Peripheral Blood Cytopenias Research Articles

Impact of Increased Duration of Trimethoprim-Sulfamethoxazole Prophylaxis for Pneumocystis Pneumonia After Renal Transplant.

BackgroundTrimethoprim-sulfamethoxazole (TMP-SMX) is recommended as prophylaxis against Pneumocystis pneumonia (PCP) in renal transplant recipients. The optimal duration of prophylaxis is unknown. Longer duration of prophylaxis may increase the risk of adverse effects. The aim of this retrospective observational cohort study was to assess the impact of increasing duration of TMP-SMX prophylaxis from 3 to 6 months after transplant on drug-resistant urinary tract infection (UTI), hyperkalemia, peripheral blood cytopenias, and incidence of PCP.Material/MethodsPatients transplanted over a 4.5-year period before and after a change in protocol from 3- to 6-months TMP-SMX prophylaxis in our unit were grouped according to planned duration of prophylaxis, and results were analyzed on an intention-to-treat basis. Baseline characteristics, laboratory values, and all urine microbiology results in the 6 months after transplant were analyzed.ResultsThe overall UTI incidence rate was higher in the 3-month (3-m) treatment group than the 6-month (6-m) treatment group (0.52 vs. 0.33 UTI per 100 patient days; rate ratio 1.56 [95% CI 1.27–1.95]). However, this was not attributable to TMP-SMX: the incidences were significantly different in months 0–3 but not months 4–6. Twenty-eight multi-resistant UTIs occurred in the 3-m group, but there were none in the 6-m group (p=0.004). There were no significant differences in renal function, serum potassium, or cytopenias during the first 6 months. There were 15 cases of PCP in the 3-m group, 3 cases in the 6-m group, and no cases during prophylaxis.ConclusionsExtending the duration of TMP-SMX prophylaxis was not associated with change in frequency of UTIs or multi-drug-resistant UTIs, nor was it associated with increased adverse events. TMP-SMX is an effective PCP prophylaxis, and these data support recommendations to extend the duration of prophylaxis after transplant.

Disruption of the Normal Hematopoietic Hierarchy Leading to Stem Cell Exhaustion in an Animal Model of Myelodysplastic Syndrome

The ineffective hematopoiesis that is characteristic of myelodysplastic syndrome (MDS) suggests functional defects of hematopoietic stem and progenitor cells (HSPC). NUP98-HOXD13 (NHD13) transgenic mice recapitulate many features of human MDS such as ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and transformation to acute myeloid leukemia (AML), and have been used as a pre-clinical model for human MDS. NHD13 mice universally develop signs of MDS (e.g., peripheral blood cytopenia, macrocytosis, dysplasia) at approximately 5 months of age, with median survival of 10 months. Two month old NHD13 mice do not show clear evidence of MDS such as peripheral blood cytopenia, dysplasia, or transformation to AML. Bone marrow nucleated cells (BMNC) from two month old NHD13 mice have a modest 1.3-fold increase of lineage negative (LN) BMNCs compared to age matched WT mice. The increased number of LN BMNCs appeared to be primarily due to a 3.4-fold increase of the LN Sca-1+cKit-(LS+Kˉ) cells, an early lymphoid-committed precursor. Lineage negative Sca-1+ c-Kit+ (LSK) cells, which include the most immature, undifferentiated cells, can be divided into five sub populations, based on expression of Flk2, CD150, and CD48. These populations have been designated Long-Term Hematopoietic Stem Cell (LT-HSC), Short-Term HSC, (ST-HSC), and Multi-Potent Progenitor 2, 3, and 4 (MPP2, MPP3, and MPP4) based on functional assays. Two-month old NHD13 mice had decreased MPP4 (5-fold), decreased LT-HSC (3.6-fold) and increased ST-HSC (2.3-fold) compared with the age matched WT mice. The expansion of ST-HSC two-month old NHD13 mice was associated with increased cell proliferation of ST- HSC, as assessed by bromo-deoxy-uridine (BRDU) incorporation. We next studied LSK subsets from NHD13 mice aged seven months, which coincided with peripheral blood findings consistent with MDS (e.g. anemia, thrombocytopenia, macrocytosis), BM from seven month old NHD13 mice showed significant reductions of all LSK population subsets. LT-HSCs show differential expression of the CD41 antigen, and CD41ˉ LT-HSCs are more quiescent than CD41+ LT-HSCs and are thought to reside at the apex of the hematopoietic differentiation hierarchy. Although there was no difference in the absolute number of quiescent CD41ˉ LT-HSC between two and six month old WT mice, six month old NHD13 mice show a marked decrease (4.2 fold) in CD41ˉ LT-HSCs, suggesting exhaustion of LT-HSC in NHD13 mice. Colony forming assays were used to assess function of the five LSK sub-populations in vitro. LT-HSC and ST- HSC from NHD13 BMNC did not produce any colonies in two independent experiments, whereas MPP2 and MPP3 from NHD13 BMNC produced a similar number and lineage distribution of colonies compared to WT BMNC. This result suggested that HSCs from NHD13 BMNC may be functionally impaired, and that NHD13 hematopoietic progenitor cells may instead be derived primarily from MPP2 and MPP3. To evaluate HSC self-renewal activity, the five LSK subsets from NHD13 BMNC were transplanted to lethally irradiated mice together with 5 x 105 WT BMNC competitor cells. None of the NHD13 LSK sub-populations showed evidence of engraftment. Since NHD13 LN BMNC have previously been shown to be more prone to apoptosis than their WT counterpart, it is possible that lack of engraftment of NHD13 LSK subsets was due to the ex vivo sorting procedure. However, we also considered the possibility that NHD13 lineage positive (LP) BMNC had acquired self-renewal potential, and were contributing to long term hematopoiesis in the NHD13 BM. Therefore, we transplanted LP and LN BMNC from NHD13 or WT mice into WT recipients, again with WT competitor BMNC. Almost half of the NHD13 LP recipients showed long-term (>26 weeks) myeloid engraftment, whereas none of the WT LP recipients showed long term myeloid engraftment. Taken together, these findings suggest that the primitive LT-HSC (LSK Flk2ˉ CD150+CD48ˉ CD41ˉ) from NHD13 BM become exhausted with age, corresponding to the presentation of findings consistent with MDS (peripheral blood cytopenia, macrocytosis). Furthermore, self-renewal activity of NHD13 LP BMNCs suggest the existence of a compensatory mechanism for the homeostasis of hematopoiesis in MDS. Disclosures Aplan: NIH: Patents & Royalties: royalties for the invention of NUP98-HOXD13.

Peripheral Blood Cytopenia and Subsequent Risk of Cardiovascular Disease and Mortality

INTRODUCTION: African-Americans (blacks) have higher risk of stroke and coronary heart disease (CHD) - collectively referred to here as cardiovascular disease (CVD), than Caucasian-Americans (whites). Though partly explained by traditional cardiovascular risk factors, half of the excess risk in blacks is not explained by known risk factors. Recent data suggest increased risk of CVD and mortality in individuals with clonal hematopoiesis, which often presents as cytopenia. Using peripheral blood cytopenia as a marker of clonal hematopoiesis, we examined the association between cytopenia and risk of CVD and mortality in blacks and whites. METHODS: The REasons for Geographic and Racial Differences in Stroke (REGARDS) cohort study enrolled 30,239 US black and white adults between 2003 and 2007 (41% black). Socio-demographics and medical history were obtained by telephone interview, and laboratory studies (including complete blood count [CBC]) and physical exam from an in-home visit at baseline. Participants or their proxies were contacted every 6 months to ascertain CVD events, hospitalizations or deaths, and medical records were reviewed to confirm these events. Cytopenia was defined using thresholds in Table 1 as presence of 2 or more of the following: i) hemoglobin in age-, sex-, and race-specific lowest 5th percentile; ii) white cell count in race-specific lowest 5th percentile; iii) platelet count in lowest 5th percentile, and iv) macrocytosis (MCV >98fL). Participants with pre-baseline history of stroke (for analyses including stroke or CVD mortality) or CHD (for analyses including CHD or CVD mortality) and those with missing CBC were excluded. Cox proportional hazards models were used to calculate hazard ratios (HRs) of incident CVD and mortality associated with cytopenia. Models adjusted for socio-demographics (Model 1), Framingham stroke or CHD risk factors (Model 2), and estimated glomerular filtration rate and C-reactive protein (Model 3) were used. Differences in the association of cytopenia with outcomes by race were tested using cross-product interaction terms, using a p of <0.1 for interaction. RESULTS: The study included 19,544 participants who were followed for a median of ~9 years. There were 798 (4.3% of those at risk) incident stroke cases and 727 (4.3%) incident CHD cases; 1033 (5.3%) died of CVD, and 3933 (20.1%) died of all-causes. Cytopenia was present in 378 (1.9%) participants, ranging from 0.9% to 3.5% in blacks, 1.4 to 3.9% in whites, 1.6 to 3.9% in men, and 0.9 to 1.8% in women, with increasing prevalence by age. There was no association between cytopenia and stroke or CHD risk in any model. However, cytopenia was associated with increased risk of all-cause mortality (HR=1.73, 95%CI: 1.34-2.22), and CVD mortality (HR=1.56, 95% CI: 1.11-2.19) in the extended risk factor Model 3 and also in CVD risk factor adjusted model (Model 2), with little evidence of confounding (Table 2). While the race by cytopenia interaction term was not significant in any model for incident CHD or mortality, the interaction for cytopenia by race for stroke was statistically significant (p-interaction=0.08) in Model 2. The HR of stroke for cytopenia in blacks was 0.86 (95%CI: 0.46-1.61), and for whites was 1.96 (95%CI: 1.0-3.82). CONCLUSION: In this large biracial cohort, cytopenia was associated with increased all-cause and CVD mortality. Cytopenia was a race-specific risk factor for stroke affecting white Americans but not black Americans. With growing knowledge on the role of clonal hematopoiesis in CVD risk and mortality, further work is needed to determine if our phenotype of cytopenia is accurate in classifying clonal hematopoiesis and for determining the mortality risk. Given these findings, assessing clonal hematopoiesis and outcomes related to clonal hematopoiesis in diverse populations is critical to understanding the interactions between somatic mutations in hematopoietic cells and CVD/mortality risk. Disclosures Safford: Amgen: Research Funding.

Recipient Age and Marked Thrombocytopenia Are Associated with Increased Mortality in Patients with Poor Graft Function Post Allogeneic Transplantation

Introduction: Poor graft function (PGF) post allogeneic transplantation is defined as peripheral blood cytopenias in the presence of donor chimerism and is increasingly recognised as a specific clinical entity. The criteria for PGF diagnosis are variable with regards to level of donor chimerism level and duration of cytopenias. Patients with this condition are at high risk of complications from bone marrow failure and need to be differentiated from patients with persisting cytopenias that spontaneously recover without serious complications. We retrospectively analysed patients treated at our centre with complete donor chimerism and cytopenias in order to identify markers of poor prognosis and define an at-risk group that will form the basis of further clinical studies. Method: Patients who underwent allogenic stem cell transplant (alloHSCT) at our centre between 2000-2016 were included in this retrospective analysis after fulfilling the criteria for PGF (1) cytopenias in at least 2 lineages (defined as platelets <100x109/L, neutrophils <1.0x109/L, haemoglobin <100g/L)sustained for 30 days and (2) myeloid chimerism >95%. Patients with relapse of their haematological malignancy within 100 days post-alloSCT were excluded. The primary outcome variables were overall survival (OS) and death from PGF. OS was evaluated in landmark analyses from day 100 (D100) post-alloSCT. Variables were investigated for associations with OS using cox proportional hazards models. Relapse and death without PGF were treated as competing risks for the outcome of death from PGF; associations with PGF were investigated using the method of Fine and Gray. Variables statistically significant at α<0.05 were included in a forward step-wise fashion in multivariate models. Previous studies have demonstrated an excess of early mortality from PGF and hence patients were censored if alive at 24 months. Results: Of 908 transplants performed between 2000-2016, 152 patients (16%) fulfilled our criteria for PGF and were alive at day 100 post transplantation. The overall survival of the PGF cohort at 24 months was 65%. There were 102 patients alive at 24 months vs 50 who died within the 24 months period. The early mortality group had a median survival of 6 months (Range 3.3-23). Demographic details of the early mortality vs those who survived to 24 months are listed in Table 1. Univariate analysis of the PGF cohort identified the following variables were significantly associated with inferior OS: non-CMV viral infection, ICU admission during initial 30 days, age as a continuous variable, grade 2-4 acute GVHD and platelet count ≤40x109 /L (P40) at D100. Age and P40 remained significantly associated with OS on multivariate analysis. Hazard ratios and P values are summarised in Table 2. Competing risk analysis revealed, Age, non-CMV viral infections, ICU admission, MPN as an indication for transplantation, P40 at D100 and grade 2-4 GVHD as significant associations for death from PGF. Age, GVHD and P40 remained significantly associated with death from PGF in multivariate analysis. Hazard ratios and P values are summarised in Table 2. Conclusion: Increasing patient age and marked thrombocytopenia at D100 are associated with inferior survival in patients with PGF. These factors identify patients at higher risk of death from PGF which inform a stricter definition of the syndrome and provide a population for future interventional studies. Disclosures Ritchie: Sanofi: Honoraria; Novartis: Honoraria; Imago: Research Funding; Beigene: Research Funding; Takeda: Research Funding; BMS: Research Funding; Pfizer: Consultancy; Amgen: Consultancy, Honoraria, Research Funding. Koldej:NanoString Technologies: Other: Travel grant.

Clinical Characteristics of Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH): A Retrospective Chart Review Study

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disease that manifests with complement-mediated chronic intravascular hemolysis resulting in hemolytic anemia, tendency to thrombosis, and peripheral blood cytopenias. Eculizumab is a recombinant humanized IgG2/4 chimeric monoclonal antibody that is approved for the treatment of PNH, generalized myasthenia gravis (gMG), atypical hemolytic uremic syndrome (aHUS), and neuromyelitis optica spectrum disorder (NMOSD). Eculizumab specifically binds to the human C5 complement protein (C5), thereby inhibiting the complement cascade. To obtain a better understanding of PNH biology and provide insight toward improving the clinical management of patients with this disease, a retrospective chart review was performed from pre-existing medical records of patients with PNH treated with eculizumab. This analysis is expected to inform eculizumab biosimilar development; data collected on clinical characteristics of this patient population are presented here. Methods: This is a multicenter retrospective chart review study of pre-existing medical records of patients ≥18 years old with PNH who were treated with eculizumab between 01 January 2008 and 31 January 2016. Anonymized data from eligible patients were recorded by sites onto an electronic case report form (eCRF) and collected into an electronic data capture (EDC) system. The primary objective of the study was to assess the lactate dehydrogenase (LDH) concentration over time in eculizumab-treated patients with PNH, including during clinical events and after discontinuation of therapy. Secondary objectives included assessment of coexisting disease, vital signs, laboratory results, and clinical findings. Subjects' LDH time profiles and mean change from baseline of LDH in the maintenance phase were assessed. Maintenance phase was defined as the time period from the first of every 2 weeks (Q2W) eculizumab dose to the data cutoff date, or from the first Q2W eculizumab dose date to the last Q2W dose date for patients who discontinued prior to data cutoff. Baseline of the maintenance phase was defined as the last non-missing assessment taken prior to or on the same day of the first Q2W eculizumab dose. The inter- and intra-subject coefficient of variation (CV) of the 12-week area under effective curve (AUEC) of LDH was based on patients who had at least 2 LDH measurements during the maintenance treatment phase within 12 weeks of each other. Results: The medical records of 47 patients with PNH who were treated with eculizumab during the study period were reviewed; of these, 27 (57.4%) were female. At the time of the diagnosis, the mean age of all patients was 35.0 years and LDH values were reported for 5 of the 47 patients, 3 of which were ≥1.5 times the upper limit of normal. LDH values for most subjects during the study were within the expected ranges for the PNH population with stabilized hemoglobin levels on eculizumab. The mean change from baseline of LDH in the maintenance phase showed a decrease or no change for most time points. For the 26 patients who had ≥2 LDH measurements that were ≤ 12 weeks of each other, inter- and intra-subject CVs for the 12-week AUEC of LDH were 74.70% (95% confidence interval [CI] of 58.23%, 88.15%) and 34.05% (95% CI of 30.65%, 38.34%), respectively. The most commonly reported disease-related symptoms were hemoglobinuria (25 patients [53.2%]), abdominal pain (14 patients [29.8%]), thrombosis (11 patients [23.4%]), and infection (8 patients [17.0%]). Disease-related symptoms listed as 'Other' were reported in 14 (29.8%) patients and included dyspnea, fatigue, exertional dyspnea, aplastic anemia, kidney failure, and dysphagia. All reported concurrent medical conditions and symptoms have been previously well-documented in PNH patients. Ninety-two transfusions were administered to 15 (31.91%) patients within 6 weeks of the first eculizumab dose. Vital signs data did not demonstrate any clinically significant findings. Conclusions: The LDH levels for the majority of PNH patients with stabilized hemoglobin levels on eculizumab were well controlled and within the expected range; the inter- and intra-subject variability of 12-week AUEC of LDH were 74.70% and 34.05%, respectively. There were no unexpected or remarkable findings with respect to disease-related symptoms, vital signs, clinical findings, or laboratory data. Disclosures Hanes: Amgen Inc.: Employment. Pan:Amgen Inc.: Employment. Chow:Amgen Inc.: Employment.

No Evidence for Hematopoietic Stem Cell Self-Renewal in-Vivo Following Inflammatory Challenge

Elevated levels of inflammation have been previously linked to both inherited and acquired bone marrow failure (BMF) syndromes, as well as to normal aging, suggesting a role in the etiology of these conditions. One potential explanation for this phenomenon is that repeated inflammation can promote the suppression of hematopoietic stem cell (HSC) function.We have previously demonstrated that interferon-α can accelerate HSC attrition by driving HSCs out of quiescence, leading to the development of BMF in a mouse model of Fanconi anemia (Walter et al. Nature, 2015). To more broadly address the impact of repetitive inflammatory challenge on HSC regeneration, we challenged C57BL6 wild type (WT) mice with polyinosinic:polycytidylic acid (pI:C), a TLR3 agonist that mimics viral infection. Injection with 1-3 rounds of pI:C (8 injections per round) in WT mice had no sustained impact on hematopoiesis, since peripheral blood (PB) and bone marrow (BM) counts were within normal ranges at 5 weeks (5wk) post-treatment. However, in vitro analysis of the clonal proliferation potential of 411 individual sorted long-term (LT)-HSCs revealed a 2-fold reduction (p<0.0001) in the total number of progeny produced per HSC. Additionally, cell fate tracking experiments showed accelerated entry into first division and differentiation following treatment. In line with this data, competitive repopulation assays demonstrated a progressive depletion of functional HSC numbers, with an approximate 2-fold decrease in multi-lineage competitive repopulating activity with each additional round of inflammatory challenge (p<0.01). In order to assess in vivo recovery of HSCs following inflammatory challenge, competitive and limiting dilution transplantation assays were used to quantify HSC frequencies using BM harvested from mice at 5, 10 or 20wk after 3 rounds of pI:C treatment. In both assays we observed a sustained ~18 fold decrease in functional HSCs, with no evidence of recovery within the 20wk window. To exclude microenvironment effects on HSC function, we performed reverse transplantation experiments in which pI.C challenged WT mice were injected with saturating doses of LT-HSCs from non-treated WT donors, in the absence of additional irradiation conditioning. We observed a durable suppression of endogenous HSCs that was sufficient to facilitate robust engraftment of donor LT-HSCs up to 20wk post-treatment. We next used the inducible transgenic Scl-tTA;H2B-GFP mouse model (Wilson et al., Cell, 2008) in order to prospectively segregate quiescent label retaining LT-HSCs (LRCs) from LT-HSCs that proliferate in vivo in response to pI:C (nonLRCs). Following a single round of pI:C challenge, label retention was reduced as a result of LT-HSC proliferation (Table 1). Importantly, the clonal proliferative potential of individual LRCs was preserved upon pI:C challenge while that of nonLRCs was more than halved. This suggests that LT-HSCs fail to undergo self-renewal divisions in vivo under these conditions but rather are functionally compromised in line with increasing proliferative history. We hypothesized that this apparent progressive irreversible depletion of functional HSCs may eventually lead to compromised hematopoiesis. We therefore assessed the hematologic parameters of aged mice that had been exposed to repetitive pI:C treatment in early to mid-life. While these mice had normal PB counts at 5wk post-treatment, upon reaching 2 years of age, treated mice demonstrated mild PB cytopenias, BM hypocellularity and a relative expansion of BM adipocytes (Table 2). Taken together, our data contradict the canonical view that HSCs demonstrate extensive self-regenerative capacity following injury. Rather, in the context of inflammatory challenge, HSCs are progressively and irreversibly depleted as they are driven out of their quiescent state. These findings have broad implications regarding the role of inflammation in the suppression of hematopoiesis that are likely relevant to BMF and also normal aging. Disclosures Lipka: InfectoPharm GmbH: Employment. Frenette:Pfizer: Consultancy; Cygnal Therapeutics: Equity Ownership; Ironwood Pharmaceuticals: Research Funding; Albert Einstein College of Medicine, Inc: Patents & Royalties.

Hairy Cell Leukemia Presenting as Presternal Soft Tissue Mass: A Case Report With Review of Literature

Abstract Hairy cell leukemia (HCL) is an uncommon chronic lymphoproliferative disorder. Initial presentation frequently includes symptoms related to peripheral blood cytopenias. It can sometimes have atypical manifestations and can present at unusual sites. We report an unusual presentation of HCL as a presternal soft tissue mass in a 45-year-old man. His peripheral blood counts showed pancytopenia. There were no palpable lymph nodes or hepatosplenomegaly. CT-scan revealed a middle mediastinal mass with extension into the presternal soft tissue. Tissue core biopsy was taken and histopathologic findings confirmed the diagnosis of HCL. To our knowledge, this is the second reported case of HCL presenting as a presternal soft tissue mass. Although rare, HCL should be considered in the differential diagnosis of tumors involving extramedullary/extranodal sites including the soft tissue and bone, so the patient can get the utmost benefit for early diagnosis of a treatment responsive disease.

The impact of missense mutation in PIGA associated to paroxysmal nocturnal hemoglobinuria and multiple congenital anomalies-hypotonia-seizures syndrome 2: A computational study

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal blood disorder that manifests with hemolytic anemia, thrombosis, and peripheral blood cytopenias. The disease is caused by the deficiency of two glycosylphosphatidylinositols (GPI)-anchored proteins (CD55 and CD59) in the hemopoietic stem cells. The deficiency of GPI-anchored proteins has been associated with the somatic mutations in phosphatidylinositol glycan class A (PIGA). However, the mutations that do not cause PNH is associated with the multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2). To best of our knowledge, no computational study has been performed to explore at an atomistic level the impact of PIGA missense mutations on the structure and dynamics of the protein. Therefore, we focused our study to provide molecular insights into the changes in protein structural dynamics upon mutation. In the initial step, screening for the most pathogenic mutations from the pool of publicly available mutations was performed. Further, to get a better understanding, pathogenic mutations were mapped to the modeled structure and the resulting protein was subjected to 100 ns molecular dynamics simulation. The residues close to C- and N-terminal regions of the protein were found to exhibit greater flexibility upon mutation. Our study suggests that four mutations are highly effective in altering the structural conformation and stability of the PIGA protein. Among them, mutant G48D was found to alter protein's structural dynamics to the greatest extent, both on a local and a global scale.

The evolving role of next generation sequencing in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are clonal haematological disorders characterized by haematopoietic cell dysplasia, peripheral blood cytopenias, and a predisposition for developing acute myeloid leukaemia (AML). Cytogenetics have historically been important in diagnosis and prognosis in MDS, but the growing accessibility of next generation sequencing (NGS) has led to growing research in the roles of molecular genetic variation on clinical decision-making in these disorders. Multiple genes have been previously studied and found to be associated with specific outcomes or disease types within MDS and knowledge of mutations in these genes provides insight into previously defined MDS subtypes. Knowledge of these mutations also informs development of novel therapies in the treatment of MDS. The precise role of NGS in the diagnosis, prognosis and monitoring of MDS remains unclear but the improvements in NGS technology and accessibility affords clinicians an additional practice tool to provide the best care for patients.

Abstract 3026: Rigosertib (RIG) modulates MAPK and hematopoiesis signaling and synergizes Azacitidine (AZA) altering viral mimicry pathway in myelodysplastic syndrome (MDS)

Abstract MDS is a heterogeneous stem cell disorder characterized by hyperproliferative bone marrow (BM) and peripheral blood cytopenias involving one or more lineages. AZA, a hypomethylating agent (HMA) is considered 1st line therapy for higher-risk disease. About 50% of MDS patients (pts) respond to AZA, with a median response of 14-24 month (Silverman Cancer Med 9th ed). For pts responding to AZA, most either relapse or progress with worsening BM failure and have a median survival of 4-6 months. Both primary and secondary resistance remains a significant challenge and results in poor survival. We previously reported that RIG, a ras-mimetic identified as a novel anticancer drug, inhibits cell cycle progression, induces apoptosis and acts as a HDAC inhibitor with chromatin modifying activity. In a Phase I/II study the combination of RIG+AZA produced an 85% overall response rate in pts who were HMA naive and 62% in HMA failures (Navada EHA 2017). The ability to reverse the resistance phenotype is a novel observation with clinical implications. In this study, we investigated the effect of AZA and RIG alone or in sequential combination (RIG/AZA; AZA/RIG) (SC) on MDS-L cell line to identify the mechanism of action of the drugs. QPCR array was used to identify the differential gene expression profile. Maximum gene alteration was observed (±2 fold change) on treatment with RIG alone, AZA/RIG and RIG/AZA with differential expression of 20 (14up+6dn), 22 (16up+6dn) and 21 (16up+5dn) genes, respectively. However, AZA alone showed minimal effect with dysregulation, the expression of only 2 genes (1up+1dn). Further, gene expression with 1.5 fold change was used for biological and functional analysis. Interestingly, hematopoiesis cell lineage and JAK-STAT signaling were the pathways most affected in RIG alone, and both the SCs. MAPK signaling is impacted in cells treated with RIG alone and AZA/RIG. This suggests that RIG plays a crucial role in modulating these pathways. However, cytokine-cytokine receptor interactions were impacted only in cells treated with both SCs. RIG-I like receptor signaling was specifically observed only in RIG/AZA. This pathway along with Toll-like receptor (TLR) signaling in RIG/AZA suggests the involvement of viral mimicry. Moreover, TLR signaling dysregulation with AZA also suggests association with viral mimicry and impacts the cell cycle pathway. These results indicate that RIG either alone or in combination acts via the MAPK signaling pathway and impacts hematopoietic signaling. In contrast, AZA had no impact on these signaling pathways. This suggests that RIG in combination with AZA affects important signaling pathways impacting hematopoiesis and may explain the synergy seen in MDS pts. Further study is needed to understand the mechanism of resistance to AZA in MDS patients and identify the potential target for reversal of drug resistance. Citation Format: Stella M. Melana, Richa Rai, Shyamala C. Navada, Rosalie Odchimar-Reissig, Erin P. Demakos, E. Premkumar Reddy, Lewis R. Silverman. Rigosertib (RIG) modulates MAPK and hematopoiesis signaling and synergizes Azacitidine (AZA) altering viral mimicry pathway in myelodysplastic syndrome (MDS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3026.

Biological versus Clinical Risk Factors in Acute Myeloid Leukemia: Is There a Winner?

We present a case of a patient with a three-month history of peripheral blood cytopenia without a confirmed diagnosis of myelodysplastic syndrome, who developed a favourable-risk acute myeloid leukemia (AML), according to the European Leukemia Net (ELN) criteria. The patient achieved a complete remission with incomplete platelet recovery (CRi) after induction. The patient achieved the morphological CR after the first consolidation and completed the first-line treatment with a syngeneic stem cell transplantation (SCT). A disease relapse occurred after one year of CR (blast cell count in the bone marrow 15%), and the patient was offered a haplo-SCT, which he refused due to personal reasons. In this paper, we discuss the interplay between clinical and biological risk factors in non-high-risk AML patients and speculate that some old clinical risk factors (e.g., age of the patient, achievement of CR after induction, and previous history of myelodysplastic syndrome) may still impact on the treatment decision algorithm of some of these patients.

PB1864 MORE THAN 2 YEARS DELAY IN DIAGNOSIS OF PNH DUE TO GASTROINTESTINAL MASK OF BONE MARROW INSUFFICIENCY

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is an ultrarare bone marrow insufficiency of childhood consisted of the triad of diagnostic symptoms: hemolytic anemia, thrombosis, and peripheral blood cytopenias. Acute abdominal pain is one of the most important prodrome of PNH mostly due to venous thrombosis of intra-abdominal veins, including hepatic, portal, mesenteric, and splenic blood vessels. Aims: To show how a complicated course of diseases due to “abdominal mask” resulted in more than 2 years delay in diagnosis might be fatal to a PNH patients. Methods: We report a boy of 15th, suffered from PNH, who presented long before the diagnosis with seasonal (spring and autumn only) paroxysmal acute and severe (8-9 VAS score) abdominal pain with cramps and tenderness and no other hematological presentation of disease. There were a couple attacks observed during the season with complete resolution of abdominal symptoms in between. He had been diagnosed as acute appendicitis, peptic ulcer disease, pancreatitis or cholecystitis especially when hyperbilirubinemia emerge, Crohn and atypical celiac disease for more than 2 years. Since a peripheral pancytopenia followed by severe anemia required erythrocyte transfusions during the last 2 attacks occurred, hematologist had been asked for consultation. Due to severe anemia with reticulocytosis, hyperbilirubinemia, highly elevated LDH, meaningful splenomegaly and almost nondetectable concentration of serum haptoglobin and a dark urine with confirmed hemoglobinuria, with a weak bone marrow aplasia, the diagnosis of PNH was feasible. The cytometric results, which revealed no expression of GPI proteins in 93.8% of type III PMNs, confirmed the diagnosis (percentage of RBC lacking GPI was not persuasive, as he has been previously transfused). Up to this point of his disease, except of transient, he had no fixed postembolic lessons in liver and kidney, and a negligible bowel signs revealed for the several time in CT/MR-angiography. He had no other embolic events in other organs as well. Results: As the results of HSCT are questionable in PNH, especially in children, we decided to start eculizumab. The complete clinical remission assessed as complete symptoms resolution had been achieved with the 2nd dose, and complete biochemical remission, based on LDH normalization, was confirmed with the 3rd dose of a MoAb therapy. The presence and percentage of PNH clones tested twice since diagnosis in both PNMs and RBCs did not change till now. We have not observed any complications during a year and half of treatment up to date of publication. Summary/Conclusion: The insidious and hidden course of thrombosis of intra-abdominal veins even accompanied by hemolysis may mask the real reason and significantly prolong the diagnosis of ultrarare disease such as a PNH, especially in children. Eculizumab is a treatment of choice of childhood PNH.

PS1333 OVEREXPRESSION OF PROTEIN TYROSINE PHOSPHATASE NON-RECEPTOR TYPE1 CAUSES STAT5 DEPHOSPHORYLATION, RESULTING IN SUPPRESSION OF CELL GROWTH SIGNAL IN K562 HUMAN LEUKEMIA CELLS

Background: The Myelodysplastic Syndrome (MDS) is a disorder derived from an aberrant pluripotent stem cell, characterized clinically by hyperproliferative bone marrow and peripheral blood cytopenias involving one or more lineages. Azacitidine (AZA), a hypomethylating agent (HMA) is consideredfirst line therapy for patients (pts) with higher-risk disease. AZA is associated with a response rate of 50% and is associated with an increase in overall survival of pts with higher-risk MDS with a median survival of 14–24 months (Silverman et al. JCO 2002; Fenaux et al. Lancet Oncol 2009). Almost all pts treated with AZA develop resistance. Those pts who do not respond initially (primary resistance), and, almost all pts who do respond to AZA relapse (secondary resistance) and develop either bone marrow failure or transform to acute myeloid leukemia. Median survival after HMA failureis only 4–6 months (Prebet et al., 2011). Both primary and secondary resistance remain a significant challenge and result in poor survival. Rigosertib (RIGO) is a Ras-mimetic that interferes with Ras/Raf signaling (Athuluri-Divakar et al, 2016) and also acts as a histone deacetylase inhibitor with chromatin modifying activity (Chaurasia ASCO 2016). The combination of RIGO/AZA had been shown to have a synergistic effect in a sequence dependent manner in vitro(Skiddan et al 2006). A phase I/II study demonstrated that the combination RIGO/AZA can reverse the clinical resistance phenotype in pts failing an HMA with an overall response rate of 90% in HMA naïve pts and 54% in pts failing an HMA (Navada et al, ASH 2018). The ability to reverse the clinical resistance phenotype is a novel observation with clinical implications. Aims: To investigate the effect of AZA and RIGO alone or in sequential combination (RIGO/AZA; AZA/RIGO) (SC) on the MDS-L cell line to identify the mechanism of action of the drugs. Methods: QPCR array was used to identify the differential gene expression profile. Results: Maximum gene alteration was observed (±2 fold change) following treatment with RIGO alone, AZA/RIGO and RIGO/AZA with differential expression of 20 (14up+6dn), 22 (16up+6dn) and 21 (16up+5dn) genes, respectively. AZA alone showed minimal effect on gene expression with altered expression of only 2 genes (1up+1dn). Gene expression with 1.5 fold change was used for the biological and functional analysis. Hematopoietic cell lineage and JAK-STAT signaling pathways were most affected by RIGO alone, and the SCs. MAPK signaling is impacted in cells treated with RIGO alone and by AZA/RIGO suggesting that RIGO plays a crucial role in modulating these pathways. Cytokine-cytokine receptor interactions were impacted only in cells treated by both SCs. The Retinoic acid inducible gene-1 (RIG-1)like receptor signaling was upregulated only by RIGO/AZA sequence. Activation of this pathway along with upregulation of Toll-like receptor (TLR) signaling by RIGO/AZA suggests the involvement of viral mimicry. Moreover, TLR signaling dysregulation with AZA also suggests an association with viral mimicry and AZA also impacts the cell cycle pathway. Summary/Conclusion: These results indicate that RIGO either alone or in combination with AZA may act via the MAPK signaling pathway to impact hematopoietic signaling. RIGO in combination with AZA activates the anti-viral defense pathway and affects TLR and RIG-1 signaling which may explain the synergy seen in MDS pts. Further in-depth study is needed to understand the mechanism of action of resistance to AZA in MDS pts and identify potential targets for the reversal of drug resistance.

PS1332 RIGOSERTIB MODULATES HEMATOPOIESIS GENE SIGNALING PATHWAYS ALONE OR IN SEQUENCED COMBINATION WITH AZACTIDINE ON MDS CELLS IN VITRO

Background:The Myelodysplastic Syndrome (MDS) is a disorder derived from an aberrant pluripotent stem cell, characterized clinically by hyperproliferative bone marrow and peripheral blood cytopenias involving one or more lineages. Azacitidine (AZA), a hypomethylating agent (HMA) is consideredfirst line therapy for patients (pts) with higher‐risk disease. AZA is associated with a response rate of 50% and is associated with an increase in overall survival of pts with higher‐risk MDS with a median survival of 14–24 months (Silverman et al. JCO 2002; Fenaux et al. Lancet Oncol 2009). Almost all pts treated with AZA develop resistance. Those pts who do not respond initially (primary resistance), and, almost all pts who do respond to AZA relapse (secondary resistance) and develop either bone marrow failure or transform to acute myeloid leukemia. Median survival after HMA failureis only 4–6 months (Prebet et al., 2011). Both primary and secondary resistance remain a significant challenge and result in poor survival.Rigosertib (RIGO) is a Ras‐mimetic that interferes with Ras/Raf signaling (Athuluri‐Divakar et al, 2016) and also acts as a histone deacetylase inhibitor with chromatin modifying activity (Chaurasia ASCO 2016). The combination of RIGO/AZA had been shown to have a synergistic effect in a sequence dependent manner in vitro(Skiddan et al 2006). A phase I/II study demonstrated that the combination RIGO/AZA can reverse the clinical resistance phenotype in pts failing an HMA with an overall response rate of 90% in HMA naïve pts and 54% in pts failing an HMA (Navada et al, ASH 2018). The ability to reverse the clinical resistance phenotype is a novel observation with clinical implications.Aims:To investigate the effect of AZA and RIGO alone or in sequential combination (RIGO/AZA; AZA/RIGO) (SC) on the MDS‐L cell line to identify the mechanism of action of the drugs.Methods:QPCR array was used to identify the differential gene expression profile.Results:Maximum gene alteration was observed (±2 fold change) following treatment with RIGO alone, AZA/RIGO and RIGO/AZA with differential expression of 20 (14up+6dn), 22 (16up+6dn) and 21 (16up+5dn) genes, respectively. AZA alone showed minimal effect on gene expression with altered expression of only 2 genes (1up+1dn). Gene expression with 1.5 fold change was used for the biological and functional analysis. Hematopoietic cell lineage and JAK‐STAT signaling pathways were most affected by RIGO alone, and the SCs. MAPK signaling is impacted in cells treated with RIGO alone and by AZA/RIGO suggesting that RIGO plays a crucial role in modulating these pathways. Cytokine‐cytokine receptor interactions were impacted only in cells treated by both SCs. The Retinoic acid inducible gene‐1 (RIG‐1)like receptor signaling was upregulated only by RIGO/AZA sequence. Activation of this pathway along with upregulation of Toll‐like receptor (TLR) signaling by RIGO/AZA suggests the involvement of viral mimicry. Moreover, TLR signaling dysregulation with AZA also suggests an association with viral mimicry and AZA also impacts the cell cycle pathway.Summary/Conclusion:These results indicate that RIGO either alone or in combination with AZA may act via the MAPK signaling pathway to impact hematopoietic signaling. RIGO in combination with AZA activates the anti‐viral defense pathway and affects TLR and RIG‐1 signaling which may explain the synergy seen in MDS pts. Further in‐depth study is needed to understand the mechanism of action of resistance to AZA in MDS pts and identify potential targets for the reversal of drug resistance.

Genetic abnormalities and pathophysiology of MDS.

Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid malignancies characterized by peripheral blood cytopenia and dishematopoiesis and frequently progress to acute myeloid leukemia. Genetic defects play a major role in pathogenesis of MDS, including cytogenetic abnormalities, gene mutations, and abnormal gene expression. Chromosomal abnormalities have been detected in approximately 50-60% of MDS patients, including the deletions of chromosome 5q and 7q, trisomy 8, and complex karyotypes. Newer genomic technologies, such as single-nucleotide polymorphism array and next-generation sequencing, revealed the heterozygous deletions resulting in haploinsufficient gene expression (e.g., CSNK1A1, DDX41 on chromosome 5, CUX1, LUC7L2, EZH2 on chromosome 7) involved in the pathogenesis of MDS. In addition, recurrent somatic mutations in more than 50 genes have been identified in 80-90% of MDS. The most recurrent genetic mutations are involved in the RNA splicing (e.g., SF3B1, SRSF2, U2AF1, ZRSR2, LUC7L2, DDX41) and epigenetic modifications, such as histone modification (e.g., ASXL1, EZH2) and DNA methylation (e.g., TET2, DNMT3A, IDH1/IDH2). TP53 mutation is associated with aggressive disease and frequently coincides with deletion of chromosome 5q. This review summarizes the recent progress in molecular pathogenesis of MDS. A better understanding of the specific subgroups of MDS patients will also aid in the development of new therapeutic approach for MDS.

Flow cytometric analysis of neutrophil myeloperoxidase expression in peripheral blood for ruling out myelodysplastic syndromes: a diagnostic accuracy study.

Suspicion of myelodysplastic syndromes (MDS) is one of the commonest reasons for bone marrow aspirate in elderly patients presenting with persistent peripheral blood (PB) cytopenia of unclear etiology. A PB assay that accurately rules out MDS would have major benefits. The diagnostic accuracy of the intra-individual robust coefficient of variation (RCV) for neutrophil myeloperoxidase (MPO) expression measured by flow cytometric analysis in PB was evaluated in a retrospective derivation study (44 MDS cases and 44 controls) and a prospective validation study (68 consecutive patients with suspected MDS). Compared with controls, MDS cases had higher median RCV values for neutrophil MPO expression (40.2% vs. 30.9%; P<0.001). The area under the receiver operating characteristic curve estimates were 0.94 [95% confidence interval (CI): 0.86-0.97] and 0.87 (95%CI: 0.76-0.94) in the derivation and validation studies, respectively. A RCV lower than 30% ruled out MDS with 100% sensitivity (95%CI: 78-100%) and 100% negative predictive value (95%CI: 83-100%) in the prospective validation study. Neutrophil MPO expression measured by flow cytometric analysis in PB might obviate the need for invasive bone marrow aspirate and biopsy for up to 29% of patients with suspected MDS.

Aplastic Anemia & MDS International Foundation (AA&MDSIF): Bone Marrow Failure Disease Scientific Symposium 2018

The bone marrow failure (BMF) syndromes are a group of rare disorders characterized by ineffective hematopoiesis resulting from deficiencies in the hematopoietic stem cell compartment. Although these diseases are typically acquired, some forms (e.g., Fanconi anemia, dyskeratosis congenita, Diamond Blackfan anemia, and Shwachman Diamond syndrome) are inherited. Patients with BMF syndromes can develop peripheral blood cytopenias and pancytopenia, and their disease can ultimately progress to acute myelogenous leukemia (AML).Research around the world is shedding light on the biology of the BMF syndromes, their clinical effects, and novel treatments. The Aplastic Anemia and MDS International Foundation (AAMDSIF) is an independent nonprofit organization whose mission is to help patients and family members cope with BMF syndromes. This report summarizes presentations on the latest scientific discoveries in BMF syndromes from the Sixth International Bone Marrow Failure Disease Scientific Symposium sponsored by AAMDSIF on March 22–23, 2018, in Rockville, Maryland.

CIRCULATING microRNA EXPRESSION IN MYELODYSPLASTIC SYNDROME

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis and cytopenia in peripheral blood, where about a third of patients may develop acute myeloid leukemia (AML). The diagnosis of MDS requires the analysis of peripheral blood and bone marrow. Depending on the percentage of blasts in the bone marrow, the number of cytopenias and cytogenetic abnormalities, determination of the prognostic indices is possible (IPSS – „International Prognostic Scoring System“, R-IPSS-„Revised International Prognostic Scoring System“, WPSS – „WHO Prognostic Scoring System“). Until today, numerous studies have been conducted on the molecular mechanisms and epigenetic pathways in myelodysplastic syndrome, and their prognostic and therapeutic importance, but there are few studies analyzing the importance of microRNAs (miRNAs) in MDS. In the last few years, there have been numerous results on the impact of aberrant miRNA expression in malignant disorders where the miRNA represent tumor suppressor genes or oncogenes. Several miRNAs have been recognized as diagnostic and prognostic parameters and possible therapeutic targets. In this paper, we present the overview of recent results on the role of miRNA in MDS.

FLOW CYTOMETRY IN MYELODYSPLASTIC SYNDROME. STEP FORWARD.

Myelodysplastic syndrome (MDS) represents a heterogeneous group of clonal diseases originating from altered (malignant) hematopoietic stem cells. Clinically, it manifests as cytopenia in peripheral blood, dysplastic changes in one or more cell lines in bone marrow and increased risk of evolution to acute myeloid leukemia (AML). Acquired chromosomal aberrations are detected in 40-50% of MDS patients. According to WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (Lion 2008), MDS diagnosis is based on the morphological interpretation of found dysplastic changes, the number of blasts, the presence of ring sideroblasts in bone marrow and the established cytogenetic abnormalities. The progress made in recent years in immunophenotyping of hematopoietic progenitor cells and mature cells of dysplastic bone marrow gives to multiparameter flow cytometric analysis (MFC), although initially included as an optional technique, an opportunity to become a standard part of the integrated MDS diagnosis and prognosis.

Clinicohematological profile of patients with peripheral blood cytopenias in clinical practice

BACKGROUND: In clinical practice, the cytopenias may result from relatively benign causes such as viral infections and Vitamin B12/folic acid deficiency to more sinister causes such as bone marrow failure. In this study, we looked into the clinicohematological profile and etiological factors of bicytopenia and pancytopenia. OBJECTIVES: To study the etiology and clinicohematological profile in patients of peripheral blood cytopenias. MATERIALS AND METHODS: This was a cross-sectional study conducted at a tertiary care hospital over a period of 1 year. Cytopenias were defined as pancytopenia when there was simultaneous presence of hemoglobin RESULTS: A total of 204 patients (103 males and 101 females) were diagnosed to have cytopenias. Pancytopenia was observed in 69/204 and bicytopenia was seen in 135/204 cases. The various causes of cytopenias included infections (n = 126 [61.76%]), megaloblastic anemia (MA) (n = 48 [23.52%]), drugs (n = 12 [5.8%]), hypersplenism (n = 8 [3.9%]), bone marrow failure syndromes such as aplastic anemia and myelodysplastic syndrome (n = 7 [3.4%]) and leukemias (n = 3 [1.4%]). We found a significant association between MA and pancytopenia (odds ratio [OR] = 2.47, P CONCLUSION: The present study concluded that infections and MA are the most common cause of bicytopenia and pancytopenia, respectively. The more serious disorders affecting the bone marrow constitute only