ALBERT

Description: [Background] Olfactomedin 4 (OLFM4), a member of the olfactomedin-related protein family, is constitutively expressed in bone marrow (BM) cells and many gastrointestinal organs and is involved in a variety of cellular functions, including proliferation, differentiation, apoptosis, and cell adhesion in tumor cells. Previous studies have suggested that OLFM4 supports the survival of leukemic stem cells derived from iPS cells of patients with chronic myeloid leukemia. Our recent study revealed a somatic mutation of OLFM4 in HLA allele-lacking granulocytes of a patients with acquired aplastic anemia (AA) in long-term remission (Blood advances, 2(9):1000-1012, 2018). The OLFM4 gene is located at chromosome 13q14.3, which is a commonly deleted region in AA patients with del(13q) who show a good response to immunosuppressive therapy and a high prevalence of increased glycosylphosphatidylinositol-anchored protein(GPI-AP)-deficient cells (Haematologica, 97(12):1845-9, 2012). There may be common mechanisms underlying the preferential commitment between GPI-AP- and del(13q) hematopoietic stem and progenitor cells (HSPCs), like insensitivity to inhibitory cytokines such as TGF-β, in immune-mediated BM failure. To confirm this hypothesis, we studied the effect of OLFM4 knockout on the erythroid differentiation of erythroid leukemia cell lines induced by TGF-β. [Methods] We established OLFM4 knockout (KO) K562 and TF-1 cells using a CRISPR-Cas9 system. OLFM4-knockdown (KD) cells were also prepared using siRNA to validate the results of OLFM4-KO cells. The OLFM4 mRNA and protein levels were determined using quantitative polymerase chain reaction, flow cytometry (FCM), Western blotting, immunocytochemistry, and immunofluorescence methods. The erythroid differentiation was assessed by measuring the expression of glycophorin A (GPA) with FCM, GATA-1 protein expression using Western blotting, and iron staining of the cells. [Results] The OLFM4 KO cells showed slower proliferation than wild-type (WT) cells. Both OLFM4-KO cells and OLFM4-KD cells showed a higher GPA expression than WT cells (median fluorescence intensity [MFI] of K562: 2924 and 2143 vs. 1469 and TF-1: 950 and 870 vs. 694, respectively). OLFM4-KO cells showed erythroid morphology, an elevated expression of GATA-1, and positivity for iron granules, suggesting that OLFM4 KO promoted the erythroid differentiation of K562 and TF-1 cells in RPMI1640 containing 10% fetal bovine serum (Figure 1). When WT cells were cultured in a serum-free culture medium (Steampro34) with or without TGF-β (6 ng/ml) for 8 days, the GPA expression was induced in both TF-1 (MFI: 4358 vs. 883), and K562 cell lines (33440 vs. 25655). The OLFM4 protein levels in these cell lines were significantly decreased by the TGF-β treatment in a dose-dependent manner, suggesting that TGF-β directly downregulated the OLFM4 expression in WT cells; the relative expression of OLFM4 was 1, 0.64, and 0.12 while that of TF-1 was 1, 0.12, and 0.05 at 0, 2, and 6 ng/ml of TGF-β, respectively (Figure 2). [Conclusion] OLFM4 prevents K562 and TF-1 cells from differentiating into erythroid cells in response to TGF-β. The erythroid differentiation of these leukemic cells may be mediated by the downregulation of OLFM4 induced by TGF-β. Haploinsufficiency of OLFM4 due to either a loss of function mutation or del(13q) may be related to the mechanisms underlying the preferential commitment of the mutant HSPCs to erythroid cells in patients with immune-mediated BM failure where TGF-β is abundantly present. Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Takeda Pharmaceutical Company Limited: Honoraria; Novartis Pharma K.K: Honoraria; Kyowa Kirin: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; SynBio Pharmaceuticals: Consultancy.

Description: [Background] Acquired aplastic anemia (AA) is thought to be caused by cytotoxic T lymphocyte (CTL) attack specific to antigens presented by class I HLA alleles on hematopoietic stem cells (HSCs) although the target antigens on HSCs are still unknown. HLA alleles that are responsible for the auto-antigen presentation, which can be inferred from the presence of leukocytes that lack particular HLA class I alleles (HLA-lacking leukocytes [HLA-LLs]), may provide useful information on the identification of autoantigens in AA. We previously reported that deep sequencing of HLA class I genes of HLA-LLs revealed various loss-of-function mutations in alleles, including HLA-A*02:06 and B*40:02, suggesting that limited HLA-class I alleles are involved in the autoantigen presentation of AA (ASH 2018). Intriguingly, 60% of patients possessing HLA-LLs due to 6pLOH or several inactivating mutations in different HLA-A or HLA-B genes shared a nonsense mutation in exon 1 (Exon1mut) of which allelic frequency was very low (range 1.0-37.3%, median 4.5%). We hypothesized that the nonsense mutation, which efficiently lacks the corresponding HLA-allelic expression, might have been overlooked due to its low VAFs, and if we could establish a highly sensitive assay for detecting Exon1mut and determine the HLA alleles that undergo the mutation for a large number of AA patients, we might be able to define all HLA alleles that are involved in autoantigen presentation of AA. [Objectives/Methods] To test this hypothesis, we developed a highly sensitive droplet digital PCR (ddPCR) assay for precisely detecting Exon1mut in the peripheral blood (PB) of AA patient. In brief, the exon 1 regions of HLA-A and HLA-B alleles were amplified using two different sets of primer pairs that are complementary to the consensus sequences of the HLA-A and HLA-B alleles. The amplicons were subjected to a ddPCR assay using TaqMan probes complementary to wild-type (WT) and mutant-specific (MT) sequences, which were labeled with different fluorochromes (6-FAM for MT and HEX for WT). Peripheral blood leukocytes from 363 patients with AA (mean 64 [range, 11-93]) years of age, 134 with severe AA and 229 with non-severe AA; 173 males and 190 females; 84 6pLOH[+] and 279 6pLOH[-]) were subjected to the ddPCR assay. All blood samples were analyzed for 6pLOH by SNP array-based methods or a ddPCR assay as previously described. The HLA allele that underwent Exon1mut was determined by targeted deep sequencing using a unique molecular identifier (UMI), which enabled us to detect variant calling at a VAF as low as 0.1%, or deduced from the alleles contained in the lost haplotype, which are known to be the frequently lost alleles due to 6pLOH. [Results] Using 2 different ddPCR mixtures for HLA-A and HLA-B, the presence of Exon1mut was evaluable in all 363 AA patients. The sensitivity of the ddPCR assay for detecting Exon1mutwas 0.07%. 6pLOH was detected in 84 (23.1%) of the 363 AA patients. Ninety-nine (27.3%) of the 363 patients with (55 [65.5%] of 84) or without (44 [15.8%] of 279) 6pLOH were found to be positive for Exon1mut. The median allele frequency of Exon1mut in DNA from the Exon1mut(+) patients was 0.6% (range, 0.074% to 21.3%). In 17 patients whose blood samples were serially available, Exon1mutwas persistently detected in 13 and disappeared in 4 patients for 10-77 months (Figure 1). Among 43 different HLA-A and HLA-B alleles carried by the Exon1mut(+) patients, those with Exon1mutcould be identified by targeted deep sequencing in 54 patients. In 13 of the remaining 42 patients with Exon1mut, the Exon1mut-involved HLA alleleswere deduced from the alleles contained in the lost haplotype due to 6pLOH. These were 12 alleles and included A*02:06 (n=11), A*31:01 (n=3), B*13:01 (n=2), B*40:01 (n=3), B*40:02 (n=26), B*40:03 (n=1), B*54:01 (n=6), A*02:01 (n=2), A*02:07 (n=1), B*44:03 (n=1), B*55:02 (n=2) and B*56:01 (n=1) (Figure 2). The last five infrequent alleles were newly identified as "risk alleles" using the Exon1mutdetection. Two-hundred and twenty (92%) of 239 patients with PNH-type cells possessed at least 1 of the 12 alleles, while 103 (85%) of 121 patients without PNH-type cells did (P=0.045). [Conclusions] The Exon1mutdetection assay identified 12 HLA-alleles that are closely and exclusively involved in the autoantigen presentation of AA in Japanese patients. Similarity analyses of their antigen-presentation motifs may help to identify autoantigen peptides in AA. Disclosures Nakao: Takeda Pharmaceutical Company Limited: Honoraria; SynBio Pharmaceuticals: Consultancy; Ono Pharmaceutical: Honoraria; Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Celgene: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria.

Description: [Background] Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic　stem cell disorder derived from an acquired mutation of the phosphatidylinositol glycan class A (PIGA) gene in the hematopoietic stem cells which results in the expansion of glycosylphospatidylinositol-anchored protein (GPI-AP)-deficient (PNH-type) hematopoietic cells. PNH-type blood cells are also observed in patients with bone marrow failure (BMF). PNH is conventionally diagnosed when patients have 〉1% of GPI-AP-deficient erythrocytes and granulocytes determined by flow cytometry. Analyses with high resolution flow cytometry by several different groups have shown that patients with aplastic anemia (AA) or low-risk types of myelodysplastic syndromes (MDS) have small percentages of PNH-type erythrocytes, granulocytes, and/or other lineages of blood cells and that these patients respond better to immunosuppressive therapies compared with BMF patients lacking PNH-type cells. In order to determine the prevalence and clinical significance of PNH-type cells in BMF patients, we conducted a nationwide multi-center prospective observational investigation, the OPTIMA study. [Methods] From July 2011, Japanese patients with PNH, AA, MDS or BMF of uncertain origin have been prospectively enrolled into the study. Six laboratories in different cities in Japan were assigned as regional analyzing centers and measured the percentages of PNH-type cells in the study population as well as collecting clinical and laboratory data. The high-resolution flow cytometry assessments used a liquid fluorescein-labeled proaerolysin (FLAER) method and a cocktail method with anti-CD55 and anti-CD59 antibodies for the detection of PNH-type granulocytes and erythrocytes, respectively. Periodic blind cross validation tests using a standard blood sample containing 0.01% PNH-type cells and a normal control were conducted to minimize inter-laboratory variations. From analysis of 68 healthy individuals 〉0.003% of PNH-type granulocytes and 〉0.005% of PNH-type erythrocytes were considered to be abnormal (Sugimori et al, Blood, 2006). [Results] As of May 2014, flow cytometry data have been collected from 1685 patients and are included in this interim analysis. Of these patients, 65 (4%) were diagnosed with PNH, 523 (31%) with AA, 459 (27%) with MDS, and 638 (38%) with BMF of unknown etiology. Overall, 154 (9%) patients had ≥1% of both PNH-type erythrocytes and granulocytes: 63 (97%) patients with PNH; 57 (11%) with AA; 18 (4%) with MDS; and 16 (3%) with BMF of unknown etiology. In total, 545 (32%) patients had ≥0.005% PNH-type erythrocytes and ≥0.003% PNH-type granulocytes. These consisted of the followings; all 65 (100%) patients with PNH; 264 (51%) with AA; 76 (17%) with MDS; and 140 (22%) with BMF of unknown origin. Lactate dehydrogenase (LDH) levels ≥1.5 × upper limit of normal range were seen in 14/329 (4%) patients with 0.005-1% PNH-type erythrocytes, 23/62 (37%) patients with 1-10% PNH-type erythrocytes, and 69/71 (97%) patients with ≥10% PNH-type erythrocytes. Periodic blind validation tests revealed that inter-laboratory differences in absolute measurements of PNH-type cells were always within 0.02%. [Conclusion] A high-resolution flow cytometry-based method, based on the Kanazawa method, that enables the detection of very low percentages of PNH-type cells was successfully transferred to 6 laboratories across Japan. Our results demonstrated that the proportion of patients identified as having small percentages of PNH-type cells differed depending on diagnosis (PNH, AA, MDS, or unknown BMF) and that elevated LDH levels (〉1.5 x upper limits of normal range) were more frequently associated with higher percentages of PNH-type erythrocytes. Our findings suggest that the high resolution method is helpful as a diagnostic tool in BMF syndromes, including AA, MDS, and PNH, and may prove useful in understanding the pathophysiology of these disorders. Disclosures Noji: Alexion Pharma: Honoraria. Shichishima:Alexion Pharmaceuticals, Inc; and Medical Review Company: Honoraria, Research Funding. Obara:Alexion Pharma: Research Funding. Chiba:Alexion Pharma: Research Funding. Ando:Alexion Pharma: Research Funding. Hayashi:Alexion Pharma: Research Funding. Yonemura:Alexion Pharma: Research Funding. Kawaguchi:Alexion Pharma: Honoraria. Ninomiya:Alexion Pharma: Honoraria, Research Funding. Nishimura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Background: Acquired aplastic anemia (AA), the prototypical bone marrow failure syndrome, is inferred to result from immune-mediated destruction of hematopoietic progenitors, as most patients respond to immunosuppressive therapies. Clonal hematopoiesis in AA is evident in the presence of paroxysmal nocturnal hemoglobinuria (PNH) cells in as many as half of patients and by identification of uniparental disomies involving 6p (6pUPD) chromosome in 13% of cases. In addition, "clonal transformation", as defined by the development of myelodysplastic syndromes (MDS) or acute myelogenous leukemia (AML) is a serious long-term complication in 10-15% AA patients. Methods: We performed targeted deep sequencing and SNP array-based copy number (CN) analysis of peripheral blood- or granulocyte-derived DNA from 439 patients with AA (280 from US and 159 from Japanese cohorts) for a panel of 103 candidate genes, chosen because they are known to be frequently mutated in myeloid neoplasms. Germline DNA was available for 288 out of 439 patients and was used to confirm the somatic origin of mutations. Whole exome sequencing (WES) was performed in 52 cases. Where serial samples were available, the chronology of detected mutations was also investigated. Results: Targeted deep sequencing provided highly concordant results between the US and Japanese cohorts; approximately one third of AA patients had mutations in genes commonly affected in myeloid neoplasms, and about one third of patients in whom mutations were identified had multiple mutations. Multi-lineage involvement of mutations was confirmed in 6 cases using flow-sorted bone marrow samples. However, compared to myeloid neoplasms, mutations in AA were at much lower variant allele frequencies (VAFs) (

Description: [Background] In cases of immune-mediated bone marrow (BM) failure, such as acquired aplastic anemia (AA) and AA/PNH, aberrant hematopoietic stem progenitor cells (HSPCs) that acquire resistance to immune attack are thought to survive and support hematopoiesis in convalescent patients. Two representative progenies of such "escape" HSPC clones are HLA class I allele-lacking (HLA[-]) leukocytes and glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells. The mechanism underlying the immune selection of HLA(-) HSPCs is the failure of cytotoxic T lymphocyte (CTL) to recognize target antigens that are presented by particular HLA class I alleles of HSPCs. However, the mechanisms underlying the immune selection of GPI(-) HSPCs remain unclear. In addition, whether or not immune pressure that persists after immunosuppressive therapy (IST) contributes to the development and maintenance of clonal hematopoiesis by HLA(-) or GPI(-) HSPCs that are often seen in patients in long-term remission is also unknown. Phenotypical analyses of HSPCs that can be obtained from peripheral blood (PB) of AA patients who possess HLA(-) or GPI(-) leukocytes may provide a hint to elucidate these unsolved issues. [Objectives/Methods] We analyzed PB lineage-CD45dimCD34+CD38+ HSPCs of 15 AA patients who had 1%-99% HLA-A2(-) or HLA-A24(-) granulocytes (Gs) using flow cytometry (FCM). PB samples from 1 patient with severe AA were obtained before IST while the other 14 patients were in remission at the time of sampling; 10 were on cyclosporin (CsA) and eltrombopag (EPAG) (n=1), CsA and anabolic steroids (n=3), CyA (n=4), anabolic steroids (n=1) and EPAG alone (n=1); and 4 were free of therapy. We also determined the percentages of HLA(-) cells in different CD34+ subsets of BM, including HSCs (CD38-CD90+CD45RA-), MPPs, CMPs, GMPs, MEPs and CLPs for patients whose BM cells were available. Six AA/PNH patients whose GPI(-) Gs were 4-99% of the total Gs were subjected to the same PB HSPC analysis. For a separate group of seven AA patients who responded to CsA and had both HLA(-) and GPI(-) G populations, the percentages of each population were serially determined over one to 12 years. [Results] FCM identified 0.01%-0.4% (median 0.01%) CD45dimCD34+CD38+ HSPCs in the mononuclear cell population of the 15 AA patients, values that were significantly lower than those of seven healthy volunteers (0.19-0.78%, median 0.58%, P

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

Description: Abstract 4429 Background: Numerical karyotypic abnormalities of undetermined significance are occasionally detected in patients with bone marrow failure (BMF), including aplastic anemia (AA) and low-risk myelodysplastic syndrome (MDS). BMF patients with 13q deletions (13q-) are likely to respond to immunosuppressive therapy (IST) without a propensity to develop into leukemia (Ishiyama K et al, Br J Haematol; 117: 747. 2002), and they possess small populations of glycosylphosphatidyl-inositol anchored protein (GPI-AP)- deficient granulocytes and erythrocytes more frequently than patients with normal karyotypes%, 15 with AA and 8 with MDS) of 1705 patients. The proportion of 13q- clones in all metaphase cells ranged from 5% to 100% with a median of 27.5%. Fourteen of the 23 (61%) patients showed 13q- alone and 9 (39%) showed other karyotypic abnormalities in addition to 13q-. GPI-AP- cells that accounted for 0.009% to 6.7% (median 0.148%) of granulocytes were detected in all (100%) of the 14 patients with 13q- alone, while the prevalence of increased GPI-AP- cells in patients with 13q- plus other abnormalities and those with a normal karyotype was 44% and 43%, respectively. A FISH analysis of PB granulocytes in individual patients revealed apparently lower percentages of GPI-AP- granulocytes than those of 13q- granulocytes, indicating that the GPI-AP- granulocytes were derived from non 13q- HSCs. Ten patients with 13q- alone were treated with IST (ATG+cyclosporine in 4 and cyclosporine alone in 6) and all of them achieved either a partial remission or a complete remission. The SNP array analysis of 5 patients revealed common deletion of a 15 Mb (13.3 to 14.3) region containing the RB gene. Wild-type and PIG-A mutant K562 cells were incubated in the presence of TGF-beta, which can inhibit leukemic cell proliferation by down-regulating phosphorylated RB protein (pRB), to determine whether the RB gene loss in 13q- HSCs has any connection with a proliferative advantage of PIG-A mutant HSCs. PIG-A mutant K562 cells were less sensitive to TGF-beta inhibition of proliferation and down-regulation of pRB levels than wild-type K562 cells. Conclusion: The markedly high prevalence of increased GPI-AP- cells and good response to IST indicate that the presence of 13q- clones is highly associated with the immune pathophysiology of BMF. Decreased sensitivity to inhibitory signals by TGF-beta due to RB gene loss in 13q- HSCs and the loss of GPI-AP-type TGF-beta receptors in PIG-A mutant HSCs may therefore be a common mechanism that confers a survival advantage to these mutant HSCs over normal HSCs in patients with immune-mediated BMF. Disclosures: No relevant conflicts of interest to declare.

Description: Background Hematopoietic stem cells (HSCs) harboring PIGA mutations acquire a survival advantage under immune pressure compared to normal HSCs in patients with acquired aplastic anemia (AA). Cytotoxic T cells (CTLs) specific to glycosylphosphatidylinositol-anchored proteins (GPI-APs) are reportedly involved in this survival advantage, because PIGA mutant HSCs cannot present GPI-AP-derived peptides via class I HLAs. However, there is no convincing evidence that CTLs specific to GPI-AP-derived peptides are involved in the “escape” hematopoiesis by PIGA mutant HSCs. We recently demonstrated that 31.4-99.4% HLA-A allele-lacking leukocytes (HLA-LLs) were detectable in approximately 13% of AA patients as a result of escape hematopoiesis by HSCs with uniparental disomy in the short arm of chromosome 6, and that some patients possessed both GPI-AP-deficient (GPI-AP-) leukocytes and HLA-LLs (Katagiri, et al. Blood 2011). We hypothesized that if GPI-AP-derived peptides serve as a target for CTLs that elicit the development of AA, HLA-LLs may always be detectable only in the GPI-AP+ leukocyte population, because PIGA mutant HSCs do not require the lack of HLA class Is for the escape from the attack by GPI-AP peptide-specific CTLs. Objectives and Methods To examine this hypothesis, the GPI-AP expression was analyzed in various leukocyte lineages in 32 (nine at diagnosis and 23 previously treated) AA patients possessing HLA-LLs by a flow cytometry (FCM) analysis with liquid fluorescent aerolysin. Results A total of 0.01%-50% GPI-AP- granulocytes (GPI-AP- Gs) were detected in 22 (69%) of the 32 HLA-LLs (+) patients. Of the 22 patients possessing both HLA-LLs and increased GPI-AP- Gs, HLA-LLs were detectable in GPI-AP+ cells alone in 19 patients (86%). However, in the remaining three patients, HLA-LLs were shown in both GPI-AP+ and GPI-AP- populations. To determine which mutation occurs first in HSCs with a PIGA mutation and 6pUPD, the lineage diversity of GPI-AP- HLA-LLs was determined in the three patients. In two of the three patients, the lineage diversity of GPI-AP- cells (G/monocytes (M)/T cells (T)/B cells (B)/NK cells (NK)) and G/M/T) was greater than that of the HLA-A-lacking cells (G/M/B/NK and G/M) suggesting that PIGA mutations occurred earlier in the maturation of HSCs than did 6pUPD. The lineage diversity was the same in the GPI-AP- cells and HLA-LLs in one patient Conclusions The presence of HLA-LLs in the GPI-AP- leukocyte population and lower lineage diversity in HLA-LLs than GPI-AP- leukocytes suggest that CTLs specific to GPI-APs are not involved in the escape of PIGA mutant HSCs in AA, and that other mechanisms, such as a lower sensitivity to myelosuppressive cytokines than wild-type HSCs, may contribute to the survival advantage of PIGA mutant HSCs. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Acquired aplastic anemia (AA) is occasionally preceded or complicated by the other autoimmune diseases such as rheumatoid arthritis and ulcerative colitis (UC), but the pathogenetic links between these diseases remain unknown. A number of case reports attributed the development of AA in UC patients to mesalazine that had been administered before the onset of AA. However, some UC patients who were referred to our clinical because of "mesalazine-induced AA" had increased percentages of glycosylphosphatidylinositol-anchored protein-deficient (PNH-type) cells that serve as a marker for immune-mediated bone marrow (BM) failure. This suggested an immune pathophysiology of UC-associated AA similar to that of idiopathic AA. Methods: To address this issue, we analyzed clinical and laboratory data including HLA-DRB1 alleles and PNH-type cells of 14 AA patients (six males and eight females) associated with UC who were referred to our clinic from 2001 through 2015. Results: The median age of the patients was 55 (range, 22-76). Diagnoses of the 14 patients were severe AA in eight, non-severe AA in five, and hypomegakaryocytic thrombocytopenia in one. UC preceded AA in 11 patients and nine of them received mesalazine at the time of AA diagnosis, while three AA patients developed UC 12, 72, and 36 months after diagnosis of AA. Increased percentages of PNH-type granulocytes (0.35% to 3.2%) were detected in 11 (79%) patients. Of 9 PNH(+) patients with AA preceded by UC, seven had received mesalazine while two had not been treated with mesalazine. The prevalence of increased PNH-type cells in AA patients who developed UC was 67% (2/3). Six patients were positive for DRB1*15:02, a well-known allele associated with susceptibility both to UC and AA in Japanese patients (Table). Another major DR15 allele, DRB1*15:01, was possessed by four patients. Seven patients received immunosuppressive therapy with either cyclosporine (CsA) alone (two patient) or CsA+antithymocyte globulin (five patients) and six of them achieved a remission of AA. Conclusions: The high prevalence of increased PNH-type cells indicates that AA complicated by UC is a legitimate immune-mediated BM failure and that mesalazine may be irrelevant to the development of AA. The high frequency of DRB1*15:02 suggests the two diseases share an immune pathophysiology. Genome wide association studies on UC patients may help to identify genes associated with a susceptibility to AA. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.