Aplastic Marrow Research Articles

Outcomes in Patients with Acute Myeloid Leukemia Treated with CLAG (cladribine, cytarabine, filgrastim) and/or Mitoxantrone with Venetoclax

Introduction: CLAG (cladribine, cytarabine, filgrastim) with or without M (mitoxantrone) has anti-leukemic activity in newly diagnosed (ND) and relapsed/refractory (R/R) AML (Wierzbowska et al. 2008, Jaglal et al. 2014, Seiter et al. 2016). Venetoclax (Ven) with hypomethylating agents (HMA), approved for patients (pts) who are not candidates for intensive chemotherapy (IC), is increasingly being employed even for pts who are fit for IC. R/R disease remains a major cause of mortality in fit pts with AML, and thus improved IC regimen is an area of active research. Various combination of IC with Ven are ongoing with promising early findings (DiNardo et al. 2021, Reville et al. 2022). In this study, we examined the outcomes of AML patients treated with CLAG and/or mitoxantrone with Ven, CLAG(M)/Ven. Methods: This was a retrospective study of pts with AML who received CLAG(M)/Ven (Days 2-8) in 2022-2023. Clinical responses were reported using ELN 2022 criteria. Kaplan-Meier analyses were used to estimate overall survival (OS) from the date of CLAG(M)/Ven initiation. Results: This cohort of 19 pts (8M/11F with median age of 60 [23-74] at diagnosis) is comprised of 68.4% (13/19) R/R and 31.6% (6/19) ND AML who had prior MDS directed therapy. There were 57.9% (11/19) with secondary/therapy-related AML and when including de novo AML pts who had mutations highly specific for secondary AML ontogeny, it increased to 78.9% (15/19). Most of the pts (68.4%, 13/19) had adverse risk disease by ELN 2022. Common mutations being TP53 (26.3%, 5/19) followed by IDH1/2 (21.1%), ASXL1 (15.8%), and RUNX1 (15.8%). There were 36.8% (7/19) who had received allogeneic hematopoietic cell transplant (alloHCT) (Table 1). Similar number of pts received CLAG/Ven (47.5%) and CLAG-M/Ven (52.6%). Pts received on average 2.6 lines of treatment prior to CLAG(M)/Ven with 1 pt received 5 prior lines of treatment after AML diagnosis. Most of the pts, 78.9% (15/19), had prior HMA/Ven. The 30-day and 60-day morality was 5.3% (1/19) (disease status unknown at the time of death) and 26.3% (5/19) (disease status: 3 yes, 1 no, 1 unknown), respectively. Among these 5 early deaths within 60 days, 3 were ND AML that were heavily pretreated for prior MDS (average 3 lines of prior therapy) and 2 were R/R AML. All of them had prior HMA/Ven. Eighty percent (4/5) had adverse risk disease and received CLAG-M/Ven. Forty percent (2/5) had TP53 mutation. Objective response rate (ORR) of the entire cohort was 26.3% (5/19) (2 CR, 1 CRi, and 2 MLFS). Among those with evaluable response (n=10), ORR was 50%. Two CR/CRi pts achieved undetectable MRD by flow cytometry. MRD was not tested in the other 3 pts. Notably, ORR was higher in pts who received CLAG/Ven (44.4%, 4/9) compared to CLAG-M/Ven (10%, 1/10). No response in ND AML and 38.5% (5/13) ORR in R/R AML. Of the 47.4% (9/19) nonevaluable pts, 6 had aplastic marrow (2 died while getting induction, 2 transitioned to alloHCT, 1 moved onto next line of treatment, and 1 lost to follow up) and 3 had recovery marrow pending. For pts who achieved a response, 60% (3/5) underwent alloHCT (all of them alive without disease at data cutoff). The median time to count recovery was 42.5 days (27 days to never recovered). Prior HMA/Ven failure, high dose cytarabine failure, and mutations did not significantly impact response in this small cohort. The median OS from the time of CLAG(M)/Ven initiation was 5.5 months with a median follow up of 8.6 months (Figure 1). OS from the date of CLAG(M)/Ven was significantly shorter in pts with TP53 mutation vs. wild-type (2 vs. 11.2 months, p=0.031). Conclusions: Our findings showed the combination regimen CLAG(M)/Ven is feasible in pts with AML. It also provided better characterization in terms of early mortality and survivals in these pts, which can help inform future clinical trial design. ORR was similar to what was previously reported with CLAG(M); however, it is encouraging that those who achieved CR/CRi and had MRD testing also achieved MRD negativity. Outcomes of TP53-mutated subset is dismal suggesting IC-based approach in this setting is limited. Larger cohort and longer follow up is needed to confirm the impact of factors such as specific regimen, mutations, and prior therapies on outcomes.

FLUDARABINE-BASED LOW-INTENSITY CONDITIONING FOR FANCONI ANEMIA IS ASSOCIATED WITH GOOD OUTCOMES IN APLASTIC ANEMIA BUT NOT IN MDS - A SINGLE-CENTER EXPERIENCE

Background: Hematopoietic stem cell transplantation [HSCT] is the only curative option for Fanconi Anemia (FA) patients with hematological abnormalities.
 Materials and Methods: This is a retrospective analysis of patients with FA who underwent a matched-related donor HSCT.
 Results: Sixty patients underwent 65 transplants between 1999-2021 using a fludarabine-based low-intensity conditioning regimen. The median age at transplant was 11 years (range: 3-37). Aplastic anemia (AA) was the underlying diagnosis in 55 (84.6%) while 8 (12.4%) had myelodysplastic syndrome (MDS) and 2 (3%) had acute myeloid leukemia (AML). The conditioning regimen used was fludarabine with low-dose cyclophosphamide for aplastic anemia and Fludarabine with low-dose busulfan for MDS/AML. Graft versus host disease (GVHD)prophylaxis consisted of cyclosporine and methotrexate. Peripheral blood stem cells was the predominant graft source (86.2%). Engraftment occurred in all but 1 patient. The median time to neutrophil and platelet engraftment was 13 days (range: 9-29) & 13 days (range: 5-31), respectively. Day 28 chimerism analysis showed complete chimerism in 75.4 % and mixed chimerism in 18.5%. Secondary graft failure was encountered in 7.7%. Grade II–IV acute GVHD occurred in 29.2% while Grade III-IV acute GVHD occurred in 9.2%. Chronic GVHD was seen in 58.5% and was limited in most patients. The median follow-up is 55 months [range: 2-144] & the 5-year estimated overall survival (OS) is 80.2 ± 5.1%. Secondary malignancies were noted in 4 patients. The 5-year OS was significantly higher in patients undergoing HSCT for AA (86.6 + 4.7%) as compared to MDS/AML (45.7+16.6%) (p= 0.001).
 Conclusion: SCT using a fully matched donor provides good outcomes with low-intensity conditioning regimens in patients with FA who have an aplastic marrow.

A Triplet Combination of Azacitidine, Venetoclax and Gilteritinib for Patients with FLT3-Mutated Acute Myeloid Leukemia: Results from a Phase I/II Study

A Triplet Combination of Azacitidine, Venetoclax and Gilteritinib for Patients with FLT3-Mutated Acute Myeloid Leukemia: Results from a Phase I/II Study

A Phase II Study of Azacitidine, Venetoclax and Trametinib in Relapsed/Refractory AML Harboring a Ras Pathway-Activating Mutation

A Phase II Study of Azacitidine, Venetoclax and Trametinib in Relapsed/Refractory AML Harboring a Ras Pathway-Activating Mutation

Lintuzumab-Ac225 in Combination with CLAG-M Yields High MRD (-) Responses in R/R AML with Adverse Features: Interim Results of a Phase I Study

Background:Lintuzumab-Ac225 (Actimab-A) is a humanized CD33 antibody, lintuzumab, conjugated to an alpha emitting isotope, actinium (Ac225), with potent single agent activity against AML. We hypothesized that a low dose infusion of lintuzumab-Ac225, after salvage chemotherapy, would effectively eliminate residual leukemia and improve remission rates, and depth of remission.Patients and Methods:Adult patients with relapsed/refractory AML (RR-AML), and with adequate organ function and performance status were eligible for screening. On screening, we assessed CD33 expression, greater than 25% of leukemic blasts must have expressed CD33 antigen by flow cytometry for inclusion. Induction consisted of G-CSF, 300mcg/d, given D1-6, cladribine 5mg/m2, given D2-6, cytarabine 2g/m2, given D2-6, and mitoxantrone 10mg/m2, given D2-4. Lintuzumab-Ac225 was administered as a single dose on either day 7, 8, or 9. This trial enrolled to 4 cohorts, administering lintuzumab-ac225 at a dose of 0.25uCi/kg, 0.50uCi/kg, 0.75uCi/kg, and 1.0uCi/kg. Treatment consisted of one induction cycle. Disease assessment, including MRD assessment by flow, was performed between Days 29-42.Results:Sixteen patients with a median age of 64 yrs were evaluable for toxicity and response. Three pts were treated in cohort 1 (0.25uCi/kg), nine pts were treated in cohort 2 (0.5uCi/kg), three pts were treated in cohort 3 (0.75uCi/kg), and one pt was treated in cohort 4 (1.0uCi/kg). Patient characteristics include 63% (n=10) pts with adverse cytogenetics, mutations involving TP53 in 44% (n=7) pts, and secondary/therapy-related AML in 50% (n=8) of patients. Patients received a median 2 lines of therapy, and 50% (n=8) received a venetoclax combination regimen prior to enrollment. Table 1 summarizes patient characteristics.No undue non-hematologic toxicities were observed in this trial. Commonest AEs were neutropenic fever and infection. Among responders, an ANC>1000 was achieved in 9/10 patients, at a median of 33 days, and a platelet count >50k was achieved in 6/10 patients at a median of 37 days. Of note, two patients proceeded to HCT prior to platelet recovery.Overall, among patients enrolled in cohorts 1-3, CR/CRi was observed in 67% (n=10) pts. One patient enrolled in cohort 4 achieved an aplastic marrow by D+42, with subsequent stem cell boost and neutrophil recovery. Of responders, 70% (7/10) had no measurable disease (MRD(-)) by flow cytometry. Among patients receiving <4 lines of prior therapy, 71% (n=5) pts with adverse cytogenetics, 80% (n=4) pts with TP53 mutation, and 80% (n=4) pts receiving a prior venetoclax combination achieved a remission. Estimated 6-month OS was 60%.Conclusions:Lintuzumab-Ac225 at a low dose appears to be safe in combination with the salvage chemotherapy regimen CLAG-M. Furthermore, this combination has demonstrated promising efficacy results among high risk RR-AML patients, namely patients with adverse molecular/cytogenetic features and patients previously receiving venetoclax. Updated Cohort 4 results will be presented at ASH.Upon determination of MTD, we plan to explore the combination of lintuzumab-Ac225 with other salvage regimens (e.g. FLAG, FLAG-Ida, MEC) to determine whether adding this novel agent can generally improve salvage outcomes in hard to treat patients. [Display omitted] DisclosuresAbedin: Helsinn: Research Funding; Agios: Honoraria; AltruBio: Research Funding; Pfizer: Research Funding; Amgen: Honoraria; Actinium: Research Funding; Astellas Pharma Inc.: Research Funding. Guru Murthy: Guidepoint: Consultancy; Cancerexpertnow: Honoraria; Techspert: Consultancy; Qessential: Consultancy; Cardinal Health Inc.: Honoraria; TG therapeutics: Other: Advisory board. Hamadani: Sanofi, Genzyme, AstraZeneca, BeiGene: Speakers Bureau; Takeda, Spectrum Pharmaceuticals and Astellas Pharma: Research Funding; Janssen, Incyte, ADC Therapeutics, Omeros, Morphosys, Kite: Consultancy. Atallah: Pfizer: Consultancy, Research Funding; BMS: Honoraria, Speakers Bureau; Takeda: Consultancy, Research Funding; Abbvie: Consultancy, Speakers Bureau; Amgen: Consultancy; Novartis: Consultancy, Honoraria, Research Funding.

Adoptive Immunotherapy with Cord Blood (CB) Grafts Targeting Shared Inherited Paternal Antigens (IPA) and/or Non-Inherited Maternal Antigens (NIMA) during Induction Treatment for High Risk Myeloid Malignancies: Safety and Preliminary Results

BackgroundSeveral groups have reported use of haploidentical peripheral blood donor cells as adjunct to chemotherapy in remission induction of myeloid leukemia (Colvin et al, BBMT 2009, Gue et al, Blood 2011). Moreover, CB grafts have been associated with greater disease control and enhanced graft-versus-leukemia responses, as a consequence of the fetal-maternal interactions during pregnancy (van Rood et al, PNAS 2009 and PNAS 2012).We evaluated prospectively the feasibility and tolerability of infusing partially HLA matched cord blood units (CBUs) with specific alloreactive properties after salvage chemotherapy to patients (pts) with refractory myeloid leukemia.Patients and MethodsPts with confirmed diagnosis of AML or MDS, according to WHO classification (excluding acute promyelocytic leukemia) who had received two or more previous induction/re-induction regimens were eligible. All pts were treated at NYPH and all CBUs were obtained from the NCBP. The study was performed under FDA IND and was approved by the IRBs of both institutions. Pts received induction chemotherapy followed by CBU infusion (1-3 days later) without any immunosuppression.CBUs eligible for the study contained a pre-cryopreservation Total Nucleated Cell (TNC) dose of 0.5-2.5x107/kg. They were HLA matched at 4-6/6, HLA-A, -B intermediate resolution, -DRB1 high resolution. CBUs had undergone HLA typing of the mother, thus allowing determination of the paternal and maternal antigens. CBUs with shared IPA targets and/or NIMA match were selected for cell therapy.ResultsA total of 34 patients underwent CBU search. Mean CBU number per search was 134 (range: 7-250) with 58% potentially eligible CBUs per pt (range: 5-159). A total of 143 CBUs underwent testing including HLA confirmatory typing. At least one eligible CBU was identified for each pt. Higher HLA allele level match was preferred in the final selection.Of the 34 pts, 23 did not receive treatment because of progressive disease and death (n=8); other clinical trials/treatments (n=6); hematopoietic stem cell transplants (HSCT) (n=9); while one is currently undergoing evaluation. Ten pts have been treated (Table); 8 had AML, 7 with primary induction failure (PIF); one had myeloid blast phase CML and one with high risk MDS. Nine pts had poor risk karyotype and 4 had prior allografts. Median age was 52 years (range: 34-67); median number of prior treatments was 11 (2-15) and median HSCT-CI was 3 (0-7).Of the CBUs infused, six were 5/6 and four were 4/6 HLA matched. The allele level compatibility and presence of shared IPA targets and/or NIMA matches are shown in the Table. CBU median TNC was 90x107 (range: 63-151) and they tended to be “older” with a median cryopreservation time of 10 years. CBUs provided a mean cell dose of 1.66 x 107 TNC/kg (range: 1.1-2.4). Mean post-thaw CD34+ and CD3+ cell doses were 52x103/kg (range: 20-90) and 0.34x107/kg (range: 0.24-0.52; SD: 0.08), respectively, with excellent post-thaw CD34+ viability (93.5%-98.3%).No CBU infusion reactions or cytokine release syndrome were observed. One pt, recipient of a prior allograft with no history of GVHD, experienced skin rash 1 week after CBU infusion. The skin biopsy was consistent with GVHD. No GVHD was observed in the other 9 pts. One pt died of disseminated adenovirus infection early after treatment; viremia was present at the initiation of chemotherapy.Transient CBU chimerism was detected in 5 of the 10 pts as early as one week and as late as 6 months after CBU infusion. Four pts with median pre-treatment BM blast counts of 56% (range 13-94) had objective hematologic responses (Cheson B JCO 2003) with median post-treatment blast counts of 0% (range 0-12), 3 with morphologic leukemia free state and one with a partial response. One patient had aplastic marrow but with flow cytometry evidence of persistent leukemia; while 4 pts recovered with morphologic persistent leukemia. Six pts went on to receive HSCT. Two subsequently relapsed and one died of disseminated adenovirus, while 3 pts remain in remission.ConclusionUse of CBUs as adoptive immunotherapy for pts with refractory myeloid leukemia represents a safe, well tolerated treatment, in which the potential for alloreactivity can be enhanced through the selection of CBUs by targeting shared IPA and NIMA present in the patients. Further, smaller CBUs, unlikely to be adequate grafts for adult transplantation, can be utilized for cell therapy. [Display omitted] DisclosuresRoboz:Cellectis: Research Funding; AbbVie, Agios, Amgen, Amphivena, Array Biopharma Inc., Astex, AstraZeneca, Celator, Celgene, Clovis Oncology, CTI BioPharma, Genoptix, Immune Pharmaceuticals, Janssen Pharmaceuticals, Juno, MedImmune, MEI Pharma, Novartis, Onconova, Pfizer, Roche Pharmace: Consultancy. Ritchie:Celgene: Consultancy, Other: Travel, Speakers Bureau; Pfizer: Consultancy, Other: Research funding to my institution; Incyte: Consultancy, Speakers Bureau; Astellas Pharma: Other: Research funding to my institution; NS Pharma: Other: Research funding to my institution; Bristol-Myers Squibb: Other: Research funding to my institution; Novartis: Consultancy, Other: Research funding to my institution, and travel, Speakers Bureau. Lee:Amgen: Consultancy; Baxalta: Consultancy; Clinipace: Consultancy; Alexion Pharmaceuticals: Consultancy.

Phase II Trial of Vosaroxin and Infusional Cytarabine for Frontline Treatment of Acute Myeloid Leukemia (VITAL) Patients Eligible for Intensive Chemotherapy: A Planned Interim Analysis

Phase II Trial of Vosaroxin and Infusional Cytarabine for Frontline Treatment of Acute Myeloid Leukemia (VITAL) Patients Eligible for Intensive Chemotherapy: A Planned Interim Analysis

Early Assessment of Treatment Response in Acute Myeloid Leukemia Using FLT PET/CT Imaging: A Trial of the ECOG-ACRIN Cancer Research Group (EAI141)

Introduction: Patients with acute myeloid leukemia (AML) are often treated with intensive induction chemotherapy to achieve complete remission (CR). Early response to standard anthracycline plus cytarabine induction (7+3) is assessed by a day 14 nadir bone marrow biopsy. The nadir marrow has limitations, though, including sampling issues and ambiguity as to whether blasts are leukemia cells vs. regenerating marrow. In prior studies, the predictive value for remission of the nadir marrow is only 67-84%. A more accurate predictor of residual disease (RD) would give clinicians an opportunity to modify therapy earlier. Positron emission tomography (PET/CT) with 3'-deoxy-3'-[18F]fluorothymidine (FLT) is a molecular imaging modality that can assess cellular proliferation in the bone marrow compartment. A prior single-institution pilot study showed a significant difference in the marrow FLT uptake between patients with AML who achieved CR and those who had RD after count recovery. Methods: EAI141 was an ECOG-ACRIN-led, prospective, multi-center study designed to assess FLT PET/CT as a predictor of CR after AML induction. The primary objective was to evaluate the predictive value of post-treatment FLT PET/CT imaging for detecting RD, with the secondary objectives of evaluating the predictive value for detecting CR and estimating the sensitivity and specificity. Maximum standardized uptake value (SUVmax) of &amp;gt; 7 g/mL in total bone marrow compartment was chosen prospectively based on data from a pilot study as a marker for presence of leukemia cells at nadir. The predictive values, sensitivity, and specificity of the FLT PET/CT and the nadir bone marrow biopsy were calculated. Eligible subjects were 18 years or older with previously untreated AML who were to receive induction with 7+3. Subjects underwent a nadir marrow biopsy and an FLT PET/CT scan 10-17 days after starting induction, and prior to any re-induction if disease was noted on the nadir marrow. A recovery marrow was performed 28-35 days after initial treatment or re-induction to assess CR or RD. Subjects could undergo an optional pre-treatment scan. Relapse-free survival (RFS) and overall survival (OS) were additional clinical outcomes. Results: 87 subjects from 9 centers were enrolled between 2016-2018, and 61 were considered evaluable. Reasons for being not evaluable included study ineligibility (n=3), no post-treatment scan (n=13), and no remission marrow (n=10). Median age was 58 years (range 21-73); 56% were men and 44% were women. CR was achieved in 56% (34/61). Treatment was a single induction course in 79% (48/61) and re-induction in 21% (13/61). Predictive value based on FLT PET/CT was 60% (9/15, 95% CI 32%-84%) for RD and 61% (28/46, 95% CI 45%-75%) for CR. Of patients who achieved CR, 28/34 had SUVmax ≤ 7 g/mL (sensitivity of 82%) and 9/27 of those who did not had high SUVmax &amp;gt; 7 g/mL (specificity 33%). Predictive value of an aplastic marrow was 59% (26/44) for CR and 56% (9/16) for RD. Of patients who achieved CR, 26/33 had marrow hypoplasia (sensitivity 79%) and 9/27 with RD had disease in the nadir marrow (specificity 33%). Significant heterogeneity of bone marrow compartment post treatment (SUVhetero ranging from 0.24 to 1.07) was observed in FLT-PET/CT scans. Heterogeneity of bone marrow compartment decreased about 50% from pre-treatment to post-treatment FLT PET/CT. OS for all participants, stratified by post-treatment FLT PET/CT response and nadir marrow results, are summarized in the Figure. Conclusions: Although this study did not show significant advantage of FLT PET/CT compared to nadir marrow to predict RD or CR on the average, it did show signal heterogeneity in the study population that may be relevant to disease biology not appreciated by a single sampling site for the nadir or remission marrow. For example, in patients with false negative nadir biopsy, in approximately 20% of patients assessment with FLT PET/CT was correct, likely reflecting bone marrow heterogeneity and limitation of a single point sampling. Similarly, in almost 60% of patients with false positive nadir biopsy, FLT PET/CT assessment was correct. Figure 1 Disclosures Jeraj: AIQ Solutions: Current equity holder in private company. Kostakoglu:F. Hoffmann-La Roche: Consultancy. Strickland:KiTE: Consultancy; Kura: Consultancy; Astellas: Consultancy; Jazz: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Sunesis: Research Funding; ArcherDx: Consultancy; AbbVie: Consultancy. Uy:Daiichi Sankyo: Consultancy; Pfizer: Consultancy; Agios: Consultancy; Genentech: Consultancy; Jazz Pharmaceuticals: Consultancy; Astellas Pharma: Honoraria. Perlman:GE Healthcare: Research Funding; AIQ: Research Funding.

The VITAL Trial: Phase II Trial of Vosaroxin and Infusional Cytarabine for Frontline Treatment of acute Myeloid Leukemia

The VITAL Trial: Phase II Trial of Vosaroxin and Infusional Cytarabine for Frontline Treatment of acute Myeloid Leukemia

Venetoclax (VEN) and tyrosine kinase inhibitor (TKI) combinations in Philadelphia chromosome-positive (Ph+) acute myeloid leukemia (AML) and chronic myeloid leukemia myeloid blast phase (CML MBP).

e18515 Background: Ph+ AML and CML MBP have poor outcomes. VEN synergizes with BCR-ABL TKIs in vitro and may eradicate Ph+ leukemic stem cells. However, the clinical activity of VEN+TKI regimens is unknown. Methods: We conducted a retrospective study on patients (pts) with Ph+ AML and CML MBP who received regimens with VEN+TKI at our institution. Results: We identified 6 pts with relapsed/refractory (R/R) Ph+ AML and 7 with CML MBP (2 transformed from chronic phase, 5 R/R). 10 pts (77%) were refractory to prior therapy and 3 (23%) had relapsed disease. Pt characteristics are shown in the Table. 5 AML pts (83%) had complex cytogenetics. Pts with CML MBP had received a median of 2 prior TKIs (range 1-3). 7 pts received decitabine-based combinations and 6 pts received intensive chemotherapy in combination with VEN+TKI. TKIs included ponatinib (n=7), dasatinib (n=4), bosutinib (n=1) and nilotinib (n=1). Among 12 evaluable pts, the overall response rate (CR + CRi + MLFS) was 50% (Table). In addition, 1 pt with AML developed aplastic marrow, underwent stem cell transplantation, and is still alive. Pts with CR/CRi vs. those without CR/CRi had higher median number of Ph+ metaphases (100% vs. 32%, p&lt;0.01) and higher baseline BCR-ABL1 PCR (100% vs. 0.5% p=0.01). The only AML pt who achieved CR/CRi had 100% Ph+ metaphases. After a median follow-up of 10.9 months (mo), 5 pts are alive (1 AML, 4 CML MBP). The median overall survival for pts with AML and CML MBP were 2 mo and not reached, respectively. The median relapse-free survival was 8.2 mo. Conclusions: VEN+TKI combinations show encouraging activity in pre-treated, advanced Ph+ leukemias, particularly CML MBP. Clonal burden of Ph+ cells correlated with response. Prospective trials are needed to evaluate VEN+TKI combinations in Ph+ leukemias. Pt characteristics and outcomes (n/N [%] / median [range]). [Table: see text]

Aplastic and Hypoplastic Anemias.

1. Suzie A. Noronha, MD* 1. *Division of Pediatric Hematology/Oncology, University of Rochester, Rochester, NY * Abbreviations: AA: : aplastic anemia AML: : acute myeloid leukemia ANC: : absolute neutrophil count ARC: : absolute reticulocyte count CBC: : complete blood cell DBA: : Diamond-Blackfan anemia DC: : dyskeratosis congenita FA: : Fanconi anemia GPI: : glycosylphosphatidylinositol HSCT: : hematopoietic stem cell transplant MCV: : mean corpuscular volume MSD: : matched sibling donor MUD: : matched unrelated donor OS: : overall survival PNH: : paroxysmal nocturnal hemoglobinuria PRCA: : pure red cell aplasia SDS: : Shwachman-Diamond syndrome TEC: : transient erythroblastopenia of childhood 1. Anemia is a common finding in general pediatrics, but knowing when to refer a patient can be challenging. 2. Pediatricians do not always order reticulocyte counts as part of the evaluation of anemia. The reticulocyte count, interpreted in the context of the mean corpuscular volume, is very useful in the development of differential diagnoses for anemia and determination of urgency of referral. 3. Pediatricians (and subspecialists) may not realize that diagnosis of malignancy, particularly head and neck cancers, at an unexpectedly early age may be associated with an inherited bone marrow failure. After completing this article, readers should be able to: 1. Assess the need for referring a patient with anemia based on whether anemia is isolated or accompanied by other hematologic or physical anomalies. 2. Recognize that anemia in the setting of congenital anomalies may signify an inherited bone marrow failure disorder. 3. Understand that inherited bone marrow failure disorders confer a higher risk of malignancy. Children with anemias often initially present to their pediatricians. Anemia may result from blood loss, a destructive process (ie, hemolysis), nutritional deficiency, or poor production (eg, ineffective erythropoiesis or hypoplastic or aplastic marrow) (Table). Hemolytic and nutritional anemias have been discussed elsewhere and are referenced briefly in this article. (1)(2)(3)(4) This review focuses on the broad differential diagnosis for anemia associated with poor production. These anemias can lead to chronic morbidity and require subspecialty care, whereas others may be managed effectively in primary care. This review offers a framework to guide evaluation and the need for subspecialty investigation. View this table: TABLE . Classification of Anemias by Mechanism and Notable Associated Laboratory Data Aplastic and hypoplastic anemias are characterized …

Outcome of Age-Adapted Approach to HLA-Identical Related Hematopoietic Stem Cell Transplantation in Severe Sickle Cell Disease: Saudi Experience

▪Sickle cell disease (SCD) is one of the most common inherited disorders in Saudi Arabia with nearly 60,000 affected individuals (Alsultan et al. Ped. Transplant. 2016). Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment in SCD. A recent international survey showed excellent long-term survival of SCD patients after HLA-identical sibling HSCT; however, patients aged 16 years or older had higher risk of GVHD and mortality (Gluckman et al. Blood 2017). HLA-identical related HSCT in adult SCD patients using non-myeloablative (NMA) conditioning consisting of alemtuzumab, 300 cGy total body irradiation (TBI), and sirolimus was successful in establishing long term stable donor chimerism with minimal toxicity and GVHD (Hsieh et. al. JAMA 2014). In this study, we reviewed the outcome of SCD patients who underwent transplant at our institution using standard protocols (NMA regimen in patients ≥14 years and myeloablative regimen in < 14 years) to address whether age remains a risk factor that influences HSCT outcome in SCD.Children (<14 years) with severe SCD received myeloablative conditioning using one of two regimens: first regimen was cyclophosphamide (Cy) 200mg/kg, busulfan (Bu) 16mg/kg, and thymoglobulin (ATG) 10mg/kg and recently we use thiotepa 8mg/kg, Bu 16mg/kg, and fludarabine (Flu) 160mg/m2. Bu pharmacokinetics was performed to target AUC of 900-1350 µmol/L.min. GVHD prophylaxis included cyclosporine and methotrexate 10mg/m2 on day +1, +3, and +6. Bone marrow was the source of stem cells in all pediatric patients. Cyclosporine was continued during the first-year post HSCT and tapered slowly afterward based on donor chimerism. Adult patients (≥14 years) received hydroxyurea at maximum tolerated dose and hypertransfusion for 2-3 months with iron chelation if indicated prior to HSCT followed by NMA conditioning consisting of alemtuzumab (1mg/kg divided over 5 days on day -7 to -3) and TBI 300 cGy with testicular shielding in males on day -1. Sirolimus was used as GVHD prophylaxis starting day -2 and continued for at least one-year post HSCT with subsequent taper based on donor chimerism. Stem cells source was GCSF mobilized peripheral blood stem cells in all adult patients targeting cell dose of CD34 10X106/kg based on recipient weight. All SCD patients underwent exchange transfusion prior to HSCT to achieve HbS <30%. Hemoglobin was maintained between 9-11 g/dL and platelet count > 50,000/uL during HSCT. Supportive care included penicillin V, acyclovir, antifungal, and Pneumocystis jiroveci pneumonia prophylaxis. Levetiracetam was used as seizure prophylaxis. Strict monitoring of blood pressure and magnesium was performed. Graft failure and death from any cause were considered as events.A total of 51 patients with severe SCD were transplanted at our center, 17 children and 34 adults. Patient and transplant characteristics are summarized in Table. Indications for HSCT included recurrent severe pain crisis (n=33), stroke (n=14), avascular necrosis (n=2), sickle cell hepatopathy (n=1), and priapism (n=1). All patients engrafted successfully for both neutrophil and platelets. There was no acute or chronic GVHD among adult patients. Two pediatric patient had mild grade I acute GVHD in the skin that was controlled with topical treatment and none of pediatric patients had chronic GVHD. Three adult patients developed secondary graft failure on day 61, 170, and 225 post transplant with aplastic marrow in two patients despite full donor chimerism and autologous recovery in the third patient. Among the two patients who had aplastic anemia, one died secondary to sepsis and one underwent successful second transplant using cyclophosphamide, Flu, and ATG. Thirty-one adults have stable donor chimerism. Event free survival (EFS) at 2 years was 90% in adult patients and 100% in children (P=0.3). Overall survival (OS) at 2 years was 97% in adults and 100% in children (P=0.4). Median follow up duration was 416 days (range, 61-1256) in adults and 203 (range, 30-2505) days in children.We demonstrated that age-adapted approach in guiding the choice of conditioning regimen intensity in severe SCD is associated with excellent outcome and minimal risk of graft rejection, GVHD, and transplant-related mortality. This real world data is important to encourage patients and transplant physicians to consider this curative treatment. Longer follow up duration is needed to confirm our findings. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Epigenetic and microenvironmental alterations in bone marrow associated with ROS in experimental aplastic anemia

Aplastic anemia or bone marrow failure often develops as an effect of chemotherapeutic drug application for the treatment of various pathophysiological conditions including cancer. The long-term bone marrow injury affects the basic hematopoietic population including hematopoietic stem/progenitor cells (HSPCs). The present study aimed in unearthing the underlying mechanisms of chemotherapeutics mediated bone marrow aplasia with special focus on altered redox status and associated effects on hematopoietic microenvironment and epigenetic status of hematopoietic cells. The study involves the development of busulfan and cyclophosphamide mediated mouse model for aplastic anemia, characterization of the disease with blood and marrow analysis, cytochemical examinations of bone marrow, flowcytometric analysis of hematopoietic population and microenvironmental components, determination of ROS generation, apoptosis profiling, expressional studies of Notch-1 signaling cascade molecules, investigation of epigenetic modifications including global CpG methylation of DNA, phosphorylation of histone-3 with their effects on bone marrow kinetics and expressional analysis of the anti-oxidative molecules viz; SOD-2 and Sdf-1. Severe hematopoietic catastrophic condition was observed during aplastic anemia which involved peripheral blood pancytopenia, marrow hypocellularity and decreased hematopoietic stem/progenitor population. Generation of ROS was found to play a central role in the cellular devastation in aplastic marrow which on one hand can be correlated with the destruction of hematopoiesis supportive niche components and alteration of vital Notch-1 signaling and on other hand was found to be associated with the epigenetic chromatin modifications viz; global DNA CpG hypo-methylation, histone-3 phosphorylation promoting cellular apoptosis. Decline of anti-oxidant components viz; Sdf-1 and SOD-2 hinted towards the irreversible nature of the oxidative damage during marrow aplasia. Collectively, the findings hinted towards the mechanistic correlation among ROS generation, microenvironmental impairment and epigenetic alterations that led to hematopoietic catastrophe under aplastic stress. The findings may potentiate successful therapeutic strategy development for the dreadful condition concerned.

Early Blast Clearance Evaluation after Induction Chemotherapy for Acute Myeloid Leukemia By Multiparameter Flow Cytometry and WT1-RNA Quantification: A Single Center Experience

Introduction: in acute myeloid leukemia (AML) the response to treatment is evaluated upon full recovery of peripheral blood counts by bone marrow (BM) assessment using morphology and cytogenetics, if appropriate number of metaphases is obtained. Real time quantitative PCR (RQ-PCR) and multiparametric flow citometry (MFC) are sensitive techniques to assess minimal residual disease (MRD) mostly used to refine risk stratification and to guide therapy. Evaluation of response to induction chemotherapy (CT) during aplasia (around day 14 from start of induction) has shown a significant prognostic impact, the presence of residual disease predicting a worse prognosis. All reported studies on early BM blast clearance evaluation rely on morphology as a single technique. Unfortunately, at day 14 BM is often not evaluable for cytology and morphologyc blast count. Unpredictability and intra-observer variability must also be considered when assessing blast count by morphology alone. No studies analysing early BM blast clearance by means of MFC and/or PCR have been published and the correlation between the two assays is unknown in this setting. Moreover, it is to be established if early intensification (around day 15 from start of induction) with a second cycle of CT could increase the CR rate in pts with disease persistence in the BM evaluated at day 14.Matherial and methods: we retrospectively evaluated data of 23 newly diagnosed AML pts who received induction CT at our center between 02/2009 and 05/2014, and for whom analysis of BM both at day 14 and after hematologic recovery (around day 28) was performed. The aim of our study was to define the prognostic value of early MRD quantification by MFC (d14-LAIP) and WT1 quantification by PCR (d14-WT1) in predicting the response to induction. Firstly we compared d14-LAIP and d14-WT1 to identify the more sensitive and specific assay in predicting the response to induction, in particular for cases not evaluable for morphologic blast count. Then we compared the outcome of pts who received or did not receive an early intensification for persistence of disease at d14 BM evaluation.Results: 20 pts received the 3+7 induction regimen, 3 pts the ICE induction regimen. After BM evaluation at day 14, 7 pts received early reinduction CT (FLAG-IDA regimen) starting at day 16 (median), 16 pts did not receive further therapy before BM evaluation at day 28. Overall CR rate was 70% (16 pts), PR/NR 30% (7 pts), TRM 4% (1 pt). At day 14, leukemic blast percentage was not evaluable by morphology in 8 (35%) cases due to marrow aplasia, in 1 (4%) case blasts were <5%, in 14 (61%) cases blasts were ≥5%; by MFC (23 cases), in 9 (39%) cases blasts were ≤2%, in 14 (61%) were >2%; by PCR (17 cases), in 4 (24%) cases WT1 was <250 cp/10e4 ABL, in 13 (76%) WT1 was ≥250 cp/10e4 ABL. At day 28 BM evaluation, of the 8 pts with aplastic marrow at day 14, 6 (75%) were in CR and 2 (25%) in PR/NR, of the 14 pts with blasts ≥5% at day 14, 9 (64%) were in CR and 5 (36%) in PR/NR. Analysis of the paired results from nadir to recovery revealed that d14-LAIP 2% had a positive predictive value (PPV) of CR at day 28 of 71% and negative predictive value (NPV) of 89%, with a sensitivity of 83% and a specificity of 80%, d14-WT1 250 had a PPV of 50% and NPV of 25%, with a sensitivity of 57% and a specificity of 20%. The d14-LAIP analysis was strongly associated with CR after induction (p 0.034). Considering the 14 pts with blasts ≥5% at day 14, 6/7 (86%) who received early reinduction CT and 3/7 (43%) who did not, obtained the CR at day 28 (p ns).Discussion: in our series d14-LAIP proved to be more predictive of response after induction CT than d14-WT1. Although one-third of pts had an early morphologic response not evaluable due to marrow aplasia MFC proved to be a useful assessment tool with a 100% applicability. Moreover, correlation between D14-LAIP and d14-WT1 was > 90%. Our data confirm the prognostic value of day 14 BM evaluation and suggest that MRD detection, also in aplasia, could drive early reinduction CT which probably could increase the CR rate, without significantly clinical complications. Anyway, this last point must be confirmed with a larger study. DisclosuresBordignon:MolMed S.p.A: Chairman and CEO Other.

Alteration of hedgehog signaling by chronic exposure to different pesticide formulations and unveiling the regenerative potential of recombinant sonic hedgehog in mouse model of bone marrow aplasia.

Chronic pesticide exposure-induced downregulation of hedgehog signaling and its subsequent degenerative effects on the mammalian hematopoietic system have not been investigated yet. Howevera number of concurrent studies have pointed out the positive correlation between chronic pesticide exposure induced bone marrow failure and immune suppression. Here, we have given an emphasis on the recapitulation of human marrow aplasia like condition in mice by chronic mixed pesticide exposures and simultaneously unravel the role of individual pesticides in the said event. Unlike the effect of mixed pesticide, individual pesticides differentially alter the hedgehog signaling in the bone marrow primitive hematopoietic compartment (Sca1+compartment) and stromal compartment. Individually, hexaconazole disrupted hematopoietic as well as stromal hedgehog signaling activation through inhibiting SMO and facilitating PKC δ expression. On contrary, both chlorpyriphos and cypermethrin increased the sequestration and degradation of GLI1 by upregulating SU(FU) and βTrCP, respectively. However, cypermethrin-mediated inhibition of hedgehog signaling has partly shown to be circumvented by non-canonical activation of GLI1. Finally, we have tested the regenerative response of sonic hedgehog and shown that in vitro supplemented recombinant SHH protein augmented clonogenic stromal progenitors (CFU-F) as well as primitive multipotent hematopoietic clones including CFU-GEMM and CFU-GM of mixed pesticide-induced aplastic marrow. It is an indication of the marrow regeneration. Finally, our findings provide a gripping evidence that downregulated hedgehog signaling contribute to pesticide-mediated bone marrow aplasia but it could be recovered by proper supplementation of recombinant SHH along with hematopoietic base cocktail. Furthermore, SU(FU) and GLI1 can be exploited as future theradiagnostic markers for early marrow aplasia diagnosis.

Morphological Spectrum of Paroxysmal Nocturnal Hemoglobinuria (PNH).

Morphological Spectrum of Paroxysmal Nocturnal Hemoglobinuria (PNH).

Evaluation of Danazol, Cyclosporine, and Prednisolone as Single Agent or in Combination for Paroxysmal Nocturnal Hemoglobinuria

Objective: The responses of 32 patients with paroxysmal nocturnal hemoglobinuria (PNH) were assessed after the patients were put on various combinations of danazol, prednisolone, and cyclosporine.Materials and Methods: Nineteen males and 13 females aged between 14 and 60 years with confirmed diagnosis of PNH were treated with danazol (4), danazol + cyclosporine (7), cyclosporine (1), and prednisolone + danazol (20). Response to these interventions was assessed regularly. Danazol was added to cyclosporine in patients with aplastic bone marrow after 3 months of cyclocporine use only unless the former therapy was successful. Four patients with aplastic marrow received only danazol because they had renal insufficiency at presentation. Patients were evaluated with regular complete blood count and routine liver and renal function tests. Results: One patient responded to cyclosporine only. Thirteen of 32 patients (40%) had complete response, 12/32 patients (37%) had partial response leading to freedom from red cell transfusion, and 2/32 (7%) had no response. Five patients (16%) died due to thrombosis or hemorrhage within 3 months of therapy before their response to therapy could be assessed. The median period of review of the cases was 4 years and 6 months.Conclusion: Danazol is a useful addition to PNH therapy both in combination with cyclosporine for hypoplastic PNH and with prednisolone for other forms of PNH, and this therapy could be a good alternative where eculizumab and anti-lymphocyte globulin cannot be used for various reasons.Conflict of interest:None declared.

G-CSF Induced Atypical Monocytic Proliferation Mimicking Acute Myelomonocytic Leukemia in Post-Induction Chemotherapy AML Patient

Abstract 4283 Introduction:Granulocyte colony-stimulating factor (G-CSF) is a major hematopoietic cytokine involved in control of neutrophil production, proliferation and survival.1 Nowadays, recombinant G-CSF is widely used in patients with hematopoietic malignanies receiving chemotherapy to increase leukocyte count.2 Studies have showed that G-CSF use in these settings is safe and does not increase relapse rate.3,4 However, A phenomena called transient atypical monocytosis, or pseudoleukemia, induced by G-CSF administration in AML patients with chemotherapy have been reported.5,6 This entity mimics recurrent/relapsed acute leukemia and causes confusion in pathologic diagnosis and potential problems in clinical management. The case presented in this report is an example of this entity. Pathology:A 55-year old male patient presented with leukocytosis and anemia. Peripheral blood smear examination revealed a blast population and monocytosis. Flow cytometry on the blood sample showed 37% blasts with expression of CD33, CD13, CD117, HLA-DR, CD34, CD14, CD15 and CD64. FISH study revealed a chromosomal abnormality of inversion 16. A diagnosis of acute myelomonocytic leukemia was established. The patient was treated with induction chemotherapy and was given Neupogen from day 6 through day 20. Bone marrow examination on the day 14th showed an aplastic marrow. However, examination of the patient's day 21 bone marrow revealed marked hypercellularity with sheets of large atypical monocytic cells and brisk mitotic figures. These cells showed round to ovoid nuclei, basophilic cytoplasm and prominent nucleoli, morphologically indistinguishable from blastic cells. Immunohistochemical studies confirmed that most of these atypical cells were lysozyme, CD163 and CD68 positive monocytes, some of them were positive for CD117. These findings raised concerns of recurrent/relapsed AML. However, FISH study was negative for inversion 16. These results indicate that the findings in the patient's day 21 marrow were most likely G-CSF-induced changes. The Neupogen was discontinued soon after. The patient was followed-up with periodic bone marrow examination and FISH study, which showed negative results. Currently, the patient is in complete remission. Discussion:We report one case of G-CSF induced atypical monocytic proliferation in an AML patient with induction chemotherapy. The findings could have created diagnostic problems if FISH study data had not available since those atypical cells were morphologically indistinguishable from blastic cells. Studies have shown that administration of recombinant G-CSF induces a rapid and sustained elevation in absolute peripheral neutrophil counts, and its use in leukemic patients during or after induction chemotherapy is safe and does not increase relapse rates.4 G-CSF shortens the transit time of developing granulocytes in bone marrow and accelerates the release of neutrophils that have undergone recent cell division.1 Our case of transient atypical monocytosis and a few similar cases reported in literatures showed an unusual response to G-CSF. This phenomenon was mostly seen in AML-M4, AML-M5 and AML-M3 cases7 and the mechanism is not entirely clear. A possible explanation is that G-CSF does not only act on neutrophils but also acts on progenitor hematopoietic cells including monocytes.8 One problem is that this entity can mimic recurrent AML causing confusion or potential misdiagnosis if clinical information or cytogenetic studies are not available. Therefore, it is important that both clinicians and pathologists be aware of this phenomenon in hematology/oncology settings. Appropriate discontinuation of G-CSF and following-up these patients with morphologic examination and cytogenetic studies is necessary. Meanwhile, a thorough understanding of exogenous G-CSF effects in cancer patients is essential to reduce any potential risks caused by its administration. Disclosures:No relevant conflicts of interest to declare.

The Frequency of Red Cells with a Spontaneously Appearing XK-Null (McLeod-like) Phenotype Is Normal In Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

AbstractAbstract 816Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a large population of blood cells with an acquired somatic mutation in the X-linked PIG-A gene. This results in an inability to synthesize glycosylphosphatidylinositol (GPI) and an inability to express on the cell surface any of the proteins that require this structure. Although there are case reports of progression to leukemia or high grade myelodysplasia, in large series of patients, this occurs only rarely. It is therefore hypothesized that in PNH, the large GPI (-) population results from selection in favor of a mutant stem cell clone in an aplastic marrow environment, without inherent predisposition to mutations. In favor of this hypothesis is the observation that the GPI(-) population is often found to have a single PIG-A mutation. An alternative hypothesis, however, is that there may be inherent hypermutability in patients with PNH; indeed, it is not unusual that 2 or even several PIG-A mutant clones can be shown to coexist in the same patient by careful sequence analysis, and cytogenetic abnormalities are not uncommon in PNH. We have previously reported a normal rate of spontaneously arising GPI (-) cells among established GPI (+) B lymphocyte lines from patients with PNH, arguing against this model, but this does not rule out the possibility that hypermutability somehow spares the lymphoid lineage. We have therefore now analyzed red blood cells from patients with PNH to determine the frequency of spontaneously arising cells with a mutation in another gene, XK, which is associated with McLeod syndrome. Like PIG-A, the XK gene resides on the X-chromosome, a large spectrum of mutations can inactivate the gene, and its associated mutant phenotype is detected by flow cytometry—as a loss of Kell antigen expression on red cells. As for PIG-A, we have previously demonstrated that healthy individuals harbor very small populations of blood cells with a spontaneously appearing XK-null (McLeod-like) phenotype, in this case at a frequency of about 39 × 10−6 in healthy adults and 9 × 10−6 in cord blood samples. In order to determine the frequency of McLeod-like cells, approximately 3 ul of whole blood is first incubated on ice with a hybridoma supernatant (MIMA 91) which has specificity for a common non-polymorphic Kell antigen. The cells are then washed twice again and stained with a secondary PE-conjugated rabbit anti-mouse antibody, washed twice again, and then stained with a FITC-conjugated anti-glycophorin A (GPA) antibody, and then washed again. At least 2 million GPA (+) events are then collected on a FACScan instrument. Stored cells from patients with the McLeod syndrome and obligate heterozygote carrier females serve as a control. In general, spontaneously appearing McLeod-like cells in normal donors form a distinct population that is strongly GPA (+) and dim for Kell antigens, typically having <20% of the mean PE fluorescence of the overall population. Here we have analyzed samples from 17 patients with PNH, 13 of whom would be classified as “classic PNH”. Among the 17 patients, the median blood counts were 2100/ul for the ANC, 10.8 gm/dl for the HGB, 138,000/ul for the platelet count, and 125,000/ul for the absolute reticulocyte count. The median % PNH clone size was 31% for the RBC and 77% for the PMN's. 7 of the patients had a history of Aplastic Anemia, 6 had a history of thrombosis, and 10 were receiving Eculizumab infusions. We have shown that among these 17 patients, the median frequency of spontaneously arising McLeod-like red cells was 46 × 10−6 (range 14 to 204 × 10−6). In comparison, among 15 normal healthy adults, the median frequency of McLeod-like cells was 48 × 10−6 (range 14 to 113 × 10−6), whereas the median frequency was 15 × 10−6 (range 11 to 37 × 10−6) among 5 cord blood samples. We conclude that there is no evidence for hypermutability in stem cells or in progenitors of the red cell lineage among patients with PNH using the XK gene as a sentinel for spontaneous somatic mutations. Disclosures:No relevant conflicts of interest to declare.

Correction of Fanconi Anemia Group C Hematopoietic Stem Cells Following Intrafemoral Gene Transfer

The main cause of morbidity and mortality in Fanconi anemia patients is the development of bone marrow (BM) failure; thus correction of hematopoietic stem cells (HSCs) through gene transfer approaches would benefit FA patients. However, gene therapy trials for FA patients using ex vivo transduction protocols have failed to provide long-term correction. In addition, ex vivo cultures have been found to be hazardous for FA cells. To circumvent negative effects of ex vivo culture in FA stem cells, we tested the corrective ability of direct injection of recombinant lentiviral particles encoding FancC-EGFP into femurs of FancC −/− mice. Using this approach, we show that FancC −/− HSCs were efficiently corrected. Intrafemoral gene transfer of the FancC gene prevented the mitomycin C-induced BM failure. Moreover, we show that intrafemoral gene delivery into aplastic marrow restored the bone marrow cellularity and corrected the remaining HSCs. These results provide evidence that targeting FA-deficient HSCs directly in their environment enables efficient and long-term correction of BM defects in FA.