bone marrow (BM) failure, in particular myelodysplastic syndromes (MDS); in such cases a BM examination is usually performed. It is known that thrombopoietin (TPO) serum levels are low in patients with ITP, while they are increased in case of amegakaryocytic thrombocytopenia. Furthermore, some platelet indices such as immature platelet fraction (IPF) may help to discriminate between hyporigenerative and consumption thrombocytopenia. Aim: The aim of this study was to compare TPO serum levels and platelet indices in patients with thrombocytopenia of different etiologies. Based on pathogenesis, we chose 3 groups of patients: ITP, amegakaryocytic thrombocytopenia (aplastic anemia (AA) or post-chemotherapy (CHT)) and thrombocytopenia in MDS and acute myeloid leukemia (AML). Although the diagnosis of amegakaryocytic thrombocytopenia and AML is straightforward with BM examination, we included patients with these diseases as a proof of concept and training set for our findings. Methods: This was a monocentric, retrospective, explorative study. Blood samples of patients with a platelet count < 100x109/L were collected. IPF, mean platelet volume (MPV), platelet distribution width (PDW), plateletcrit (PCT), platelet large cell ratio (P-LCR) were obtained using Sysmex XN-1000 hemocytometer (Sysmex Corporation). TPO serum levels were measured with a quantitative sandwich ELISA (R&D Systems). Microsoft Excel software was used for descriptive statistical analysis. Anova and Student's t-test were used for comparisons between groups. A p-value <0.05 was considered statistically significant. Results: We enrolled 84 patients: 50 ITP, 22 AML/MDS (13 AML, 9 MDS), 12 AA/post-CHT (4 AA, 8 post-CHT). Results are reported in Table 1. Neither mean platelet count (p=0.20) nor PCT (p=0.44) were significantly different between the three groups. Mean TPO serum levels were higher in patients with AA/post-CHT compared to ITP and AML/MDS groups (p<0.001). All patients in AA/post-CHT group had a TPO serum level >500 pg/mL, this value was never reached in patients with ITP and AML/MDS. TPO serum levels were higher in AML/MDS patients compared to ITP patients (p<0.001). While TPO serum levels were available for all patients, platelet indices were not, due to technical issues. IPF was available in 46/50 ITP patients, 20/22 AML/MDS and 8/12 AA/post-CHT, and it was higher in ITP patients if compared to AA/post-cht (p=0.021) and AML/MDS (p=0.0045). MPV, PDW and P-LCR were available in 27/50 ITP patients, 12/22 AML/MDS and 9/12 AA/post-CHT: they were higher in ITP patients than in the other groups (AML/MDS p=0.0185, AA/post-CHT p=0.017). In our cohort, patients with TPO ≥250 pg/mL and IPF <12% had a higher probability to belong to AML/MDS group (TPO: OR 0.08, 95% CI 0.02-0.32; IPF: OR 0.19, 95% CI 0.06-0.59). These findings were confirmed by multivariate analysis (IPF: p-value = 0.0185, CI 0.8647-0.9868; TPO: p-value = 0.004, CI 1.002-1.011). The other platelet indices were not included in this analysis due to the paucity of data. No correlation was found between platelet count, TPO and IPF in ITP patients. Of note, no differences were observed in terms of platelet count, TPO serum levels ad IPF between patients with AML and MDS. Conclusions: Our findings confirm that, in patients with the same platelet count, those with consumptive thrombocytopenia have higher IPF and lower TPO levels, while patients with amegakaryocytic thrombocytopenia have higher TPO levels and lower IPF. While TPO levels alone can distinguish amegakaryocytic thrombocytopenia from other etiologies, these preliminary data suggest that by combining IPF and TPO, patients with consumptive thrombocytopenia can be distinguished from MDS patients. This could be useful to spare BM examination in selected cases, or as an additional tool when the diagnosis remains uncertain also with BM examination. These data need to be confirmed in larger cohorts. The role of the other platelet indices, and the technical issues in their determination, needs further studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation for Congenital Amegakaryocytic Thrombocytopenia, a PDWP/EBMT Study

Background: Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare inherited bone marrow failure syndrome, which is characterized by a severe thrombocytopenia at birth without predictive stigmata and by a risk for progression into aplastic anemia and myeloid malignancy. CAMT is caused by a mutation of the thrombopoietin (THPO) receptor, c-Mpl. Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only potentially curative treatment option. The outcomes of transplanted patient for CAMT have not been well described. Design/Method We conducted an EBMT (European Society for Blood and Marrow Transplantation) registry-based retrospective study in patients diagnosed with CAMT receiving an allogeneic HSCT. Results 66 patients with CAMT could be included which were transplanted between 1998 and 2020. Median age at diagnosis was 1.3 years (0.1-2.8). The mutation was identified in 18 patients and absent in 7 patients. For the remaining patients, the genetic testing was not performed. The median age at transplant was 3.2 years (0.3-13). The median year of transplant is 2013. Bone marrow (BM) was the main stem cell source (59%) followed by peripheral blood (PB) (27%) and cord blood (CB (12%)) with 1 BM+CB from the same donor. The predominant donor type was HLA-matched (65% with matched-sibling donor (MSD) 34.8%, unrelated donor (UD) 10/10 22.7%, matched other relative 4.5% and umbilical cord blood (UCB) 6/6 3%)), followed by UD < 10/10 (15.1%), then the UCB< 6/6 (9%) then haploidentical (7.5%) and UD missing HLA (3%). The most frequently used conditioning regimen included busulphan and cyclophosphamide in 30% of cases, followed by the busulfan-fludarabine (14%) and treosulfan-fluradarabine-thiotepa (14%). Considering death and second transplant as competing event, platelet recovery (>= 20 000/mm3) occurred in 69.8% (95% confidence interval [CI] 56.6-79.7%) at day 60 and 71.4% (95% [CI] 58.2-81.1%) at day 180. Grade II-IV acute graft-versus-host disease (GvHD) was 20% (95% CI 11.3-30.5%), grade III-IV was 3.1% (95% CI 0.6-9.6%), and the 6-year cumulative incidence (CI) of chronic GvHD was 14.3% (95% CI 6.9-24.3%). The 6y-CI was 17.2% (95% [CI] 9.1-27.5%). Six-year overall survival (OS) was 85.6% (95% [CI] 74-92.3%) with a transplant related mortality of 8.0% (95% [CI] 2.9-16.6%). 6y-GVHD/-graft failure-free survival was 65.7% (95% [CI] 52.6-76%). OS was significantly lower after CB transplantation (6y OS: 37.5% with 95% [CI] 8.7-64.4%) than after BM (6y OS: 95% [CI] 81.5-98.7%) or PB (6y OS: 88.9% 95% [CI] 62.4-97.1%) transplantation (p<0.001). Conclusion: This study demonstrates that HSCT, the only available potentially curative option, has an acceptable 6-year overall survival and an acceptable rate of chronic GvHD. While the number of patients in such a rare disease does not allow to perform multivariable analyses, we can confirm that OS is better with the use of BM as the main stem cell source compared to the CB.

Abstract 9440: Acs in a Patient With Amegakaryocytic Thrombocytopenia: A Management Dilemma

Introduction: The management of Acute Coronary Syndrome (ACS) in thrombocytopenia is challenging. This abstract highlights the management dilemma of a patient who presented with high-risk ACS and was found to have Amegakaryocytic thrombocytopenia (AMT). Case Presentation: A fifty-five-year-old man with no cardiovascular risk factors was admitted with high-risk ACS with recurrent ischemic ECG changes and high troponin-T. He was found to have a low platelet count of 9 x10^3/uL. The initial impression was ACS and ITP. Course: No antiplatelet or anticoagulation was commenced. CT coronary angiography showed ostial left main (LM) severe stenosis. The patient received steroids and IVIG. A week later, cyclosporin and eltrombopag were added as the thrombocytopenia was refractory. Bone marrow biopsy showed AMT. His chest pain was persistent with ECG ischemic changes. A multidisciplinary team (MDT) decided that revascularization is urgently required, but the surgical option would have an extremely high bleeding risk. Alternatively, the Percutaneous option using radial access would have significantly less bleeding risk. However, he will need DAPT for at least the first week, which if interrupted, he will be at risk for stent thrombosis. The final decision was for platelet transfusion, then to proceed with the PCI once the platelet count is 50,000 or more, and to maintain the count &gt; 15,000 by transfusion whenever required. Management: After the targeted transfusion, the patient was started on aspirin and clopidogrel, then successful IVUS guided PCI to ostial LM with new generation DES was done via radial access and low therapeutic ACT range. Post PCI, the patient remained asymptomatic; he was observed as an inpatient for 14 days, during which he required platelet transfusion once when his platelet count dropped &lt; 15.000. DAPT was given for a week and then he was maintained on clopidogrel. In 3 months of follow-up, the patient had no cardiac complaints, no bleeding, and platelets were maintained between 15 to 30 on clopidogrel. Conclusion: ACS with severe thrombocytopenia is a management dilemma. Platelets transfusion, radial access, targeting low ACT, and using new generation stents for shorter DAPT duration might decrease the bleeding risk and thrombotic events.

MECOM-associated syndrome: a literature review and case reports

Mutations in the MECOM gene (MDS1 and EVI1 complex locus) may be one of the causes of a rare combination of congenital radioulnar synostosis resulting in extremely limited forearm pronation and supination, and amegakaryocytic thrombocytopenia. The clinical spectrum of the disease can range from isolated radioulnar synostosis with or without hematologic manifestations to severe bone marrow failure without skeletal abnormalities. Other phenotypic manifestations include clinodactyly, brachydactyly, cardiac and renal malformations, presenile hearing loss, and B-cell deficiency. In view of the heterogeneity of phenotypic manifestations of the disease, the term “MECOM-associated syndrome” was proposed for all patients with mutations in the MECOM gene. Here we report 3 pediatric cases of MECOM-associated syndrome with different clinical manifestations, diagnostic approaches, therapeutic options, and outcomes. The patient’s parents agreed to use the information, including the child’s photo, in scientific research and publications.

Defective binding of ETS1 and STAT4 due to a mutation in the promoter region of THPO as a novel mechanism of congenital amegakaryocytic thrombocytopenia.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a recessive disorder characterized by severe reduction of megakaryocytes and platelets at birth, which evolves toward bone marrow aplasia in childhood. CAMT is mostly caused by mutations in MPL (CAMT-MPL), the gene encoding the receptor of thrombopoietin (THPO), a crucial cytokine regulating hematopoiesis. CAMT can be also due to mutations affecting the THPO coding region (CAMT-THPO). In a child with the clinical picture of CAMT, we identified the homozygous c.-323C>T substitution, affecting a potential regulatory region of THPO. Although mechanisms controlling THPO transcription are not characterized, bioinformatics and in vitro analysis showed that c.-323C>T prevents the binding of transcription factors ETS1 and STAT4 to the putative THPO promoter, impairing THPO expression. Accordingly, in the proband the serum THPO concentration indicates defective THPO production. Based on these findings, the patient was treated with the THPO-mimetic agent eltrombopag, which induced a significant increase in platelet count and stable remission of bleeding symptoms. Herein, we report a novel pathogenic variant responsible for CAMT and provide new insights into the mechanisms regulating transcription of the THPO gene.

Acquired Amegakaryocytic Thrombocytopenia Progressing to Aplastic Anaemia.

Acquired amegakaryocytic thrombocytopenia (AAMT) is a rare disorder of the bone marrow characterized by a lack of megakaryocytes and preservation of other cell lines. It can occur due to an intrinsic stem cell defect or secondary to viral infections, autoimmune disorders, lymphoproliferative disorders or environmental toxins. With time, it can progress to aplastic anaemia (AA) and can have a poor prognosis. No standard guidelines exist for the treatment of AAMT progressing to AA. Herein, we report a rare case of AAMT leading to AA and review the handful of cases previously published in the literature.LEARNING POINTSAcquired amegakaryocytic thrombocytopenia can present as isolated severe thrombocytopenia which can initially be misdiagnosed as immune thrombocytopenia.Lack of response to steroids and intravenous immunoglobulin should raise suspicion for acquired amegakaryocytic thrombocytopenia.Over time, acquired amegakaryocytic thrombocytopenia can progress to aplastic anaemia, which confers a worse prognosis.

Pembrolizumab-Induced Acquired Amegakaryocytic Thrombocytopenia and Successful Combination Treatment With Eltrombopag, Romiplostim and Cyclosporine: A Brief Communication.

Pembrolizumab is a humanized antibody directed against the programmed death-1 receptor on the cell surface, resulting in a T-cell-mediated immune response against tumor cells that can be complicated by immune-related adverse events. Here we present a novel case of pembrolizumab-induced acquired amegakaryocytic thrombocytopenia (AAT) in a patient with metastatic nasopharyngeal carcinoma. AAT is an incredibly rare and challenging entity for which there is no standardized treatment. We discuss our therapeutic approach using a calcineurin inhibitor and thrombopoietin receptor agonists for successful treatment of AAT and resumption of multiagent cytotoxic chemotherapy.

A Retrospective Multicenter Case Study Evaluating the Characteristics, Management and Outcome of Acquired Amegakaryocytic Thrombocytopenia

A Retrospective Multicenter Case Study Evaluating the Characteristics, Management and Outcome of Acquired Amegakaryocytic Thrombocytopenia

Role of Next Generation Sequencing in the Diagnosis of Unexplained Thrombocytopenias- an Experience at a Tertiary Care Hospital

▪BACKGROUNDNext Generation Sequencing (NGS) has been enormously rewarding in the field of diagnostic hematology. In particular, the diagnosis of inherited disorders has progressed in leaps and bounds. These patients tend to remain undiagnosed for a long period of time not only because of unavailability of molecular diagnostics but also due to lack of cognizance and atypical presentations.Thrombocytopenia (TCP) is a common hematological presentation and can lead to chronic hospital visits to life-threatening bleeds. Most of these patients have acquired disorders such as immune TCP, malignancies, liver disease etc. However, some of them are likely to have unidentified inherited causes. We thus intended to study the utility of NGS in the definitive diagnosis of unexplained TCPs with or without other cytopenias to understand the clinicopathologic characteristics of these patients.METHODOLOGYThis was a retrospective descriptive study done at two centres over three years from May 2018 to May 2021. Patients with TCP with one of the following: (a) positive family history (b) clinical/ laboratory clues to an inherited cause (c) chronic TCP with no response to conventional therapies and sent for clinical exome sequencing done by NGS were included in the study. Patients who were negative for germline mutations were excluded.Sequencing of targeted genes was performed on the Illumina platform with a mean coverage of >80-100X. Mutations annotated as pathogenic, likely pathogenic and variant of uncertain significance (VUS) were considered clinically significant. VUS are mutations that are difficult to classify as pathogenic and require clinical validation and family testing.RESULTSOur cohort included 18 patients and were divided into two groups- cases of isolated TCP and cases of TCP with anemia and/or neutropenia.Patients presenting with isolated thrombocytopeniaWe had nine cases of isolated TCP out of which there were three cases of X-linked macrothrombocytopenia with MYH9 mutation, two cases of Wiskott Aldrich syndrome (WAS) and one case each of congenital thrombotic thrombocytopenic purpura (TTP), atypical Hemolytic Uremic Syndrome (HUS), Fanconi anemia (FA) and grey platelet syndrome. The demographic and mutational characteristics are described in Table 1. Clinical and laboratory clues were present in 7 cases, such as chronic kidney disease, micro/ macrothrombocytopenia, neutrophil inclusions etc. Bone marrow examination was carried out in 4 cases- the significant dyspoiesis in FA and myelofibrosis in grey platelet syndrome mislead to a diagnosis of MDS and myelofibrosis respectively. Seven patients had received treatment with steroids, immunosuppressants, splenectomy, danazol and TPO mimetics before the NGS diagnosis.Patients presenting with thrombocytopenia and other cytopeniasThis group consisted of nine cases with two cases each of Dyskeratosis Congenita (DKC) and WAS and one case each of TTP, Ghosal hematodiaphyseal dysplasia, Congenital Amegakaryocytic Thrombocytopenia (CAMT), B-cell immunodeficiency with hypogammaglobulinemia type-25 and double homozygous for FA and DKC. The majority of patients in this group were young and had lower platelet counts (Table 2). The most common associated cytopenia was anemia. Phenotypic clues to diagnosis were present in cases of DKC and WAS. The common differentials considered in this group were inherited bone marrow failure syndromes (IBMFS), congenital immunodeficiency syndromes etc. Bone marrow examination was done more frequently in these patients and showed hypocellular marrow in IBMFS and absent megakaryocytes in CAMT. These patients have also been treated with steroids, IVIg, danazol and TPO mimetics.CONCLUSIONPatients with inherited isolated TCPs have a chronic course and heterogenous causes therefore tend to be diagnosed later in life. However, patients with TCPs and other cytopenias tend to present at a younger age with infrequent family history. IBMFS was the most common disorder identified in this latter group of patients. Positive family history, clinical and laboratory clues and absence of response to conventional therapies should prompt workup of inherited causes by NGS to avoid long term ineffectual treatment. Further, NGS mutations, in particular VUS have to be interpreted with caution with the help of parental study, clinical presentation, in-silico analysis and inputs from molecular and genetic experts. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Anti Thymocyte Globulin-Based Treatment for Acquired Bone Marrow Failure in Adults.

Idiopathic acquired aplastic anemia can be successfully treated with Anti Thymocyte Globulin (ATG)-based immune suppressive therapy and is therefore considered a T cell-mediated auto immune disease. Based on this finding, several other forms of idiopathic acquired bone marrow failure are treated with ATG as well. For this review, we extensively searched the present literature for evidence that ATG can lead to enduring remissions in different forms of acquired multi- or single-lineage bone marrow failure. We conclude that ATG-based therapy can lead to an enduring hematopoietic response and increased overall survival (OS) in patients with acquired aplastic aplasia. In patients with hypocellular myelodysplastic syndrome, ATG can lead to a hematological improvement without changing the OS. ATG seems less effective in acquired single-lineage failure diseases like Pure Red Cell Aplasia, Amegakaryocytic Thrombocytopenia and Pure White Cell Aplasia, suggesting a different pathogenesis in these bone marrow failure states compared to aplastic anemia. T cell depletion is hypothesized to play an important role in the beneficial effect of ATG but, as ATG is a mixture of polyclonal antibodies binding to different antigens, other anti-inflammatory or immunomodulatory effects could play a role as well.

Outcomes in Hematopoietic Stem Cell Transplantation for Congenital Amegakaryocytic Thrombocytopenia

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare, inherited bone marrow failure syndrome. Hematopoietic stem cell transplantation (HSCT) is considered a curative treatment option, but existing descriptions of patient and transplant characteristics and outcomes after related and unrelated donor HSCT are sparse. We describe outcomes after HSCT for congenital amegakaryocytic thrombocytopenia (CAMT; n = 86) from 2000 to 2018. We conducted an analysis of data collected by the Center for International Blood and Marrow Transplant Research on patients with CAMT receiving therapeutic allogeneic HSCT. The predominant donor type was HLA-matched or mismatched unrelated donors (n = 58, 67%). The remaining included HLA-matched sibling (n = 23, 27%) and HLA-mismatched relative (n = 5, 6%). The predominant graft types were bone marrow (n = 53, 62%) and cord blood (n = 25, 29%). The median age at transplantation was 3 years, with 82 of 86 patients being transplanted aged ≤10 years. The 5-year graft failure-free and overall survival were 83% (95% confidence interval [CI], 74-90) and 86% (95% CI, 78-93), respectively. An examination for risk factors confirmed mortality was higher after HLA-mismatched relative and mismatched unrelated donor HSCT compared to HLA-matched sibling and matched unrelated donor HSCT (hazard ratio 3.52, P = .04; 75% versus 93%). The 1-year incidence of graft failure was 19% after HLA-mismatched HSCT (n = 32) compared to 7% after HLA-matched HSCT (n = 54, P = .15). Day-100 grade II-IV acute graft-versus-host disease was 13%, 26%, and 30% after HLA-matched sibling, HLA-matched and mismatched unrelated donor HSCT. The 5-year incidence of chronic graft-versus-host disease was 33% with 24 of 28 patients having received grafts from HLA-matched (n = 13) and mismatched unrelated (n = 11) donors. Although HLA-matched donors are preferred, HLA-mismatched donors also extend survival for CAMT.

English

BACKGROUND This is a clinic-haematological study, conducted in Sidhanta Hospital, Bhopal, to identify the causes of pancytopenia. Out of total 1200 cases of complete hemograms, 56 cases were categorized as pancytopenias, and out of these, 24 cases were subjected to bone marrow examination. The remaining 32 cases of pancytopenia, were put on periodic follow up with peripheral blood smear examination, based on clinical data. Pancytopenia is the simultaneous presence of anaemia, leucopenia and thrombocytopenia that may result from various disease processes, involving the bone marrow primarily or secondarily. Pancytopenia is reduction in all the three cellular components of the blood, namely red blood cells (RBCs), leucocytes (WBCs) and platelets. It is a common entity which is encountered by practitioners. The presentation is in the form of cytopenias leading to infections, anaemia, or bleeding manifestations. All the cases of pancytopenia need a through approach to reach to the cause of the same so that it can be managed in the best possible manner. Cytopenias are reduction in any of the three cellular components of the blood i.e. RBCs, WBCs or platelets. It can be reduction in two cellular components (bicytopenia) or a reduction in all the three cellular components (pancytopenia). In bicytopenia, the most common combination to be seen is anaemia and thrombocytopenia, whereas the least common is leucopenia with thrombocytopenia. (1) For practical purposes, it should have haemoglobin &lt; 10 g%, absolute neutrophil count &lt; 1,500/cumm and platelets &lt; 1,00,000/cumm. It is labelled as severe when the three values are &lt; 7 g%, &lt; 500/cumm and &lt; 20,000/cumm respectively. The purpose of this study was to find out different causes of pancytopenia and the use of bone marrow examination in evaluation of pancytopenia. METHODS This is a cross sectional study, conducted exclusively in the Department of Medicine, at a tertiary care hospital, in Bhopal from August 2019 to December 2019. A total of 56 cases of pancytopenia were analysed with clinico-haematological features. Criteria for diagnosis of pancytopenia were: Haemoglobin less than 10 gm/dl, TLC less than 4000/mm3 and platelet count less than 1,00,000/mm3. We have correlated the complete hemogram findings with bone marrow examination (if required) and peripheral smear examination in order to analyse the root cause of every case of pancytopenia. Pancytopenia is a haematological entity, we have to analyse the cause of it in order to find out the correct diagnosis and treat the patient accordingly. Bone marrow examination is useful in the investigation of PUO (pyrexia of unknown origin), as it leads to an etiological diagnosis in many of the cases. RESULTS In these 56 cases, only 24 cases (42.85 %) were subjected for bone marrow examination. Commonest cause of pancytopenia was episode of viral fever constituting 28 cases (50 %). CONCLUSIONS In cases of PUO, bone marrow examination is a very useful investigation. In cases diagnosed as idiopathic thrombocytopenic purpura (ITP), when the patient does not show improvement in counts, a repeat bone marrow examination should be done, as very rarely; acquired amegakaryocytic thrombocytopenia may be the cause. Though bone marrow examination is an absolute indication in cases of pancytopenia, it is important to wait for at least 2 – 3 weeks, and do a repeat hemogram, especially in cases of viral fever where the counts usually improve after fever subsides. KEY WORDS Pancytopenia, Bone Marrow, Viral Fever

Inducible SBDS Deficiency and Germline FANCC/FANCG Deficiency Differentially Impact the Capacity of Bone Marrow Niches to Engraft Hematopoietic Stem Cells after Transplantation

Strategies to optimize interactions between the recipient bone marrow (BM) microenvironment and donor hematopoietic stem cells (HSC) could improve hematopoietic stem cell transplantation (HSCT) outcomes by reducing graft failure rates, improving blood and immune reconstitution efficiency, and reducing intensive cytotoxic conditioning requirements. Such methods are particularly relevant for patients with inherited bone marrow failure syndromes (iBMFs), rare genetic disorders for which HSCT is required to cure bone marrow failure (BMF), but for which HSCT is also associated with a high incidence of delayed donor engraftment, graft failure and morbidity due to chemotherapy and radiation sensitivity.In previous studies of the MPL-/- model of congenital amegakaryocytic thrombocytopenia, we discovered that decreased numbers of megakaryocytes and/or long-term (LT)-HSC caused by absent thrombopoietin/MPL signaling resulted in diminished capacity of the MPL-/- BM niche microenvironment to efficiently engraft wildtype (WT) donor HSC after HSCT. This observation led us to hypothesize that pre-existing BMF, regardless of cause, may generally alter HSC niche elements critical for engraftment after HSCT. We now present data testing this hypothesis in mouse models of Fanconi Anemia (FA) and Shwachman-Diamond Syndrome (SDS).To model FA, we used double mutant FANCC-/-;FANCG-/- mice, which have previously been demonstrated to develop manifestations of BMF. In our facility, FANCC-/-;FANCG-/- mice did not develop evidence of BMF, as measured by blood counts, BM cellularity, and HSC quantitation out to 1 year of age even with induction of HSC cycling by polyinosinic-polycytidilic acid (pIpC). Using SCT assays in which GFP+ WT donor BM was transplanted into FA recipients (FANCC-/-, FANCG-/-, FANCC-/-;FANCG-/-) or WT controls after 800-1100 cGy total body irradiation (TBI), efficiency and durability of donor engraftment was assessed at 3 weeks and 5 months, respectively, after primary SCT by competitive secondary HSCT. Unexpectedly, all FA recipients demonstrated more rapid BM reconstitution and enhanced early hematopoietic progenitor engraftment of WT donor BM compared with WT controls. However, there was no significant difference in LT-HSC engraftment efficiency or durability in the FA recipient groups versus WT controls.To model SDS, we first attempted to generate a model of osteoblast-specific SBDS deletion by crossing Col1A1Cre mice with mice carrying floxed SBDS alleles (SBDSfl/fl). However, this Col1A1CreSBDSExc model proved to be embryonic lethal at E12-E15 due to profound developmental abnormalities. We thus generated an inducible model by crossing SBDSfl/fl mice with Mx1Cre+ mice, and inducing SBDS deletion in Mx1-inducible BM hematopoietic and osteolineage niche cells by pIpC administration. SBDS deletion induced by pIpC was confirmed with PCR methods.Within 4 weeks of pIpC induction, Mx1CreSBDSExc mice show evidence of BMF, including a 40% reduction in platelet counts, inverted myeloid/lymphoid cell ratio in blood, and up to 80% reduction in immunophenotypic BM LT-HSC. When we attempted HSCT with GFP+ WT donor BM in Mx1CreSBDSExc and littermate control recipients following 1100 cGy TBI, 90% of Mx1CreSBDSExc recipients died by day 9 after HSCT, whereas all littermate controls survived. BM sections from Mx1CreSBDSExc recipients demonstrated persistent aplasia out to 1 week post-HSCT, suggesting that Mx1CreSBDSExc died from primary graft failure. Competitive secondary HSCT performed from BM harvested 1 week after primary HSCT demonstrated severe deficits in GFP+ WT donor HSC engraftment in Mx1CreSBDSExc recipients. Our initial studies as to why donor engraftment fails in Mx1CreSBDSExc recipients have identified that compared to WT, Mx1CreSBDSExc mice show significantly reduced expansion of osteolineage niche cells after TBI, a response that we have previously shown to be critical for efficient donor engraftment after HSCT.Taken together, our data demonstrate that pre-existing BMF and SBDS deficiency in BM osteolineage cells, but not FANCC or FANCG deficiency without co-existent BMF, significantly reduce the capacity of the BM niche to engraft donor HSC. Future studies defining specific niche deficits in our inducible SBDS deficiency model will provide critical insights into cellular and molecular pathways required for efficient engraftment after HSCT. DisclosuresNo relevant conflicts of interest to declare.

CALR mutant protein rescues the response of MPL p.R464G variant associated with CAMT to eltrombopag

AbstractCongenital amegakaryocytic thrombocytopenia (CAMT) is a severe inherited thrombocytopenia due to loss-of-function mutations affecting the thrombopoietin (TPO) receptor, MPL. Here, we report a new homozygous MPL variant responsible for CAMT in 1 consanguineous family. The propositus and her sister presented with severe thrombocytopenia associated with mild anemia. Next-generation sequencing revealed the presence of a homozygous MPLR464G mutation resulting in a weak cell-surface expression of the receptor in platelets. In cell lines, we observed a defect in MPLR464G maturation associated with its retention in the endoplasmic reticulum. The low cell-surface expression of MPLR464G induced very limited signaling with TPO stimulation, leading to survival and reduced proliferation of cells. Overexpression of a myeloproliferative neoplasm–associated calreticulin (CALR) mutant did not rescue trafficking of MPLR464G to the cell surface and did not induce constitutive signaling. However, it unexpectedly restored a normal response to eltrombopag (ELT), but not to TPO. This effect was only partially mimicked by the purified recombinant CALR mutant protein. Finally, the endogenous CALR mutant was able to restore the megakaryocyte differentiation of patient CD34+ cells carrying MPLR464G in response to ELT.

Selective T-cell depletion targeting CD45RA as a novel approach for HLA-mismatched hematopoietic stem cell transplantation in pediatric nonmalignant hematological diseases.

Severe aplastic anemia and congenital amegakaryocytic thrombocytopenia are rare bone marrow failure syndromes. Treatment for aplastic anemia consists of hematopoietic stem cell transplantation (HSCT) from a matched sibling donor or immunosuppressant drugs if there is no donor available. Congenital amegakaryocytic thrombocytopenia is a rare autosomal recessive disease that causes bone marrow failure and has limited treatment options, except for transfusion support and HSCT. In the absence of a suitable matched sibling donor, matched-unrelated, haploidentical, or mismatched donors may be considered. A 2-step partial T-cell-depletion strategy can remove CD45RA+ naïve T cells responsible for graft-versus-host disease (GvHD) while preserving memory T cells. Five patients underwent transplantation using this strategy with rapid neutrophil and platelet recovery. Acute and chronic GvHD ≥ grade 2 appeared in two and one patient, respectively. No severe infections were observed before day + 100. A high (60%) incidence of transplant-associated microangiopathy was observed. Three patients (60%) remain alive, with a median follow-up of 881 (range 323-1248) days. CD45RA-depleted HSCT is a novel approach for patients lacking a suitable matched donor; however, further improvements are needed.

Hematopoietic Stem Cell Transplantation During the Era of COVID-19 in Queen Rania Children's Hospital.

Background:Corona virus disease 2019 (COVID-19) is causing a health crisis nowadays, and all countries are following the recommendations of the WHO to decrease the spread of the disease. Till now, few data are available regarding the clinical course, severity of the disease and the duration of infectivity of COVID-19 in patients received Hematopoietic Stem Cell Transplantation (HSCT).Objective:To evaluate the medical protocols and outcome of patients who underwent HSCT during the pandemic of COVID-19.Methods:A retrospective review of the medical files of patients who underwent hematopoietic stem cell transplantation during the era of COVID-19. The following data were reviewed for all patients: age, gender, primary disease, viral screening protocols for donors and recipients, COVID-19 status and outcome. The European society for blood and marrow transplantation (EBMT) guidelines were applied strictly on all of our patients, donors and bone marrow transplant unit staff.Results:A total of 10 children were transplanted, 8 of them received allogenic transplant from matched donor and two patients received autologous transplant. Regarding allogenic transplants, all of our patients except two were transplanted as an emergency, 2 of them were Aplastic anemia, 2 patients were Fanconi anemia, one patient was Amegakaryocytic thrombocytopenia, and one patient was Acute myeloid leukemia. Only two patients were not an emergency as one of them had Thalassemia major and the other one was Sickle cell anemia. The autologous transplant was done for two patients with Neuroblastoma stage 4 as part of their treatment protocol. At a median follow up of 5.5 months (range, 2 month-7 months) two patients (20%) developed COVID-19, which was asymptomatic in both of them. One of our patients (10%) died due to cytomegalovirus (CMV) pneumonia. No one of our patient was affected by the emergency regulations applied by the country and hospitals during the pandemic of COVID-19 virus.Conclusion:Hematopoietic stem cell transplantation can be performed safely for emergency cases, if we strictly follow the guidelines of EBMT.

Secondary juvenile myelo-monocytic leukemia in a patient with congenital amegakaryocytic thrombocytopenia

Secondary juvenile myelo-monocytic leukemia in a patient with congenital amegakaryocytic thrombocytopenia

Haploidentical stem cell transplantation with post-transplant cyclophosphamide for osteopetrosis and other nonmalignant diseases.

Allogeneic hematopoietic stem cell transplantation (HSCT) is curative for a variety of nonmalignant disorders including osteopetrosis, bone marrow failures, and immune deficiencies. Haploidentical HSCT is a readily available option in the absence of a matched donor, but engraftment failure and other post-transplant complications are a concern. Post-transplant cyclophosphamide (PT-Cy) regimens are gaining popularity and recent reports show promising results. We report our experience with nine pediatric patients with nonmalignant diseases who were transplanted from a haploidentical donor with PT-Cy. From 2015 to 2019, nine children with nonmalignant diseases underwent haploidentical HSCT with PT-Cy, two as a second transplant and seven as primary grafts after upfront serotherapy and busulfan-based myeloablative conditioning. Patient’s diseases included osteopetrosis (n = 5), congenital amegakaryocytic thrombocytopenia (n = 2), hemophagocytic lymphohistiocytosis (n = 1), and Wiskott Aldrich syndrome (n = 1). Two patients failed to engraft following upfront PT-Cy transplants, one was salvaged with a second PT-Cy transplant, and the other with a CD34+ selected graft. None of the patients suffered from graft-versus-host disease. Three patients died from early posttransplant infectious complications and six patients are alive and well. In conclusion, haploidentical HSCT with PT-Cy is a feasible option for pediatric patients with nonmalignant diseases lacking a matched donor.

Desenlace de los pacientes pediátricos con falla medular tratados en un centro de alta complejidad

Los síndromes de falla medular (SFM) son trastornos infrecuentes, con una incidencia anual de 2-4 casos por millón. Las opciones de tratamiento incluyen terapia de inmunosupresión (TIS) y restauración de la hematopoyesis con trasplante de progenitores hematopoyéticas (TPH).Objetivo: Analizar los desenlaces de pacientes pediátricos diagnosticados con SFM tratados en una institución de alta complejidad.Pacientes y Método: Estudio retrospectivo de pacientes pediátricos con diagnóstico de SFM que consultaron a la Fundación Valle del Lili, Cali. Se realizo análisis estadístico descriptivo según SFM adquirida (SFMA) y SFM congénita (SFMC). Los desenlaces incluyeron: tratamiento, complicaciones, supervivencia global (SG) en los trasplantados, calculada con el método KaplanMeier.Resultados: Se incluyeron 24 pacientes con SFM, edad 6,5 ± 4 años, 50% mujeres. El 58% fueron SFMC, 9 con anemia de Fanconi, 2 disqueratosis congénita, 2 trombocitopenia amegacariocítica congénita, uno anemia Diamond-Blackfan. Doce pacientes con TPH tuvieron SG a 5 años de 83%. SFMA correspondió al 42%, 6 recibieron TIS-TPH, 3 TIS y 1 TPH, la SG del grupo con TIS-TPH fue 86%. Seis pacientes fallecieron, 4/6 relacionadas con infección.Conclusiones: En esta serie fue mayor el número de casos con SFMC. La SG de los pacientes llevados a TPH es comparable con la reportada en estudios recientes. La causa de muerte predominante fue infecciosa que también se ha reportado previamente. El tratamiento instaurado en los pacientes de esta serie mostró resultados favorables en un centro de alta complejidad en un país latinoamericano.