The Influence of Precursor Depletion and dose Regimens on Resistance to Erythropoiesis-Stimulating Agents: Insights from Simulations with Instantaneous Dose-Adaptation Algorithm.

Erythropoiesis-stimulating agents (ESAs) have been a common treatment for anemia associated with chronic kidney disease (CKD), while 10-20 % of patients continue to suffer from persistent anemia despite receiving ESA treatments. Our previous findings suggested that intensive ESA usage can cause resistance by depleting the erythroid precursor cells. Here, we used a mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model of ESAs and conducted simulations to evaluate the influence of dose regimens and other factors (such as administration route, individual PK/PD parameters, types of ESAs, and disease status) on ESA resistance with instantaneous dose adaptations in healthy populations and anemic patients. The simulated results show that instantaneous dose-adaptation can reduce ESA resistance, but up to 30 % of subjects still ended up developing ESA resistance in healthy populations. The Smax is markedly higher in hypo-responders than in normal-responders, while hypo-responders possess fewer precursors and experience a faster decline compared to normal-responders. There is a ceiling effect of increasing ESA dosage to improve HGB responses and reduce ESA resistance, and the limit is lower in anemic patients compared to healthy populations. Subcutaneous administrations and ESAs with longer half-lives lead to stronger HGB responses and less resistance at equivalent doses. Taken together, this study indicates that precursor depletion contributes to ESA resistance and dose regimens can greatly influence the occurrence of ESA resistance. Furthermore, ESA treatment for patients showing ESA resistance should avoid continuously increasing doses and instead consider stimulating the renewal of precursors.

Thrombopoietin treats erythropoietin resistance by correcting EPO-induced progenitorcell depletion.

Recombinant human erythropoietin (rHuEPO) is a prevalent treatment for anemia in patients with chronic kidney disease. However, up to 10% of these patients exhibit EPO resistance or hyporesponsiveness, which may be caused by the depletion of erythroid progenitor cells. Thrombopoietin (TPO) has the potential to promote the growth of early progenitor cells and correct the depletion. In this study, we investigate the efficacy and the underlying mechanism of the combination therapy of TPO and EPO to EPO resistance. First, the in vivo studies suggested that intensive EPO treatment induced progenitor cell depletion in the bone marrow, where the depletion was corrected by TPO. Then, colony assays showed that EPO and TPO synergistically enhanced the burst-forming unit-erythroid (BFU-E) production but antagonistically boosted the colony-forming units of megakaryocytes (CFU-MK) production. Also, we found TPO promoted hematopoietic stem and progenitor cells (HSPCs) production, while EPO drove HSPCs toward the erythroid lineage. Additionally, EPO induced more megakaryocytic-erythroid progenitors (MEPs) toward the erythroid output. Model-based simulations indicate the efficacy of this combination therapy for treating EPO-resistant anemia in rats. In conclusion, our study demonstrated the efficacy of combination therapy in addressing EPO-resistant anemia by correcting EPO-induced erythroid progenitor depletion.

Novel Combination of Erythropoietin and Romiplostim to Treat Chemotherapy-Induced Anemia and Thrombocytopenia via Pharmacodynamic Interaction on Hematopoietic Stem and Progenitor Cells.

Chemotherapy-induced anemia and thrombocytopenia (CIAT) in cancer patients are often caused by the damage of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. We have previously shown that romiplostim, a thrombopoietin receptor agonist that could stimulate the expansion of HSPCs, could synergize with recombinant human erythropoietin (rHuEPO) to promote erythropoiesis in addition to stimulating platelet production, whereas rHuEPO could influence the platelet count through stem cell competition. Therefore, we hypothesize that a combination of romiplostim with rHuEPO can alleviate CIAT simultaneously, while minimizing the risk of thrombosis. In this study, we demonstrated that rHuEPO and romiplostim exhibit no stimulatory effects on the growth and invasion of LA-7 cancer cells both in vitro and in vivo. Using a rat model with carboplatin-induced anemia and thrombocytopenia, we showed that the red blood cells and hemoglobin concentration recovered faster, and the secondary thrombocytopenia was alleviated in the rHuEPO and romiplostim combination therapy groups compared with the corresponding rHuEPO monotherapy groups. The rebound phenomenon of platelets was inhibited compared with the romiplostim monotherapy group. In vitro study further demonstrated that romiplostim expands HSPCs and synergizes with rHuEPO to promote erythropoiesis, while rHuEPO inhibited megakaryopoiesis. Furthermore, we developed a mechanism-based pharmacokinetic-pharmacodynamic model to quantify the effects of the two drugs. This study suggests that rHuEPO and romiplostim combination therapy can treat CIAT simultaneously in rats while minimizing the risk of thrombosis, indicating that combination therapy might be superior to monotherapy in the supportive therapy of cancer patients undergoing chemotherapy.

SPINK2 Protein Expression Is an Independent Adverse Prognostic Marker in AML and Is Potentially Implicated in the Regulation of Ferroptosis and Immune Response.

There is an urgent need for the identification as well as clinicopathological and functional characterization of potent prognostic biomarkers and therapeutic targets in acute myeloid leukemia (AML). Using immunohistochemistry and next-generation sequencing, we investigated the protein expression as well as clinicopathological and prognostic associations of serine protease inhibitor Kazal type 2 (SPINK2) in AML and examined its potential biological functions. High SPINK2 protein expression was an independent adverse biomarker for survival and an indicator of elevated therapy resistance and relapse risk. SPINK2 expression was associated with AML with an NPM1 mutation and an intermediate risk by cytogenetics and European LeukemiaNet (ELN) 2022 criteria. Furthermore, SPINK2 expression could refine the ELN2022prognostic stratification. Functionally, an RNA sequencing analysis uncovered a potential link of SPINK2 with ferroptosis and immune response. SPINK2 regulated the expression of certain P53 targets and ferroptosis-related genes, including SLC7A11 and STEAP3, and affected cystine uptake, intracellular iron levels and sensitivity to erastin, a specific ferroptosis inducer. Furthermore, SPINK2 inhibition consistently increased the expression of ALCAM, an immune response enhancer and promoter of T-cell activity. Additionally, we identified a potential small-molecule inhibitor of SPINK2, which requires further characterization. In summary, high SPINK2 protein expression was a potent adverse prognostic marker in AML and might represent a druggable target.

Scaling Pharmacodynamics from Rats to Humans to Support Erythropoietin and Romiplostim Combination Therapy to Treat Erythropoietin-Resistant Anemia.

Recombinant human erythropoietin (rHuEPO) is one of the most effective drugs for the treatment of anemia in patients with chronic kidney disease. However, EPO-resistance is an important contributor to the increased risk of adverse effects. We previously showed that EPO treatment could induce precursor cell depletion, resulting in EPO-resistance. We further found that the combination of EPO with romiplostim, a thrombopoietin receptor agonist that can stimulate the expansion of hematopoietic stem cells, can treat EPO-resistance. In this study, we performed interspecies pharmacodynamic (PD) scaling of this combination therapy for human dose prediction. The pharmacokinetic parameters of both rHuEPO and romiplostim in humans were obtained from previous studies. The PD parameters obtained in rats were scaled to humans using allometric equations. The relationship between PD parameters of the megakaryocyte lineage from rats, monkeys, and humans was in agreement with those from the literature on allometric scaling. The PD response was translated to humans based on allometric scaling and agreed with the observed data. These parameters were used to simulate hemoglobin and platelet response in humans. RHuEPO 50 IU/kg thrice weekly and romiplostim 1 µg/kg once every 4 weeks from the second week is the recommended combination dosing regimen according to the model prediction. Our work successfully scaled the PD of rHuEPO and romiplostim monotherapy from rats to humans. The predicted dosing regimen of each drug in the combination therapy is less intensive than the approved starting dose of each drug, which supports additional evaluations of the combination therapy in humans.

Pharmacogenomic Profiling of Pediatric Acute Myeloid Leukemia to Identify Therapeutic Vulnerabilities and Inform Functional Precision Medicine.

Despite the expanding portfolio of targeted therapies for adults with acute myeloid leukemia (AML), direct implementation in children is challenging due to inherent differences in underlying genetics. Here we established the pharmacologic profile of pediatric AML by screening myeloblast sensitivity to approved and investigational agents, revealing candidates of immediate clinical relevance. Drug responses ex vivo correlated with patient characteristics, exhibited age-specific alterations, and concorded with activities in xenograft models. Integration with genomic data uncovered new gene-drug associations, suggesting actionable therapeutic vulnerabilities. Transcriptome profiling further identified gene-expression signatures associated with on- and off-target drug responses. We also demonstrated the feasibility of drug screening-guided treatment for children with high-risk AML, with two evaluable cases achieving remission. Collectively, this study offers a high-dimensional gene-drug clinical data set that could be leveraged to research the unique biology of pediatric AML and sets the stage for realizing functional precision medicine for the clinical management of the disease. SIGNIFICANCE: We conducted integrated drug and genomic profiling of patient biopsies to build the functional genomic landscape of pediatric AML. Age-specific differences in drug response and new gene-drug interactions were identified. The feasibility of functional precision medicine-guided management of children with high-risk AML was successfully demonstrated in two evaluable clinical cases. This article is highlighted in the In This Issue feature, p. 476.

Mutational spectrum and prognosis in Chinese patients with prefibrotic primary myelofibrosis.

Prefibrotic primary myelofibrosis (Pre-PMF) has been classified as a separate entity of myeloproliferative neoplasms (MPNs). Pre-PMF is clinically heterogeneous but a specific prognostic model is lacking. Gene mutations have emerged as useful tools for stratification of myelofibrosis patients. However, there have been limited studies comprehensively investigating the mutational spectrum and its clinicopathological significance in pre-PMF subjects. In this study, we addressed these issues by profiling the mutation status of 141 genes in 172 Chinese MPN patients including 72 pre-PMF cases. Our findings corroborated the clinical/molecular distinctiveness of pre-PMF and suggested a refined risk classification strategy for this entity.

A Novel Combination Therapy of Erythropoietin and Thrombopoietin to Treat Erythropoietin-Resistance anemia.

PURPOSE: Treatment with recombinant human erythropoietin (rHuEPO) may correct anemia in patients with chronic kidney disease. However, up to 10% of these patients exhibit EPO resistance or hyporesponsiveness, which may be caused by the depletion of erythroid progenitor cells. Thrombopoietin (TPO) stimulates the self-renewal of stem cells and promotes the growth of early erythroid progenitor cells. The objective of this study was to determine whether the combination of recombinant human TPO (rHuTPO) and rHuEPO could correct the depletion of erythroid precursor cells to treat EPO-resistant anemia. METHODS: To test our hypothesis, pharmacokinetic (PK) and pharmacodynamic (PD) studies of rHuEPO and rHuTPO were performed in healthy rats. Rats received rHuEPO or rHuTPO alone or in combination. Plasma concentrations of rHuTPO and rHuEPO were measured. PD responses, including erythropoietic and thrombopoietic responses, were assessed in peripheral blood. RESULTS: On one hand, the results demonstrated the synergistic effect of the combination of rHuEPO and rHuTPO on erythropoiesis. Compared with rHuEPO monotherapy, the combination therapies further stimulated the production of red blood cells and hemoglobin. On the other hand, rHuEPO inhibited the platelet production induced by rHuTPO and mitigate the risk of blood clots. Furthermore, we successfully developed a mechanism-based PD model to simultaneously characterize the responses of the two molecules. CONCLUSIONS: Overall, our study indicated that a combination therapy of rHuTPO and rHuEPO could be used to treat EPO-resistant anemia and provided a quantitative basis for further optimizing the combination therapy for clinical use.

Fate Determination Role of Erythropoietin and Romiplostim in the Lineage Commitment of Hematopoietic Progenitors.

Erythropoietin (EPO) and thrombopoietin (TPO) have long been known to promote erythropoiesis and megakaryopoiesis, respectively. However, the fate-changing role of EPO and TPO on megakaryocyte-erythroid progenitors (MEPs) to develop along the erythroid versus megakaryocyte lineage remains unclear. We have previously shown that EPO may have a fate-changing role because EPO treatment could induce progenitor cells depletion and result in EPO resistance. Therefore, we hypothesize that a combination of romiplostim, a TPO receptor agonist that could stimulate the expansion of progenitors, with EPO can treat EPO resistance. Using rats with anemia due to chronic kidney disease, we demonstrated that romiplostim synergized with EPO to promote red blood cells production whereas EPO inhibited platelet production in a dose-dependent manner to reduce the risk of thrombosis. Corroborating findings from in vivo, in vitro experiments demonstrated that romiplostim expanded hematopoietic stem cells and stimulated megakaryopoiesis whereas EPO drove the progenitors toward an erythroid fate. We further developed a novel pharmacokinetic-pharmacodynamic model to quantify the effects of EPO and romiplostim on megakaryopoiesis and erythropoiesis simultaneously. The modeling results demonstrated that EPO increased the differentiation rate of MEPs into burst-forming unit-erythroid cells up to 22-fold, indicating that the slight increase of MEPs induced by romiplostim could be further amplified and recruited by EPO to promote erythropoiesis. The data herein support that romiplostim in combination with EPO can treat EPO resistance. SIGNIFICANCE STATEMENT: This study clarified that erythropoietin (EPO) drives the fate of megakaryocyte-erythroid progenitors (MEPs) toward the erythroid lineage, thus reducing their megakaryocyte (MK) lineage commitment, whereas romiplostim, a thrombopoietin receptor agonist, stimulates megakaryopoiesis through the MK-committed progenitor and MEP bifurcation pathways simultaneously. These findings support an innovative combination of romiplostim and EPO to treat EPO-resistant anemia because the combination therapy further promotes erythropoiesis compared to EPO monotherapy and inhibits platelet production compared to romiplostim monotherapy.

Clofarabine, cytarabine, and mitoxantrone in refractory/relapsed acute myeloid leukemia: High response rates and effective bridge to allogeneic hematopoietic stem cell transplantation.

Clofarabine is active in refractory/relapsed acute myeloid leukemia (AML). In this phase 2 study, we treated 18- to 65-year-old AML patients refractory to first-line 3 + 7 daunorubicin/cytarabine induction or relapsing after 3 + 7 induction and high-dose cytarabine consolidation, with clofarabine (30 mg/m2 /d, Days 1-5), cytarabine (750 mg/m2 /d, Days 1-5), and mitoxantrone (12 mg/m2 /d, Days 3-5) (CLAM). Patients achieving remission received up to two consolidation cycles of 50% CLAM, with eligible cases bridged to allogeneic hematopoietic stem cell transplantation (allo-HSCT). The mutational profile of a 69-gene panel was evaluated. Twenty-six men and 26 women at a median age of 46 (22-65) years were treated. The overall response rate after the first cycle of CLAM was 90.4% (complete remission, CR: 69.2%; CR with incomplete hematologic recovery, CRi: 21.2%). Twenty-two CR/CRi patients underwent allo-HSCT. The 2-year overall survival (OS), relapse-free survival (RFS), and event-free survival (EFS) were 65.8%, 45.7%, and 40.2%, respectively. Multivariate analyses showed that superior OS was associated with CR after CLAM (P = .005) and allo-HSCT (P = .005), and superior RFS and EFS were associated with allo-HSCT (P < .001). Remarkably, CR after CLAM and allo-HSCT resulted in 2-year OS of 84.3% and 90%, respectively. Karyotypic aberrations and genetic mutations did not influence responses or survivals. Grade 3/4 neutropenia/thrombocytopenia and grade 3 febrile neutropenia occurred in all cases. Other nonhematologic toxicities were mild and uncommon. There was no treatment-related mortality and the performance of allo-HSCT was not compromised. Clofarabine, cytarabine, and mitoxantrone was highly effective and safe in refractory/relapsed AML. This study was registered at ClinicalTrials.gov (NCT02686593).

Topologic Analysis of Plasma Mitochondrial DNA Reveals the Coexistence of Both Linear and Circular Molecules.

BACKGROUND: Cellular mitochondrial DNA (mtDNA) is organized as circular, covalently closed and double-stranded DNA. Studies have demonstrated the presence of short mtDNA fragments in plasma. It is not known whether circular mtDNA might concurrently exist with linear mtDNA in plasma. METHODS: We elucidated the topology of plasma mtDNA using restriction enzyme BfaI cleavage signatures on mtDNA fragment ends to differentiate linear and circular mtDNA. mtDNA fragments with both ends carrying BfaI cleavage signatures were defined as circular-derived mtDNA, whereas those with no cleavage signature or with 1 cleavage signature were defined as linear-derived mtDNA. An independent assay using exonuclease V to remove linear DNA followed by restriction enzyme MspI digestion was used for confirming the conclusions based on BfaI cleavage analysis. We analyzed the presence of BfaI cleavage signatures on plasma DNA ends in nonhematopoietically and hematopoietically derived DNA molecules by sequencing plasma DNA of patients with liver transplantation and bone marrow transplantation. RESULTS: Both linear and circular mtDNA coexisted in plasma. In patients with liver transplantation, donor-derived (i.e., liver) mtDNA molecules were mainly linear (median fraction, 91%; range, 75%-97%), whereas recipient-derived (i.e., hematopoietic) mtDNA molecules were mainly circular (median fraction, 88%; range, 77%-93%). The proportion of linear mtDNA was well correlated with liver DNA contribution in the plasma DNA pool (r = 0.83; P value = 0.0008). Consistent data were obtained from a bone marrow transplantation recipient in whom the donor-derived (i.e., hematopoietic) mtDNA molecules were predominantly circular. CONCLUSIONS: Linear and circular mtDNA molecules coexist in plasma and may have different tissue origins.

Oral arsenic trioxide incorporation into frontline treatment with all-trans retinoic acid and chemotherapy in newly diagnosed acute promyelocytic leukemia: A 5-year prospective study.

BACKGROUND: Strategies using oral arsenic trioxide (As2 O3 ) are efficacious in relapsed acute promyelocytic leukemia (APL), but they have not been examined in newly diagnosed cases. METHODS: Sixty-two consecutive patients (24 men and 38 women) with a median age of 52 years (range, 22-85 years), 36% of whom had high-risk features, underwent induction with all-trans retinoic acid at 45 mg/m2 /d, oral As2 O3 at 10 mg/d, and ascorbic acid at 1 g/d (the all-trans retinoic acid-arsenic trioxide-ascorbic acid [AAA] regimen) for 6 weeks (with patients younger than 70 years additionally receiving daunorubicin at 50 mg/m2 /d × 3); they then underwent consolidation with 2 monthly cycles of daunorubicin (50 mg/m2 /d × 2) and cytarabine (100 mg/m2 /d × 5) and received AAA maintenance (2 weeks every 8 weeks) for 2 years. A contemporaneous cohort of 37 newly diagnosed patients (15 men and 22 women) with a median age of 51 years (range, 23-78 years), not consenting to oral As2 O3 induction but receiving similar induction, consolidation, and AAA maintenance, served as a comparator group; 46% of these patients had high-risk features. RESULTS: The oral As2 O3 induction cohort showed a complete remission (CR) rate of 100%. After a median of 37 months (range, 13-82 months), there were no relapses, so conventional risks (age, leukocyte and platelet counts, and Fms-like tyrosine kinase 3 [FLT3] mutations) were not relevant. The leukemia-free survival (LFS) and overall survival (OS) rates were 100% at 3 years and 94.1% at 5 years. The non-As2 O3 induction cohort showed a CR rate of 100%. After a median of 52 months (range, 14-77 months), there were 3 relapses (8%). Comparable patients in the oral As2 O3 induction and non-As2 O3 induction cohorts showed similar OS, but LFS was significantly superior in the oral As2 O3 induction cohort. CONCLUSIONS: The incorporation of oral As2 O3 into induction for newly diagnosed APL was safe and decreased relapses.

Prevalence and Clinicopathologic Significance of BRAF V600E Mutation in Chinese Multiple Myeloma Patients.

BACKGROUND: Previous studies in Western countries demonstrated BRAF V600E mutation only in a small subset of multiple myeloma (MM) patients. However, the prevalence and clinicopathologic significances of this mutation remain unclear in Chinese MM patients. PATIENTS AND METHODS: We studied diagnostic bone marrow samples from 205 Chinese MM patients by allele-specific PCR to detect BRAF V600E mutation and by high-resolution melting assay to detect KRAS and NRAS mutations. The mutations were confirmed by independent assays. RESULTS: BRAF V600E mutation was found in 9.3% of the cases, the highest prevalence hitherto reported. In addition, the mutation was significantly associated with hypercalcemia and a male predominance but not with aggressive extramedullary diseases or a high serum creatinine level as reported in Western studies. Importantly, BRAF V600E mutation was an adverse prognostic factor for overall survival in younger MM patients by subgroup analysis. Concurrent analysis of RAS mutations highlighted differential alteration spectrum of RAS signaling between Chinese and Western MM, which may suggest a unique myeloma-related genetic profile in Chinese patients. CONCLUSION: Our study revealed a higher prevalence of BRAF V600E mutation in Chinese MM patients. The associated prognostic impacts on younger patients could be beneficial to risk stratification and potential application of BRAF-targeted therapies in Chinese MM management. This is the first large-scale study revealing the prevalence and clinicopathologic significances of BRAF V600E mutation in Chinese myeloma.

Eltrombopag for advanced myelodysplastic syndromes or acute myeloid leukaemia and severe thrombocytopenia (ASPIRE): a randomised, placebo-controlled, phase 2 trial.

BACKGROUND: Thrombocytopenia is a life-threatening complication in patients with advanced myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). In this study (ASPIRE), we aimed to assess eltrombopag, an oral thrombopoietin receptor agonist, for thrombocytopenia (grade 4) treatment in adult patients with advanced MDS or AML. METHODS: ASPIRE consisted of an open-label, double-blind phase for 8 weeks and a randomised, double-blind phase (parts 1 and 2, reported here) for 12 weeks, and an open-label extension (part 3). Eligible patients were men and women aged 18 years or older, with intermediate-2 or high-risk MDS or AML, with bone marrow blasts of 50% or less, and had either grade 4 thrombocytopenia due to bone marrow insufficiency (platelet counts <25 × 109 per L) or grade 4 thrombocytopenia before platelet transfusion, with 25 × 109 platelets per L or greater after transfusion. Additionally, eligible patients had at least one of the following within the screening period of 4 weeks: platelet transfusion, symptomatic bleeding, or platelet count of less than 10 × 109 per L. During part 1, patients received eltrombopag, and dose-escalation criteria for part 2 were determined. In part 2, we randomly allocated patients 2:1 using an interactive voice-response system to eltrombopag or placebo, stratified by baseline platelet count (<10 × 109 platelets per L vs ≥10 × 109 platelets per L) and disease (MDS vs AML). In parts 1 and 2, patients received supportive standard of care and initiated eltrombopag or placebo at 100 mg per day (50 mg per day for patients of east-Asian heritage) to a maximum of 300 mg per day (150 mg per day for patients of east-Asian heritage). The part 2 primary objective was assessed by a composite primary endpoint of clinically relevant thrombocytopenic events (CRTE) during weeks 5-12, defined as one of the following events, either alone or in combination: grade 3 or worse haemorrhagic adverse events; platelet counts of less than 10 × 109 per L; or platelet transfusions. Efficacy analyses were based on intention to treat; clinically meaningful efficacy was defined as 30% absolute difference between groups. This trial is registered with ClinicalTrials.gov, number NCT01440374. FINDINGS: In part 1, 17 patients received eltrombopag and 11 patients completed treatment; four experienced significantly increased platelet counts, and ten had reduced platelet transfusion requirements. In part 2 we randomly allocated 145 patients to receive supportive care plus eltrombopag (n=98) or placebo (n=47); similar proportions had MDS (50 [51%] patients to eltrombopag, 22 (47%) patients to placebo) or AML (48 [49%] patients to eltrombopag, 25 [53%] patients to placebo). Average weekly CRTE proportions from weeks 5-12 were significantly lower with eltrombopag (54% [95% CI 43-64]) than with placebo (69% [57-80], odds ratio [OR] 0·20, 95% CI 0·05-0·87; p=0·032) although the difference between treatment groups was less than 30%. The most common grade 3 and grade 4 adverse events were fatigue (six [6%] in the eltrombopag group and one [2%] in the placebo group), hypokalaemia (six [6%] and two [4%]), pneumonia (five [5%] and five [11%]), and febrile neutropenia (five [5%] and six [13%]). Serious adverse events were reported in 56 (58%) eltrombopag-treated patients and 32 (68%) placebo-treated patients. Seven eltrombopag recipients and two placebo recipients had serious adverse events that were suspected to be study drug-related (eltrombopag: acute kidney injury, arterial thrombosis, bone pain, diarrhoea, myocardial infarction, pyrexia, retinal vein occlusion, n=1 each; placebo: vomiting, white blood cell count increased, n=1 each). Two eltrombopag recipients (arterial thrombosis n=1; myocardial infarction n=1) and no placebo recipients experienced fatal serious adverse events suspected to be study drug-related. INTERPRETATION: No new safety concerns were noted with eltrombopag and the trial met the primary objective of a reduction in CRTEs; eltrombopag might be a treatment option for thrombocytopenic patients with AML or MDS who are ineligible for other treatment and who are not receiving disease-modifying treatment. FUNDING: Novartis Pharma AG.

Unicentric Castleman Disease.

Unicentric Castleman disease (UCD) is a rare lymphoproliferative disorder that manifests typically as proliferation of a single lymph node or region of lymph nodes. Histologically, hyaline vascular variant is found in a majority of UCDs. UCD commonly presents in younger patient populations. Patients with UCD may be asymptomatic or present with symptoms related to mass effects on surrounding structures. It is difficult to achieve a definitive diagnosis by imaging alone. Histologic examination of the lesion remains the gold standard for diagnosis. Complete surgical resection is the best primary treatment modality for UCD resulting in excellent long-term survival and low recurrence rates.

Safety and efficacy of long-term treatment of chronic/persistent ITP with eltrombopag: final results of the EXTEND study.

In phase 2/3 trials, eltrombopag treatment of 6 months or less in patients with chronic/persistent immune thrombocytopenia (ITP) increased platelet counts and reduced bleeding. The open-label EXTEND study evaluated long-term safety and efficacy of eltrombopag in adults with ITP who had completed a previous eltrombopag study. For the 302 patients enrolled, median duration of eltrombopag treatment was 2.37 years (2 days-8.76 years). Median platelet counts increased to 50 × 109/L or more by week 2 and were sustained throughout the treatment period. Overall, 259 patients (85.8%) achieved a response (platelet count ≥50 × 109/L at least once in the absence of rescue), and 133 (52%) of 257 patients achieved a continuous response of 25 weeks or longer. Responses in patients with platelet counts lower than 15 × 109/L, more previous therapies, and/or splenectomy were somewhat lower. Thirty-four (34%) of 101 patients receiving concomitant ITP medication discontinued 1 or more medication. In patients with assessments, bleeding symptoms (World Health Organization grades 1-4) decreased from 57% at baseline to 16% at 1 year. Forty-one patients (14%) withdrew because of adverse events. Hepatobiliary adverse events (n = 7), cataracts (n = 4), deep vein thrombosis (n = 3), cerebral infarction (n = 2), headache (n = 2), and myelofibrosis (n = 2) occurred in more than 1 patient; the remaining adverse events occurred only once. Rates of thromboembolic events (6%) and hepatobiliary adverse events (15%) did not increase with treatment duration past 1 year. EXTEND demonstrated that long-term use of eltrombopag was effective in maintaining platelet counts of 50 × 109/L or more and reducing bleeding in most patients with ITP of more than 6 months' duration. Important adverse events (eg, thrombosis, hepatobiliary, and bone marrow fibrosis) were infrequent. (ClinicalTrials.gov:NCT00351468).

DNA of Erythroid Origin Is Present in Human Plasma and Informs the Types of Anemia.

BACKGROUND: There is much interest in the tissue of origin of circulating DNA in plasma. Data generated using DNA methylation markers have suggested that hematopoietic cells of white cell lineages are important contributors to the circulating DNA pool. However, it is not known whether cells of the erythroid lineage would also release DNA into the plasma. METHODS: Using high-resolution methylation profiles of erythroblasts and other tissue types, 3 genomic loci were found to be hypomethylated in erythroblasts but hypermethylated in other cell types. We developed digital PCR assays for measuring erythroid DNA using the differentially methylated region for each locus. RESULTS: Based on the methylation marker in the ferrochelatase gene, erythroid DNA represented a median of 30.1% of the plasma DNA of healthy subjects. In subjects with anemia of different etiologies, quantitative analysis of circulating erythroid DNA could reflect the erythropoietic activity in the bone marrow. For patients with reduced erythropoietic activity, as exemplified by aplastic anemia, the percentage of circulating erythroid DNA was decreased. For patients with increased but ineffective erythropoiesis, as exemplified by ß-thalassemia major, the percentage was increased. In addition, the plasma concentration of erythroid DNA was found to correlate with treatment response in aplastic anemia and iron deficiency anemia. Plasma DNA analysis using digital PCR assays targeting the other 2 differentially methylated regions showed similar findings. CONCLUSIONS: Erythroid DNA is a hitherto unrecognized major component of the circulating DNA pool and is a noninvasive biomarker for differential diagnosis and monitoring of anemia.

From chronic immune thrombocytopenia to severe aplastic anemia: recent insights into the evolution of eltrombopag.

Thrombopoietin (TPO) is the most potent cytokine stimulating thrombopoiesis. Therapy with exogenous TPO is limited by the formation of antibodies cross-reacting with endogenous TPO. Mimetics of TPO are compounds with no antigenic similarity to TPO. Eltrombopag is an orally-active nonpeptide small molecule that binds to the transmembrane portion of the TPO receptor MPL. Initial trials of eltrombopag have centered on immune thrombocytopenia (ITP), which is due to both increased destruction and decreased production of platelets. Eltrombopag at 25-75 mg/day has been shown to be highly effective in raising the platelet count in ITP with suboptimal response to immunosuppression and splenectomy. These successful results led to the exploration of eltrombopag in other thrombocytopenic disorders. In hepatitis C viral infection, eltrombopag raises the platelet count sufficiently enough to allow treatment with ribavirin and pegylated interferon. Because MPL is expressed on hematopoietic cells, eltrombopag use in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) might enhance leukemic proliferation. Clinical trials of eltrombopag in MDS and AML, however, have shown amelioration of thrombocytopenia without promoting disease progression. In severe aplastic anemia (SAA) not responding to immunosuppression with anti-thymocyte globulin (ATG) and cyclosporine, eltrombopag as a single agent at 150-300 mg/day results in an overall response rate of 40-70%. At high doses, adverse effects including pigmentation, gastrointestinal upset and hepatic derangement have become evident. Current studies have examined the first-line use of eltrombopag in combination with ATG in SAA. In a recent study, eltrombopag used at 150 mg/day with horse ATG resulted in an overall response rate of 90% in newly diagnosed SAA patients, with a complete response rate of about 50%. Clonal karyotypic aberrations are, however, found in 10-20% of SAA patients treated with eltrombopag. The safety and efficacy of eltrombopag in SAA require further evaluation, particularly when it is used with less intensive immunosuppression.

International, evidence-based consensus diagnostic criteria for HHV-8-negative/idiopathic multicentric Castleman disease.

Human herpesvirus-8 (HHV-8)-negative, idiopathic multicentric Castleman disease (iMCD) is a rare and life-threatening disorder involving systemic inflammatory symptoms, polyclonal lymphoproliferation, cytopenias, and multiple organ system dysfunction caused by a cytokine storm often including interleukin-6. iMCD accounts for one third to one half of all cases of MCD and can occur in individuals of any age. Accurate diagnosis is challenging, because no standard diagnostic criteria or diagnostic biomarkers currently exist, and there is significant overlap with malignant, autoimmune, and infectious disorders. An international working group comprising 34 pediatric and adult pathology and clinical experts in iMCD and related disorders from 8 countries, including 2 physicians that are also iMCD patients, was convened to establish iMCD diagnostic criteria. The working group reviewed data from 244 cases, met twice, and refined criteria over 15 months (June 2015 to September 2016). The proposed consensus criteria require both Major Criteria (characteristic lymph node histopathology and multicentric lymphadenopathy), at least 2 of 11 Minor Criteria with at least 1 laboratory abnormality, and exclusion of infectious, malignant, and autoimmune disorders that can mimic iMCD. Characteristic histopathologic features may include a constellation of regressed or hyperplastic germinal centers, follicular dendritic cell prominence, hypervascularization, and polytypic plasmacytosis. Laboratory and clinical Minor Criteria include elevated C-reactive protein or erythrocyte sedimentation rate, anemia, thrombocytopenia or thrombocytosis, hypoalbuminemia, renal dysfunction or proteinuria, polyclonal hypergammaglobulinemia, constitutional symptoms, hepatosplenomegaly, effusions or edema, eruptive cherry hemangiomatosis or violaceous papules, and lymphocytic interstitial pneumonitis. iMCD consensus diagnostic criteria will facilitate consistent diagnosis, appropriate treatment, and collaborative research.

A 2-Year, Longitudinal, Prospective Study of the Effects of Eltrombopag on Bone Marrow in Patients with Chronic Immune Thrombocytopenia.

BACKGROUND: The long-term effects of eltrombopag on bone marrow (BM) reticulin and/or collagen deposition in previously treated adults with chronic immune thrombocytopenia (ITP) were assessed. METHODS: Three BM biopsies were collected at baseline and after 1 and 2 years of eltrombopag treatment. Specimens were centrally processed, stained for reticulin and collagen, independently reviewed by 2 hematopathologists, and rated according to the European Consensus 0-3 scale of marrow fibrosis (MF). RESULTS: Of 162 patients enrolled, 93 completed all 3 protocol-specified BM biopsies. All patients with a baseline assessment were negative for collagen. Of 159 patients assessed at baseline, 150 (94%) had normal reticulin (MF-0) and 9 (6%) had minimally increased reticulin (MF-1). After 2 years, 83/93 patients (89%) with BM biopsies had MF-0, 10 (11%) had MF-1, and none had MF-2 or MF-3. Five out of 127 patients (4%) at 1 year and 1 out of 93 (1%) at 2 years had collagen deposition. None of the patients had clinical symptoms typical of BM dysfunction or abnormalities of clinical concern based on white blood cell count or peripheral blood smear. CONCLUSION: For most patients with chronic ITP, eltrombopag is not associated with clinically relevant increases in BM reticulin or collagen formation.