PNH Clone Research Articles

Impact of Eltrombopag on Clonal Evolution in Refractory Aplastic Anemia

Impact of Eltrombopag on Clonal Evolution in Refractory Aplastic Anemia

The Importance of Eculizumab in Paroxysmal Nocturnal Hemoglobinuria: A Cohort Study

IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is a rare, often progressively debilitating and life-threatening acquired disease characterized by chronic, complement-mediated intravascular hemolysis. PNH is characterized by the deficiency of the endogenous glycosyl phosphatidylinositol-anchored complement regulatory proteins CD55 and CD59 and other proteins on the surface of blood cells, which results in chronic uncontrolled activation of the complement system at the site of the PNH cells and intravascular hemolysis and thrombosis. PNH may present in the absence of an underlying bone marrow disorder (BMD), as a condition secondary to BMDs, such as aplastic anemia (AA) or myelodysplastic syndrome, or as sub-clinical PNH. In this cohort study we will report clinical findings, management options and eculizumab response of 38 PNH cases in our clinic.Materials and MetodWe followed 38 PNH cases in this cohort study. We used flow cytometric PNH clone detection test since 1993 but HAM's test and sucrose lysis test were the only tests for the PNH diagnosis in older cases. We used eculizumab in 20 of our patients who had transfusion dependent chronic intravascular hemolysis and/or thrombosis. All of of the patients were vaccinated for Neisseria meningitides, Pneumococcus and Hemophilus influenza type B prior to 2 weeks of initiation of Eculizumab. Eculizumab was administered 600 mg/day for 4 weeks and dosage was elevated to 900 mg/day at week 5, then 900 mg/day every other week as maintenance therapy. The response to therapy was evaluated by LDH, blood cell counts and haptoglobin levels. Clinical and laboratory findings, management options and treatment effect on survival curves were evaluated.FindingsThirty-eight PNH patients who were followed in our clinic since 1985 were enrolled in this study. Twenty of 38 were female and the median age was 35 years (ranging from 18 - 62). The oldest one has been followed for 32 years. Chronic hemolytic anemia and at least one lineage of cytopenia were seen in each patient during diagnosis.Fifteen patients were diagnosed as AA and were treated with immunosuppressive therapy before PNH diagnosis. Following AA diagnosis, PNH was diagnosed on average 35 months (three to 192 months) . One of the patients was diagnosed as MDS and followed-up for five years with this diagnosis. Twenty-two of the cases were de-novo PNH disease. Thrombotic complications were seen at the diagnosis in two patients. Unfortunately, we also have seen thrombotic complications in 10 more patients during follow-up period before eculizumab availability.Five of our patients were lost on eight, 14, 60, 86 and 260 months after their diagnosis. We lost two of them with infection and two of them with thrombotic complications. Fifth patient that we lost at 260 months of her follow-up had complete response with eculizumab and was deceased following a fall from height. Two of our patients went to abroad for treatment. Six of them were lost to follow-up. We currently follow 25 of 38 patients. We still use eculizumab in 20 of our patients. We used eculizumab in median 8 months after PNH diagnosis (One to 360 months). In 15 patients, transfusion dependent chronic intravascular hemolysis was the main indication and for the remaining 5 patients thrombosis was the main indication. Quality of life parameters changed significantly in all of the patients and we didn't observe any new thrombotic episodes. We used eculizumab as an important treatment option for a median 63 months (eight to 98 months) and we didn't encounter any side effects other than mild headache and flu-like symptoms in 5 of the cases.Discussion and conclusionPNH is a life-threatening disease and four of the patients in our cohort died in median 37 months of their diagnosis. Although thrombosis was the main mortality and morbidity reason in our cohort, we didn't see any thrombotic episodes under eculizumab treatment. Even in induction period of the therapy, both clinical and laboratory findings improved significantly with Eculizumab. None of our patients needed transfusion on eculizumab treatment. The survival of our PNH patients on eculizumab is excellent and we didn't lose any of our patients since induction of eculizumab therapy.In conclusion, eculizumab is the first disease-modifying, targeted therapy for PNH and its availability has radically changed the management of patients with this rare disease. DisclosuresYenerel:Alexion: Honoraria, Speakers Bureau.

Heterozygous CTC1 Variants in Acquired Bone Marrow Failure

Germ line (GL) alterations of telomerase machinery genes may lead to inherited telomeropathies, but recent analysis of large control populations revealed that some of the previously assumed pathologic variants are present in comparable frequencies in healthy individuals. Pathogenic telomerase gene variants can be found, but are rare in idiopathic aplastic anemia (AA) and are associated with excessive telomere attrition. Previously, in a cohort of patients with MDS, pathogenic germ line variants of telomerase genes were very extremely rare.Recently a patient with bone marrow failure and liver cirrhosis presented with a biallelic CTC1 gene mutation. CTC1 is a member of the CTC complex, located on Chr 17p13.1, and critical for telomere replication. GL alterations of this gene were found to cause inherited disease, including Coats plus (CP), dyskeratosis congenital (DC) and cerebroretinal microangiopathy with calcifications and cysts (CRMCC) in a compound heterozygous manner. Our patient (age 28) had compound heterozygous CTC1 germline mutations (p.Lys242Leufs*41 and p.Cys985del) resulting in early childhood presentation of DC, subsequent severe AA at the age of 19, and a significant shortening of telomeres. Both parents as confirmed CTC1 variant carriers showed normal blood counts.Based on this index case and literature reports, manifest CTC1 diseases follow a recessive trait, however, but it is possible that heterozygous carriers may indeed have also an increased, albeit attenuated risk of BMF leading to a later manifestation or incomplete penetrance. To test this possible scenario we screened a cohort of acquired BMF (n=538) with AA/PNH (172), MDS/AML (n=366), with deep NGS of all coding regions of CTC1. In total, we identified 10 heterozygous CTC1 variants in 10 unrelated patients (8 AA/PNH and 2 MDS); 4/10 variants (1 stop gain R1202X and 3 frameshift deletions D405fs, P999fs, E454fs) were fulfilled the criteria of Tier-1 lesions and were found in AA/PNH patients (4/172, 2.3%), The remaining 6 missense variants were of uncertain significance or likely benign(Tier-2), 2 of which (H1092G and E1136K) were found in a compound heterozygous configuration in a AA/PNH patient. K438N, as a novel missense variant, was recurrently present in 2 MDS pts. Given the expected frequency of the CTC1 variants found in controls (104/33370, 0.3%), CTC1 variants appear to be enriched in AA/PNH subgroup (p<0.001). Of note is that none of the carriers of pathogenic CTC1 mutations showed any physical signs of inherited congenital BMF syndromes or any family history of leukemia, BMF and any other cancers. Interestingly, patients with sAA/PNH syndrome and biallelic CTC1 variant eventually evolved to MDS, while the other monoallelic CTC1 carriers showed stable disease and responsed to immunosuppression. Flow cytometric telomere length measurement (adjusted for age) showed a markedly shortened telomere in the index case, as well as 1 SAA case carrying biallelic CTC1 variants and 1 AA case with co-concurrent POT1 and CTC1 variants when compared with age matched controls. A significant difference was seen when comparing telomere length between CTC1 variants carriers and age-matched normal controls, while no difference was seen among CTC1 variants carriers, AA/PNH group without any variants.Because of the curious co-incidence of heterozygous CTC1 variants and AA/PNH, we further analyzed this subgroup for the presence of other telomerase gene mutations, 11/172 Tier-1 variants, while 6 Tier-1 GLVs detected in MDS. Most frequent SNVs were found in POT1 with 5/172 (3%) in AA/PNH and 3/366 (0.8%) in MDS/AML. Notably, in one AA/PNH patient POT1 stop gain variant (Q364X) was co-concurrent with CTC1 heterozygous frame shift deletion (P999fs). The enrichment of variants in TELO-related genes in AA/PNH subgroup was found in comparison to MDS/AML (p<.01) and ExAC database controls (p<.01). Further clinical phenotype analysis indicated that 3 AA/PNH cases harboring POT1, TINF2 and TERT Tier-1 variants had family history of cancer. Family history of cancer was also found in a MDS patient with a TINF2 variant.In sum, our results indicate that heterozygous CTC1 variants were associated with otherwise typical AA with clonal outgrowth of PNH clone and the presence of this variant does not seem to preclude response to immunosuppression. DisclosuresMaciejewski:Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

A Rare Cause of Elevated Liver Tests in a Pregnant Patient

Case: 26-year-old female at 22 weeks gestation with history of anemia presented with nausea, vomiting and sharp, epigastric, non-radiating abdominal pain for 4 days. She denied any bleeding. Surgical and medication history were notable for a previous dilation and curettage and a daily vitamin. Vital signs were normal, physical exam revealed pallor, gravid abdomen with epigastric tenderness to palpation. Hemoglobin was 6.8 g/dL and platelets 84 K/mcL. Liver panel showed AST 87 U/L, ALT 42 U/L and total bilirubin 3.2 mg/dL (direct 0.50 mg/dL). Reticulocytes were 8%, LDH 1142 U/L, haptoglobin undetectable with negative direct antiglobulin test. Peripheral smear showed macrocytosis, thrombocytopenia, anisopoikilocytosis, and no schistocytes. Acute viral hepatitis panel and parvovirus serologies were negative. Doppler ultrasound showed splenomegaly, normal fetus and liver with near occlusive thrombus of the middle hepatic vein with reversal of flow in the right portal vein. CT abdomen/pelvis revealed heterogeneous enhancement of liver with filling defects in multiple hepatic veins suggestive of thrombi. ADAMST13 level, protein C and S, antithrombin, glucose-6-phosphate dehydrogenase levels, factor V Leiden, Beta-2 glycoprotein, ANA, ASMA, SPEP and AMA were all negative. Endoscopy was negative for portal hypertensive gastropathy or varices. Duodenal biopsies were normal. Flow cytometry showed PNH clone percent of 84% in granulocytes, 68% in monocytes, and type III RBC (complete CD59 deficiency) of 8% confirming diagnosis of PNH. The patient was transfused, started on anticoagulation and eculizumab. Her anemia improved and she delivered a healthy, full-term baby. Discussion: PNH is characterized by a triad of intravascular hemolysis, thrombosis and bone marrow failure. Thrombosis occurring in atypical locations should prompt consideration of PNH. The hepatic vein is the most frequently involved vessel. PNH is associated with increased fetomaternal morbidity and mortality and thrombosis is the leading cause of death. Unexplained hemolytic anemia in a pregnant patient with concomitant abnormal liver tests may initially evoke diagnosis of HELLP (hemolysis, elevated liver enzymes, and low platelets), however, management of the two condition differs vastly. Anticoagulation is recommended for pregnant women with PNH thrombosis unless contradicted.Eculizumab reduces the risk of thrombosis. BMT is the only curative therapy for PNH with success rates of up to 100%.

Clonal hematopoiesis in aplastic anemia

Aplastic anemia (AA) is an autoimmune-mediated bone marrow failure syndrome. While AA is not a malignant disease, clonal hematopoiesis is commonly detected via next-generation sequencing and single nucleotide polymorphism (SNP) array. Clonal hematopoiesis in AA has been confirmed by the detection of classic X chromosome skewing, PNH clones, UPD6p, and various mutations. The most frequent genetic events in AA are UPD6p and somatic mutations in BCOR/BCORL1, PIGA, DNMT3A, and ASXL1. While some mutations are common between patients with AA and healthy elderly donors, UPD6p and PIGA mutations are specific to clonal cells in AA, which need to manage their highly autoimmune extrinsic environment. During the evolution of AA into myelodysplastic syndrome (MDS), additional genetic events are frequently acquired that provide MDS cells with intrinsic survival benefits. Hematopoietic cells in AA appear to achieve clonal expansion by their escape from recognition and cytotoxicity by CD8 T-cells, accounting for the distinct landscape of genetic events observed in AA.

HLA-mismatched bone marrow transplantation in severe aplastic anemia

Hematopoietic stem cell transplantation (SCT) can be curative for patients with severe aplastic anemia (SAA).1, 2 Concerns about graft failure and graft-versus-host disease (GVHD), more so for human leukocyte antigen (HLA) -mismatched donors, have impeded the utility of SCT for these patients. The introduction of post-SCT cyclophosphamide (PTCy) has been shown to decrease alloreactivity in the host-versus-graft and graft-versus-host directions and could be particularly well suited for SAA patients.3 Only a few patients in two recent reports have documented outcomes of patients with SAA treated with a haploidentical transplant and PTCy-based GVHD prophylaxis, both using non-myeloablative (NMA) conditioning with fludarabine, cyclophosphamide and 2 Gy total body irradiation (TBI).4, 5 Concern for higher rejection rate, both primary and secondary, has prompted us to use a more intense conditioning regimen, as previously described.6 Here, we report clinical outcomes in transfusion dependent (n=6, 5 were refractory to first line immunosuppressive therapy, one patient received hypomethylating agent for hypoplastic MDS subsequently diagnosed as AA) SAA patients who underwent HLA-mismatched bone marrow SCT from haploidentical (n=2) and 9/10 matched unrelated donors (n=4) at our center between January 2010 and May 2013. Conditioning regimen consisted of melphalan (140 mg/m2) on day-8, thiotepa (5 mg/kg) on day-7 and fludarabine (160 mg/m2) on days −6 to −3. GVHD prophylaxis consisted of cyclophosphamide (50 mg/kg) on days +3 and +4, tacrolimus for 180 days and mycophenolate mofetil for 100 days post transplant. Median age of patients at the time of SCT was 37 years (range 21–50), median Karnofsky performance score was 90 (range 80–100) and median time from diagnosis to transplant was 9 months (range 2–15 months). Of the six patients, two of them had a concomitant PNH clone>20% prior to SCT. All patients engrafted the donor cells with 100% chimerism noted for both myeloid and T-cell lineage at day +30 post SCT. Of the four-long-term survivors, 100% donor (lymphoid and myeloid) chimerism was noted starting from day 30 until 1 year post SCT in three patients and for up to 6 months for one patient (follow-up care was transferred to an outside institution and was found to be disease-free at 1 year post SCT). Median time to neutrophil and platelet engraftment was 16 and 13 days, respectively (Table 1). Acute GVHD was seen in three patients (grades-1, 2 and 4 each) and led to death in one patient who had a 9/10 MUD SCT (Table 1). No chronic GVHD (cGVHD) or disease relapse was noted. After a median follow-up of 949 days (range 735–1617), 4/6 (66.6%) patients (two haploidentical and two 9/10 MUD) were alive, fully engrafted without cGVHD.

ICCS ガイドラインに準拠した高感度PNH 血球測定の性能評価

ICCS ガイドラインに準拠した高感度PNH 血球測定の性能評価

Thrombin Generation in Paroxysmal Nocturnal Haemoglobinuria

INTRODUCTION: PNH is a pathology in which the uncontrolled activity of the complement system leads to systemic complications. It is characterized by an acquired hemolytic anemia with intermittent hemoglobinuria, bone marrow failure and thromboembolic events (TEV). Venous thrombosis is the main cause of death. However its physiopathological mechanism is only partially understood. The large number of patients with thrombocytopenia affects the management of primary and secondary antithrombotic prophylaxis. We choose to demonstrate the hemostatic unbalance associated with PNH clone through a global coagulation assay. METHODS: We used a fluorogenic thrombin-generation assay, in platelet-poor plasma, with and without thrombomodulin (TM). Analysis of the efficiency of TM in reducing the endogenous thrombin potential, or ETP (ETP ratio) and of the upper limit of thrombin concentration, or peak (peak ratio) was done to identify the hypercoagulable state. Subjects were divided in three groups: PNH patients (if PNH clone >= 10%) (n=23, PNH), patients with acquired idiopathic aplastic anemia or clone-associated (clone < 10%) (n=25, AA), and controls (n=39, NN). Patients and controls were investigated for thrombophilia (TB) and PNH clone, underwent blood tests, and regular exams to evaluate hemostasis. Patients were evaluated for the presence of risk factors for TEV through questionnaires. Results were analyzed in two steps: the first included only patients negative for TB and with no risk factors for TEV (PNH n=14, AA n=12 and NN n=33); the second step, done only in patients, included individuals using hormonal contraceptives, with positive TB tests, diagnosis of any asymptomatic infection or TEV associated to temporary risk factors that occurred in a period longer than one year since inclusion in the study (PNH n=23, AA n=25). The aim of the second step was to gather the largest possible number of patients in these low prevalence pathologies. The study was approved by the local Ethics Committee, and performed in accordance with the Helsinki Declaration. RESULTS: The presence of the PNH clone >= 10% was associated with TM inefficiency in reducing the ETP [ETP ratio PNH 0,68 (0,32-0,93), AA 0,49 (0,20-0,81) and controls 0,48 (0,19-0,89) p <0,05, PNH x AA p=0,032, PNH x NN p=0,004)] and in reducing the peak [peak ratio PNH 0,84 (0,46-1,03), AA 0,65 (0,28-0,96) and controls 0,60 (0,25-0,99) p <0,05, PNH x AA p= 0,041, PNH x NN p= 0,001)]. In the first analysis, which had greater clinical relevance, we observed a positive correlation between ETP ratio and the activity of the von Willebrand factor (FvW:RCo) (Spearman rank correlation = 0,59, p<0,05), whereas a negative correlation was observed between ETP ratio and the levels of protein C (PC) (Pearson correlation coefficient = -0.59, p<0,05). The PNH group presented the shortest time to peak (min) [PNH 5,9 (5,3-7,3), AA 6,8 (5,0-8,8), NN 6,1 (4,7-8,7) p<0,05, PNH x AA p=0,037). CONCLUSION: The thrombin-generation assay effectively detects the prothrombotic phenotype associated to PNH. The positive and negative correlations found respectively between ETP ratio, FvW:RCo and PC suggests that endothelial activation, and the PC system as well, may be deficient in these patients. Inflammation secondary to activation of the complement system may lead to lower endothelial expression of TM and of the PC receptor, leading to lower PC activation. Our findings, together with recent descriptions of a reduced expression of the TM activity secondary to nitric oxide depletion (observed in studies on statins) may explain the aggressive nature of thrombophilia in PNH and the development of TEV even in those taking oral anticoagulants. DisclosuresNo relevant conflicts of interest to declare.

Efficacy and Safety of Unrelated Cord Blood Transplantation in Patients with Acquired Refractory Aplastic Anemia: A Phase II Study on Behalf of Eurocord and the Francophone Society of Bone Marrow Transplantation and Cellular Therapy

▪Background: While therapeutic outcomes in patients (pts) with acquired severe aplastic anemia (SAA) has greatly improved in recent years, results remain poor for pts who are refractory to first-line immunosuppressive therapy (IST) and lack a matched unrelated donor. We conducted a prospective multi-center phase II uncontrolled trial to assess the efficacy and safety of cord blood transplantation (CBT) in refractory SAA pts (NCT 01343953).Patients and methods: Pts with SAA refractory to IST with anti-thymocyte globuline (ATG) and ciclosporin (CsA) were eligible in case of no existing matched unrelated donor but available one or two unrelated CB units containing alone or both together more than 4 x 107/Kg frozen nucleated cells/Kg (no more than 2 out of 6 HLA mismatches allowed between each CB unit and the pts). Pts with isolated bone marrow cytogenetic abnormality (absence of morphologic evidence of myelodysplastic syndrome) were also eligible. The conditioning regimen consisted of fludarabine 30mg/m2 (D-6 to D-3), cyclophosphamide 30mg/kg (D-6 to D-3), ATG (thymoglobulin) 2.5mg/kg at D-3 and D-2 (5mg/Kg total dose) and total body irradiation (2 Gray) on D-2. All pts received one injection of anti-CD20 (150 mg/m2) to prevent EBV reactivation (D+5). CsA was given alone as prophylaxis for graft versus host disease (GVHD). The trial used the Fleming's Single Stage Phase II Design, with sample size computed to demonstrate an improved overall survival (OS) rate at one year from 20% up to 50%, with type I and type II error rates set at 0.05. A minimum sample of 25 pts was required, with a minimum number of 9 pts alive at one year needed to indicate treatment efficacy. Secondary endpoints included cumulative incidences (CumI) of engraftment, acute and chronic GVHD (aGVHD and cGVHD), infections, relapse and late cause of death.Results: 29 pts were included between June 2011 and October 2015. One pt was diagnosed with dyskeratosis congenita and a matched unrelated donor was identified for another pt between inclusion and anticipated date of CBT (exclusion criteria). One pt died before CBT due to a septic shock (D+4 after inclusion). Analyses were conducted in the remaining 26 pts. Median time between SAA diagnosis and CBT was 12 months (interquartile range (IQR) 8.7 to 17.8). All pts received at least one course of IST before CBT (2 courses, n=8). Twenty-three pts received 20 or more transfusions before CBT. BM karyotyping showed 2 pts respectively with monosomy 7 in 4 and 15 mitoses as well as one pt with trisomy 8 in 4 mitoses before CBT. Eight pts (31%) have a PNH clone (median 4.5% [IQR: 3.1-9.7]). Median age at time of CBT was 16 years (IQR 8.25 to 23) and median follow-up is 13.2 months (IQR 4.1 - 23.5). Three out of 26 pts incorrectly received twice the dose of ATG (10mg/Kg total dose) due to protocol violation. Sixteen pts received one CB and 10 received two CB units. Median number of nucleated cells infused was 3.7 x 107/Kg (IQR, 3-4.9) and CD34+ cells infused was 1.4 x 105/Kg (IQR, 1.0-1.9). Myeloid engraftment occurred in 23 pts, with a 60 day-CumI of neutrophil engraftment of 88.5% with full chimerism for all of them. Three pts did not engraft (2 deaths at D71 and D92 and one successful second HSCT). The 100 day-CumI of grade II-IV acute GVHD was 40% (95% CI, 20-60) (8 grade II; 0 grade III; 2 grade IV). Among the 23 pts alive at day 100, five developed cGVHD leading to a one-year CumI of cGVHD at 27% (95% CI, 7-47) (severe cGvHD in 2 pts). During follow-up, 8 pts experienced a total of 18 grade III infections with a 6-month CumI of 32% (95% CI, 13-51). None of the pts presented post-transplant EBV-related lymphoproliferative disorder. Immune reconstitution is shown in Figure 1. Three pts died before 1 year due to non-engraftment (n=2) and infection (n=1, one of those who received twice the dose of ATG). With 23 pts alive at 1 year, the primary objective of the study was thus reached. The one-year treatment related mortality (TRM) was 15% (95%CI, 4-35). One additional pt who had also received twice the dose of ATG died (severe cGvHD) after one year (at 13.6 months), resulting in a 14-month OS of 81% (95%CI, 66-100) (Figure 2).Conclusion: CBT with at least 4 x 107/Kg frozen nucleated cells/Kg after a FLU-CY-TBI-ATG conditioning regimen gave rise to very encouraging results in this particular high risk SAA refractory population. Pediatric and young adult SAA pts who are refractory to first-line IST should now be considered either way for matched unrelated donor or CBT. [Display omitted] [Display omitted] DisclosuresPeffault de Latour:Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria, Research Funding; Amgen: Research Funding.

Clonal Dynamics of Refractory Aplastic Anemia in Patients Treated with Eltrombopag

Despite documented success of immunosuppressive therapy (IST) in the treatment of AA, a minority of patients remain refractory, most responses are incomplete, and use of hematopoietic cell transplantation (HCT) is limited in older patients or those with significant comorbidities. While the introduction of the cMpl agonist eltrombopag (EPG) as salvage therapy or in conjunction with IST has revolutionized treatment for refractory AA. It may be effective in improving primary response rates to IST, engaging growth factor receptors with agonistic therapeutics (such as EPG) and also has the potential to promote evolution/expansion of mutant clones, thereby increasing the rate of progression to secondary myelodysplastic syndromes (MDS), a serious complication of AA occurring in 10-20% of patients. Clonogenic somatic mutations typical of MDS in patients with AA and PNH may increase the risk of progression to MDS.DNA from marrow samples of primary refractory AA patients was subjected to analysis before and after initiation of EPG to evaluate clonal expansion or evolution using a targeted multi-amplicon deep NGS panel of all ORFs of the top 60 most commonly mutated genes in MDS. In addition to the EPG treatment group, a case control cohort matched for age and duration from AA diagnosis to last clinical follow up (who did not receive EPG), was studied.Among 210 AA patients treated at Cleveland Clinic, we identified 26 who were treated with EPG for IST-refractory AA; median duration of treatment was 56 wks. The overall response rate after 12 weeks of therapy was 58% (15/26), while 31% of patients (8/26) showed stable disease with intermittent transfusions (one of whom underwent HCT). In 3 non-responders, one developed PNH, one had refractory AA/PNH, and one progressed to AML (see below). Expansion of PNH granulocytes after EPG treatment was observed in 23% of patients (6/26). In addition, 15% (4/26) had atypical subclonal chromosomal abnormalities.Prior to EPG, at least a single somatic event was found in 31% of patients (8/26), with 2 patients harboring 2 mutations. Events included CEBPA, EZH2, BCOR/BCORL1, ASXL1, U2AF1/2, TET2, and DNMT3A mutations. Following EPG therapy, acquisition of new somatic mutations was observed in 23% of cases, including RUNX1, U2AF1, BCOR, RIT1, and CEBPA. In cases with pre-existing clones, 6 clones expanded (e.g., BCOR or ASXL1 from VAF of 8 to 21% and 9 to 29%, respectively) despite clinical hematologic response, while in 2 cases clones disappeared (e.g., U2AF2 and BCORL1). In 54% of cases (14/26), we found detectable levels of a PNH clone at the time of diagnosis. Six of those cases had PNH clonal expansion post-EPG treatment, of which two developed clinically significant PNH clonal burden requiring eculizumab therapy.In the case-control cohort, 26 AA patients who received IST but were not treated with EPG, were followed for comparable time periods, and no evidence of progression to MDS was recorded. One patient was noted to have trisomy 15 on cytogenetics at diagnosis. "MDS type" molecular mutations were present in 10 patients similar to EPG cohort. Among these patients, 3 had persistent clones of U2AF1, DNMT3A, and STAT3 over one year without acquisition of any new molecular mutations. . PNH granulocytes expanded in 50% of AA cases, decreased in 30% and stayed stable in 20%. Thus, we did not observe any difference in expansion of PNH clones between those treated and untreated with EPG (p=0.73). Unlike for PNH clones, accounting for both new evolution and expansion of preexisting molecular mutations, the frequency of these clonal events was significantly higher in the EPG treated group (p=0.009).In conclusion, we observed occasional expansion of clones with potentially leukemogenic mutations during treatment with EPG in pts with AA. While higher rates of MDS evolution were not observed in this cohort of EPG treated patients, we found that serial evaluation of somatic mutations can inform clonal evolution and can potentially be used as abiomarker for evaluation of risk for post-AA MDS. Continued use of EPG in such patients should be judicious. DisclosuresCarraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Subcutaneous Low Dose Alemtuzumab: Role As a Salvage Therapy in Immune -Mediated Marrow Failure Conditions

▪Aplastic anemia (AA), pure red cell aplasia (PRCA), pure white cell aplasia (PWCA), idiopathic neutropenia (IN) and T cell large granular lymphocytic leukemia (LGL) are bone marrow failure (BMF) conditions unified by immune-mediated pathogenesis leading to cytopenias. Intravenous alemtuzumab, a CD52 monoclonal antibody approved for the treatment of chronic lymphocytic leukemia (CLL) and used for relapsed AA, has a response rate in AA of 37-58%. However, dosing and route was likely based on those used in CLL regimens and resulted in significant immunosuppressive complications.Here we summarize our experience with sc alemtuzumab given at the maximum dose of total 100 mg as etiologic treatment (either salvage therapy or preferred therapy) to patients with various BMF. We investigated the therapies administered to patients seen at the Cleveland Clinic or at the University of Naples (NCT00895739) between years 2002-2015.Of 484 patients with BMF conditions (Cleveland: AA: 204, LGL: 174, PRCA: 56; Naples: AA: 24, LGL: 2, PRCA: 14), 65 patients with AA (N=22), PRCA (N=22), LGL (N=18) and 3 cases of IN/PWCA were treated sc alemtuzumab, given according 2 distinct schedules: i. initial dose of 3 mg sc followed by 10 mg once or twice weekly for at least 8 consecutive weeks; ii. 4-5 day dose escalation (3-10-30-30-30 mg) regimen. In both schedules, some patients received a much longer course with maintenance treatment or occasional boosts to prevent or treat relapse. Patients received bactrim and acyclovir prophylaxis, IVIG for hypoglobulinemia and underwent CMV monitoring. The median age of patients was 57.5 years (range: 31-84 years). Alemtuzumab was used as salvage therapy in Cleveland (except for 3 AA cases with contraindication to anti-thymocyte globulin or cyclosporine), whereas in Naples untreated patients were allowed per protocol. As a result, patients had a median of 2 lines of therapy (range: 0-6), had either untreated (35%), relapsed (22%) or refractory (43%) BMF condition. Among the 22 AA patients 12 were untreated, or had relapsed (N=4), or refractory disease (N=6), with concomitant PNH clone in 8/22 patients. LGL patients (N=18) treated with alemtuzumab were younger, had a median of 2 prior therapies, 50% were STAT3 mutants, with comparable degree of cytopenias but excess splenomegaly (60% vs. 38%, p=.012) at presentation, compared to the remainder LGL cohort. Amongst these patients, 18% had relapsed LGL while the rest had refractory disease. PRCA cases treated were either idiopathic (N=18, of whom 3 associated with thymoma) or LGL-associated (N=4); this group was very heterogeneous, including 9 untreated patients (in Naples) and a subgroup of heavily pretreated patients (5 with refractory disease) with a median of 5 prior therapies, with mean hemoglobin of 7.5 g/dL. Among the group of PWCA/IN, one case of PWCA was LGL-associated.The treatment was well tolerated in 92% of the patients, except in four patients who did not complete treatment course due to adverse events. The main adverse events include infectious complications with 7/65 developing CMV reactivation, 4/65 HSV reactivation, and more rarely other viral reactivation (VZV and HBV, 1/65 each). Two fatal infections were recorded: 1 fungemia (heavily pretreated with other therapies) and 1 JCV-related PML (in a patients also receiving chemotherapy for refractory thymoma).The overall response rate was 55%; by disease subtype 54% of AA, 44% of LGL, 59% of PRCA and all three cases of PWCA/IN responded. The median time to response in AA was 12 weeks (range: 6-24 weeks), and responses lasted a median of 6 months (range: 2-72 months). In AA, we observed responses in 72% of the cases treated with almetuzumab upfront and 45% of the relapsed/refractory disease. Similarly in PRCA cases, 77% of the alemtuzumab upfront and 46% of the relapsed/refractory disease responded. With a median follow up >4 years, no late treatment-related complication emerged; treatment failures were mostly due to relapses (sometimes refractory to further immunosuppressive treatment), with clonal evolution around 15% (mostly in non-responders).In summary, low-dose sc alemtuzumab is a reasonable alternative option for patients with BMF conditions, especially in relapsed/refractory cases; patients need to be monitored with a heightened vigilance for infectious complications, as well as for possible recurrence of their underlying disease. DisclosuresRisitano:Alexion Pharmaceuticals: Other: lecture fees, Research Funding; Alnylam: Research Funding; Rapharma: Research Funding; Novartis: Research Funding.

Nationwide Survey on the Use of Eltrombopag in Patients with Severe Aplastic Anemia: Report on Behalf of the French Reference Center for Aplastic Anemia

▪ BACKGROUNDFew therapeutic options are currently available for patients (pts) with severe aplastic anemia (SAA) refractory to antithymocyte globulin (ATG) plus ciclosporine (CsA) and not eligible for allogeneic stem cell transplantation. It has recently been reported that eltrombopag (ELT), a TPO receptor agonist, is efficient to improve tri-lineage blood counts in this setting. However, real-life use of this drug is still largely unknown. In pts with SAA refractory to ATG, physicians can accede to ELT in France through a compassionate use program. We took advantage of this program to assess the efficacy and safety of ELT in SAA pts. PATIENTS AND METHODSThe French National Reference Center for Aplastic Anemia conducted this study in pts who received ELT for the treatment of SAA. Latest guidelines were used to define disease severity, treatment indication, and response rates (Marsh et al, BJH 2014). The diagnosis of SAA was confirmed by marrow biopsy and Camita criteria for all pts. Pts were eligible if they received at least 2 months of ELT alone or in combination with other treatment in case of relapsed/refractory disease or front-line therapy for pts not eligible to the association of ATG and CsA. Pts were identified through a national e-mailing on behalf of the French reference center for SAA and the French Society of Hematology. All data presented here were collected at the reference date of June 26th, 2016. The study was conducted according to Helsinki's Declaration. RESULTSForty-six pts (male, 54%) who received ELT between July 2012 and February 2016 were identified in 17 French centers. Indications for ELT were relapsed/refractory SAA in 35 pts (76%) after 1 (49%), 2 (29%) or 3 (9%) courses of CsA+ATG. Eleven pts considered unfit for ATG also received the drug as first line therapy. The characteristics of the pts according to ELT indications are shown in Table 1. Median age at time of ELT initiation was 61 years [IQR 40 to 70]. 44 pts had idiopathic SAA including 17 (37%) with a detectable PNH clone (median size 7%). Two pts (4%) with dyskeratosis congenita also received ELT. ELT was introduced 17 months [8-50] after the initial diagnosis of SAA and with a median of 6 months [3-14] after the last course of immunosuppressive therapy. The maximal dose was 150 mg/day [100-150] for a median duration time of 6 months [4-12]. At last follow-up 22(48%) pts were still on treatment, 4(9%) pts stopped because of good hematological response, 1(2%) and 15(33%) after limited toxicity and failure to improve hematological status. Before treatment, median neutrophils count was 790/mm3 [500-1215] and pts received a median number of 4 red blood cells packs [2-4] and 3 platelets apheresis units [2-4] every month. Neutrophils counts were 765 [515-1475], 1100 [600-1800], 1200 [670-1915] and 1200/mm3 [757-2300] at 1, 3, 6 months and at last follow-up respectively. The rates of transfusion independence for both red cells and platelets were 7%, 33%, 46% and 46% at 1, 3, 6 months and at last follow-up. In pts achieving transfusion independence, hemoglobin and platelets level improved of 3 gr/dL [1.4, 4.5] and 42 G/L [11, 100], respectively. The rate of transfusion independence was not different among first line and refractory pts (p=0,5). We did not observe any response in the 2 patients with dyskeratosis congenita. No factor associated with hematological response to ELT was identified.Liver toxicity (cytolysis) occurred in 11 pts (1 grade 3 that required withdrawal of the treatment and 10 grade 1 who responded to dose reduction). 2 pts had a grade 2 intestinal toxicity which improved after dose reduction. Other side effects where related to SAA (28% infections, 13% hemorrhages). Bone marrow karyotype analysis after ELT was done in twelve pts (26%) (median time 14 months [5-22] after ELT start). In 10 pts the karyotype was normal, in one, trisomy 8 was identified (already seen at SAA diagnosis), and karyotype was a failure in 1 pt. CONCLUSIONWe report here the first real-life multicenter study about the use of ELT in SAA. In a particular severe pts population with no other treatment possibility, we confirm a 40% rate of hematological improvement with transfusion independence. Some of the pts who were not eligible to ATG plus CsA (comorbidities) also received ELT first line with similar response rates. Elderly pts unfit for ATG may thus benefit from this treatment which at the best should be given through prospective clinical trials. [Display omitted] DisclosuresAdes:Celgene, Takeda, Novartis, Astex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Terriou:Novartis: Consultancy; amgen: Consultancy. Peffault de Latour:Alexion: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding.

Extended Duration of Anti-Thymocyte Globulin (ATG) Administration May Reduce the Risk of Graft-Versus-Host Disease in Patients with Aplastic Anemia Undergoing Matched-Related Allogeneic Peripheral Blood Stem Cell Transplantation

Bone marrow is the preferred stem cell source for allogeneic transplantation in the treatment of severe aplastic anemia (AA) due to a higher risk of GVHD associated with peripheral blood stem cell allografting. Higher doses or longer durations of administration of lympholytic agents such as antithymocyte globulin (ATG) or alemtuzumab may prevent GVHD and reduce graft failure rate. We treated a 19 y/o AA woman with transfusion and antibiotic dependent severe AA associated with a small PNH clone using 12/12 HLA-match allogeneic peripheral blood stem cells (allo-PBSCT) from her brother. Conditioning regimen consisted of Cyclophosphamide (CY) at 50 mg/kg/day intravenously on day -5 through day -2 before transplant. Twelve hours after each doses of CY, she was given horse ATG (Atgam; Upjohn, Kalamazoo, MI) at 30 mg/kg IV per dose infused over a period of 10 hours. ATG was given for 4 days, instead of 3 days as in the original study (R. Storb et al, Blood 1994;84:941-9). She received premedication with oral acetaminophen, intravenous diphenhydramine, and methylprednisolone at 100 mg, 30 minutes prior to each dose of ATG. During the first infusion of ATG, the patient received hydrocortisone 50 mg IV twice for infusion-related reactions, but tolerated all subsequent infusions.She received prophylaxis with acyclovir, levofloxacin, and fluconazole. Unmanipulated, G-CSF-mobilized allo-PBSCs at dose of 4.125 x 106 CD34+cells/kg were transplanted fresh in 48 hours after the last dose of CY. She received GVHD prophylaxis with tacrolimus and low dose methotrexate (5 mg/kg/day) on Days +1, +3, +6, and +11. Her transplant course was notable for breakthrough vaginal spotting despite leuprolide administration, and neutropenic fever with negative infectious workup. On day +14, the patient was given a single dose of G-CSF to expedite WBC recovery. Based on CIBMTR criteria, neutrophil and platelet engraftments occurred on day +15 and day +20, respectively. She was started on steroids with a quick taper for suspected engraftment syndrome with ongoing fevers with negative cultures. She was discharged on day +16 after transplant with a total length of hospital stay of 22 days. Her outpatient course was completely uneventful with no GVHD, infection or any other transplant-related complications. The patient was never readmitted. Her blood counts remained within normal range and blood chimerism analysis showed 100% donor in myeloid and lymphoid on day+30 and day+100 evaluations. She is currently day+120 on continuous tacrolimus. This case illustrates a successful early outcome after peripheral blood allogeneic stem cell transplantation in a young patient with aplastic anemia using modified ATG protocol. The concept of T cell in vivo purging after allo-PBSCT with extended administration of ATG in aplastic anemia warrants a prospective trial. DisclosuresSeddon:Sanofi: Speakers Bureau.

Clonal Events of Aplastic Anemia Related to the Evolution to Myelodysplastic Syndrome

Somatic mutations constitute clonal markers now amenable to monitoring by deep NGS. While transient and low frequency clones have been described in AA, their pathophysiologic link to the overtly clonal complication of AA, secondary MDS following AA (sMDS), has not been established. Clarification of this relationship may provide clues as to the genesis of sMDS. Identification of predictive markers for AA patients at risk for this complication is necessary. The etiology of sMDS within AA may include either expansion of a preexisting clone or truly late clonogenic events. In both instances, progression may result in clonal escape. To address these questions we studied 258 AA and 60 PNH patients, identifying 35 patients (11%) who evolved to sMDS. Cytogenetic analysis showed abnormal karyotype in 76% cases; 5% had complex karyotype and -7/del(7q) was present in 67% of cases. The presence of a PNH clone was detected in a similar proportion of cases that transformed to sMDS vs. those that did not (P=.76). For comparison, we have also analyzed primary de novo cases of MDS (pMDS) with (N=19) and without (N=161) -7/del(7q). In contrast to sMDS, -7/del(7q) was present in 12% of cases of pMDS. Using WES on 8 cases and a 60 gene targeted panel on 15 cases, confirmed mutational events and chromosomal aberrations were found in 21/23 patients with sMDS; 18/23 cases of sMDS had at least 1 confirmed somatic mutation.By comparison of mutational profiles ASXL1, RUNX1, PIGA, SETBP1, and CBL were most common in sMDS (26.1%, 21.7%, 18%, 13% and 13%, respectively). Because sMDS included a high proportion of patients with -7/del(7q), we compared sMDS with -7/del(7q) to pMDS with -7/del(7q) for coexisting mutational events. Mutations in RUNX1 appeared to be more frequent in sMDS vs. pMDS (27% vs. 0%, P=.03). In contrast, TP53 was more common in pMDS (7% vs. 32%, P=.1). Similarly, there were several other distinctive differences between all sMDS and pMDS irrespective of cytogenetics: mutations in U2AF1 were common in pMDS, mutations in RUNX1 appeared to be more frequent in sMDS vs. pMDS (22% vs. 5.5%, P=.02). Mutations in PIGA gene constituted a marker for sMDS derivation from AA. To discern a possible biological relationship we have also compared mutational profiles of hypocellular pMDS and sMDS, but no significant differences aside from PIGA prevalence were found.If sMDS is derived from mutations present at the AA stage, one would expect overlaps in the mutational spectrum of AA before and after evolution to MDS. DNMT3A, BCOR/BCORL1, and PDGFR family mutations were found at higher frequency in AA while ASXL1, RUNX1, SETBP1, PIGA, and CBL were higher in sMDS. Thus, cross-sectional analysis suggests that most of the clonal events occurring during the course of AA do not initiate sMDS. To further examine these findings we performed serial sequencing analyses: in 7 patients with sMDS WES was performed and clonal architecture was analyzed. We then queried whether mutations present in MDS were detected in archival samples at presentation using deep targeted NGS (depth 5-10x104 rds. In 4/7 cases the alterations appeared to be ancestral events for sMDS evolution. When an additional 68 AA cases were studied by deep NGS, somatic mutations were present in 31% of AA patients at presentation. Patients with clonal events at presentation tended toward worse progression free survival compared to patients without mutations (P=.1). Mutations found at both initial presentation and upon evolution were suggestive of a slow expansion of previously cryptic clones (ASXL1, CUX1, TET2, CBL, RUNX1, and SETBP1). Patients with these gene mutations (n=21) before immunosuppressive therapies (IST) had worseoverall survival compared to patients without these mutations (n=47; P=.009). To assess the potential impact of IST, we also investigated a subset of 37 patients (25 responders/ 12 refractory) following IST. Clonal somatic events were identified in 42 of them, but there was no association between the response to IST and somatic mutations at presentation (P=.7).Our results demonstrate that while subclonal mutations indicative of oligoclonal hematopoiesis are frequent in AA, the presence of specific permissive ancestral somatic events at the outset of AA predisposes patients to sMDS, a feature that has diagnostic and prognostic implications. DisclosuresMakishima:The Yasuda Medical Foundation: Research Funding. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau.

Long-Term Outcome after Immunosuppressive Therapy with Rabbit ΑΤG for Pediatric Aplastic Anemia: A Single-Center Retrospective Study in China

▪Background: Antithymocyte globulin (ATG)-based immunosuppressive therapy (IST) has been successfully used as the first-line treatment for severe / very severe aplastic anemia (SAA/VSAA) patients if no HLA-matched sibling donor was eligible for HSCT as a first choice. It was reported rabbit ATG (rATG) produced more profound immunosuppressive activity compared to horse ATG (hATG). However, recent clinical studies indicated that the stronger lympholytic activity did not mean that rATG was more effective. Most experiences from adult SAA/VSAA implied the efficacy of rATG was worse than hATG. However, susceptibility of children to intensive IST might not be exactly the same as adult patients, long-term efficacy of rATG in historic studies for children with SAA/VSAA was still elusive.Purpose: This study includes the largest cohort of pediatric AA patients treated with first-line rATG+CSA regimen published to date after a median follow-up of 69 months, aiming to assess the long-term outcome of rATG for children, and to identify the significant prior factors in clinical decision making.Methods: We reviewed 231 SAA/VSAA patients under 18 years old assigned to rATG+CSA from February 2000 to May 2014 in Department of Pediatrics, the Blood Diseases Hospital & Institute of Hematology, CAMS & PUMC. Response was evaluated 3, 6, 9, 12 24, 36 and 60 months after IST. We separately defined SAA-II as a specific type of gradually progressed SAA from a NSAA status within a longer period for at least 6 months. Multivariate logistic regression models were used to evaluate the effects of variables on the responses at different time points. Multivariate Cox model analysis of overall survival (OS) and failure-free survival (FFS) was calculated for variables with a log rank P value less than 0.1 in Kaplan-Meier analysis.Results: Of the overall patients, the total responded patients were 79(34.3%), 110(51.6%), and 129 (60.6%) at 6, 9, 12 months following IST, respectively. Intriguingly, 22 patients achieved delayed response between 12 months and 24months after IST, which increased the overall response rate by 10.2%, afterwards the rate reached a plateau by 3 years with the best response rate of 74.6% (Figure 1). Differences in baseline clinical parameters pre-IST were associated with response to IST. Absolute neutrophil count (ANC) less than 0.1*109/L was associated with an unfavorable early response rate at 6 months (P=0.009); absolute lymphocyte count (ALC) less than 1.6*109/L was a significant predictor for better response by 6 months and 12 months in multivariate analysis [6 months, P=0.033 vs. 12 months, P=0.021]. Lower absolute reticulocyte count (ARC no more than 18.5*109/L) predicted worse late IST response by 2 years and 3 years. In our large series of cohort, 5-year OS and FFS were 82.7% and 61.9%. Patients with VSAA as a significantly unfavorable prognostic factor had a much lower probability of 5-year survival when compared to patients diagnosed with SAA (76.4% vs. 87.2%, P<0.001, Figure2A). In multivariate analysis, SAA-II (P=0.021, Figure2B), and a pretreatment lower ARC (P=0.020, Figure2C) were independent unfavorable prognostic factors for FFS, but moderate PNH clone size (more than 5%) was verified as a good predictor for FFS (P=0.006, Figure 2D). At the last follow-up, twelve of the 135 responders relapsed after IST, meanwhile eight patients in responders and seven patients in non-responders experienced clonal evolution after IST, corresponding to cumulative incidences at 5.2% of relapse and 6.5% of evolution, which were obviously lower than previous reports.Conclusions: The combination of rATG and CSA was confirmed as an effective first-line therapy for children with SAA/VSAA in our cohort. We discerned a protracted recovery but an ultimately comparable long-term outcome of rATG. Baseline blood parameters (ANC, ALC, ARC) were predictive factors of response rate. Intensive supportive care may be necessarily pivotal to survival in cases of VSAA. Importantly, moderate PNH clone might be beneficial to FFS. Besides, for those who experienced gradually progressed disease course, early HSCT might be a more preferable option than receiving IST although further validation remains to be done. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High sensitivity flow cytometry to detect small population of PNH clone in bone marrow failure syndrome in Japan

High sensitivity flow cytometry to detect small population of PNH clone in bone marrow failure syndrome in Japan

FLAER を用いた高精度フローサイトメトリー法による本邦PNH 型血球陽性骨髄不全例の解析

FLAER を用いた高精度フローサイトメトリー法による本邦PNH 型血球陽性骨髄不全例の解析

Clinical and laboratory characteristics in patients of myelodysplastic syndrome with PNH clones

目的分析伴阵发性睡眠性血红蛋白尿症克隆的骨髓增生异常综合征（MDS-PNH）患者的临床特征及PIG-A基因突变等实验室特征。方法回顾性分析2013年8月至2015年8月诊治的218例MDS患者，其中经流式细胞术检出MDS-PNH 13例，选取同期17例再生障碍性贫血（AA）-PNH综合征患者及14例PNH患者，采用直接测序法分析PIG-A基因突变情况，比较各组患者的临床及实验室特征。结果MDS-PNH患者在MDS患者中的比例为5.96%（218例中13例），其中9例MDS-PNH患者使用环孢素A（CsA）治疗，8例获得血液学改善（HI）。MDS-PNH患者粒、红细胞PNH克隆大小及LDH水平[分别为19.2%（1.0%~97.7%）、4.3%（0~67.2%）、246（89~2 014）U/L]与PNH患者[分别为60.2%（3.1%~98.0%）、27.9%（2.5%~83.6%）、1 137（195~2 239）U/L]比较差异均有统计学意义（P值分别为0.026、0.007、0.049），与AA-PNH综合征患者[分别为23.2%（1.5%~96.0%）、14.4%（1.1%~62.8%）、406（192~1 148）U/L]比较差异均无统计学意义（P值均>0.05）。7例MDS-PNH患者检测到PIG-A基因突变，错义突变6例，移码突变1例，4例患者有相同的突变，为c.356G>A（R119Q）；9例AA-PNH综合征和11例PNH患者PIG-A基因突变，均存在c.356G>A（R119Q）。3组患者的PIG-A基因突变均为小克隆突变，以错义突变为主，59%（19/32）的突变发生在2号外显子。结论MDS-PNH患者CsA治疗效果好，PNH克隆较小，基因突变主要集中在2号外显子，可能存在突变热点。

Relationship Between Myelodysplastic Syndrome and Paroxysmal Nocturnal Hemoglobinuria: Spanish Erythropathology Group and Spanish Paroxysmal Nocturnal Hemoglobinuria Working Group Experience

▪INTRODUCTIONPNH clones appear frequently in patients with myelodysplastic syndrome (MDS) and aplastic anemia (AA) which may even evolve into classic PNH. It has been described isolated cases of clonal evolution from AA, through classic PNH and MDS; and at the end, acute leukemia (AL) has rarely been described. These patients show a progressive replacement of the PNH clone by a dysplastic one carrying a monosomy of chromosome 7.OBJECTIVEWe designed a retrospective study of patients included in the database of the Spanish PNH Registry in search of patients with a MDS and a PNH clone or those with a classic PNH who evolved into MDS. Our aim was to study the characteristics of patients who showed some of these entities and AA history.RESULTSOur search retrieved 6 patients, of whom 3 were MDS cases with a small PNH clone (Table 1). One of the patients had had a previous classic PNH followed by a MDS. The remaining two patients had been diagnosed with AA, developing classical PNH and MDS later; of these, one (patient 2) died with an acute leukemia.The median follow-up time was 83 (range 56-480) months, while the follow-up time from diagnosis of MDS was 68 (range 6-108) months. The patient characteristics are describe in Table 1.There are two remarkable features, diagnosis of a RAEB-1 in two cases and the frequency of trisomies of chromosome 8 and 21 and deletion of chromosome 7. These chromosomal abnormalities appeared sequentially as MDS evolved in 3 patients. The PNH clone disappeared in those patients who developed a MDS after an AA/classic PNH and persisted in those MDS cases who showed a PNH clone at diagnosis of MDS.Table 1Patients CharacteristicsPrevious diagnosisFollow-up timeMDS typeKaryotypeCase 1None70RARS+ 8Case 2AA + PNH56RCMD-7 evolution to -7, + 21Case 3Classical PNH83RAEB-1+ 8Case 4None83RAEB-1+21 evolution to +21, +8Case 5AA + classical PNH480RCMD+ 8 evolution to +8, -7Case 6None108RCMDNormalCONCLUSIONSPatients with a diagnosis of AA, classic PNH or MDS with a PNH clone must be followed up for life, with bone marrow studies with karyotype and measurements of the PNH clone, to rule out the development of other hematological entities. Our series is a small one and only studies with a larger number of patients will allow us to establish definitive conclusions. The inclusion of these patients in the International PNH Registry is strongly advised. DisclosuresNo relevant conflicts of interest to declare.

Single-Center Experience of Immunosuppressive Therapy with or without Eltrombopag in Patients with Aplastic Anemia

BackgroundImmune-mediated destruction of hematopoietic stem (HSC) and progenitor cells, leading to their depletion, has been implicated as the pathophysiology behind bone marrow (BM) failure in aplastic anemia (AA). Immunosuppressive therapy (IST) using antithymocyte globulin (ATG) and cyclosporine has been successfully used in the treatment of cytopenias in patients (pts) with severe aplastic anemia (AA). Recently, the thrombomimetic eltrombopag (E-PAG) has been shown to have activity in refractory AA, presumably by increasing the numbers of BM HSCs. To simultaneously target both immune destruction and HSC depletion, we studied the combination of E-PAG with IST in pts with newly diagnosed severe AA.MethodsThis is a single-arm phase II study evaluating the following combination in pts with AA: hATG 40 mg/kg/day (or 35 mg/kg/day for age ≥ 55 yrs) intravenously (IV) for 4 days, methylprednisone 1mg/kg/day IV daily for 5 days (prior to each dose of ATG), followed by PO prednisone tapered off over 1 month, G-CSF (filgrastim or pegfilgrastim) SQ for up to 3 months, and cyclosporine (5 mg/kg) daily for up to 6 months. After the results of E-PAG in refractory AA, the protocol was amended to study the addition of E-PAG to IST. It was started at a dose of 50mg PO daily with increase by 25mg Q2 weeks to a max of 150mg PO daily in the absence of platelet response. Responses were assessed at 3 and 6 months after initiating therapy per the standard criteria.ResultsA total of 31 pts with untreated AA were enrolled; the first 17 (55%) were treated with IST alone and the next 14 (45%) with IST + E-PAG. The characteristics of pts in the 2 cohorts are listed in Table 1. Pts receiving E-PAG were older, had significantly lower blood counts, and were less likely to have T-cell receptor clonality (27% vs. 60%, P=0.06). Twenty-one of the 31 evaluable pts responded (ORR=68%), including 7 CR (23%). Pts receiving IST + E-PAG had a higher ORR rate vs. IST alone (79% vs. 59%, P=0.28) and a longer median time to response (TTR, 46 vs. 37 days, P=0.14). Table 2 outlines the pretreatment pt characteristics of responders vs. non-responders. Overall, responding pts had significantly higher pretreatment WBC (P=0.04) and ANC (P=0.05); were more likely to have a detectable PNH clone (P=0.07); and less likely to have oligoclonal T-cells. Figure 1 shows OS by therapy and response. The 12-month OS for pts receiving IST vs. IST+ E-PAG was 86% vs 94% (P=0.90), respectively. The 12-month OS for responders vs. non-responders was 100% vs. 58% (P=0.01), respectively. The treatment was tolerable, with no additional toxicities noted after the addition of E-PAG. The most common grade 3/4 non hematologic toxicities were infection (3), musculoskeletal pain (3), transaminase elevation (1), fatigue (1), intracranial bleed (1), chest pain (1), headache (1), and serum sickness (1). One pt had an allergic reaction (grade 3) and was taken off protocol.Despite including older pts and those with significantly lower blood counts, the cohort receiving E-PAG had a higher ORR than IST alone, albeit with a longer TTR. A higher WBC count and ANC as well as the presence of a PNH clone were associated with response and response was associated with an improved OS. The combination of hATG, cyclosporine, steroids, G-CSF and E-PAG is a well-tolerated and effective regimen in the treatment of aplastic anemia and should be studied further. Table 1CharacteristicISTIST + Eltrombopagp-valueN1714Median Age49 (21-71)62 (22-84)0.14Median WBC2.1 (0.7 - 6.4)2.1 (0.2 - 4.3)0.78Median Platelet32 (2 - 77)15 (5 - 34)0.02Median Hb9.3 (7.5 - 10.8)8.7 (7.1 - 10.4)0.04Median ANC0.6 (0 - 5.2)0.3 (0 - 0.7)0.01CytogeneticsDiploid (%)10 (59)13 (93)0.05Insuff (%)7 (41)1 (7)0.05PNH Clone Present (%)9/14 (64)10 (71)1TCR Clonality Present (%)9/15 (60)3/11 (27)0.06Responses [CR+PR] (%)10 (59)11 (79)0.28Complete Response (%)4 (24)3 (21)1Med Time to Response, Days (range)37 (10 - 111)46 (21 - 200)0.14Table 2CharacteristicRespondersNon-Respondersp-valueN2110Median Age56 (21 - 84)57 (23 - 80)0.5Median WBC2.2 (0.6 - 6.4)1.7 (0.2 - 2.5)0.04Median Platelet20 (2 - 63)29 (11 - 77)0.24Median Hb9 (7.9 - 10.8)9.1 (7.1 - 10.7)0.78Median ANC0.6 (0 - 5.2)0.1 (0 - 0.8)0.05CytogeneticsDiploid (%)18 (86)5 (50)0.07Insuff (%)3 (14)5 (50)0.07PNH Clone Present (%)16/20 (80)3/8 (38)0.07TCR Clonality Present (%)7/17 (41)5/9 (56)0.68Monoclonal (%)7/17 (41)2/9 (22)0.42Oligoclonal (%)0/17 (0)3/9 (33)0.03 [Display omitted] DisclosuresOff Label Use: Use of eltrombopag in the frontline treatment of aplastic anemia is off-label. Daver:ImmunoGen: Other: clinical trial, Research Funding. Cortes:Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Teva: Research Funding; BerGenBio AS: Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. DiNardo:Novartis: Research Funding. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria.