PNH Clone Research Articles

Hematological Response to Avatrombopag Switch in Thrombopoietin Receptor Agonist-Refractory Aplastic Anemia

ATG combined with CsA is the first-line treatment for patients with SAA who cannot choose HLA-matched HSCT because of various conditions. The HR rate is only approximately 60-80%, and the CR is not high. The lack of residual HSC in patients is an important reason for this unsatisfactory effect. RhTPO/TPO-RA combine with HSC membrane receptors to promote their proliferation and differentiation, expand multipotent hematopoietic progenitor cells in vivo. Studies have found that TPO-RA eltrombopag (EPAG) combined with IST can significantly increase the HR rate and improve the quality of the HR. From 40-50% hematologic improvement can also be obtained with refractory SAA. However, TPO-RA treatment is ineffective in some patients. Avatrombopag (APAG) acts on the transmembrane region of the TPO receptor, has a stronger effect than EPAG, and has no obvious liver toxicity. We analyzed the efficacy and safety of IST combined with rhTPO and/or EPAG in patients with SAA of switching to APAG. We retrospectively analyzed the data of 10 patients who were hospitalized between January and December 2021. All patients have been diagnosed with V/SAA. They received IST combined with rhTPO/TPO-RA treatment, failed to obtain a HR, and were converted to APAG. The patients were treated with IST for at least 6 months, combined with rhTPO and/or EPAG for at least 3 months, and did not get HR. None of the patients included in this study was suitable for undergo allogeneic HSCT. APAG was administered orally at a dose of 40 mg/day once a day for at least 6 months.The efficacy criteria for refractory SAA were defined as follows: one-line response included platelet response (HI-P), neutrophil response (HI-N), and erythroid response (HI-E). The 2-line HR could be a combination of any two of the aforementioned one-line responses. A trilineage response was defined as a patient with simultaneous platelet, erythroid, and neutrophil responses, and the quality of the trilineage response was the same as that of the initial treatment with SAA/TDNSAA. Ten patients were included in the study, with a median age of 54 (14-68) years, including five males and five females. All patients received IST for a median of 8.5 (6-40) months. Four patients received rhTPO for 1-2 months followed by EPAG for 2-8 months. Six patients received only EPAG for 3-4 months. All the patients received rhTPO/TP-RA for a median of 3.5 (3-10) months. Five patients with PNH clones were positive by flow cytometry, and there was no hemolytic or biochemical evidence of hemolysis. Two patients had clonal chromosomal abnormalities: one with 47,XY; +8 [2/20] and the other with 45,XY; -Y [10/20]. Ten patients were converted to APAG treatment for a median of 6 (6-10) months. After 3 months of follow-up , seven patients obtained a HR (7/10, 70%), of which two patients obtained a HI-P and HI-N, and five patients (5/10, 50%) obtained a three-line HR (1-CR, 3-GPR, 1-PR). Seven patients (7/10, 70%) obtained HR after 6 months of follow-up, of which one patient obtained HI-P/N and six patients (6/10, 60%) obtained three-line HR (3-CR, t2-GPR, 1-PR). The patient with two-line HR further improved after 8 months of APAG, reaching a three-line blood cell response to PR. At the end of the last follow-up, the HR rate of all patients with APAG conversion therapy was 70%, and all of them achieved three-line blood cell responses (4-CR, 1-GPR, 2-PR). During the follow-up period, no APAG-related adverse events of grade 2 or above were found in any patient, no thrombotic events occurred, and no fibrous tissue hyperplasia was found in the bone marrow pathology review after 6 months of treatment. Among the five patients with PNH clones before APAG conversion therapy, three patients did not change significantly. Otherwise one patient's granulocyte PNH clone decreased from 16.2% at baseline to 6.3%, and the other patient's PNH clone increased from 9.8% at baseline to 19.6%, whereas there was no symptomatic hemolysis in all patient. One patient (1/10, 10%) with normal chromosomal before therapy get a new clonal chromosomal abnormality after 3 months. It was 46XX, t(X;4 )(p11.4;q21) [4/20]; the HR was assessed as GPR. In conclusion, patients with SAA refractory to TPO-RA, drug conversion therapy can be based on the safety, pharmacological, and pharmacodynamic characteristics of TPO-RA drugs. Therefore, APAG is a better choice for switch therapy in these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Rare PNH Cells May be Considered As Genuine PNH Clone: Insights from Analysis of the 5-Year French Nation-Wide Multicenter Observational Study

Introduction The successive International Guidelines (Borowitz. et al. Cytometry B Clin Cytom 2010; Illingworth. et al. Cytometry B Clin Cytom 2018) recommend both detection and follow-up of rare PNH cells using high-resolution flow cytometry (FCM) analysis. These rare PNH cells, defined as GPI-deficient cells ranging from 0.01% to <0.1%, are distinguished from minor PNH clone, ranging from 0.1% to 1%. However, clinical relevance as well as long-term evolution of such rare PNH cells are still unknown. Objective We aimed to compare clinical and biological presentation of patients with rare PNH cells or a minor PNH clone along with long-term follow-up using data provided by the 5-year French nation-wide multicenter observational study, the Observatory of PNH Clones. Methods All patients of any age with a newly or previously diagnosed PNH clone or GPI-deficient cells ≥0.01%, detected in France by FCM from February 29th, 2016 to February 28th, 2021 were eligible for inclusion in this Observatory, provided that the center had participated in the inter-laboratory comparison program ongoing since 2013 (Debliquis et. al. Am J Clin Pathol 2015). For each patient, the baseline assessment was always considered as the initial PNH clone detection, even if it occurred before the initiation of the Observatory. Thus, this strategy allowed the collection of cases with long-term follow-up. A referent cytometrist at each center included patients in the e-CRF available on the CytHem-HPN website providing clinical and biological information as well as FCM raw data files. This study was approved by the national research ethics board. Results As of February 28th, 2021, 41 registered flow cytometry laboratories across France enrolled 637 patients with a PNH clone or GPI-deficient cells ≥ 0.01%. Selection of patients displaying rare PNH cells (i.e., o.o1% to <0.1%) or a minor PNH clone (i.e., 0.1% to 1%) on neutrophils led to the re-examination of 163 patient FCM files (provided by 26 centers) by the two principal investigators. FCM data and the e-CRF filling were both thoroughly reviewed leading to the update of roughly half of the submitted files. This enabled the validation of 138 patients at diagnosis and the exclusion of 18 (11%) patients who did not display a genuine PNH clone according to the international recommendations (at least 20 clustered events). For 7 of 163 selected patients, exchanges with centers are still ongoing for FCM data validation or due to missing clinical or biological data. Sixty-nine of the 138 validated patients displayed at least one follow-up point (Figure 1). Thus, we were able to compare 49 patients with rare PNH cells and 89 patients with a minor PNH clone with a significantly different clone size on the 3 lineages (median size on neutrophils: 0.03 [0.01 - 0.09] vs 0.34 [0.1 - 0.97] ; p<0.0001, on monocytes: 0.03 [0 - 7.88 ] vs 0.50 [0 - 7.08] ; p<0.0001, on red blood cells: 0.01 [0 - 1.36 ] vs 0.08 [0 - 1.24]; p= 0.0096). The patient median age at diagnosis was similar for both groups, 59 years [6 - 91] with 4 pediatric cases (<18y) for "rare PNH cells” group vs 49 years [10 - 87] with 9 pediatric cases for "minor PNH clone” group and a M/F sex ratio of 1.45 vs 0.98. Diagnoses were made between 2010 and 2021 with clinical information available in 98% of cases (n=47 and 88). Ninety-two to 94% of patients from both groups displayed BM failure including mainly aplastic anemia (n=30/47 vs 61/88), myelodysplastic syndrome (n=2 vs 6) and unexplained cytopenia(s) (n=11 vs 16). Only 4 cases of thrombosis for "rare PNH cells” group vs 3 for "minor PNH clone” group were observed, and the 2 cases of hemolytic anemia belong to the latter group. A more severe thrombopenia was observed in the "minor PNH clone” (68 [2-356] vs 44 [2-308] G/l) while all other biological parameters showed no significant difference. Long-term follow-up up to 7.0 years showed that one third of patients from both groups displayed an increase in clone size (above 0.1% for "rare PNH cells” and above 1% for "minor PNH clone", see Figure 2), most remained stable and a few decreased, the disappearance being always due to BM transplantation. Conclusion This multicenter study based on robust FCM analysis showed no differences between the clinical and biological presentation of patients with either rare PNH cells or a minor PNH clone. Long-term follow-up showed that rare PNH cells can evolve into minor PNH clone, suggesting that these cases may be considered as genuine minor clones. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Predictors, Risk Factors and Outcomes of Patients with SAA with Anti-HLA Class I Antibodies Treated with Immunosuppressive Therapy

Introduction: Platelet refractoriness, defined as persistently inadequate post-transfusion count increments, can be immune or non-immune. Human leukocyte antigen (HLA) Class I antibodies are the usual immune cause. The presence of HLA platelet antibodies (Abs) in hematologic disease has been associated with poorer clinical outcomes, graft failure in hematopoietic stem cell transplant, increased bleeding, and decreased survival. In immune severe aplastic anemia (SAA), literature addressing prophylactic transfusion of platelets is lacking, but most recommend transfusion with bleeding at a count <10 or <20 K/ µL if febrile or during ATG (anti-thymocyte globulin) administration. Chronic platelet transfusion is a risk for development of HLA Abs; nevertheless, some chronically transfused patients do not become alloimmunized while others do so rapidly, even after a single platelet transfusion. We analyzed our SAA patients to document the rate of HLA alloimmunization and its possible association with disease characteristics and outcomes after immunosuppressive therapy (IST). Methods: Clinical records from 444 SAA patients treated at the NIH Clinical Center from 2000 to 2022 were retrospectively reviewed. Demographics included: age, sex, HLA Class 1 Abs status, platelet refractoriness, peripheral blood counts, PNH clone, treatment and relapse, evolution to myeloid malignancy or chromosome abnormality, and overall survival (OS). Patients were tested for HLA Abs if they met 1 or more of 3 clinical criteria: 1) history of HLA Abs; 2) history of or current platelet transfusion refractoriness; 3) work up for bone marrow transplantation. Clinical refractoriness was defined as lack of platelet increment of >5 K/µL or a corrected count increment (CCI) of >5 K/µL. Patient lacking evidence of refractoriness were presumed anti-HLA negative and not tested, and those that tested negative for HLA Ab were transfused platelets unmatched for any HLA antigens. Logistic regression was used to analyze associations between age, sex, blood counts, PNH clone, and disease severity, and Kaplan Meier curves and Cox proportional hazards model for survival. Data were analyzed using R, and p<0.05 was considered statistically significant. Results: Demographic data were collected for 444 patients (Table.1). HLA Ab testing was performed in 155 patients while 289 didn't meet criteria for testing. Of 444 patients, 99 (22%) tested positive for HLA Class 1 Abs. Seventy-three of 99 patients responded to HLA matched platelets, whereas, 17 remained refractory and for 9 data were unavailable. Univariate analysis predicted male sex to be protective against HLA Abs formation (OR=0.59,p= 0.02), while more severe disease (VSAA, OR= 2.51, p<0.001) and lower baseline blood counts predicted alloimmunization. Low platelet counts (p<0.001) were most significant followed by absolute reticulocyte (ARC) and absolute neutrophil counts (ANC) with p=0.005. PNH was not associated with refractoriness, although data were incomplete. Only HLA Class 1 Ab status and age, and sex were strong predictors of survival in both univariate and multivariate analysis. ANC, ARC, and disease severity were significant predictors of survival in the univariate but not multivariate model. Median time to relapse was 2.4 years with a relapse rate of 36% at median follow-up. Rate of relapse was 46% in alloimmunized patients vs.34% in the Ab negative group, but this difference was not statistically significant (p=0.2). OS for the entire cohort was 84% at median follow up 4 years; HLA Ab-negative patients had a significantly higher OS compared with positive (86% and 75% respectively; p = 0.02). Rates of clonal evolution were also higher in the HLA Ab positive group but not statistically significant (25% vs. 13%, p=0.08). Conclusions: We report a large cohort of IST-treated SAA patients, comparing outcomes with and without HLA alloimmunization. Although SAA patients are usually heavily transfused platelets, we observed a rate of HLA alloimmunization of 22%. OS was decreased in HLA alloimmunized patients. Presence of HLA ab and age were strong predictors of survival. ANC, ARC and disease severity were predictors of survival only in the univariate model. Relapse and clonal evolution may be increased when HLA Abs are present. Overall, the presence of HLA Abs is associated with poorer outcomes in SAA patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparable Long-Term Outcomes of Low-Dose Versus Standard-Dose Equine Anti-Thymocyte Globulin with Cyclosporine in Aplastic Anemia

Background: Immunosuppressant therapy (IST) with equine anti-thymocyte globulin (eATG) and cyclosporine is the current standard treatment for transplant-ineligible aplastic anemia (AA) patients. Recently eltrombopag has been added to this IST backbone. The standard dose of eATG is 40mg/kg/day, however, the ideal dose of eATG is not known. We studied the efficacy of 25 mg/kg/day x 4 days (low-dose) with standard-dose eATG in transplant-ineligible AA patients at our tertiary care centre. Methods: AA patients (age >12 years) who received eATG and cyclosporine from 2003 to 2021 were retrospectively analysed.The choice of eATG dose was based on patients'finances. Patients developing nephrotoxicity with cyclosporine were switched to oral mycophenolate mofetil. Overall response rates (ORR) [complete response (CR) and partial response (PR)] were analysed at 3 months, 6 months and 12 months after eATG.Kaplan-Meier survival analyses were performed to analyse overall survival (OS) and event free survival (EFS). Results: Ninety-three AA patients received eATG during the study period. Their median age was 30 years (range 13-65 years); 60 (64.5%) were males. PNH clone was present in 33 (35.5%) patients. Median duration from symptom onset to diagnosis was 3 months (range 1-100 months). 62 patients (66.7%) received low-dose eATG; 31 patients (33.3%) received standard-dose eATG. 17 patients (18.3%) received eltrombopag in addition to eATG plus cyclosporine. The number of patients eligible for response assessment at 3 months, 6 months and 12 months were 93 (100%), 86 (92.4%) and 80 (86%), respectively. ORRs in the total study population at 3 months, 6 months, and 12 months were 49.5%, 66.3%, and 72.5%, respectively; respective CR rates were 5.4%, 14% and 20%. The differences between ORRs among low-dose versus standard-dose eATG groups at 3 months (50% vs 48.4%; p = 0.88), 6 months (63.8% vs 71.4%; p = 0.67) and 12 months (69.6% vs 79.2%; p = 0.167) were statistically insignificant. Median duration of oral cyclosporine after eATG was 25 months (range 3-144 months); 23 patients (24.7%) who were having suboptimal response at 6 months (PR 15, NR 8) and 12 months (PR 18, NR 5) achieved CR after 12 months with the continuation of cyclosporine. The median duration of follow up was 52 months (range 3-229 months). 11 patients (11.8%) relapsed after eATG (7 in low-dose group, 4 in standard-dose group) after a median duration of 32 months (range 12-84 months). Two patients (2.1%) underwent clonal evolution to AML and MDS-EB2 at 83 months and 35 months, respectively (both in standard-dose group). 15 patients (16.1%) died during follow up and 18 (19.3%) were lost to follow up. 10-year OS and 10-year EFS of the total study population were 80% and 64%, respectively. There was no significant difference between low-dose and standard-dose groups in terms of 10-year OS (82.4% vs 74.8%; p = 0.43) and 10-year EFS (64.5% vs 63.1%; p = 0.64), respectively. Conclusion: This study revealed that low-dose eATG and standard dose eATG have comparable response rates and long-term survival. CR rates continue to improve with continuation of oral cyclosporine beyond 12 months in patients who have partial or no response at 6 and 12 months. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Role of Heterozygous Variants in SAMD9/9L and Recessive Bone Marrow Failure-Related Genes in Adults with Severe Aplastic Anemia

Background Genetic reports of patients with severe AA (SAA) often identify heterozygous variants of unknown significance in recessive genes associated with inherited bone marrow failure syndromes (IBMFS), such as Fanconi anemia (FA) and telomere biology diseases (TBD). More recently, heterozygous SAMD9 and SAMD9L (SAMD9/9L) variants have also been reported in children and young adults with BMF, but whether all variants are gain-of-function (GoF) and pathogenic is unknown. In a large cohort of SAA patients, we investigated the prevalence of heterozygous variants in SAMD9/9L and FA/TBD genes and correlated them to disease characteristics and clinical outcomes. Methods SAA patients (n=198; median age [range]: 32 [3-82]) enrolled on a phase II trial for treatment with horse anti-thymocyte globulin, cyclosporine, and eltrombopag (NCT01623167) at the National Institutes of Health from 2012 - 2022 were included (Table 1). All patients were screened for germline mutations in IBMFS related genes. SAMD9/9L variants were segregated by rarity according to frequency in the gnomAD database as common (<0.1% in overall population, including variants with maximum population frequency [MaxPopFreq] >1%), rare (MaxPopFreq <0.1%), very rare (MaxPopFreq <0.01%), ultra-rare (MaxPopFreq <0.005%), and novel (absent in gnomAD). Results We first investigated whether patients had an undiagnosed SAMD9/9L disorder. Although 40/198 patients (20%) had a heterozygous SAMD9/9L variant, including 8 (20%) with 2 variants, these were mostly classed as common (73%; n=35), followed by rare, very rare, ultra-rare and novel variants seen in 17% (n=8), 4% (n=2), 2% (n=1), and 4% (n=2), respectively. Most variants were missense (n=39; 81%), followed by nonsense (n=6; 13%) and frameshift mutations (n=3; 6%). Except for ultra-rare and novel, SAMD9/9L often co-occurred with typical features of immune AA (6p-LOH, PNH clones, HLA and PIGA mutations). None of the 40 patients with germline SAMD9/9L variants had primary somatic genetic reversions (SGR) such as somatic loss of function (LoF) mutations (VAF>0.5%) in SAMD9/9L or UPD7q. Since GoF SAMD9/9L variants are located on chr7 and they associate with development of monosomy 7, we investigated whether SAMD9/9L variants in our cohort were associated with clonal evolution after treatment. However, only 2/40 patients (5%) with chr7 abnormalities had these variants, both common. Patients with SAMD9/9L variants, both common and rare, were significantly younger (35 years vs. 24 years, p=0.026) and more frequently had VSAA (defined as absolute neutrophil count <0.2 K/uL) than those without these variants (62% vs. 41%, p=0.02); they had lower median neutrophil counts (p=0.038), reticulocyte counts (p=0.024), and platelet counts (p=0.047). The presence of rarer (MaxPopFreq<0.1%; n=11) or common SAMD9/9L variants (n=29) did not impact disease severity, or correlate with molecular characteristics of immune AA, response to immunosuppressive therapy, and overall survival (Figure 1). In addition to SAMD9/9L variants, 82 patients (41%) had 1 or more heterozygous variants in FA/TBD, but these were not associated with disease severity or with clinical outcomes, even when co-occurring with SAMD9/9L variants. Twenty five patients (13%) had co-occurring heterozygous mutations in both SAMD9/9L and FA/TBD genes, but these were not correlated with disease severity or response to therapy when compared to patients without co-occurring mutations. Conclusions As opposed to classical SAMD9/9L disorders in pediatric cohorts, SAMD9/9L variants found in our primarily adult patients with SAA are not disease-causing germline GoF mutations, even the rarest variants. Most patients with variants responded to immunosuppressive therapy, and the less rare variants were associated with molecular features of immune AA but not SGR, both findings suggestive of immune pathophysiology. Interpretation of genetic reports, including SAMD9/9L, in adults appears to be different from mostly pediatric cohorts with SAMD9/9L disorders. Heterozygous SAMD9/9L variants do affect AA severity but are not predictive of response to therapy or prognosis. FA/TBD heterozygous variants did not impact disease characteristics or outcomes. We did not observe a higher rate of clonal evolution in patients with SAMD9/9L after EPAG treatment compared to patients carrying FA/TBD variants and others without variants. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcomes of Older Patients with Aplastic Anemia Treated with Frontline Anti-Thymocyte Globulin (ATG)-Based Immunosuppressive Therapy

Background: Advances in the treatment of aplastic anemia (AA), such as the addition of eltrombopag (EPaG) to immunosuppressive therapy (IST) and the expanded use of unrelated and haploidentical stem cell transplant (SCT) have steadily improved outcomes. However, little is known about the characteristics and outcomes of patients (pts) ≥ 60 years (yrs) of age with newly diagnosed AA who have been treated with an ATG-based regimen. Presumed low bone marrow (BM) reserves, higher frequency of underlying clonal hematopoiesis, intolerance to therapy, comorbidities, and challenges in proceeding to salvage SCT have led to the speculation that response and survival of these pts to IST may be inferior than their younger counterparts. Methods: We retrospectively analyzed the characteristics and outcomes of pts ≥ 60 yrs of age (OLDER) at the time of diagnosis of AA treated with frontline (F/L) ATG based therapy at our center and compared them to pts < 60 yrs of age (YOUNG) treated with F/L ATG over the same time period. We captured their clinical characteristics, cytogenetic abnormalities, mutations in DNMT3A (D), ASXL1 (A) and TET2 (T), exposure to EPaG, response, time to response (TTR) and survival. Overall response included complete and partial response per the standard definitions. Relapse free survival (RFS) was tabulated from the time of best response to loss of response, 2nd ATG infusion, salvage SCT or death (whichever was earlier) and overall survival was tabulated from the time of response to death from any cause. Results: From Jan 2011-May 2012, 43 OLDER pts were treated as F/L for AA of whom 41 (95%) received ATG based therapy; 68 YOUNG pts were treated as F/L for AA over the same time period of whom 62 (91%) received ATG based regimen. Four YOUNG pts vs. no pts in the OLDER group received upfront SCT, and 2 pts in each group were treated with non ATG based therapy: single agent CSA, EPaG, or alemtuzumab. Comparative baseline characteristics are depicted in Table 1. There were no differences in the baseline WBC, peripheral blood lymphocyte percentage and platelet counts between the ATG treated OLDER and YOUNG groups. Both groups had similar frequencies of cytogenetic abnormalities and PNH clones, though OLDER pts had mutations in D-A-T genes compared to the YOUNG group (62% vs. 38%, p=0.05). Both groups had equal concurrent exposure to EPaG with ATG. Cumulatively 23/41 (56%) pts in the OLDER group had an overall response (OR) compared to 43/62 (70%) pts in the YOUNG group (p=0.1); 11/23 (48%) and 23/43 (53%) responders in each group attained CR respectively. There were no differences in OR rates in both groups based on concurrent EPaG exposure: 61% vs. 46% with and without EPaG, respectively, in the OLDER group (p=0.5) and 74% vs 60%, respectively, (p=0.38) in the YOUNG group. On logistic regression analysis using baseline platelet counts, PB lymphocyte (%), D-A-T mutations, karyotype, and EPaG use as covariates, none affected the odds of OR in the 2 groups. The median TTR was 111 days (range, 10-323 days) and 177 days (51-410) in YOUNG AND OLDER groups, respectively; and this was not affected by EPaG use in either group. At a median follow of 38 months (mos) from ATG initiation (53 mos for the OLDER group and 32 mos for the YOUNG group), the median RFS was 49 mos and 39 mos (p=0.6) and median OS 55 vs. not reached (NR) (p <0.0001) in OLDER and YOUNG groups respectively (Fig.1). Significantly more pts in the YOUNG group underwent a subsequent SCT (19% vs. 5%) due to loss of response/ no response, possibly accounting for the higher OS rates. Rates of transformation to MDS or AML was 5% in both groups. Conclusion: Frontline ATG based IST in older pts with AA is a feasible and safe treatment option with promising response rates of 56% and median RFS more than 4 years; figures which are comparable to younger pts with AA treated with IST. The overall risks of transformation to MDS/AML on long term follow-up remain low (≈5%). Further studies assessing the response of the ATG+EPaG combination in these pts and the possibilities of clonal evolution with time needs to be conducted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anti-Human T Lymphocyte Porcine Immunoglobulin Combined with Cyclosporine As First-Line Immunosuppressive Therapy for Severe Aplastic Anemia in China: A Large Single-Center, 10-Year Retrospective Study

Immunosuppressive therapy (IST) based on antithymoglobin (ATG) and cyclosporin (CsA) is the first-line treatment for severe aplastic anemia (SAA) patients who are ineligible for transplantation. Horse ATG is recommended as the standard preparation for first-line immune suppression in SAA, but it has been unavailable in China since the year 2000. The anti-human T lymphocyte porcine immunoglobulin (p-ATG, provided by Yujin Bio-Pharma Wuhan CNBG Co., Ltd., Wuhan, China) was made available in 1983 and approved for the treatment of aplastic anemia by the Sino Food and Drug Administration in 2004. It is a commonly used ATG preparation in IST for SAA in China since 2009. To date, only a few small retrospective cohort studies reported that p-ATG combined with cyclosporine (CsA) is a useful immunosuppressive regimen for SAA in China. This study retrospectively analyzed the data of 1023 consecutive patients diagnosed with SAA that aimed to evaluate the early hematologic response and long-term outcomes of this large cohort of patients with SAA who received p-ATG plus CsA as first-line therapy from 2010 to 2019. Patients received p-ATG for 5 consecutive days (day 1-5), at a dose of 20 mg/kg/day. And CsA was administered orally from day 1 of p-ATG treatment at two separate doses starting from 3 to 5 mg/kg/day and adjusted to maintain the trough concentration at 150-250 ng/ml and the peak concentration at 800-1000 ng/ml. One thousand twenty-three aplastic anemia were included in this study, with a median age of 24 (4-75) years, with 500 males and 473 females. The median follow-up was 57.2 months (3 days-137.5 months). Twenty-nine patients died within 3 months after IST, with an early death rate of 2.8% (29/1023); the median death time was 0.9 (0.1-2.9) months. The overall hematologic response rate (ORR) was 40.6% (415/1023) at 3 months, including 5.9% (60/1023) of patients with CR. The ORR was 53.2% (544/1023) at 6 months, including 13.5% with CR. Of the 395 patients who did not respond at 6 months, 26.1% (103/395) had an overall response at 12 months without any rescue treatment (10 had a CR, 93 had a PR). By the last follow-up, 9.2% (64/697) of the patients had experienced a relapse. The 5-year cumulative incidence of relapse was 9.0% [95% confidence interval (CI), 4.2 to 16.0%]. The 5-year cumulative incidence of clonal evolution was 4.5% (95% CI, 1.4 to 10.6%). The 5-year cumulative incidence of hPNH was 4.7% (95% CI, 1.2 to 12.4%). In patients with PNH clones at diagnosis, the 5-year cumulative incidence of hPNH was 22.5% (95% CI, 10.1 to 37.8%). The 5-year OS rate was 83.7% (95% CI, 81.1 to 86.0%). The 5-year event-free survival rate was 50.4% (95% CI, 47.1 to 53.5%). In conclusion, p-ATG combined with CsA for the treatment of AA is effective and safe, and p-ATG can be used as an alternative ATG preparation for the standard IST regimen in areas where h-ATG is not available.

Successful Use of Granulocyte Transfusion Therapy As a Bridge to Hematopoietic Stem Cell Transplant in a Child with Aplastic Anemia and Refractory Perirectal Abscess

Background: Idiopathic aplastic anemia (IAA) is a rare, life-threatening acquired form of bone marrow failure characterized by multilineage cytopenias and hypocellular bone marrow. Profound neutropenia associated with very severe aplastic anemia (vsAA) is an important risk factor for development of life-threatening fungal and bacterial infections. Perirectal cellulitis and perianal abscess are one such infection and are associated with high rates of morbidity. The ability to eradicate infections in IAA is limited by the lack of imminent neutrophil recovery prior to treatment with either immune suppressive therapy (IST) or hematopoietic stem cell transplant (HSCT). We describe the successful use of granulocyte transfusion to treat antibiotic refractory proctitis with perianal abscess in a profoundly neutropenic child with vsAA. Case: A 7yoF presented with fever and pancytopenia in the setting of Human Metapneumovirus and Parainfluenza. Bone marrow biopsy showed hypocellularity (60-70%) with granulocytic and megakaryocytic hypoplasia, favoring viral suppression. Ultimately, she progressed to transfusion dependence. Repeat bone marrow aspirate and biopsy ultimately revealed bone marrow aplasia, DEB chromosomes, telomere lengths, and PNH clones were negative, confirming the diagnosis of vsAA. She was subsequently admitted for febrile neutropenia (F&N) and was found to have mild respiratory COVID-19, but began to complain of rectal pain that was initially attributed to her history of constipation. She defervesced on IV antibiotics (abx) and was discharged home with negative cultures. Within a week she represented with hematochezia and F&N and was found to have proctitis with perianal fistula on MRI (figure 1); GI PCR panel was + Enteroaggregative E. coli, no resistant organisms were identified. She clinically improved and discharged on IV piperacillin-tazobactam (pip-tazo). Despite 2 weeks of IV abx, she developed new fever and imaging confirmed persistent proctitis with peri-rectal abscess. As she had limited response to abx alone we trialed a short course of granulocytes with improvement in anorectal inflammation by CT. She was discharged with a plan to continue IV pip-tazo until achieving radiographic cure; however, interval imaging after an additional 2 weeks of abx showed rebound of her proctitis and she was again admitted with F&N and rising inflammatory markers. Her failure to eradicate her peri-rectal infection despite prolonged antibiotics (>4weeks), along with her previous response to a short course of granulocyte transfusion prompted us to prescribe a prolonged 10-day course of granulocytes with continued antibiosis. Her post-granulocyte CT showed resolution of her perirectal abscess and proctitis (figure 2). With resolution of her infection, she was able to proceed to HSCT which she completed without recurrence of her anorectal infection. Discussion: Conventional granulocyte transfusions are considered when patients have severe neutropenia (<500/uL), bacterial or fungal infection refractory to antimicrobials, and reasonable expectation of neutrophil recovery, but evidence for efficacy is limited. In our case, there was no expectation of imminent bone marrow recovery and our patient's infection limited her ability to proceed to IST or HSCT for treatment of her vsAA. Thus, in conjunction with infectious disease and transfusion medicine, and given her known response to granulocytes, we successfully re-attempted a prolonged, but limited, 10-day course of granulocyte transfusions and were able to eradicate a case of antibiotic-refractory proctitis with peri-anal fistula. We suggest an expanded application for the use of granulocytes in profoundly neutropenic patients without imminent recovery for severe, anti-microbial refractory infection. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Allogeneic Peripheral Blood Stem Cell Transplantation for Bone Marrow Failure Syndromes Using CD34+-Selected Progenitor Cells Co-Infused with a Reduced Number of Non-Mobilized T-Cells

Background Severe aplastic anemia (SAA) patients who fail immunosuppressive therapy (IST) may be candidates for curative allogeneic hematopoietic stem cell transplantation (HCT). Patients with SAA are often heavily transfused with underlying immune-mediated disease, which increases their risk for graft rejection when bone marrow (BM) is used as a graft source. Peripheral Blood Stem Cell (PBSC) grafts have been associated with better engraftment but at a cost of a higher incidence of graft-versus-host disease (GVHD). We developed a novel protocol combining high numbers of GCSF-mobilized CD34+ peripheral blood stem cells with a BM transplant-equivalent dose of T-cells (collected from donors before GCSF administration) to improve engraftment and decrease the risk of GVHD associated with a conventional, unmanipulated PBSC HCT. Methods In this phase II trial, eligible patients included those age 4-80 years with a diagnosis of SAA or other bone marrow failure (BMF) syndromes with ≥ 9/10 HLA-matched related (sibling/family) donors (MRD) or 10/10 matched unrelated donors (MUD). All patients received conditioning with cyclophosphamide (day -7 to -6), anti-thymocyte globulin (day -5 to -2), and fludarabine (day -5 to -1). For MUD patients, low dose total body irradiation (200 cGy) was added to the regimen on day -1. GVHD prophylaxis included cyclosporin (day -4 until at least day +30) and methotrexate (5 mg/m2 , day +1, +3 and +6). The primary outcome of the study was chronic GVHD at 1 year. Secondary outcomes included mortality, engraftment and chimerism, acute and chronic GVHD, immune reconstitution, and comparisons of MRD vs. MUD patients. Results Thirty-five patients (27 with MRD and 8 with MUD) were transplanted between February 2011 and December 2021. Patients had a median age of 30 (range 6-70) years, 46% were males and 54% were females, including 31 having SAA, 3 with SAA transformed to myelodysplastic syndrome (MDS), and one with MDS; 11 patients also had PNH clone pre-transplant. The median numbers of transplanted CD34+ and CD3+ T-cells were 8 ´ 106 cells/kg and 2 ´ 107 cells/kg, respectively. After HCT transplants, all patients had neutrophil recovery with a median 14 (10-32) days. Except for 2/35 (6%) patients having late graft failure, all other 33 (94%) patients achieved sustained engraftment. Platelet recovery was reached in 29 (83%) patients at a median 19 (13-321) days. A total of 97% of patients achieved >95% donor myeloid chimerism (median 14 days), and 94% of patients achieved >95% donor T-cell chimerism (median 30 days). Reactivation of CMV occurred in 21/29 (72%) patients at risk with none developing CMV disease. Full immune reconstitution occurred for most patients by 1-year post-transplant. With a median follow-up of 5 years, the estimated 200-day survival and overall survival were 89% and 85%, respectively. The cumulative incidence of acute grade II-IV GVHD was 23%, and 1-year chronic GVHD rate was 11%, which was significantly lower than our hypothesis that this regimen would lower the chronic GVHD rate to 50% (P < .001). The subgroups of patients with MRD and MUD were compared (Table 1): patients with MUD showed a slower time to achieve neutrophil recovery (P = .001) and platelet recovery (P = .03), and a slightly high graft failure incidence (2 cases), but most other outcomes were similar, compared to patients with MRD. Conclusion For patients with SAA and other BMF syndromes, transplanting high doses of mobilized CD34+ selected cells from MRD and MUD donors combined with a bone marrow transplant equivalent of non-mobilized donor T-cells (a partially T-cell depleted PBSC transplant) achieves rapid and durable donor engraftment and excellent survival. The cumulative incidence of both acute and chronic GVHD with this regimen was significantly lower than that historically reported with conventional PBSC transplants. Notably, low acute and chronic GVHD rates were also achieved in SAA patients with disease refractory to conventional IST who received a graft from a 10/10 HLA-matched MUD donor, although hematopoietic recovery was slightly delayed compared to recipients of a partially T-cell depleted graft from a MRD donor. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MDS-504 Mutation Profiling and Characteristics of Hypoplastic MDS

Myelodysplastic syndromes (MDS) are heterogenous spectrum of myeloid neoplasms. A subset of patients with MDS have hypoplastic marrow (h-MDS) and is being recognized more as a separate entity in the upcoming 2022 WHO classification. We aim to study characteristics and the molecular profiling of hypoplastic (h-MDS) in comparison with normo-/hypercellular MDS (n-MDS). This was a retrospective analysis of all patients diagnosed with MDS from Moffitt Cancer Center MDS database. Patients were divided into two groups: those with h-MDS, defined as cellularity less than 30%, and those with n-MDS, cellularity greater than 30%. Individuals with MDS/MPN were excluded. A total 4037 patient with MDS were identified, 403 (10%) were h-MDS. The median age at diagnosis was 65 and 68 for h-MDS and n-MDS respectively (P=0.03); h-MDS was observed more in females compared to n-MDS (41% vs 35%, P=0.008). There was no difference in terms of IPSS-R, LGL PNH clones, and history of autoimmune diseases. MDS-RS was more observed in n-MDS. Interestingly, t-MDS was more observed in h-MDS (29% vs 20%, P<0.005). Patients with h-MDS had more thrombocytopenia, neutropenia and were more likely to be platelet transfusion dependent. The following somatic mutations were detected more frequently in n-MDS compared to h-MDS: SF3B1 (P<0.005), SRSF2 (P<0.005), TET2 (P=0.004), and ASXL1 (P=0.02). There was a better response to ATG/CSA among h-MDS 46% compared to 21% for n-MDS (P=0.019). On the contrary, patients with n-MDS were more likely to respond to lenalidomide 46% when compared to patients with h-MDS in which 22% responded (P=0.001). There was no statistically significant difference in response to hypomethylating agents. The median overall survival (OS) for h-MDS was 41.6 months compared to 35 months in patients with n-MDS (P=0.05). The HR for OS was 0.88 (P=0.057) adjusted for IPSS-R. There was no difference in rate of AML transformation. h-MDS accounted for 10% of MDS cases. The OS for h-MDS is better than n-MDS, which tended to have more frequency of splicing and epigenetic somatic mutations.

PB1947: ELTROMBOPAG COMBINED WITH IST AS A MULTICENTER STUDY OF THE FIRST LINE TREATMENT OF CHILDREN WITH SAA.

Background: Eltrombopag and Improved Hematopoiesis in Refractory Aplastic was published in the New England Journal of Medicine in 2012 The research found that Eltrombopag monotherapy showed significant hematologic reaction in adult patients with refractory SAA, of which 44%(11/25) patients had clinically significant hematologic performance. Important progress has also been made in the treatment of SAA with Eltrombopag combined with IST: A nonrandomized, historically-controlled study funded by the NHLBI intramural Research Program of the National Institutes of Health compared the efficacy and safety of conventional IST therapy with Eltrombopag in combination with IST for SAA. The research results were published in Eltrombopag Added to Standard Immunosuppression for Aplastic Anemia in 2017 in the New England Journal of Medicine. In this study, a total of 92 SAA patients aged 2 years and above were enrolled, and the results showed that the overall effective rate and complete response rate of Eltrombopag combined with IST in the treatment of SAA were higher than that of traditional IST standard treatment. However, there is no research on Eltrombopag therapy for children SAA in China. The aim of this study was to improve the hematologic response rate and quality, and to prevent late complications such as recurrence and clonal progression, by adding Eltrombopag to standard IST therapy. Aims: To evaluate the efficacy and safety of attriboma combined with IST as first-line treatment for SAA patients, and to study the clonal transformation and the incidence of PNH cloning in attriboma combined with IST patients. Methods: This study was a non-randomized, multi-center, historical controlled study, with traditional IST standard therapy as the historical control. All patients in the experimental group received “IST+ Attribopal” combined therapy, in which ATG dose was 3.5mg. Kg-1.d-1 for a total of 5 days. The initial dose was 1.25 mg · kg-1 · d-1 for 2-5 years old children, 37.5mg · d-1 for 6-11 years old, 75mg · d-1 for 12-18 years old, and continued for 6 months. A total of 31 children were included in the Altrupalpa group and 136 in the historical control group. Results: There were no differences in gender, severity of disease, co-infection before ATG, co-infection within 1 month after ATG, incidence of serum disease, CD3, CD4, CD8, TREG cell count, hemoglobin level, platelet count, neutrophil count and ferritin level between the attripopa group and the historical control group. In terms of the effective rate, the effective rate of attriboma group was 62.5% vs 68.8% (P= 0.548) in June and 86.4% vs 76%(P=0.285) in September, showing no statistical significance. Among them, neutrophil count recovery was better in the attriboma group than in the historical control group at 1 month, 3 months, 6 months, 9 months and 12 months after the application of ATG, the difference was statistically significant (P < 0.05), while platelet and hemoglobin recovery was not different. 2-year EFS 80.4% VS 83.4% (P=0.799) and 2-year OS 93.3% VS 91.3% (P=0.548) in the attriboma group and the historical control group. None of the patients with clone transformation or PNH clone were found in all the follow-up of attriboma group. Summary/Conclusion: There was no difference in infection rate, serum disease rate and overall effective rate between the two groups within 1 month, but the neutrophil count level of the attriboma groups was better than that of the historical control group.

Bone Marrow Multipotent Mesenchymal Stromal Cells from Patients with Aplastic Anemia Retain Their Ability to Support Hematopoietic Precursors despite Pronounced Changes in Gene Expression.

The properties of bone marrow-derived multipotent mesenchymal stromal cells (MSC) of patients with aplastic anemia at the onset of the disease are studied insufficiently. The aim of this work was to test the ability of MSC from patients with aplastic anemia to maintain hematopoietic precursors and to analyze the expression of genes associated with hematopoiesis and immune response. The ability of MSC to maintain hematopoietic precursors was determined by counting cobblestone area-forming cells; gene expression was analyzed by quantitative PCR. It was shown that MSC of patients with aplastic anemia preserve their ability to maintain hematopoietic precursors. Pronounced changes in the expression of the VEGFA and ANGPT1 genes were found. MSC from aplastic anemia patients with PNH clone significantly differ from those from aplastic anemia patients without PNH clone in terms of the expression of the SDF1, IL1R, and VEGFA genes. Changes in gene expression can be associated with the pathogenesis of the disease.

Clinical observation of low-dose combination chemotherapy in refractory/recurrent paroxysmal nocturnal hemoglobinuria patients: A single-center retrospective analysis.

BackgroundWe performed a retrospective analysis to investigate the clinical characteristics and therapeutic strategies of 20 refractory/recurrent PNH patients, including the clinical efficacy of chemotherapy treatment, safety, and survival.MethodsThe clinical data of 20 classic PNH patients who were refractory/recurrent or had glucocorticoid dependence in our hospital were analyzed, including clinical manifestations, laboratory examinations, treatment efficacy, and survival.ResultsSeventeen patients had a marked improvement in anemia after chemotherapy, 14 patients acquired blood transfusion independence, and the Hb of 3 patients increased to normal levels. Although 6 patients still needed blood transfusion, the transfusion interval was significantly prolonged. The percentages of LDH, TBIL, and RET, which are indicators of hemolysis, were significantly lower than those before chemotherapy. The dosage of adrenal glucocorticoids was reduced by more than half compared with that before chemotherapy.ConclusionsChemotherapy can reduce PNH clones, promote normal hematopoiesis, and control hemolytic attack. It is a promising and widely used therapeutic method.

Immunogenetic, Molecular and Clinical Determinants of Clonal Evolution in Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

Immunogenetic, Molecular and Clinical Determinants of Clonal Evolution in Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

Total Oral Anticoagulation for Classic PNH As Primary Prophylaxis Strategy in a Complement Inhibitor Restricted Scenario: A Retrospective Analysis

Introduction.Paroxysmal nocturnal hemoglobinuria (PNH) is associated with thrombosis and bone marrow failure. Thrombosis is the leading cause of death and morbidity, with 29-44% of patients presenting at least one thrombotic episode during their disease's natural course. Treatment with complement inhibitors is a strategy associated with reducing up to 80% of thrombosis risk, and improving survival of PNH patients. However, the high cost of these therapies limited their access in Low and Middle-Income Countries (LMIC).Mexico, like other LMIC, experience low access to complement inhibitors. Hence, we established a primary thromboprophylaxis strategy in classic PNH. The primary objective of the present study is to analyze thrombosis-free survival and all-cause mortality in patients with classic PNH subjected to this prophylaxis strategy compared to historical controls.Methods.Single-center, retrospective, case-control study. We included patients treated from 1991 to 2021, diagnosed with classic PNH (clone size ≥50% determined by flow cytometry). We defined the treatment group as patients who received total oral anticoagulation as primary prophylaxis. We excluded patients who had presented thrombosis prior to classic PNH diagnosis, sub-clinic PNH, bone marrow failure, platelets <50x10 3/µ, and patients who failed to maintain a therapeutic (2 to 3) INR value three months after starting total oral anticoagulation with vitamin K antagonists (VKA). We collected data regarding clone type (II, III, or mixed), clone size, LDH values (<1.5 vs ≥1.5 x ULN), platelet count, thrombosis occurrence, thrombosis site, bleeding cases and their severity according to the international society on thrombosis and haemostasis (ISTH) classification, and causes of death.Results.We found 44 patients with classic PNH diagnosed from 1990 to 2021. Median age at diagnosis was 39 years (28 -51), 50% were female, median LDH value 1669 U/L (926-2,241), and median clone size 95% (80-99). Clone type III was most prevalent, with 61.36% cases, followed by mixed type II-III with 38.64%; we found no type II clone patients. Median follow-up was 21.03 years. Four patients developed a site of thrombosis; three cases (75%) presented simultaneous venous and arterial thrombosis, and one (25%) venous only. The most common site for thrombosis was the portal vein (n=3), followed by supra-hepatic veins (n=1).A total 19 patients (43.2%) received primary prophylaxis with total oral anticoagulation, 17 (89.5%) used VKA and 2 (10.5%) direct oral anticoagulants. We found no differences regarding age, LDH value, clone size and clone type between the treatment and control groups. There were no cases of thrombosis in the treatment group, while 5 patients (20%) in the control group developed thrombosis at some point (p=0.038, Table 1). Thrombosis-free survival was not reached in the treatment group, vs 12.78 years (IC95%,6.19-19.37) in the control group (p=0.026, Figure 1). Bleeding episodes occurred in 15.8% (n=3) of the treatment group patients, all of them minor according to the ISTH classification.The global survival was 85% at 15 years in the anticoagulation group vs 74% at 15 years in the control group, (p=0.071, figure 2). Mortality was 15.8% (n=3) in the treatment group vs 28.0% (n=7) in the control group, p=0.338. The only cause of death in the treatment group was progression to aplastic anemia. Causes of death in the control group were thrombosis (28.6%, n=2), aplastic anemia (28.6%, n=2), acute leukemia (14.2%, n=1), sepsis (14.2%, n=1) and digestive tract bleeding (14.2%, n=1).Discussion and conclusions.Thrombosis is the most frequent cause of morbidity and mortality in classic PNH patients. In 2003, Hall et al. found that primary prophylaxis with full-dose oral anticoagulation drastically reduced the thrombotic risk in these patients (0 vs. 36.5%, p=0.01). Meanwhile, complement inhibitor has become the mainstay therapy, thromboprophylaxis with oral anticoagulation has virtually stopped in countries where this therapy is accessible. The present study replicated the Hall et al. results, and demonstrated that primary thromboprophylaxis is safe and effective in reducing the risk of thrombosis. In LMIC, where access to complement inhibitors is limited, the use of thromboprophylaxis could abate thrombosis and should be taken in consideration in all patients. Hence, the clonal progression would become the leading cause of death. [Display omitted] DisclosuresApodaca Chavez: Abbvie: Speakers Bureau; Asofarma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rangel-Patiño: Abbvie: Speakers Bureau; Bristol: Consultancy. Demichelis: Jazz: Consultancy; Novartis: Consultancy, Research Funding, Speakers Bureau; ASH: Research Funding; Astellas: Consultancy; Gilead: Consultancy; Abbvie: Consultancy, Speakers Bureau; Bristol/Celgene: Consultancy, Speakers Bureau; AMGEN: Consultancy, Speakers Bureau.

Efficacy and Safety of Eltrombopag with or without Immunosuppressant in Patients with Relapsed/Refractory Acquired Aplastic Anemia: A Real-World Experience

Background: Eltrombopag (EPAG) with or without immunosuppressant (IST) has been applied in acquired aplastic anemia (AA), yet data of EPAG+IST in relapsed/refractory AA was limited, and no study has compared efficacy and safety between EPAG+IST and EPAG monotherapy in relapsed/refractory AA patients.Aims: To evaluate and compare the efficacy and safety between EPAG+IST and EPAG monotherapy in relapsed/refractory AA patients in a real-world setting.Methods: Data from patients diagnosed as acquired AA in our center were retrospectively collected. All the enrolled patients were refractory/relapsed to the standard IST for at least 6 months before EPAG. All patients had been treated with EPAG, which was started at 25 mg/day and increased every 2 weeks to a maximum of 150 mg/day until a best response was achieved. Meanwhile, some patients were treated with cyclosporin A (CsA) or tacrolimus (FK506) at the same time. EPAG had to be prescribed for at least 6 months before evaluation. Complete response (CR), overall response (OR) and relapse rate, as well as adverse events and factors which could affect efficacy were analyzed.Results: Totally 99 patients (83 non-severe AA (NSAA) and 16 SAA) were included in the study. The median age at EPAG initiation was 46 (13-88) years old, the median time of EPAG treatment was 11 (6-41) months and the median time of follow-up was 18 (6-41) months. 72 patients were treated with EPAG+IST, including 41 (56.9%) treated with EPAG+FK506 and 31 (43.1%) treated with EPAG+CsA. 27 patients were treated with EPAG alone. No significant difference was found between EPAG+IST group and EPAG group in patient baseline characteristics like age, male proportion, NSAA proportion, presence of PNH clone, proportion of previous ATG+CsA / CsA treatment, previous IST duration and dosage. With compatible follow-up time, EPAG exposure duration and dosage, there was no significant difference in OR/CR rate at 3 rd/6 th/12 th month between patients who was treated with EPAG+FK506 and EPAG+CsA. Under similar compatible baseline conditions, the OR rate was 33.3% vs 22.2% (P=0.284) at 3 rd month, 61.1% vs 37.0% (P=0.032) at 6 th month, and 67.2% vs 42.1% (P=0.051) at 12 th month for patients treated with EPAG+IST and EPAG alone, respectively, but no significant difference was found in time to response (3 (1-12) vs 3 (1-7) months, P=0.679) or CR rate at 3 rd/6 th/12 th month (6.9%/12.5%/20.7% vs 3.7%/7.4%/5.3%, P>0.05) between the two groups. Relapse occurred at 6 th to 12 th month of EPAG treatment, and the relapse rate at 12 th month was 9.8% and 27.3% (P=0.154) for patients treated with EPAG+IST and EPAG alone, respectively. For patients treated with EPAG+IST, responders had a significantly higher baseline reticulocyte count (60.25 (11.5-230.5)×10 9/L vs 16.7 (6.6-56.6)×10 9/L, P=0.040) compared with non-responders. No predictive factors for the overall response were found for patients treated with EPAG alone. Adverse events which led to dosage regulation were gastrointestinal disorders (2.8% vs 3.7%, P=1.000), elevated creatinine (2.8% vs 0, P=0.599), elevated ALT (1.4% vs 0, P=1.000) and arthralgia (0 vs 3.7%, P=0.280) for patients with EPAG+IST and EPAG, respectively. No deaths were found in either group, while the clone evolution rate was 2.8% and 3.7% (P=1.000) in EPAG+IST and EPAG monotherapy group, respectively.Conclusion: EPAG+IST had higher OR rate than EPAG monotherapy with similar side effects for patients with relapsed/refractory acquired AA. Those with higher baseline reticulocyte count were more likely to respond to EPAG+IST.Key words: relapsed/refractory, aplastic anemia, eltrombopag, immunosuppressant, efficacy DisclosuresNo relevant conflicts of interest to declare. OffLabel Disclosure:In the presented study, eltrombopag was prescribed in relapsed/refractory aplastic anemia patients.

AN UNUSUAL SURVIVING HISTORY: MULTISYSTEM INVOLVEMENT UNTIL ADULT LIFE WITH NIEMANN PICK TYPE B

Niemann-Pick disease (NPD) occurs with the storage of lipids including sphingomyelin and cholesterol due to acid sphyngomyelinase deficiency. Based on genetic cause and clinical picture NPD are divided in four main types. The type B is called as non-neuronopathic variant in which many patients may survive several decades. Infiltration by lipid-laden foam cells of tissues contribute to life-threatening complications. We here present a case who has been diagnosed as having NPD in the adulthood. A 46-year-old male patient with peripheral edema and dyspnea and abdominal distention was investigated. He has a medical history of aortic and tricuspid valve regurgitation with severe pulmonary hypertansion, decreased ejection fraction as 35% and acsending aort aneurism on 30 years old. He experienced three years later ascending aortic replacement and aortic valve replacement. He developed dyspnea, bleeding gums, and alveolar hemorrhage was diagnosed on 40s. Pancytopenia associated massive splenomegaly and hepatomegaly contibute reassesment of the disease. Bone marrow revealed moderate hypercellularity T lymphocytosis, focal mild dysplasic changes, and mild reticulin fiber increase. No cytogenetic abnormality and PNH clone was detected. He had developed congestive heart failure and massive proteinuria. Also he had medically controlled hyperlipidemia and interstitial lung disease. A storage disease investigation was started. Plasma Chitotriosidase was found to be increased and leukocyte sphingomyelinase activity was decreased. A genetic screening for NPD revealed homozygote (SMPD1 p.V36A (c.107T> C) (rs1050228) and heterozygote G508R (c.1522G> A) (rs1050239).NPD type was diagnosed with probable kidney involevement and cardiac cirrhosis. Supportive treatment was decided. He succumbed in a short time on sepsis atack unfortunetaly. NPD type B is a rare storage disease. It is a multisystemic disease characterized by its clinical variability and could be overlooked until adulthood life with various differential diagnosis option. It should be considered.

Study of the outcome of pediatric patients with aplastic anemia treated with immunosuppressive therapy-Single center experience

Background: Immunosuppressive therapy is the Initial therapy for children with severe Aplastic anemia (SAA) and lacking a matched related donor. Cyclosporine A (Cy A) alone has been tried as single agent in resource poor Countries. Patients and Methods: This retrospective study was done to assess the outcome of immunosuppressive therapy (IST) in SAA children. It included 23 patients treated at Ain Shams University Pediatrics Hospitals between 2011- 2019 with IST, they received rabbit anti-thymocyte globulin (r ATG) plus (Cy A) (Group I, 8 patients) or Cy A alone (Group II, 15 patients). The median follow- up duration was 59 months. Results: one patient (4.3%) developed AML clone. another patient (4.3%) developed PNH clone. There was no significant difference in response between the two groups, Complete response was 34.7%, 43.5%, and 47.8% and partial response was 34.7%, 26.1%, 21.7% at 3, 6, and 12 months, respectively in the whole cohort. NR was present in 30.4% at 3, 6, and 12 months. Hypertension in 62.5% and higher levels of ALT, total and direct bilirubin were observed in group I as compared to group II. HAAA was clinically evident in 7 patients; 4 of them were hepatitis A, 1 was EBV, and 2 were seronegative for all viruses. At presentation they had significantly higher level of ALT, total bilirubin, and direct bilirubin. Conclusion: IST is an effective therapy in patients with SAA with overall response of 70%. Cy A alone can be an effective therapy in developing countries.

Paroxysmal Nocturnal Hemoglobinuria Results from Initial Clonal PIG-a Mosaicism

The pathogenesis of PNH involves alteration of at least one HSC clone, characterized by a somatic mutation in the PIG-A gene. The resulting phenotype appears to offer a growth advantage over other unaffected HSCs, yet the failure of PIG-A mutant clones to expand in animal models suggests that additional factors are necessary for the development of PNH. The association of PNH with aplastic anemia (AA) is evidence of selection pressure by immune attack, but secondary hits in additional genes may also be clonally acquired to render PNH clones competitive irrespective of immune pressure. With the advent of NGS, clonal architecture analysis has been revolutionized. Yet, despite increased resolution, NGS is hampered by the presence of normal/non-clonal cells at varying frequencies in the sequenced sample, often decreasing the detection threshold and precluding recognition of subclonal mosaicism.To study these issues in PNH, 133 patients with PNH (N=33), AA (N=33), and AA/PNH (N=67) were sequenced using deep targeted NGS covering all exons of the PIG-A gene. All patients had a detectable PNH clone by flow cytometry, with the mean WBC clone size of 0.22% in AA, 27.4% in AA/PNH, and 77.1% in PNH. 223 PIG-A mutations were identified in this cohort; 35% were frameshift mutations, 34% missense, 14% splice site, 13% nonsense, and 3% non-frameshift. In unpurified blood, PIG-A mutations were found in 27/33 (82%) of PNH patients, 36/67 (54%) of AA/PNH, and 2/33 (6%) of AA, with a clear association between PNH clone size and the ability to detect mutations by NGS (P<0.0001). In DNA extracted from cells flow sorted by FLAER negativity, detection rates were 26/29 (90%), with an average of nearly 3 mutations per patient, indicating that mechanisms such as microdeletions may affect as many as 10% of PNH or AA/PNH patients and that clonal mosaicism is common. Our results also show that indeed type III and II PNH cells are derived from 2 distinct clones, each with a distinct mutation.To examine disease pathogenesis, we studied 47 patients longitudinally using both PNH flow and deep sequencing. Serial testing illustrates possible modes of evolution: mutations can fluctuate in the contribution to clonal burden over time with the variant allelic frequency of some mutations increasing while others decrease. Linear regression of the number of mutations vs . time from diagnosis suggests that in AA/PNH (N=29), the number of mutant clones detected increases over time (P=.012) while in classic PNH (N=18) the trend is reversed, as the number of mutations detected decreases (P<.0001) and a dominant clone emerges to supply a majority of blood cells.Traditionally, PNH has been considered a monoclonal disease in which a PIG-A mutant HSC clone can outcompete normal hematopoiesis within the context of permissive conditions. Using deep NGS, our results clearly indicate that while a single clone ultimately may dominate, PNH begins as oligoclonal process with the eventual emergence of the most competitive HSC clone(s) populating hematopoietic space. Initially, an environment that “favors” a state of GPI-anchor deficiency thereby allows multiple clones to evolve. Over time, the most “fit” PNH HSC clones dominate, resulting in monoclonality. In addition, our results confirm the presence of distinct mutations with corresponding phenotype through sequencing of sorted type II and type III PNH cells. Clonal drift may also be observed, likely through a newly acquired advantage of one clone within the available pool of GPI-deficient PIGA mutant clones. Our previous work indicated that acquisition of additional enabling mutations results in a more complex clonal architecture comparable to that seen in MDS, an intrinsic mechanism of the selection process that involves mutations in genes altered in MDS. Additional intrinsic hits also explain patient-specific kinetics of PNH evolution and apparent independence of established PNH from the effect of immunosuppression. Thus, while immune selection may provide initial conditions favorable for oligoclonal PNH evolution, once pressure is relieved, intrinsic factors can lead to PNH monoclonality. Alternative mechanisms could include loss of dominance by exhaustion, senescence, or telomere shortening.In sum, our study clearly demonstrates initial polyclonality early in the pathogenesis of PNH with selection of dominant clones during disease duration. DisclosuresMakishima:Yasuda Medical Foundation: Research Funding.

Surveillance of the Immune Repertoire of Aplastic Anemia Patients Using Deep Sequencing

Cytotoxic T cells (CTL) responses have been implicated in the pathogenesis of idiopathic aplastic anemia (AA). In the past, CDR3 region of variable portion of B-chain (VB) of TCR has been analyzed by capillary electrophoresis and later by sequencing to identify clonal expansions or oligoclonal skewing of otherwise polyclonal TCR VB repertoire. CTL clonal expansions, most likely, a result of aberrant antigenic drive, lead to increased numbers of identical CDR3 sequences which can serve as clonal barcodes for individually expanded T cell clones. In AA oligoclonal skewing of VB TCR has been detected at presentation using manual clonotyping and interpreted as an overheated cellular response to putative autoimmune targets located within hematopoietic stem cell compartment. With the advent of NGS, the process of analysis of TCR repertoire has been improved: while manual sequencing allowed for characterization of only hundreds of VB CDR3 clonotypes, deep TCR sequencing affords incredible sequencing depth and therefore precision and resolution. We hypothesized that deep TCR sequencing (NGS clonotyping) can be used to identify changes in the T cell repertoire, including clonal expansions which would either qualitatively or quantitatively enable to us to predict subsequent responsiveness to immunosuppression (IST), propensity of relapse or potentially monitor the IST intensity.We have identified 26 AA patients (56% severe AA) from whom samples were obtained at presentation and following IST with ATG/CsA including at recovery and relapse. In 10 patients serial samples were analyzed to allow for comparisons between clonal repertoire at presentation and in intervals following IST including when hematologic response was achieved. A hematologic response was seen in 88% of patients. PNH clone was present in 15/23 AA (AA/PNH).Genomic DNA was sequenced according to the ImmunoSeq NGS protocols. A group of 587 controls with an average sequencing depth of 30± 11 x 104 reads was used as reference. The average sequencing depth in AA patients was 74± 53 x103 productive reads. The healthy individuals showed a Simpson's Diversity value (SDV) of .998 (i.e ., number of unique clonotypic reads/number of total clonotypic reads). A total of 341/587 (58%) healthy controls had >1 rearrangement >1% of total repertoire with a mean productive frequency of 2.4%± 2.1%.The average SDV for AA was 0.993. Analyzing the frequency of productive rearrangements in patients at diagnosis, 22/26 patients had >1 rearrangement >1% of total repertoire with a mean productive frequency of 2.9%± 2.1%.We then analyzed the homology of the amino acid sequence of each expanded TCRB clone >1%. If the corresponding CDR3 amino acid sequence was absent in any other individuals (AA patients or controls), we referred to it as a “private” clone. In contrast, “public” clones had CDR3 sequences, which were also present in other individuals; to that end, a clone was defined as “public expanded” if the corresponding productive frequency was >1% in any other control or patient. We estimated that of all the immunodominant clones found in AA patients, 41% were private, likely recognizing patient specific antigens, while 33% were shared only between AA patients (42% in 2, 32% between 3 and 5, 26%>5 patients, N=22) and thus were AA-related. Public specificities constituted 59% of expanded clonotypes. The public clonotypes likely recognize ubiquitous antigens, which also exist in a tolerant form in healthy individuals. Any expanded clonotypes present in controls even if also found in patients (at any frequency) are the least likely to be pathogenic.We subsequently analyzed serial samples to assess the changes in size of the individual clones and establish a correlation between response and contraction of pathogenic clones. Average clonal contraction by 10.9 fold was observed in 50% of responders, and thus the remaining stable or expanding clones are unlikely pathogenic. In contrast, in non-responders only 33% of clones contracted by an average of 2.4 fold. Reappearance of originally expanded clones as well as new expanded clonotypes was observed upon relapse.In sum, our studies indicate that deep TCR sequencing will provide intricate insights into the dynamics of autoimmune process in AA and in the future may be used for monitoring of the efficiency of IST in the clinic and for the study of potentially pathogenic clones in the laboratory. DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees.