PNH Clone Research Articles

Molecular Characterization of Leukemia Evolving from Paroxysmal Nocturnal Hemoglobinuria

Myeloid malignancies can evolve from prior hematologic disorders, most commonly AML evolving from MDS. AA and PNH are not malignant conditions but they can evolve to myeloid malignancies with a lower frequency compared to MDS and other myeloid and myeloproliferative diseases. PNH can evolve to myeloid disorders with an incidence rate of 4-10%. Here we aim to revisit the subject of malignant evolution of PNH to myeloid disorders by analyzing the molecular background of PNH using modern NGS technologies. Clinical characteristics, demographics and mutational profiles of patients were collected at The Cleveland Clinic Foundation. In total a cohort of 243 patients evaluated for hemolytic PNH (n=83), AA (39) and AA/PNH (n=121) followed for a period of 22 years was evaluated. Inclusion criteria were: complete flow cytometric panels of PNH cells, patients without antecedent AA (pPNH, 45), patients evolving from AA with PNH clone >20% (sPNH, 38), karyotype (at diagnosis of PNH and transformation), clinical parameters including time to malignant progression, and molecular characterization resulting from NGS performed using various library preparation systems (TruSeq, TruSight, Nextera), comparison of molecular mutations with control cohorts (AA, 160; MDS, 835). The incidence rate of myeloid disorders in our PNH cohort was 3% (7/243). Among hemolytic PNH patients, 7 patients progressed to AML (n=1), MDS (n=5) or myelofibrosis (n=1). Median age was 48 yr (range, 24-80); M/F, 5/2. Median PNH clone size was 71% (range, 29-99). Three progressors were in the pPNH and 4 in the sPNH group. Time to malignant diagnosis was <1-22 yr: 4 yr for 3 patients, 5 yr for 1 patient, <1 yr for 2 patients and 22 years for 1 patient. At the time of transformation, combination of karyotypic abnormalities and molecular lesions showed that 4 cases had abnormal karyotype (+8, -7, del13q) and 5 cases carried myeloid mutations (ASXL1, BCOR, NPM1, TET2, U2AF1, WT1). Normal karyotype was seen in 3 patients with ASXL1+U2AF1, U2AF1 and BCOR+NPM1+WT. In 2 patients, del13q and -7 were associated with BCOR and TET2. In 2 others, del(13q) and +8 were the only detected aberrancies. A total of 45 somatic mutations equally distributed in the 2 groups (pPNH, 22; sPNH, 23) and with similar VAFAVG (pPNH vs. sPNH 38% vs. 33%) were found. PNH showed a higher proportion of individuals with mutations compared to AA/PNH+AA (42 vs. 22% of cases with ≥1 mutation; P=.002) with a median VAF percentage significantly higher in PNH vs. AA/PNH+AA (40 vs. 19%; P<.0001). FLT3 (n=2), JAK2 (n=1), LUC7L2 (n=1), NPM1 (n=1), SRSF2 (n=1) and ZRSR2 (n=2) were exclusively mutated in PNH with the presence of some of them (FLT3, NPM1) possibly representing an early onset of clonal evolution. BCOR/BCORL1 core was more mutated in PNH as compared to AA/PNH+AA patients (11 vs. 3%; P=.01) as did TET2 and U2AF1. This pattern of mutations had a strong similarity with that of MDS. VAF comparison between myeloid mutations and PIGA mutations showed that accessory mutations in patients with PNH were secondary hits, with PIGA mutations being the founder lesions. We then analyzed the clonal hierarchy of mutations using VAF-adjusted for X-chromosomal loci in males and inferred mutational rank (founder/ co-dominant) vs. secondary (sub-clonal). PIGA mutations were founder lesions in 70% of the cases, co-dominant in 2% and secondary in 28% suggesting that additional mutations represented mainly cooperative events without over representing PIGA. In fact, comparison of VAF and PNH clonal size showed that 70% of the hits were present in the same clone (VAFSUM >55), 17% were equivocal (possibly hits biclonal with a VAFSUM between 45-55), while 13% were more likely a result of clonal chimerism (hits present in different clones; VAFSUM <45). Clonal evolution of hemolytic PNH to MDS/AML is rare, but still occurs and it is accompanied by mutations in typical myeloid genes (BCOR, NPM1, TET2, U2AF1) which in permissive circumstances are capable to change the cell's fate favoring clonal evolution. Our results suggest that most PNH cases can carry additional mutations in the same clone and these mutations can be secondary hits, with PIGA mutations being the founder lesions. However even when mutations in myeloid genes are dominant, the phenotype of the patients is inferred by PIGA. This observation supports the nature of PNH as a monogenic disease with clinical manifestations resulting by PIGA mutations rather than by myeloid genes. Disclosures Sekeres: Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

The Path to Cure: Using Haploidentical (haplo) Donors and High-Dose Post-Transplant Cyclophosphamide (PTCy) for Treatment-Naïve and Refractory Severe Aplastic Anemia (SAA)

SAA is a life-threatening hematopoietic stem cell disorder that is often treated with bone marrow transplantation (BMT) to reconstitute hematopoiesis and eliminate clonality. Our group has shown that PTCy after related HLA-haplo BMT can facilitate engraftment and yield low rates of relapsed and refractory (R/R) SAA. Given the risk of relapse or secondary clonal disease following immunosuppressive therapy (IST) is > 50% at five years, we decided to implement this treatment paradigm in untreated SAA. Here, we review the haplo outcomes in SAA, with an update in R/R SAA patients and new results in treatment-naïve (TN) SAA patients using haplo donors and PTCy. Patients were eligible with SAA. Fanconi anemia and short telomere patients were excluded. R/R patients had ≥ one course of IST. The conditioning regimen included standard published haplo backbone of antithymocyte globulin, low dose CY, and total body irradiation (TBI). All R/R patients received 200cGy. The TBI dose for the TN cohort was modified after 3 graft failures (GF) in initial 7 patients treated at 200cGy; thus, the last 8 TN patients received 400cGy. After the marrow graft was infused on day 0, cyclophosphamide 50mg/kg/day IV on days +3,+4 post-transplant was administered. All received mycophenolate mofetil beginning day +5 through 35 and tacrolimus day +5 to 365. Engraftment definitions include: absolute neutrophil count >1.0 x10E9/L for three consecutive days; days from last red blood cell transfusion for hemoglobin and platelet count >50,000 for 7 days without transfusion. Thirty-five SAA patients received haplo BMT: 20 R/R and 15 TN. The median follow-up is 31 months (90% CI: 26.0, 45.4). The median age was 25 (range, 4-69) years, with 23 patients > age 20 and 57% were males. The median related haplo donor age was 34 years (range, 19-57), including three non-first degree relatives. Table 1 shows data by cohort. Median time to neutrophil recovery was 17 days (range 15-88), to red cell recovery 23 days (range 6-58) and to platelet recovery 28 days (range 15-108). At the time of BMT, 25 of 35 patients had evidence of clonality (mostly PNH clones). Four of 35 patients (11.4%) (95% CI: 3.2, 26.7%) experienced GF. There was one GF out of 20 patients in the R/R group (5%) (95% CI: 0.1, 24.9%) and 3 of 15 in the TN group 20% (95% CI: 4.3,48.1%, p= 0.20). OS for all patients (Figure 1) is 94% (90% CI: 88,100%) at one and two years, 100% in the R/R group and 86% (90% CI: 72, 100%) in the TN group. The cumulative incidence of grade II-IV aGVHD at day 100 is 9% (90% CI: 1, 17%). The cumulative incidence of chronic GVHD at 2 years is 9% (90% CI: 1,17%). All R/R patients are alive and well, and fully engrafted with 100% donor chimerism; patient with primary GF is 100% chimeric after 2nd transplant from different haplo donor. One patient has extensive cGVHD. Of the first 7 TN patients who received 200cGy TBI, 4 are fully engrafted and well; of the 3 with GF, 2 died from infection and one is now 100% chimeric using second haplo donor. After increasing the TBI dose to 400cGy, the subsequent 8 patients are alive with full donor chimerism. Nonmyeloablative haplo BMT with PTCy represents a potential cure in SAA, with all 20 R/R currently alive, disease-free (including eradication of clonal diseases), and no evidence of active GVHD. Extending this approach to TN patients was associated with higher GF rates. Perhaps this is not surprising given that the potential beneficial effect of front-line IST on engraftment is absent. The increase in TBI dose to 400cGy in TN patients appears to achieve more durable engraftment without early greater toxicity (infection, delayed engraftment, GVHD); more patients and longer follow up are required to understand rates of fertility and other survivorship issues. Alternative donor BMT (including haplo donors) should be offered to all SAA patients with R/R SAA who are fit enough to tolerate non-myeloablative conditioning. A potential advantage of haplo donors over unrelated donors is that the transplant center has full control and oversight of allograft quality, which can be especially important with the use of the preferred allograft source for SAA, bone marrow. Non-myeloablative haplo BMT in TN SAA could lead to a paradigm-shift in the field, such that essentially all children and fit adults can proceed quickly to a relatively safe, curative BMT. Disclosures DeZern: Astex Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy. Cooke:Jazz pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Subcutaneous Alemtuzumab Has Activity in Treatment-Naïve Patients with Acquired Aplastic Anemia

Subcutaneous Alemtuzumab Has Activity in Treatment-Naïve Patients with Acquired Aplastic Anemia

Telomeric Length to Characterize and Prognosticate Bone Marrow Failure, Risk of Clonal Evolution and Multi-System Complications in Aplastic Anemia and Other Hypocellular Cytopenic Disorders, Independent of Telomere Gene Complex Mutations

Introduction Telomere length is shortened in patients with idiopathic aplastic anemia (AA) and other bone marrow failure disorders (BMFD) and predicts risk of clonal evolution (CE), relapse and overall survival (OS). Telomereopathies predominantly cause bone marrow failure, are multi-systemic disorders with variable penetrance, and may involve inter-play of other factors in disease manifestation and organ affliction. Telomere length (TL) in AA and other hypocellular BMFD, independent of mutations in telomere genes (TGC), has not been studied as a scoring tool, as well predicting the risk of affliction of other organ/systems in these disorders. We systematically review a large cohort of 472 patients in a single centre with AA/BMFD using TL and TGC analysis as a discriminator, to study risk of CE and OS, manifesting with liver/lung and skin complications, cancer predisposition and likelihood of a family member presenting with cytopenias. Methods We screened 1060 consecutive patients at a single centre from the years 2011-18, with AA or unexplained cytopenias for telomere length (TL) analysis using a multiplex qPCR methodology as described by Cawthon et al. 472 (44.5%) patients had TL less than the 25th centile, of whom 243 had <1st centile, 122 had <10th and 107 had TL between the 10-25th centile. These 472 patients underwent TGC mutation analysis on a customised panel of 12 TGC genes (TERT, TERC, DKC1, TINF2, NHP2, NOP10, RTEL1, CTC1, USB1, WRAP53, ACD and PARN) using deeply parallel sequencing and were studied alongside their clinical parameters, disease progression, treatments and OS. Results Table attached. The median age and gender across the 3 cohorts studied (TL 10-25th centile, <10th centile and <1st centile) were comparable, with a median age of 43 for the entire cohort. 127 (26.9%) of patients were detected to have a total of 142 mutation in the TGC. A third of patients with BMFD and TL<1st centile had a TGC mutation, but the prevalence did not statistically differ with the other cohorts of TL <10th centile and between 10-25th centile. Patients with TL<1st centile can have mutations in 2 telomere genes, but this is less frequently seen when TL<10th centile and not seen in the cohort with TL between 10-25th centile. TL does not correlate with the manifestation and presence of liver, respiratory and skin problems of telomere disease. TL in BMFD does not associate with increased predisposition to cancer, as their presence was not statistically significant across the 3 groups. Disease manifestation with bone marrow failure, haematological indices, need for blood transfusions, presence of a PNH clone and karyotypic abnormalities, including complex and monosomal karyotype were not dissimilar across the 3 TL cohorts. Not surprisingly, there were more family members with features of cytopenias and BMFD, on screening of patients with a confirmed TGC mutation. TL did not predict a difference in treatment strategies used across the 3 groups, although more patients with a TGC mutation received anabolic steroids. The risk of clonal evolution to PNH or MDS/AML and overall survival was again similar across the 3 TL cohorts with an OS of 93% at 1078 days follow-up for the entire cohort. Conclusion TL can reliably be used as a screening tool to investigate patients for further TGC mutation analysis in patients with AA or BMFD. Heterozygous state mutations in TERT are the commonest, and can be associated with mutations in other TGC genes, particularly RTEL1 and TERC. This may cause a compounding factor in shortening the TL further. However, TL<1st centile does not associate with more severe cytopenias (BMFD) or predict more multi-system manifestation or increased cancer pre-disposition. Indeed, in our cohort it failed to show an increased risk of clonal evolution or decreased OS. Telomere biology is a dynamic process and assessments at different time points, from diagnosis to the time point of progression and transformation, may yield better understanding in pathogenesis of BMFD. TL and TGC mutation analysis in a single centre study from 2011-18. Table Disclosures Kizilors: Incyte biosciences: Research Funding. Mufti:Celgene: Consultancy, Research Funding.

A Prospective, Cross Sectional Study of PNH Clone in MDS Patients Using High Sensitivity Flowcytometry: A Single Center Experience.

Subclinical PNH can be present in patients with bone marrow failure like aplastic anemia and myelodysplastic syndrome (MDS). Such clone may have prognostic and therapeutic implications. In literature around 1-10% MDS cases have shown a PNH clone, however, data from India is relatively scarce. A high sensitivity PNH assay was employed using a single tube combination of FLAER, CD157, CD64, CD15 and CD45 antibodies in adult patients of MDS at presentation. A clone size of > 0.01% was taken as significant. A total of 30 patients were included. PNH clone was present in 30% cases. Correlation done between PNH clone size and LDH values showed moderately positive correlation (r = 0.735, p = 0.001, r2 = 0.541). As per this study a LDH cut off of 247IU is likely to predict a PNH clone (> 1%) with moderate sensitivity and specificity. High sensitivity PNH assay is able to detect small PNH clone. Calculating the cut-off of LDH to predict PNH positivity can help us judiciously prescribe this test in MDS patients in resource constrained settings.

Methodological Study on Flow Detection of PNH Clone and Its Clinical Screening Significance

Methodological Study on Flow Detection of PNH Clone and Its Clinical Screening Significance

Paroxysmal Nocturnal Hemoglobinuria: Grasping the Flow of Diagnostic Dilemmas

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a rare life-threatening condition due to an acquired somatic mutation of the PIGA gene, leading to nonmalignant clonal expansion of hematopoietic stem cells, which are deficient in glycosyl phosphatidylinositol-anchored proteins (GPI-APs). Fluorescein-labeled proaerolysin (FLAER) and flow cytometry are key tools in the diagnosis of PNH. While clonal detection of PNH in both tests has a sensitive diagnostic threshold of 0.01% in erythrocytes and 0.05% to 1% in leukocytes, one must be cautious in ruling out the possibilities of myelodysplastic syndrome (MDS) or aplastic anemia. We propose guidelines in the differential diagnosis and evaluation of PNH from these and other hematologic disorders that can arise from GPI-AP deficient cells. These guidelines are based on a meta-analysis of five research literature sources, including four case studies. We also compare and contrast our limits of quantification of the in-house PNH assay at University of Kentucky Healthcare with those of an interlaboratory validation of 11 institutions within the United Kingdom. Our report advocates for thorough evaluation of multiple laboratory and clinical variables affecting sensitivity and accuracy of flow cytometry and FLAER in PNH. Furthermore, we recommend lowering of the in-house limit of quantification from the current 1% to 0.01%. This allows for the critical consideration of conditions such as MDS and aplastic anemia and their disease courses, all of which can present with PNH clones as low as 0.01% on flow cytometry and FLAER.

Study for the diagnostic screening of paroxsymal nocturnal hemoglobinuria in Turkey: Prospective multicentric evaluation of suspected patients

BackgroundParoxysmal nocturnal hemoglobinuria (PNH) is a rare disease presenting with variable and various clinical findings. PNH might be overlooked and diagnosis may be delayed due to low awareness about PNH. This is the first multicenter study in Turkey, investigating the efficiency of diagnostic screening of PNH by multiparameter flow cytometry (FCM) according to consensus guidelines. MethodsWe evaluate the efficiency of consensus clinical indications for PNH testing with FCM in 1689peripheral blood samples from 20 centers between January 2014 and December 2017. ResultsOverall, at the 20 centers contributing to this study, PNH clone were detected in 62/1689 samples (3.6%) by FCM test. 75.8% (n = 47) of patients with PNH clone had aplastic anemia, 3.2% (n = 2) had Coombs (-) hemolytic anemia, 6.5% (n = 4) had unexplained cytopenia, 3.2% (n = 2) had MDS with refractory anemia, 1.6% (n = 1) had hemoglobinuria and 9.7% (n = 6) had others (elevated LDH, splenomegaly, etc.). In contrast, we detect no PNH clone test in patients who were screened for unexplained thrombosis. ConclusionsOur study showed that current clinical indications for PNH testing are highly efficient and diagnostic screening of suspected patients for PNH with FCM is recommended. However, advanced screening algorithms are required for patients presenting with unexplained thrombosis and normal complete blood count.

Efficacy of Low Dose Combined Chemotherapy for Patients with Relapsed and Refractory Aplastic Anemia-Paroxysmal Nocturnal Hemoglobinuria Syndrome

To evaluate the clinical efficacy of low dose combined chemotherapy（LDCC） for patients with relapsed and refractory aplastic anemia-paroxysmal nocturnal hemoglobinuria（AA-PNH） syndrome, and to analyze the advantages of LDCC in the treatment of AA-PNH syndrome. The clinical characteristics and the curative effect of LDCC in 9 patients with relapsed and refractory AA-PNH syndrome were retrospectively analyzed. Five patients were treated with MP therapy［melphalan 2 mg/(m2·d); prednisone 0.5 mg/(kg·d)］, and the other 4 patients were treated with HA therapy(HHT 2 mg/d iv drip, for 5 days; Ara-C 100 mg/d iv drip, for 5 days). The changes of PNH clone, dosage of corticosteroid, hemolysis and the relapse of disease, hematological parameters and adverse reactions were compared before and after therapy. All patients were treated for 1-2 courses. Seven out of 9 patients responded well, the dosage of corticosteroid and the bilirubin concentration decreased significantly and anemia was relieved in 7 patients (P<0.05). One patient relapsed in one year. PNH clone of 3 patients turned negative. Five patients did not rely on blood transfusion in 1 year. There was no bone marrow failure to be found in all patients. The LDCC has better efficacy and safety in the treatment of patients with AA-PNH syndrome, moreover, the patients is more tolerant to LDCC, thus the LDCC may be a selection for treatment of patients with relapsed and refractory AA-PNH syndrome.

PB1859 PROSPECTIVE COHORT STUDY OF KOREAN PATIENTS WITH BONE MARROW DISORDERS WITHOUT LARGE PNH CLONE TO DEFINE FACTORS ASSOCIATED WITH PNH CLONE EXPANSION

Background:Factors associated with PNH clone evolution is not well described in the Asian population.Aims:In this prospective cohort study, we recruited Korean patients with bone marrow disorder to define factors associated with an increase in PNH clone size.Methods:We included patients with aplastic anemia (AA) or myelodysplastic syndrome (MDS) if PNH clone is not absent at the time of screening. We also included patients who have small PNH clones (&lt;10%) regardless of bone marrow pathology except for hematologic malignancies. Patients should be free of complement inhibitor treatment for at least 8 weeks before study enrollment. PNH clone size testing was performed using high‐resolution flow cytometry and FLARE.Results:We enrolled 227 patients (Male:Female 121:106, mean age 53 years) from 6 centers in Korea. PNH clone was ≥ 1% in 28 patients, 0Table1 Characteristics of 5 patients who experienced more than 10% of PNH clone expansionSummary/Conclusion:We constructed a prospective cohort of Korean patients with AA, MDS or small PNH clones. PNH clone distribution had a distinctive pattern according to underlying bone marrow pathology. We expect to construct a precise model for PNH clone expansion using this cohort.image

PB1867 USE OF THERAPY WITH COMPLEMENT INHIBITOR AND THROMBOPOIETIN RECEPTOR AGONISTS IN TREATMENT OF PATIENT WITH SAA/PNH. CLINICAL CASE.

Background:Aplastic anemia (AA) is severe blood system disease, in which more than half of patients may have PNH clone, and there may occur associated variants AA with PNH (AA/PNH). The most frequently observed is the development of classical hemolytic PNH at AA remission, but possibly a combination of the two diseases. For such cases, there is a positive experience of AA combined treatment with immunosupressive therapy (IST) and targeted therapy using complement activation inhibitors. With resistant AA a therapeutic optionis possible using thrombopoietin receptor agonists.Aims:To treat of patient with SAA/PNH in absence of possibility of full use of ISTMethods:A clinical observation of a 53‐year‐old female with AA/PNH in the course of the disease is presented. Since 1994 patient, aged 53 yrs, has been under our supervision. The diagnosis was established according to a comprehensive examination, with severity determination in accordance to international criteria. The examination used morphological, histological, immunological (including highly sensitive flow cytometry), biochemical methods. No compatible donor for bone marrow transplantation was found. Anti‐lymphocyte globulin (ALG), cyclosporin‐A (CyA), Eculizumab and thrombopoietin receptor agonists were used in treatment.Results:ALG and cyclosporine treatment gave only a temporary effect – an improvement in hemogram parameters and a reduction in transfusion dependence. CyA doses were reduced (about 3 mg per day) due to adverse effects. The possibilities of active IST were also limited by comorbidities, including chronic hepatitis B. Since 1999, signs of chronic intravascular hemolysis and hemolytic crises have been observed. Flow cytometry have detected PNH clone. Subsequently, according to ICNN protocol testing data, from 2011 to the present, the clone size was determined 96‐99% with a predominance of type III erythrocytes. According to repeated bone marrow assessments, severe aplasia with pancytopenia in the peripheral blood was maintained. Anemia persisted up to 65 g/dL with transfusion dependence (up to 12‐16 doses of red blood cells per year), leukopenia within 0.9‐1.5 x109/l and thrombocytopenia 5‐15 x109/l with cutaneous hemorrhagic syndrome. On 2013, after vaccination with meningococcal vaccine, Eculizumab treatment was started according to the standard scheme with achievement of an adequate control of hemolysis. Clinical manifestations of hemolysis are absent; the level of LDH does not exceed 1.5 ULN. Therapy continues to the present. Until 2015, CyA therapy was periodically resumed due to the persistence of SAA criteria. In 2013‐2015 the patient additionally received thrombopoetin agonist therapy: Romiplostimum (4‐6 ug/kg once a week) with breaks every 3‐4 months. Two months after the start of therapy, a clinical effect was obtained – a decrease in hemorrhagic syndrome. After 3 months, there was a trend towards an increase in platelet levels. At present, severe leukopenia (1.0 – 2.2 x109/l) and neutropenia (0.5‐0.7x109/l) persist. At the same time, the platelet level is 30‐70 x109/l without spontaneous hemorrhages, since January 2018 the patient does not receive blood transfusions and hemoglobin stays at 10.2‐11.0 g/dL. Also there has been a significant improvement in the quality of life.Summary/Conclusion:This case demonstrates the possibility of a significant improvement in the course of sAA/PNH (including quality of life) in the absence of a sufficient effect of IST, but adequate target control of hemolysis and the use of thrombopoietin receptor agonists.

PB1855 UPFRONT HAPLOIDENTICAL TRANSPLANT IN YOUNG SEVERE APLASTIC ANEMIA PATIENTS ‐ RESULTS OF A PILOT STUDY FROM SOUTH INDIA

Background:Haploidentical bone marrow transplant is one of the second line treatment option in severe aplastic anemia patients failing immunosuppressive therapy. In younger patients, allogeneic bone marrow transplant from a matched sibling donor gives more than 80% long‐term remission rates. But unfortunately, only 30% patients will have a matched sibling donor. In a middle‐income country like India, the availability and affordability of patients to an unrelated donor is limited. Most of our patients have got a single chance for treatment and many relapsed patients go into palliative care.Aims:Aim of the present study is to evaluate the efficacy and safety of upfront Haploidentical bone marrow transplant in less than 40 years old severe aplastic anemia patients not having a 10/10 matched donor.Methods:This is a prospective non‐randomized single center study conducted during the period from January 2017 to June 2018. Eleven patients less than 40 years old underwent upfront Haploidentical bone marrow transplantation for severe aplastic anemia during the above period. Peripheral blood stem cell was used in all patients.FLU‐ATG‐PTcy was the conditioning regimen used in all patients with Cyclosporine + MMF based GVHD prophylaxis. This study got clearance from institutional review board and the transplant was sponsored by government.Results:In six patient's donors were parents (Mother in four and father in two) and rest of the donors were siblings. Two were Fanconi positive and none had a PNH clone. Seven were 5/10 matched, three were 6/10 and one was 7/10 matched. All patients had engraftment at a median of 11 days (range 8 to 13 days). One patient had fatal grade IV acute GVHD and succumbed to GVHD on day +36. Seven patients (64%) had grade I/II GVHD and three patients (27%) had grade III GVHD. 64% patients had grade I/II chronic GVHD at date cut off of January 2019. 10 patients (89%) attained complete remission and were alive till January 2019.The overall survival rate at one year post transplant was around 89% which is better than immuno‐supressive therapy in this age group. No differnces were observed in engraftment dates or survival outcomes based on type of donor and degree of mismatch. The study patients are very small to get statistically significant rates.Summary/Conclusion:With nearly 90% overall remission and survival rate at one year, upfront haploidentical transplant seems to be a safe and effective option in young patients with severe aplastic anemia. This will help to reduce the cost of Matched unrelated donor stem cells significantly in resource poor countries and increase the number of patients opting for a curative treatment.

Aplastic anemia in children: How good is immunosuppressive therapy?

Aplastic anemia (AA) is an uncommon disorder in children, with hematopoietic stem cell transplant being the 1st line therapy; immunosuppressive therapy (IST) is the alternative therapy and is the most commonly used modality of treatment. There is paucity of data from the developing countries regarding treatment outcome with IST. We aimed to assess the outcome of IST in children with AA. Data for 43 children treated with IST from January 2012 to January 2017 (5 years) were retrieved from clinic records. IST included equine antithymocyte globulin (ATG) along with cyclosporine A. Complete response, partial response and nonresponse was seen in 9 (21%), 14 (32.5%) and 20(46.5%) patients, respectively. The median time to best response in the whole cohort was 19.1 months. However, complete response occurred nearly 2-year post-IST. There was no difference in outcome related to severity of AA, the presence of PNH clone, higher ALC or different available brands of ATG. There was a significantly better rate of response (p value: .03) at 6 months in patients who went on to achieve a CR vs patients who achieved a final PR only. An overall response rate, including partial and complete response, of 53.5% was seen in our cohort with a 3-year OS of 63%. This is not at par to the outcome reported from developed nations. The available brands of ATG were similar in terms of response kinetics as well as survival. A delayed time to complete response with prolonged requirement of cyclosporine therapy was seen in the cohort.

Assessment of PNH clone by multiparameter flow cytometry in cytopenia and hematologic malignancies

Assessment of PNH clone by multiparameter flow cytometry in cytopenia and hematologic malignancies

Luigi Del Vecchio 1955–2018

Luigi Del Vecchio 1955–2018

Prognostic and Predictive Impact of Small PNH Clones in a Large Cohort of Patients with Myelodysplastic Syndromes and Aplastic Anemia: A Single-Center Experience

▪Background: the use of high sensitive FLAER in the last 10 years improved the detection of very small PNH clones (<1%) in various hematologic conditions, including aplastic anemia (AA)/myelodysplastic syndromes (MDS). Although generally considered unremarkable, the recent observation of asymptomatic end-organ damage due to undiagnosed thrombosis even in patients without overt hemolysis questions the assertion that small PNH clones are of subclinical value.Aim: To evaluate the prevalence of PNH clones ≥0.01% in AA/MDS patients tested at a single tertiary center, and to assess their impact on disease prognosis (leukemic evolution, death), occurrence of thrombosis, and response to current therapies.Methods: We retrospectively collected clinical (diagnosis, stage, therapy, complications and outcome) and laboratory features (complete blood counts, LDH, PNH clone size) of 869 MDS and 531 AA patients tested from March 1998 till October 2017.Results: Figure 1 shows clinical and laboratory characteristics of MDS and AA patients, divided according to the presence/absence of PNH clones. A PNH clone was less frequently found in MDS cases versus AA (20.3% vs 61%). Focusing on MDS, PNH+ cases were significantly more hypoplastic, mainly displayed IPSS low/int-1 score, and showed deeper cytopenias (significantly for Hb and PLT) and higher LDH levels. Dividing patients according to clone size (negative, 0.01-1%, 1.01-10%,10.01-50%, and >50% on granulocytes), we observed a significant worsening of cytopenia and raise of LDH along with clone size increase. Likewise, lower IPSS risk patients more frequently displayed a greater clone size. As regards therapy, PNH+MDS showed significantly higher response rates to immunosuppressive therapies (ATG and CyA, 84% vs 44.7%, p=0.01) and to HSCT (71% vs 56.6%, p=0.09) compared to PNH-, and the cumulative probability of response to any treatment significantly improved along with clone size increase (from 52 to 100%, p=0.03). In addition, PNH+MDS showed lower rate of progression and AML evolution, and a longer OS [mean 11.9+0.7 years (10.5-13.3) vs 7.3+0.3 (6.6-7.9), p<0.0001] compared to PNH- ones. Interestingly, we observed a significant reduction of death frequency along with clone size increase (56% in PNH- vs 24% in PNH+ with clone size 0.01-1%, p<0.0001). However, PNH+MDS had a higher incidence of thrombotic events, with greater frequency along with clone size increase (from 5% in PNH- to 50% in PNH+ with clone size >50%, p<0.0001). As expected, worse OS also significantly correlated with older age, male gender, transfusion dependence, MDS progression/AML evolution, higher IPSS score, and non-response to therapy.Regarding AA, together with a higher frequency of PNH clones, we also found a higher frequency of large clones (>10%) compared to MDS (p=0.04, bars chart). PNH+AA showed deeper thrombocytopenia, higher reticulocyte counts and LDH values. As observed for MDS, we found a significant worsening of cytopenia and raise of LDH along with clone size increase. In addition, PNH+ AA showed higher response rates compared to PNH- (97 vs 77% for HSCT, p=0.01; 78 vs 50% for IST, p<0.0001; and 88% vs 65% considering any treatment, p<0.0001). PNH+AA also showed lower rate of MDS progression and death (p=0.01 and p<0.0001), and longer OS [mean 15.8+0.43 years (14.9-16.7) vs 6.5+0.35 (5.8-7.21), p<0.0001]. Also in AA, we observed a significant reduction of death frequency along with clone size increase (32.1% in PNH- vs 9.2% in PNH+ with clone size 0.01-1%, p<0.0001). Worse OS also correlated with older age, male gender, presence of cytopenia, and non-response to therapy.Conclusions: Prevalence of PNH clones of any size is high in patients with MDS and AA. We firstly show a positive impact of PNH clone positivity on response to IST and HSCT in MDS. The presence of a PNH clone also correlated with lower disease progression and better OS. Furthermore, we confirm the known favourable prognostic and predictive value of PNH clones in a large AA cohort. Clone size analysis suggests that even small clones (0.01-1%) have a clinical and prognostic significance. [Display omitted] DisclosuresKassam:AbbVie: Equity Ownership. Yallop:Pfizer: Consultancy; Servier: Other: Travel funding. Mufti:Celgene: Research Funding. Kulasekararaj:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support .

Bystander Proliferation of Piga-Mutated Hematopoietic Progenitor Cells in Acquired Aplastic Anemia Patients Possessing HLA Class I Allele-Lacking Leukocytes

[Background] Hematopoietic stem progenitor cells (HSPCs) with PIGA mutations are thought to acquire a survival advantage over normal HSPCs under immune attack against HSPCs and produce glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells in patients with acquired aplastic anemia (AA). Various underlying mechanisms of the survival advantage of PIGA-mutated HSPCs have been proposed; however, it remains still unclear how PIGA-mutated HSPCs are immunologically selected in AA. Approximately 15% of AA patients with increased GPI(-) cells possess another aberrant leukocyte subset that lacks the expression of the HLA-class I allele due to a copy number-neutral loss of heterozygosity of the HLA haplotype, which occurs in the short arm of chromosome 6 (6pLOH) as a result of uniparental disomy, or HLA allelic mutations. The presence of HLA-class I allele-lacking leukocytes (HLA-LLs) is considered to be the most compelling evidence to support the involvement of cytotoxic T lymphocytes (CTLs) in the development of bone marrow failure. Charactering GPI(-) leukocytes and platelets in AA patients with HLA-LLs may provide an insight into the mechanism underlying the immune selection of PIGA-mutated HSPCs.[Patients and Methods] We investigated the presence of GPI(-) leukocytes, erythrocytes, and platelets in 63 patients with AA using high-sensitivity flow cytometry (FCM). For the platelet analysis, platelet rich plasma (PRP) was obtained by centrifuging anticoagulated blood at 1000 rpm for 7 minutes with the brake turned off. Thirty microliters of PRP was incubated with monoclonal antibodies specific to CD55-PE, CD59-PE, CD41a-APC and HLA-A2 or A24-FITC for 20 minutes at room temperature in the dark. To prevent doublets, samples were diluted 1 to 100 in PBS and filtered with mesh immediately before the FCM analysis. Thirty of the 63 patients were heterozygous for the HLA-A allele with A24 and A2, and thus the presence of both HLA-LLs and HLA-A allele-lacking platelets could be evaluated by FCM. The lack of the HLA-A allele due to 6pLOH or allelic mutations in all HLA-LL(+) patients was confirmed by a droplet digital PCR or deep sequencing.[Results] Increased GPI(-) granulocytes, which accounted for 0.01-99.8% of the total granulocytes, were detected in 37 (58.7%) patients while HLA-A24 or A2-lacking granulocytes accounted for 0.39-98.3% of the total granulocytes in 20 (66.7%) of the 30 patients. Eight patients possessed both GPI(-) cells and HLA-LLs. In all 8 of these patients, the two aberrant cell populations were mutually exclusive. The analyses of different cell lineages revealed HLA-A allele-lacking cells in all lineages of cells, including granulocytes (Gs), monocytes (Ms), T cells (Ts), B cells (Bs), NK cells (NKs), and platelets (Ps) in 7 of the 8 patients; the remaining one patient had the GMTP pattern. In contrast, the lineage diversity of GPI(-) in the 8 patients was more restricted; GMTBNKP was only detected in 2 patients; the combinations in the other 6 patients were GT (n=1), GMBNKP (n=2), GMTNKP (n= 1) and GMTBP (n= 2). In Case 1, GPI(-) cells were not detected in T cells while HLA-A24(-) cells were detected in all lineages of cells including T cells (Figure 1). The limited lineage diversity of GPI(-) cells was also evident in 6 patients who did not possess HLA-LLs (GMP, GMBP, GMBNKP, GMTNKP, GMTBP) with GPI(-) granulocytes>10% while the GMTBNKP pattern was common in 10 HLA-LL(+) patients who did not possess GPI(-) cells, regardless of their percentage of HLA-A allele-lacking granulocytes. Longitudinal follow-up of 5 patients over a period of 8-27 years showed a decline in the percentage of GPI(-) granulocytes (39.2 to 0.00%, 11.4 to 0.04%, 3.50 to 0.30%, 1.77 to 0.00% and 0.79 to 0.11%) and a reciprocal increase in the percentage of HLA-A allele-lacking granulocytes (80.0 to 95.2%, 92.0 to 99.1%, 24.0 to 24.4%) in 3 patients who had been placed under observation; in two patients (Cases 2 and 3) whose GPI(-) granulocyte percentages had been >10%, the PNH clones were completely replaced by HLA-LL clones during 6 and 8 years, respectively (Figure 2).[Conclusions] The limited diversity of the blood cell lineage and spontaneous decline of GPI(-) cells that coexisted with HLA-LLs suggest that GPI(-) cells are derived from the PIGA-mutated hematopoietic progenitor cells that were allowed to proliferate as a bystander in the environment where the CTL attack against HSPCs is taking place. [Display omitted] DisclosuresNakao:Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria.

Clinical and Molecular Heterogeneity of Moderate Aplastic Anemia

Clinical and Molecular Heterogeneity of Moderate Aplastic Anemia

The Effectiveness of Eltrombopag at the Different Stages of Aplastic Anemia and Probability of Clonal Expansion

Background. New insight into pathogenesis of aplastic anemia (AA) in conjunction with effectiveness of Eltrombopag allowes to recommend it for initial treatment of de novo AA. However, eltrombopag was an attractive option for patients at different stages of the therapy.Aim. In our study we present various application points of eltrombopag as a therapeutic option in patients (pts) at different stages of the disease.Materials and patients. We are presenting our single-center experience, that include 11 pts with acquired aplastic anemia (SAA/NSAA = 10/1) on different stages of the disease. All pts were divided into four groups. In the 1st group we included refractory pts after two (n=3) or three (n=2) lines of antithymocyte globulin (ATG) therapy. The 2nd group included two pts who received the second ATG course in combination with eltrombopag, after faild first ATG. In the 3rd group included 2 pts with refractoriness after 1 course of ATG, who couldn't receive the second ATG/CsA because of severe infections. The 4th group included two pts with one lineage thrombocytopenia but transfusion independent. The median time from the start of immunosuppressive therapy (IST) to beginning of eltrombopag was Me(min-max): 56(12-115), 51(8-94), 1(1-1), 69(37-100) months respectively. The daily dose of eltrombopag for pts was 100-150 mg.Results. Hematological response are present in 3 out of 5 pts in the 1st group. Median duration of eltrombopag treatment was 8,5 (3-12) months. It is necessary to note that responding pts were refractory to 2nd (n=1) or 3rd (n=2) ATG and transfusion dependence. All pts in the 2nd group achieve PR (Me duration eltrombopag treatment was 12 months). All pts from 3rd group achieved granulocytic response, that allowed to recover get out from infections and to hold the 2nd ATG. However in 1 pt monosomy 7 was detected by FISH in bone marrow after therapy. It was noted that before eltrombopag administration there were not cytogenetic aberrancies but there were myelodysplastic features by flow cytometry (low index of granularity in granulocytes, low HLA-DR expression of monocytes and high CD56 expression in both granulocytes and monocytes) and PNH-clone. After eltrombopag treatment and monosomy 7 detection we performed a FACS sorting and FISH-study for PNH+ and PNH- granulocytes. Monosomy 7 was found in PNH- but not in PNH+ granulocytes. From the 4th group thrombocytopenia resolved in 1 pt (11 months eltrombopag duration), in over case present threelineage relapse subsequently.Conclusion. Eltrombopag can be used in pts with long-term ineffective IST and as a bridge to the next step of IST. It is necessary to remain cautious about the earlier development of clonal complications, even in patients from the so-called favorable risk group with the PNH clone. DisclosuresNo relevant conflicts of interest to declare.

Eltrombopag Rescue for Childhood Severe Acquired Aplastic Anemia after Poor Response to Immunosuppressive Treatment: A Nationwide Study on Behalf of the French Reference Center for Aplastic Anemia

▪Introduction: Thrombopoietin receptor agonist eltrombopag (ELT) provides hematologic improvement in up to 50% of adults with acquired aplastic anemia (AAA) refractory to immunosuppressive therapy (IST). However, current data for ELT efficacy and tolerance in childhood AAA is very limited.Methods: We conducted a multicenter nationwide retrospective study on behalf of the French Reference Center for Aplastic Anemia. Patients under 18 treated with ELT after IST for AAA were included. Lineage responses were defined as follows: red cells transfusion independency or hemoglobin increase of 1.5 g/dL, platelets transfusion independency or a 20 G/L increase in platelets counts, doubling of the neutrophil count or an increase above 0.5 G / L.Results: We identified 12 children (2-17 years old, mean 9.5±5.9 years) treated for severe (7) /very severe (5) AAA with horse ATG and cyclosporine as first-line treatment between 2014 and 2018 in five different centers. Minor PNH clone was identified at diagnosis in two patients. All patients had a normal bone-marrow karyotype and FISH. None had a matched related donor. Median time between IST onset and eltrombopag initiation was 3.5 months [1-46]. All patients still met severe aplastic anemia criteria and needed RBC and platelet transfusions at ELT initiation. Mean hemoglobin, reticulocytes, platelets and neutrophils values were 7.3±0.8 g/dL, 24.5±24 G/L, 11±5.9 G/L and 0.8±0.7 G/L. Seven patients had received IST for more than 3 months and were therefore considered refractory to this treatment. ELT was indicated for an uncontrolled bacterial infection in 2 patients one and two months after IST initiation. One patient received an excessive dose of 12.5 mg/kg/day. Average ELT dosage for the other patients was 2±0.6 mg/kg/d. The median duration of treatment with ELT was 5.5 months [1-10]. After ELT initiation, nine patients (75%) reached hematological response for at least one cell line with no additional treatment. In these patients, hemoglobin increased on average by 3±1.3 g/dL, neutrophils by 2.5±3.5 G/L and platelets by 89.8±39.7 G/L. Seven patients (58%) achieved a trilineage response and were both RBC and platelets transfusions independent after 1 month (1), 3 months (3) and six months (7). Five patients (41.6%) reached a robust response (platelets > 50 G/L, hemoglobin > 10 g/dL, neutrophils > 1 G/L). All these trilineage responders were still on cyclosporine at the time of last assessment. ELT was withdrawn in 4 trilineage responders. Two of them still fulfilled robust response criteria 3 and 43 months after ELT withdrawal while the 2 others required occasional transfusions. The 3 others trilineage responders who were still receiving ELT remained transfusion independent 6 (2) and 11 (1) months after ELT initiation. Two patients (16.6%) had a unique lineage response: one was lost of view and one developed a myelodysplasia with RUNX1 mutation 3 months after ELT initiation. He is alive 8 months after HSCT. No clonal evolution was reported in the 5 other patients for whom cytogenetics follow-up was available. No response was achieved in 3 patients (25%), all of whom were treated for less than 3 months: ELT was withdrawn for deemed inefficiency in 2 patients (16.6%) after 1 and 2 months respectively and for toxicity (disseminated intravascular coagulation) in another patient after 2 months. These patients underwent HSCT. One patient died of graft failure 3 months after HSCT. Overall, eleven patients (91%) were alive 13.4±5.6 months after eltrombopag onset. Disseminated intravascular coagulation occurred in a 2 years patient who received a large ELT dosage (12.5 mg/Kg). No other grade III-IV toxicity was reported.Discussion/Conclusion: This is the first report of ELT for childhood severe AAA after IST. Eltrombopag was overall well tolerated. We observed a high rate of sustained trilineage response as reported in adult patients and only 1 case of clonal evolution which is a known complication in non-responsive AAA. Onset of the trilinear hematologic responses is progressive and achieved 3-6 months after ELT initiation. A longer follow-up of this cohort is mandatory to assess response durability and clonal evolution final risk. Nevertheless, we propose that ELT should be considered for severe AAA non-responsive to IST after 3 months of treatment especially if only an alternative donor is available. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.