Severe Pancytopenia Research Articles

Systemic chemotherapy combined with thrombopoietin receptor agonist for the treatment of diffuse large B-cell lymphoma complicated with aplastic anemia

Immunosuppressive therapies, including antithymocyte globulin and cyclosporine (CsA), are used for the treatment of aplastic anemia, but they reportedly cause lymphoproliferative diseases. Here, we report two cases of aplastic anemia in which diffuse large B-cell lymphoma developed during treatment with CsA. In both the cases, CsA was discontinued and combination therapy with R-CHOP (rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine sulfate, and prednisolone) plus the thrombopoietin receptor agonist eltrombopag was initiated. Furthermore, supportive care, including blood transfusion and granulocyte colony-stimulating factor, was provided. After six or eight courses of R-CHOP therapy, a complete metabolic response was achieved without serious adverse events. These cases illustrate the safety of combining R-CHOP with eltrombopag therapy in patients at a high risk of severe pancytopenia.

Methotrexate-induced toxidermia and pancytopenia in a patient with ectopic pregnancy: a\xa0case report

BackgroundMethotrexate is an anticancer drug from the antimetabolite class. It is also used in gynecology and obstetrics and is the molecule of choice for the medical treatment of ectopic pregnancies. We report a case of toxidermia associated with severe pancytopenia induced by methotrexate for ectopic pregnancy.Case presentationA 30-year-old Malagasy (African) woman was admitted to the Emergency and Intensive Care Department for probable toxidermia following injection of 75 mg of methotrexate for an ectopic pregnancy. She had developed generalized erythema, which started 48 hours after the injection. The secondary onset of phlyctenular maculopapular skin lesions, generalized purpura, and erosions of the oral mucosa in a context of febrile jaundice prompted her hospitalization. On admission, the patient presented with febrile neutropenia, pancytopenia, renal failure, and hepatic cytolysis. She received transfusions of fresh whole blood, erythromycin, and amphotericin B. The course was fatal within 2 days of hospitalization. The patient died of multiple organ failure.ConclusionsOur case is mainly distinguished by the lack of use of granulocyte growth factors and folinic acid. In the event of severe reactions to methotrexate, the management should be multidisciplinary and as much as possible within an intensive care unit.

Case Series: Bone Marrow Failure in Teen Siblings with Unique RPS19 Variant

Aplastic anemia occurs when there is a lack of hematopoiesis in the bone marrow leading to peripheral pancytopenia. It is a rare entity and is often idiopathic. However, upon presentation, inherited bone marrow failure syndromes and acquired etiologies must be considered. Investigating causality is particularly important when multiple family members are affected, especially in a short amount of time. It is also essential to identify novel causative genetic variants of bone marrow failure in order to direct treatment in these patients.In our case series, we describe two siblings who presented two weeks apart with severe pancytopenia. The first patient is a 13 year-old non-binary female (prefers pronouns they/them/theirs) who presented from clinic after getting routine labs with white blood cell (WBC) 1.83 x10(3)/mcL, absolute neutrophil count (ANC) 0.16 x10(3)/mcL, hemoglobin (Hgb) 4.8 gm/dL, platelet 13 x10(3)/mcL. Bone marrow biopsy revealed marked hypocellularity (0-10%) with hypoplasia. The second patient is their 16 year-old brother who presented two weeks later with new-onset petechial rash and was found to have WBC 4 x10(3)/mcL, ANC 2.19 x10(3)/mcL, Hgb 6.3 gm/dL, platelet 16 x10(3)/mcL. His bone marrow biopsy demonstrated variable cellularity (10-70%), but after months of transfusion-dependence he met criteria for severe aplastic anemia. Laboratory evaluation for acquired etiologies such as infection was negative. Both patients had a shared medical history of depression briefly treated with fluoxetine, but otherwise no potential medical triggers or environmental exposures were identified. Telomere length analysis was normal and chromosomal breakage analysis was negative. Upon genetic evaluation, both patients were found to have a heterozygous variant of unknown significance of RPS19 (c.-163>T), which substitutes a moderately conserved nucleotide in the noncoding exon 1 in 5‘ untranslated region of RPS19. Although this variant has not been classified as pathogenic, three other variants in the 5‘ untranslated region of RPS19 have been reported in patients with Diamond-Blackfan Anemia (DBA). In contrast to classic DBA, these patients did not present in infancy or early childhood. Likewise, they lacked congenital anomalies or other classic phenotypic characteristics of disease. Lab studies showed normal erythrocyte adenosine deaminase levels, though it should be noted that these levels could not be obtained prior to transfusion in both patients. Given lack of other identified etiologies, however, an inherited bone marrow failure syndrome, possibly due to this variant, was presumed. With suspected but potentially unidentifiable genetic predisposition, matched sibling donor transplant was deferred and both patients underwent a matched unrelated donor bone marrow transplant with reduced-intensity conditioning.With these cases, we aim to share a unique presentation of aplastic anemia that reveals a potentially novel pathogenic variant as well as to provide our approach to medical management in pediatric aplastic anemia in the setting of uncertainty. Identification of other patients with bone marrow failure and this genetic variant will be important to determine its pathogenicity. DisclosuresNo relevant conflicts of interest to declare.

Efficacy and Safety of Eltrombopag Combined with Cyclosporine As First-Line Therapy in Adults with Severe Acquired Aplastic Anemia: Results of the Interventional Phase 2 Single-Arm Soar Study

Efficacy and Safety of Eltrombopag Combined with Cyclosporine As First-Line Therapy in Adults with Severe Acquired Aplastic Anemia: Results of the Interventional Phase 2 Single-Arm Soar Study

Severe Pancytopenia and Stomatitis Case due to the Treatment with High Dose Methotrexate

Abstract: Objective: Methotrexate is used to suppress inflammation in many rheumatologic conditions. Here we present an elderly patient who developed serious side effects due to methotrexate. A 78 year old male admitted to emergency department with oral mucosal bleeding, skin rash, decrease in oral nutrition intake and weakness. He had been using 5 milligrams of methotrexate as 2 tablets twice a day for 10 days, after he was diagnosed with psoriatic arthritis 15 days ago. A diagnosis of methotrexate intoxication established with history, physical examination and laboratory analysis, which revealed pancytopenia. His signs and symptoms, as well as pancytopenia were recovered on 6th day of the hospitalization. He had been treated with folinic acid and filgrastim along with supportive care. Although methotrexate treatment and toxicity is well established it is still a clinical challenge that all clinicians must be aware of. In conclusion, methotrexate intoxication is a clinical entity that can lead to serious clinical consequences, and it is essential to diagnose and initiate appropriate treatment without delay to prevent morbidity and mortality. Keywords: Methotrexate toxicity, Pancytopenia, Treatment, Folinic acid, Filgrastim, Inflammation, Psoriatic arthritis.

Effective elimination of high-dose methotrexate by repeated hemodiafiltration and high-flux hemodialysis in patients with acute kidney injury.

Acute kidney injury (AKI) after high dose methotrexate (HD-MTX) is associated with delayed MTX-excretion and life-threatening toxicity. Glucapridase, the recommended therapy, is expensive and not always available. We describe 3 cases (69, 67, 73 years) with diffuse large B-cell lymphoma who developed AKI and early-onset severely delayed MTX elimination after HD-MTX. MTX serum concentrations were 101 and 69 μmol/L at 24 h after administration in two patients and 34 μmol/L at 32 h in the third. Since glucarpidase was unavailable, we performed daily high-flux hemodialysis (HF-HD) or online hemodiafiltration (HDF) sessions (median duration, 6 h). The median serum MTX elimination half-life during HDF/HF-HD sessions was similar in all patients (median, 4.4 h; IQR, 3.8-5.3 h), but serum MTX concentrations rebounded after each dialysis by a median of 40% of the trough concentrations. The three patients underwent multiple dialysis sessions, until MTX serum concentrations remained sufficiently low to be neutralized by leucovorin. Only 1 patient developed severe pancytopenia, and renal function normalized in all patients after 3-6 weeks. In conclusion, when glucarpidase is unavailable or delayed, early, repeated and prolonged HDF/HF-HD effectively enhance MTX elimination and prevent toxicity in patients with AKI and severely delayed MTX elimination after HD-MTX.

Neurocysticercosis in a Japanese woman with lung cancer who repeatedly visited endemic countries

BackgroundTaenia solium, present in most developing countries, infects many individuals and may result in their death. Neurocysticercosis (NCC) develops after invasion of the brain by parasitic larvae. It is the most common parasitic disease of the human central nervous system. On imaging scans it can be similar to brain tumors. We report a patient with a metastatic brain tumor and NCC. The co-presence of NCC was diagnosed based on specific neuroimaging- and epidemiologic findings.Case presentationA 36-year-old non-smoking Japanese woman with a history of non-small-cell lung cancer had undergone resection of the lower lobe followed by cytotoxic chemotherapy 2 years before apparently suffering recurrence. A positron emission computed tomography (PET) scan incidentally revealed multiple intracranial cold spots exhibiting differences in their shape and size. On brain magnetic resonance imaging (MRI) scans we observed many different patterns of peripheral edema and gadolinium-enhancing effects. As she had often visited Latin America and Southeast Asia and had eaten raw pork and Kimchi, we suspected that the brain lesions were due to NCC rather than metastatic brain tumors. However, serum immunoblotting assay and DNA analysis were negative for T. solium. Rather than performing resection, we administered albendazole (ABZ) and dexamethasone because her earlier cytotoxic chemotherapy had elicited severe pancytopenia. Except for a single large lesion in the left frontal lobe, this treatment resulted in a significant reduction in the size of these lesions and a decrease in perilesional edema. She underwent resection of the residual lesion 10 months later. Histology revealed it to be a metastatic tumor. Polymerase chain reaction (PCR) assay for NCC was negative. In the course of 11-months follow-up there has been no recurrence.ConclusionThis is the first presentation of NCC in a Japanese woman with a metastatic brain tumor. NCC was incidentally discovered on PET scans and, based on her travel history and epidemiological findings; it was diagnosed and successfully treated with ABZ. NCC is endemic in most developing countries and as visits to such countries have increased, NCC must be ruled out in patients with multiple cystic nodular brain lesions.

One severe case of congenital toxoplasmosis in China with good response to azithromycin

BackgroundMost infants infected with Toxoplasma gondii are completely asymptomatic at birth, yet they may develop ocular and neurological sequelae in the first few months of life. Cases of congenital toxoplasmosis with severe jaundice early after birth combined with pancytopenia and splenomegaly are extremely rare. Here, we report on a rare case of congenital toxoplasmosis presenting with severe jaundice and hemolysis early after birth combined with pancytopenia and splenomegaly.Case presentationA male preterm infant with severe jaundice and splenomegaly was admitted to our department. Laboratory examinations revealed severe hyperbilirubinemia, increased reticulocytes, and pancytopenia. After comprehensive analysis and examination, the final diagnosis was congenital toxoplasmosis, and the infant was treated with azithromycin and subsequently trimethoprim-sulfamethoxazole. Regular follow-up revealed congenital toxoplasmosis in both eyes, which was surgically treated, while neurofunctional assessment results were unremarkable. In this case of congenital toxoplasmosis combined with severe jaundice, we treated the infant with two courses of azithromycin, followed by trimethoprim-sulfamethoxazole after the jaundice resolved. Clinical follow-up indicated that this treatment was effective with few side effects; thus, this report may serve as a valuable clinical reference.ConclusionsTimely diagnosis and adequate treatment are closely associated with congenital toxoplasmosis-related prognosis. Infants with congenital toxoplasmosis require long-term follow-up, focusing on nervous system development and ophthalmology.

A rare case report of transient pancytopenia in a patient with coronavirus disease 2019

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19) infection. The ongoing pandemic of COVID?19 has sought the attention of clinicians and researchers to investigate and describe its various pathological aspects. Infection with SARS-CoV-2 and the resulting COVID-19 can affect virtually any organ of the human body, including the bone marrow which, in turn, causes pancytopenia. Diagnosing pancytopenia in the setting of COVID-19 infection is challenging and requires ruling out all other potential causes of pancytopenia. Herein, we report a rare case of severe pancytopenia secondary to COVID-19 infection that developed severe transient pancytopenia which improved with conservative management.

T Cells Trafficking to Bone Marrow Have Different Characteristics from Those to Other Organs in an Immune-Mediated Bone Marrow Failure Mouse Model

Background and objective T-cell-mediated destruction of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) is the pathophysiology of aplastic anemia (AA). Human AA has been modeled in mice by adaptation of historic “runt disease” in which infusion of lymph node (LN) cells into recipients mismatched at MHC or minor histocompatibility (minor-H) antigen loci produced BM failure with severe pancytopenia and marrow hypoplasia that highly resemble manifestations of human AA. These models have been used to test the plausibility of immune mechanisms inferred from the study of patients and patient cells, and also to serve as a platform for testing new treatments. While much has been learned from these models, the dynamics of T cell homing, expansion and localization in target tissues have not been well examined. Thus, we employed C.B10 AA model to characterize T cells trafficking to BM and other organs. Methods C.B10 mice received 5 Gy total body irradiation (TBI) and were then infused with B6-DsRed LN cells at 5×106 cells/mouse via tail vein injection. On days 3, 6, 9, and 12, mice were sacrificed, and BM, spleen, LNs, and thymus were collected to analyze the dynamic change and activation status of donor T cells in these organs by flow cytometry. On day 12, blood was collected from retro-orbital sinus for the analyses of hematologic parameters, while donor-derived (DsRed) T cells were sorted from BM, spleen and LNs into CD3+CD4+ and CD3+CD8+ fractions for RNA isolation and gene expression analyses by PCR array. Results Relative to control animals that received TBI without LN cell infusion, AA mice developed severe BM failure with dramatic decrease in white blood cells, platelets and total BM cells on day 12 after LN cell infusion. Frequencies of DsRed T cells trafficking to BM, LNs and spleen increased with time. Surprisingly there were very few DsRed T cells in BM on days 3 and 6, significantly lower than those in spleen or LNs. On day 12, DsRed T cells increased dramatically in BM to a level much higher than those in the spleen and LNs, suggesting that donor T cells homed primarily to spleen and LNs first, expanded and differentiated, and then trafficked to BM. Frequency of DsRed T cells in thymus was the lowest during the whole process, implying that thymus is more resistant to donor T cell infiltration, which may represent an immune privilege site (Figure 1A). On day 12, CD3+CD8+ and CD3+CD4+ T cells were essentially of donor (DsRed) origin in BM, spleen and LNs of AA mice (CD3+CD8+ at 99.12±0.29%, 92.07±0.90%, and 93.59±1.04%, and CD3+CD4+ at 98.38±0.20%, 82.87±1.81%, and 39.78±4.24%, respectively) except CD3+CD4+ T cells in LNs. Transcriptome analyses showed higher expression in Cd38, Ifng, Lag3, Csf1, Spp1, and Tnfsf13b in BM CD3+CD4+ and CD3+CD8+ T cells, but lower expression in Foxp3 and Ctla4 in BM CD3+CD4+ T cells than did those in spleen and LNs, indicating that BM T cells were at a state with stronger activation but weaker immunosuppression signaling . Meanwhile, we assessed donor T cell phenotypes using CD44 and CD62L markers, and found that almost all donor CD4+ or CD8+ T cells in BM were of effector memory T cell phenotype (EM: CD44+CD62L-) on days 6 , 9 and 12, while T cells in spleen and LNs were of EM, central memory (CM:CD44+CD62L+), and naïve (NA: CD44-CD62L+) phenotypes on days 6 and 9 before converting to EM phenotype on day 12 (Figure 1B), indicating that T cells in the BM were at a more terminal differentiation stage ready for the initiation of HSPC destruction. The expansion of EM T cell phenotype in BM failure mice reflects our recent observation in AA patients from which we found clonal expansion enriched in effector memory CD8+ T cells defined by CD28-CD57+ phenotype (Giudice et al. ASH abstract, 2016). Conclusions We demonstrate here that donor CD3+CD8+ and CD3+CD4+ T cells first home to host spleen and LNs for expansion and differentiation, and then traffick to host BM where they display strong signaling of activation with a terminal effector memory phenotype. The discovery of stage-specific T cell lodging pattern provides fresh insight for the development of new treatment for AA targeting T cell trafficking intervention. [Display omitted] DisclosuresYoung:Novartis/GSK to institute: Research Funding.

Enhanced Expression of CXCR4 Facilitates the Trafficking of Anti-c-Kit Chimeric Antigen Receptor (CAR) T Cells to Bone Marrow and Achieves Effective Donor Stem Cell Engraftment

Allogeneic hematopoietic stem cell (HSC) transplantation is one of the curative therapeutic options for patients with a variety of inherited disorders of blood cells, including immune deficiencies and hemoglobinopathies. In addition, considerable progress has been made toward achieving clinical benefit from autologous HSC ex vivo gene therapy for some of these disorders. Generally, some level of conditioning using chemotherapy and/or radiation is needed to achieve the required engraftment of allogeneic HSC or gene corrected autologous HSC. There is considerable interest in finding less toxic and more focused approaches to achieve the conditioning. Promising results were observed using antibody-based approaches including anti-c-kit (CD117) or anti-CD45 which directly target HSCs, and these results can be enhanced by linking the antibody to a toxin such as saponin. Here, we explored a related approach in mouse models in which chimeric antigen receptor (CAR)-T cells targeting c-kit (c-kit CAR-T cells) were employed as conditioning to achieve significant engraftment of congenic HSCs. Our initial attempts at efficient conditioning with c-kit CAR-T cells failed because there was insufficient trafficking of CAR-T cells to bone marrow (BM) and insufficient CAR-T cell expansion in vivo and because we did not eliminate the CAR-T cells before transplanting congenic HSCs.To address these barriers, before injecting CAR-T cells (derived from C57BL/6 Thy1.1/CD45.2 mouse spleen on Day1 and transduced with CAR retrovirus on Day2) we pre-treated the recipient mice (Thy1.2/CD45.2) with low-dose cyclophosphamide (CY, 125 mg/kg; Day2) and co-transduced the c-kit CAR-T cells with a CXCR4 (CD184) expression vector (Day2), followed by injection of 5×106 CAR-T cells on Day3. We later removed these CAR-T cells by treating the mice with anti-Thy1.1 antibody (Ab) on Day11 prior to HSC transplantation from congenic Thy1.2/CD45.1 mice (Day12; 3×106 cells/mouse).After single CAR retrovirus transduction, >90% of T cells expressed CAR. When used in vitro, CAR-T cells efficiently depleted BM c-kit+ population (10.5% reduced to 1.6%) and completely suppressed colony formation in nitrocellulose medium. In initial in vivo studies, we used a luciferase marker in the CAR-T cells to allow whole body imaging in addition to flow cytometry (FACS) of BM to assess trafficking. With no pre-treatment of mice, both in vivo imaging and FACS demonstrated poor trafficking of CAR-T cells to BM (< 1% of cells), and there was no decrease of the BM c-kit+ population. In contrast, pre-treatment with low-dose CY and overexpression of CXCR4 on the CAR-T cell surface improved the CAR-T cell expansion in vivo and the migration into BM (mean Thy1.1+ cells, 11.9%; n= 5), and this was associated with significant in vivo depletion of BM c-kit+ population (9.0% reduced to 0.0%). Severe pancytopenia was confirmed in peripheral blood (Hgb 2.2 g/dL compared to the normal, 13.0 g/dL) along with aplastic BM (Figure A), and these mice died within 4 weeks without BM transplant. Subsequent injection of anti-Thy1.1 Ab completely depleted CAR-T cells in viv o. When congenic BM cells were transplanted into mice conditioned in this manner (CY+CAR-T+Thy1.1 Ab), these mice achieved complete recovery of hematopoiesis with significant engraftment of donor BM in all lineages in peripheral blood (Figure B) and long-term HSCs in BM (> 20 % donor chimerism).Our findings provide proof-of-concept that c-kit CAR-T cells can be used to achieve sufficient conditioning to allow donor chimerism after congenic transplantation without use of high-dose chemotherapy or radiation. Low-dose CY was required to achieve the reduction of host T cells and stimulate in vivo expansion of CAR-T cells. A key element to achieve BM HSC depletion was the co-transduction of CXCR4 that greatly increased trafficking of the CAR-T cells to BM. This finding may be one of the most important aspects of this study, with broader implications for the CAR-T cell field in suggesting that use of trafficking receptors or ligands might be generalizable as a method to enhance targeting to designated organs or solid tumors. In summary, our studies add to the list of tools that should be further explored in the effort to seek less toxic conditioning regimens for BM transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The WASP P460S Mutation Causes a New Phenotype of WASP Mutations Related Disorder: X-Linked Pancytopenia

Mutations of the Wiskott-Aldrich Syndrome Protein (WASP) are responsible for a rare and severe disease: Wiskott-Aldrich Syndrome (WAS), including classic Wiskott-Aldrich Syndrome (WAS), X-linked thrombocytopenia (XLT), and congenital X-linked neutropenia (XLN). Classic WAS patients present typical WAS manifestation: eczema, thrombocytopenia, small platelet and recurrent infection, while XLT patients present thrombocytopenia, and XLN patients present congenital neutropenia. Bone marrow failure (BMF) is a series diseases characterized by pancytopenia in peripheral-blood(PB), different degree of bone marrow hypocellularity. In this study, we present two unrelated boys with pancytopenia who were admitted to our hospital on account of declining blood cells and different degree of hematopoietic failure. We identified a new WAS gene mutation C1378T inducing the WASP P460S mutation in both penitents by the second generation sequencing. Furthermore, one case developed T cell acute lymphoblastic leukemia (T-ALL) after severe pancytopenia recovered for 4 years and relapsed in mediastinum transplanted with cord blood for 1 year, another case recovered after haploidentical transplantation and kept stable for 1 year. To date, the role of WAS gene mutation in aplastic anemia-like hematopoietic failure remains unknown. We further verified WASP P460S mutation in these two patients by Sanger sequencing. In patient's PB cells, normal expression level of total WASP proteins was detected. This notion was different from general WASP deficiency classic WAS or XLT patients. To test the function of WASP P460S mutation in pancytopenia, we examined the effect of WASP P460S mutation on cell proliferation. Human myelogenous leukemia K562 cell line, which lacks endogenous WASP, was transfected with lentivirus vectors carrying mutant or wild type of WAS gene. We found that the cells expressing WASP P460S grew significantly slower than the cells expressing the wild type. Moreover, actin polymerization assay showed that increased actin polymerization function in WASP P460S mutation compared with wild type. In addition, P460S mutation had no obvious effect on Daunorubicin-induced apoptosis response. Our findings revealed that the WASP P460S mutation played a role in the inhibition of cell proliferation and actin polymerization which may lead to a new phenotype for WASP mutation related X-linked pancytopenia. DisclosuresNo relevant conflicts of interest to declare.

RUNX3 Promotes the Development of MDS/MPN Overlap Syndrome Via Enhancing Expression of Myc in the Absence of Tet2

The RUNX transcription factors have a critical role in normal and malignant hematopoiesis including myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). RUNX3 is expressed in various hematopoietic cells, and the reduced expression of RUNX3 due to promoter DNA hyper-methylation is seen in patients with acute lymphoid leukemia, indicating a tumor suppressive role of RUNX3. The loss of Runx3 promotes the development of MPN-like disease in mice. However, loss-of-function mutations and/or a deletion of RUNX3 are rarely observed in patients with myeloid malignancies. In fact, given that enhanced levels of RUNX3 expression are found in a part of patients with AML and MDS, RUNX3 may play an oncogenic role for myeloid malignancies.To determine how RUNX3 over-expression promotes the development of myeloid malignancies, we generated a mouse model of RUNX3 -induced MDS/MPN by utilizing Tet2 -deficient hematopoietic stem cells (HSCs). Since TET2 loss-of-function mutations initiate the transformation of HSCs, we harvested bone marrow CD150+CD34-LSK HSCs from tamoxifen-treated Cre-ERT2 (WT) and Cre-ERT2;Tet2Δ/Δ (Tet2 KO) mice, and transduced expression of 3xFLAG-tagged RUNX3 by using a retrovirus vector in vitro. Then, we transplanted them into lethally-irradiated Ly5.1+ recipient mice together with wild-type competitor cells. We observed that Tet2 KO mice died dominantly due to MPN-like disease, whereas RUNX3 -expressing Tet2 wild-type (RUNX3/WT) HSCs neither competed wild-type bone marrow cells nor developed hematological malignancies in mice by 12 months post transplantation. In contrast, RUNX3 -expressing Tet2 KO (RUNX3/Tet2 KO) HSCs preferentially generated myeloid cells, but not lymphoid cells, and subsequently competed wild-type cells in the PB and the BM. We found that 7 out of 18 RUNX3/Tet2 KO mice developed lethal MDS/MPN diseases despite a longer latency. At the time of sacrifice, RUNX3-Tet2 KO mice showed severe pancytopenia accompanied with dyplastic blood cells including pseudo-Pelger neutrophils and micro megakaryocytes. In addition, these moribund mice significantly enlarged spleen due to the expansion of myeloid cells. FACS analysis demonstrated that the expansion of LSK hematopoietic stem/progenitor cells (HSPCs) in the BM and the spleen, consistent with the features of extramedullary hematopoiesis. Thus, RUNX3/Tet2 KO mice recapitulated the phenotypic features of human MDS/MPN overlap syndrome, indicating that RUNX3 over-expression is prerequisite to the development of MDS/MPN in the absence of Tet2, although it has an adverse effect on the function of Tet2 -present HSCs, reminiscent with a tumor suppressive function of RUNX3 as seen in Runx3 KO mice.To understand the underlying molecular mechanism in the pathogenesis of RUNX3 -induced MDS/MPN, we performed gene expression analysis in HSPCs isolated from WT, RUNX3/WT, Tet2 KO and RUNX3/Tet2 KO mice at pre-disease and MDS/MPN stages. Gene Set Enrichment Analysis (GSEA) demonstrated that RUNX3/Tet2 KO MDS/MPN cells significantly enhanced expression of c-Myc target genes, compared to either WT cells or RUNX3/Tet2 KO cells at the pre-disease stage. While RUNX3/Tet2 KO cells showed negative enrichments of differentiation-related pathway genes, compared to WT cells, we found that those MDS/MPN cells enhanced expression of cell cycle, ribosome, and mitochondria-related biological pathways, in which c-Myc may regulate target genes and contribute to the transformation. In order to determine whether RUNX3 directly activates expression of c-Myc, we performed ChIP-sequencing by using an anti-FLAG antibody in RUNX3/Tet2 KO bone marrow cells, and found that c-Myc 3' distal enhancer regions showed conserved binding sites for Runx transcription factors. In addition, we found that RUNX3 -expressing human AML cells showed higher sensitivity to JQ1, a BRD4 inhibitor, following the significant reduction of both RUNX3 and C-MYC expression. The growth inhibition by JQ1 was rescued by ectopic expression of RUNX3 .In conclusion, RUNX3 and c-Myc collaborated to activate the function of oncogenic enhancers, in which Tet2 deficiency may alter the epigenetic status for allowing the bindings of these transcription factors, to an extent not seen in RUNX3-expressing alone. We demonstrated that RUNX3 functions as either an oncogene or a tumor suppressor for myeloid malignancies in the context dependent manner. DisclosuresHarada:Celgene: Research Funding; NIPPON SHINYAKU CO.: Speakers Bureau; NOVARTIS: Research Funding.

Unresponsive Severe Aplastic Anemia in a Young Patient: Case Report and Short Review of the Literature

Abstract Aplastic anemia or medullary aplasia is a medical condition characterized by pancytopenia and is associated with a high prevalence of morbidity and mortality. In patients in whom bone marrow transplantation cannot be attempted, an immunosuppressive regimen is considered to be the first-line therapy. Also, the addition of eltrombopag from the first day of immunosuppressive treatment seems to significantly increase response rate. Unfortunately, there are a small number of patients who remain unresponsive to all these therapies. Here we present the case of a young woman who was referred by the family doctor complaining of marked physical asthenia, new onset dyspnea, and dizziness. Apart from a severe pancytopenia, no further changes have been brought to light by paraclinical investigations. After multiple secondary causes were excluded, the patient was diagnosed with idiopathic aplastic anemia. Even if bone marrow transplant was the first-line therapy in this case, because of the severe leukopenia, it was not possible to determine the HLA type. Therefore, the patient was prescribed immunosuppressive treatment. Despite the three drug-associated therapy (horse anti-thymocyte globulin, cyclosporin A, and eltrombopag), the response was unsatisfactory, with the persistence of severe pancytopenia.

The impact of Parvovirus B19 on hereditary haemolytic anaemias.

The impact of Parvovirus B19 on hereditary haemolytic anaemias.

Is simultaneous partial splenectomy during pediatric liver transplantation safe and effective for severe hypersplenism? A prospective cohort study.

Simultaneous splenectomy during liver transplantation is indicated for patients with cirrhosis complicated by severe hypersplenism, but disastrous procedure-related complications remain a special concern. Simultaneous partial splenectomy was adopted in pediatric liver transplant recipients with severe hypersplenism-related pancytopenia at our institution. A prospective, single-center analysis of 21 pediatric patients diagnosed with cirrhosis and severe hypersplenism, who underwent liver transplantation between January 2015 to December 2019, was conducted. Patient characteristics, intraoperative parameters, and postoperative outcomes were compared between patients with simultaneous partial splenectomy (n=13) and those without (n=8). Simultaneous partial splenectomy significantly increased platelet and leukocyte counts in the early postoperative period, without increasing operative time, intraoperative blood loss and postoperative hospital stay (P=0.64, P=0.44, P=0.26, respectively). No significant differences were observed between the two groups regarding the incidence of postoperative hemorrhage (P=0.38), pneumonia (P=0.33), cholangitis (P=0.38), thrombotic complications (P=1.00), cytomegalovirus infection (P=0.53), Epstein-Barr virus infection (P=0.20) and acute rejection (P=0.26). Simultaneous partial splenectomy during liver transplantation could serve as a feasible alternative to splenectomy in selected patients with severe hypersplenism, which can achieve a satisfactory long-term hematological response, but avoid untoward complications of splenectomy.

Efficacy of tocilizumab therapy in a patient with severe pancytopenia associated with a STAT3 gain-of-function mutation

BackgroundWe aimed to report the clinical characteristics, immunological features, and treatment of one patient with a de novo STAT3 gain-of-function mutation identified by next generation sequencing. We investigated the efficacy of tocilizumab therapy in immune dysregulation diseases caused by STAT3 mutation.ResultsThe patient was a 16-year-old girl. She presented with recurrent respiratory infections and chronic diarrhea after birth. She had life-threatening autoimmune pancytopenia at 14 years old. After receiving glucocorticoid therapy, she developed diabetes. However, her pancytopenia relapsed when the glucocorticoid was tapered. Next-generation sequencing showed a de novo heterozygous mutation in the STAT3 gene, c.1261G > A (p. G421R), which was previously described as a gain-of-function mutation. After tocilizumab therapy, her pancytopenia fully resolved, and insulin and glucocorticoid therapies were gradually discontinued within 12 months. She had lymphopenia and an inverted CD4/CD8 ratio before therapy. Lymphocyte subpopulation analysis indicated an expansion of effector memory CD4+, effector memory CD8+ and central memory CD4+ T cells. The proportions of memory B cells and naive CD4+ T cells were decreased, and the proportion of naïve B cells was increased. None of the abnormal lymphocytic changes improved significantly. STAT3 GOF mutations were identified by next gene sequencing in those with early-onset multi-organ autoimmunity. Including our patient, 13 patients with STAT3 GOF mutations received targeted treatment. Twelve of them were treated with tocilizumab alone or combination tocilizumab with JAK inhibitor, and ten patients improved.ConclusionsGene sequencing should be performed for patients with early-onset refractory or multiorgan immune dysregulation diseases. Targeted drugs can effectively improve the clinical problems associated with STAT3 gain-of-function mutations, while nontargeted immunosuppressive therapy is usually insufficient.

Minimal role of interleukin 6 and toll-like receptor 2 and 4 in murine models of immune-mediated bone marrow failure.

Immune aplastic anemia (AA) results from T cell attack on hematopoietic cells, resulting in bone marrow hypocellularity and pancytopenia. Animal models have been successfully developed to study pathophysiological mechanisms in AA. While we have systemically defined the critical components of the adaptive immune response in the pathogenesis of immune marrow failure using this model, the role of innate immunity has not been fully investigated. Here, we demonstrate that lymph node (LN) cells from B6-based donor mice carrying IL-6, TLR2, or TLR4 gene deletions were fully functional in inducing severe pancytopenia and bone marrow failure (BMF) when infused into MHC-mismatched CByB6F1 recipients. Conversely, B6-based recipient mice with IL-6, TLR2, and TLR4 deletion backgrounds were all susceptible to immune-mediated BMF relative to wild-type B6 recipients following infusion of MHC-mismatched LN cells from FVB donors, but the disease appeared more severe in IL-6 deficient mice. We conclude that IL-6, TLR2, and TLR4, molecular elements important in maintenance of normal innate immunity, have limited roles in a murine model of immune-mediated BMF. Rather, adaptive immunity appears to be the major contributor to the animal disease.

Advances in Diagnosis of Mitochondrial Diseases: Case Report of an Infant with Pearson Syndrome

Pearson syndrome (PS) is a mitochondrial disorder that presents in early infancy as a multisystemic disease affecting the bone marrow and pancreas. It may present with anemia, diarrhea, exocrine pancreatic dysfunction, and failure to thrive.[1] Delay in diagnosis can lead to severe morbidity and mortality in infancy. We report the case of a 9-month-old presenting with failure to thrive, severe macrocytic anemia and pancytopenia initially thought to have gastroesophageal reflux and feeding intolerance. Severe macrocytic anemia and pancytopenia prompted an early bone marrow evaluation. Abnormal bone marrow findings including vacuolated marrow precursors and ringed sideroblasts along with persistent mild lactic acidosis led to a rapid and extensive genetics workup. Whole exome sequencing including mitochondrial genome sequencing detected a 2.3 kb heteroplasmic deletion in m.12113_14421 encompassing the MT-ND5 gene consistent with the diagnosis of Pearson Syndrome. This case report highlights the advances in molecular genetic testing to diagnose patients with complex medical histories along the spectrum of mitochondrial diseases and the importance of early diagnosis to start treatment.

LOPH and D-MHC in the Treatment of FeLV Associated Acute Leukemia in a Cat

Background: Bone marrow primary malignancies are denominated leukemias, classified as myeloid or lymphoid, according to the cell lineage, and acute or chronic, according to the cell´s state of maturation. In cats, acute lymphoid leukemia is the most common form, especially in regions endemic for feline leukemia virus and / or feline immunodeficiency virus. A new treatment protocol for lymphomas, called LOPH, has been described for animals with FeLV persistent viremia. This study aimed to report a case of a cat presenting with FeLV associated acute leukemia and treated with the LOPH protocol, and, in the rescue phase, a modification of the D-MAC protocol, denominated D-MHC.Case: A 4-year-old mixed breed intact queen was attended due to lethargy and inappetence. The patient did not present any relevant abnormalities in the clinical exam and complementary exams were performed including complete blood count, biochemical profile, SNAP Feline Triple Test, chest radiographs and abdominal ultrasound. Imaging tests and biochemical values were unremarkable, but the patient presented a reagent result for FeLV and severe leukocytosis due to lymphocytosis. The morphological evaluation of the blood smear revealed the presence of blasts, in a concentration greater than 20% of the nucleated cells, which allowed the characterization of a leukemic state, probably lymphoid. First-line treatment was based on the LOPH protocol, including Lomustine, Vincristine, Prednisoloneand Doxorubicin, in four-week cycles. Nevertheless, during the third cycle, 66 days after the institution of this protocol, the patient presented a febrile condition along with marked leukocytosis due to lymphocytosis, confirming leukemia recurrence. A rescue attempt was performed with a modification of the D-MAC protocol, originally consisting of the combination of dexamethasone, melphalan, actinomycin-D and cytarabine, but with replacement of actinomycin-D by doxorubicin, and therefore nominated D-MHC. After the three cycles there was a return of the febrile condition associated with severe pancytopenia and euthanasia was elected due to poor clinical condition, resulting in a survival time of 124 days. The hematological toxicity of the induction protocol included anemia and neutropenia, with mainly grade I events, but with the occurrence of a grade IV event. The adverse effects of the rescue protocol were similar, but with a greater number of grade IV events.Discussion: FeLV is considered the most lethal retrovirus of the domestic cat, with a major impact on health and life expectancy. Persistent FeLV antigenemia increases the risk of hematopoietic neoplasms in 62.1 times due to a direct insertional mutagenesis. In endemic regions, approximately 70% of cats with acute leukemia have persistent FeLV antigenemia, as the patient in this report. The diagnosis was made through association of retroviral status and the identification of more than 20% of blasts, possibly lymphoblasts, in the blood stream, sparing the need for a myelogram. Considering the poor prognosis for acute lymphoid leukemias and the patient's retroviral status, treatment was initiated with the LOPH protocol, including lomustine, as a potent agent to induce remission, and doxorubicin, which can result in longer remission intervals. After 66 days free of the disease, the patient presented recurrence of the leukemic condition, starting the rescue protocol D-MHC. Remission was again obtained with duration similar to the first protocol, however, on the occasion of a new leukemia recurrence, euthanasia was elected. The treatment adopted for the patient in this report resulted in a longer survival time than reported in other studies. Despite the aggressiveness of the protocols, especially the D-MHC, it was possible to perform it using the monocyte and granulocyte stimulation factor to reverse neutropenia.