HLA associations, somatic loss of HLA expression, and clinical outcomes in immune aplastic anemia

Immune aplastic anemia (AA) features somatic loss of HLA class I allele expression on bone marrow cells, consistent with a mechanism of escape from T cell-mediated destruction of hematopoietic stem and progenitor cells. The clinical significance of HLA abnormalities has not been well characterized. We examined somatic loss of HLA class I alleles, and correlated HLA loss and mutation-associated HLA genotypes with clinical presentation and outcomes after immunosuppressive therapy in 544 AA patients. HLA class I allele loss was detected in 92 (22%) of the 412 patients tested, in whom there were 393 somatic HLA gene mutations and 40 instances of loss of heterozygosity. Most frequently affected was HLA-B*14:02, followed by HLA-A*02:01, HLA-B*40:02, HLA-B*08:01, and HLA-B*07:02. HLA-B*14:02, HLA-B*40:02, and HLA-B*07:02 were also overrepresented in AA. High-risk clonal evolution was correlated with HLA loss, HLA-B*14:02 genotype, and older age, which yielded a valid prediction model. In two patients, we traced monosomy 7 clonal evolution from preexisting clones harboring somatic mutations in HLA-A*02:01 and HLA-B*40:02. Loss of HLA-B*40:02 correlated with higher blood counts. HLA-B*07:02 and HLA-B*40:01 genotypes and their loss correlated with late onset of AA. Our results suggest the presence of specific immune mechanisms of molecular pathogenesis with clinical implications. HLA genotyping and screening for HLA loss may be of value in the management of immune AA. This study was registered at clinicaltrials.gov as NCT00001964, NCT00061360, NCT00195624, NCT00260689, NCT00944749, NCT01193283, and NCT01623167.

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Somatic HLA mutations expose the role of class I–mediated autoimmunity in aplastic anemia and its clonal complications

Blood Advances ◽

10.1182/bloodadvances.2017010918 ◽

2017 ◽

Vol 1 (22) ◽

pp. 1900-1910 ◽

Cited By ~ 27

Author(s):

Daria V. Babushok ◽

Jamie L. Duke ◽

Hongbo M. Xie ◽

Natasha Stanley ◽

Jamie Atienza ◽

...

Keyword(s):

Aplastic Anemia ◽

Clonal Evolution ◽

Gene Mutations ◽

Therapy Response ◽

Hla Class I ◽

Class I ◽

Key Points ◽

Inactivating Mutations ◽

I Gene

Key Points Somatic HLA class I gene mutations are frequent in aAA and define HLA class I restricted autoimmunity in aAA. HLA alleles targeted by inactivating mutations are overrepresented in aAA and correlate with poor therapy response and clonal evolution.

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Identification of an HLA Class I Allele Closely Involved in the Pathogenesis of Acquired Aplastic Anemia

Blood ◽

10.1182/blood.v128.22.729.729 ◽

2016 ◽

Vol 128 (22) ◽

pp. 729-729

Author(s):

Yoshitaka Zaimoku ◽

Hiroyuki Takamatsu ◽

Kazuyoshi Hosomichi ◽

Tatsuhiko Ozawa ◽

Noriharu Nakagawa ◽

...

Keyword(s):

T Cell ◽

Aplastic Anemia ◽

Missense Mutation ◽

Hla Class I ◽

The Other ◽

Class I ◽

Variant Allele ◽

Cytotoxic T Cell ◽

Hematopoietic Stem

Abstract [Background] The frequent loss of heterozygosity of the HLA haplotype in the short arm of chromosome 6 (6pLOH) in leukocytes is thought to offer compelling evidence of cytotoxic T cell (CTL) involvement in the development of acquired aplastic anemia (AA) because it represents the escape of hematopoietic stem/progenitor cells (HSPCs) with 6pLOH from the attack of CTLs that are specific to autoantigens presented by the lacked HLA class I allele. Although our previous study suggested that HLA-B*40:02 is the major allele involved in this phenomenon, the exact role of B*40:02 remained unclear because 6pLOH involving this allele is always associated with a lack of HLA-A and C alleles in the haplotype, and the presence of B*40:02-missing leukocytes were unable to be shown due to the lack of monoclonal antibodies (mAbs) specific to B61, the HLA-B antigen that corresponds to B*40:02. We recently succeeded in generating a mAb specific for HLA-B61 that enabled us to explore the role of B*40:02 in the development of AA. [Methods] Using the new mAb, we examined peripheral blood samples of 28 AA (12 with 6pLOH and 16 without 6pLOH) patients carrying this allele for the presence of B61(-) leukocytes using flow cytometry. HLA genes were enriched by sequence capture, a hybridization-based gene enrichment method, from genomic DNA of sorted B61(-) granulocytes, and were subjected to deep sequencing using an NGS (MiSeq). B61(+) granulocytes or T cells were used as controls. Potential mutations responsible for the B61-missing were identified when 10 or more variant reads were found only in B61(-) granulocytes. Thereafter, HLA-B alleles carrying those mutations were determined taking advantage of the nearest allele-specific SNPs. [Results] Among the 12 6pLOH(+) patients, 10 (83%) possessed 0.5%-60% B61-missing granulocytes that were not lacking HLA-A, in addition to 12% to 99% 6pLOH(+) granulocytes that lacked both B61 and an HLA-A allele on the same haplotype (Figure 1). B61(-) granulocytes that accounted for 0.5%-99% of the total granulocytes were detected in 9 (56%) of the 16 6pLOH(-) patients. The prevalence of missing B61 in the 28 AA patients was 21/28 (75%), much more frequent than those of the 3 other major alleles (A*02:01, 32%; A*02:06, 30%; A*24:02, 6%). B61(-) granulocytes were available for mutation analyses of HLA-B alleles in 15 of the 19 patients who possessed B61(-) granulocytes. The mean coverage of HLA-B gene was 426x. In total, 43 somatic mutations of HLA-B were identified in B61(-) granulocytes, all of which were present in B*40:02 but not in any of the other HLA-B alleles. Median variant allele frequency was 4.8% (range, 1.0% - 43%) and the number of mutations in each patient was 1 to 6 (Figure 2). Thirty-nine mutations were exonic while 4 were intronic. Exonic mutations included frameshift insertions (n=12), frameshift deletions (n=16), non-frameshift deletions (n=2), nonsense mutations (n=7), a missense mutation (n=1) and a start codon mutation (n=1). All four intronic mutations were considered to be a splice site mutation; two mutations deactivated 5' and 3' splice sites, whereas the other two were single base substitutions within intron 3, making alternative 5' splicing site with strong consensus sequence: GGC [A>G] TGAGT and TTC [C>G] TGAGT. Surprisingly, missense mutations in the alpha-2 and alpha-3 chain-coding region of HLA-B*40:02 were detected exclusively in the B61(+) granulocytes of two patients possessing B61(-) granulocytes, suggesting the inability of the mutant HSPCs to interact with CTLs. Variant allele frequencies of the two missense mutation were 40% and 45%, respectively. As a result of the mutation, virtually all granulocytes of the two patients were affected by B*40:02 mutations that allowed the HSPCs to escape the T cell attack. [Conclusions] The markedly high prevalence of leukocytes lacking HLA-B*40:02 as a result of either or both 6pLOH or structural gene mutations clearly indicates that antigen presentation by HSPCs to CTLs via the HLA-B allele plays a critical role in the pathogenesis of AA. Disclosures Takamatsu: Celgene: Honoraria; Janssen Pharmaceuticals: Honoraria. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

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

Blood ◽

10.1182/blood-2018-99-115623 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1308-1308 ◽

Cited By ~ 1

Author(s):

Takeshi Yoroidaka ◽

Kohei Hosokawa ◽

Tatsuya Imi ◽

Takamasa Katagiri ◽

Fumihiro Azuma ◽

...

Keyword(s):

T Cells ◽

Aplastic Anemia ◽

Progenitor Cells ◽

Hla Class I ◽

Survival Advantage ◽

Class I ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Lineage Diversity

Abstract [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.

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Telomere Shortening Promotes Chromosomal Instability and Predicts Malignant Clonal Evolution in Aplastic Anemia.

Blood ◽

10.1182/blood.v114.22.3208.3208 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3208-3208

Author(s):

Rodrigo T. Calado ◽

James N Cooper ◽

Phillip Scheinberg ◽

Colin Wu ◽

Marco A Zago ◽

...

Keyword(s):

Bone Marrow ◽

Aplastic Anemia ◽

Telomere Length ◽

Malignant Transformation ◽

Chromosomal Instability ◽

Bone Marrow Cells ◽

Clonal Evolution ◽

Monosomy 7 ◽

Marrow Cells

Abstract Abstract 3208 Poster Board III-145 In murine models, telomere erosion promotes chromosomal instability via breakage-fusion-bridge cycles, contributing to the early stages of tumorigenesis. However, direct evidence that short telomeres predispose to cancer development in humans is lacking. In acquired aplastic anemia, evolution to malignant clonal disorders is a major complication after immunosuppressive therapy, affecting up to 15 percent of patients at 10 years. We investigated whether telomere length measured at diagnosis predicted clonal evolution in these patients. Telomere length was measured from DNA extracted from peripheral blood leukocytes collected at disease presentation in 183 consecutive patients enrolled in successive clinical trials for immunosuppressive regimen as first line therapy for severe aplastic anemia at the Clinical Research Center, National Institutes of Health (ClinicalTrials.gov identifier numbers, NCT00001964, NCT00260689, and NCT00061360) and 164 healthy volunteers. Leukocyte telomere length of aplastic anemia patients at diagnosis was in the normal range and was not shorter than in healthy controls (ANOVA-F test). Telomere length was corrected for age and patients were separated into two groups: patients with short telomeres (in the lowest quartile) and long telomeres (other quartiles). Telomere length was a critical and independent predictive biomarker for evolution to myelodysplastic syndrome, especially monosomy 7, and acute myeloid leukemia (AML) in patients with acquired aplastic anemia (Multivariate Cox Proportional Hazard Model, P=0.006). Patients with short telomeres had six-fold higher probability to develop clonal malignant disease than did patients with longer telomeres. Bone marrow cells of aplastic patients were cultured in vitro for short term in the presence of cytokines and high-dose granulocyte-colony stimulating factor (G-CSF) and cells of patients with short telomeres (n=5) showed increased telomere-free chromosomal ends in comparison to cells of patients with long telomeres (n=6), by fluorescence in situ hybridization (FISH; P<0.0001). Spectral karyotyping (SKY) revealed that cultured bone marrow cells of patients with short telomeres exhibited aneuploidy and translocations, including Robertsonian translocations, which were not found in cells of patients with long telomeres. Bone marrow cells at diagnosis were further evaluated for the presence of monosomy 7 cells using interphase FISH in 73 patients. Telomere length inversely correlated with the frequency of monosomy 7 cells: the shortest the telomeres, the highest the percentage of aneuploid cells at diagnosis (Pearson r=-0.5110; P=0.0009). We further employed bone marrow cells of clinically healthy individuals carrying loss-of-function telomerase mutations and with extremely short telomeres (n=5) as a model for telomere dysfunction in hematopoietic cells in the absence of human disease. In vitro culture of these cells yielded aberrant karyotypes by SKY, including translocations and aneuploidy, and end-to-end chromosomal fusions by FISH. These results indicate that telomere length at diagnosis predicts evolution to myelodysplasia and leukemia in patients with acquired aplastic anemia treated with immunosuppression. Our findings support the hypothesis that short and dysfunctional telomeres restrain stem cell proliferation and predispose for malignant transformation by selecting stem cells that are prone to chromosomal instability. This is the first prospective study to demonstrate that short telomeres in human hematopoietic cells promote chromosomal instability in vitro and predispose to malignant transformation in humans. Disclosures Cooper: NIH-Pfizer: Research Funding.

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A brief, but comprehensive, guide to clonal evolution in aplastic anemia

Hematology ◽

10.1182/asheducation-2018.1.457 ◽

2018 ◽

Vol 2018 (1) ◽

pp. 457-466 ◽

Cited By ~ 8

Author(s):

Daria V. Babushok

Keyword(s):

Aplastic Anemia ◽

Somatic Mutations ◽

Clonal Evolution ◽

Clinical Care ◽

Hla Class I ◽

The Elderly ◽

Hematologic Malignancies ◽

Class I ◽

Clonal Hematopoiesis ◽

Immune Mediated

Abstract Acquired aplastic anemia (AA) is an immune-mediated bone marrow aplasia that is strongly associated with clonal hematopoiesis upon marrow recovery. More than 70% of AA patients develop somatic mutations in their hematopoietic cells. In contrast to other conditions linked to clonal hematopoiesis, such as myelodysplastic syndrome (MDS) or clonal hematopoiesis of indeterminate potential in the elderly, the top alterations in AA are closely related to its immune pathogenesis. Nearly 40% of AA patients carry somatic mutations in the PIGA gene manifested as clonal populations of cells with the paroxysmal nocturnal hemoglobinuria phenotype, and 17% of AA patients have loss of HLA class I alleles. It is estimated that between 20% and 35% of AA patients have somatic mutations associated with hematologic malignancies, most characteristically in the ASXL1, BCOR, and BCORL1 genes. Risk factors for evolution to MDS in AA include the duration of disease, acquisition of high-risk somatic mutations, and age at AA onset. Emerging data suggest that several HLA class I alleles not only predispose to the development of AA but may also predispose to clonal evolution in AA patients. Long-term prospective studies are needed to determine the true prognostic implications of clonal hematopoiesis in AA. This article provides a brief, but comprehensive, review of our current understanding of clonal evolution in AA and concludes with 3 cases that illustrate a practical approach for integrating results of next-generation molecular studies into the clinical care of AA patients in 2018.

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Spectrum and Clinical Significance of HLA Class I Alleles and Their Somatic Mutations in Immune Aplastic Anemia

Blood ◽

10.1182/blood-2019-127359 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3738-3738

Author(s):

Yoshitaka Zaimoku ◽

Sharon D. Adams ◽

Bhavisha A Patel ◽

Audrey Ai Chin Lee ◽

Sachiko Kajigaya ◽

...

Keyword(s):

Bone Marrow ◽

Cell Surface ◽

Aplastic Anemia ◽

Clinical Significance ◽

Somatic Mutations ◽

Clonal Evolution ◽

Hla Class I ◽

Surface Expression ◽

Class I ◽

Cell Surface Expression

Clonal hematopoiesis associated with loss of HLA class I alleles due to somatic mutations and/or 6p loss of heterozygosity (LOH) is frequent in immune aplastic anemia (AA). HLA-B*40:02 is more likely to be involved in HLA loss in Japanese AA patients, suggesting a role for this allele in immune pathophysiology (Zaimoku Y et al, Blood 2017). Mutations in non-B*40:02 HLA class I alleles have been reported in a limited number of patients from the United States (Babushok D et al, Blood Adv 2017) and Japan (Mizumaki H et al, 60th ASH meeting), but their prevalence and clinical significance are not well characterized. We investigated somatic mutations of HLA class I alleles, HLA allele frequencies, and their correlations with outcomes of therapy in a total of 532 AA patients, aged 2 years or older, treated on various Hematology Branch protocols (clinicaltrials.gov NCTs 00001964, 00061360, 00195624, 00260689, 00944749, 01193283, and 01623167). HLA allele-lacking (HLA-) monocytes from cryopreserved peripheral blood mononuclear cells were screened by flow cytometry after staining with allele-specific monoclonal antibodies for HLA-A and/or HLA-B (HLA-flow) in 172 AA patients. HLA- monocytes accounting for 0.5% to 100% (median 9.5%) of total monocytes were detected in 49 (28%) of the 172 patients and in 59 (15%) of 382 alleles analyzed (Figure 1). Loss of cell surface expression was frequent for HLA-B14 (46%), B27 (33%), B49 (33%), A68 (26%), A2 (23%), B40 (21%), and B8 (21%). One percent to 60% (median, 8.9%) of glycosylphosphatidylinositol-linked protein-negative (GPI-) monocytes were also present in 43% (21 of 49) of the patients with HLA- monocytes, but GPI- clones had normal HLA cell surface expression. Deep sequencing of HLA-A, HLA-B and HLA-C on sorted HLA- and HLA+GPI+ monocytes was performed in 42 of the 48 patients from whom adequate cells were available. Somatic mutations and/or LOH corresponding to the lacking alleles were detected in all 42 cases (Figure 1): 9 had both somatic mutations and LOH, 20 had somatic mutations only, and 13 had LOH only. Among the 13 patients who showed only LOH in the absent allele, 6 had somatic mutations in other alleles of HLA+ monocytes that was not analyzable of HLA expression, and 2 had a breakpoint of LOH between HLA-A and HLA-C, leading to loss of a single HLA-A allele. Somatic mutations or LOH involving only one allele were present in 37 patients among 6 HLA-A alleles (in 02:01 [7 patients], 02:05 [1], 02:06 [3], 02:11 [1], 68:01 [2], 68:02 [2]) and 10 HLA-B alleles (07:02 [1], 08:01 [4], 14:01 [1], 14:02 [7], 27:05 [1], 35:02 [1], 35:05 [1], 40:01 [1], 40:02 [3], 45:01 [1]), but were not found in HLA-C alleles. HLA allele frequencies in AA patients, including 271 white Americans, 120 African-Americans, and 99 Hispanics and Latinos, were compared with ethnicity-matched individuals in bone marrow donor datasets of the National Marrow Donor Program, and underwent random-effects meta-analyses. HLA-B*07, B*14, and B*40 were overrepresented in AA, while A*02, A*68, and B*08 frequencies were similar to those of healthy donors (Figure 2). In 164 severe AA patients who were initially treated with horse antithymocyte globulin (hATG), cyclosporine, and eltrombopag between 2012 and 2018, 36 and 79 were positive and negative for HLA- monocytes, respectively, and 49 were not tested by HLA-flow. There was no significant difference in overall and complete response rates at six months among the three groups (Figure 3). Clonal evolution, defined as acquisition of abnormal bone marrow cytogenetics or morphology, especially high-risk evolution to chromosome 7 abnormalities, complex cytogenetics, or morphological MDS/AML, tended to be more frequent in patients with HLA- monocytes, compared to the other two groups, but the difference did not reach statistical significance. Clinical outcomes were also assessed according to the presence of specific HLA alleles in 400 severe AA patients who were treated with hATG-based initial immunosuppressive therapy from 2000 to 2018: there was no significant differences in probabilities of response and clonal evolution according to the alleles associated with somatic mutations. Our study revealed that somatic mutations in HLA genes in AA are broadly distributed, but some alleles are preferentially affected. Inconsistent with previous studies, we found that outcomes of therapy did not significantly correlate with HLA gene mutations or with distinct HLA alleles. Disclosures No relevant conflicts of interest to declare.

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HLA class I allele-lacking leukocytes predict rare clonal evolution to MDS/AML in patients with acquired aplastic anemia

Blood ◽

10.1182/blood.2020010586 ◽

2021 ◽

Author(s):

Kohei Hosokawa ◽

Hiroki Mizumaki ◽

Takeshi Yoroidaka ◽

Hiroyuki Maruyama ◽

Tatsuya Imi ◽

...

Keyword(s):

Aplastic Anemia ◽

Clonal Evolution ◽

Hla Class I ◽

Class I

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Towards Identifying the Target of Autoimmunity in Aplastic Anemia

Blood ◽

10.1182/blood-2020-136772 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 2-2

Author(s):

Benjamin F Frost ◽

Jamie Duke ◽

Hongbo Xie ◽

Deborah Ferriola ◽

Joseph H Oved ◽

...

Keyword(s):

Aplastic Anemia ◽

Research Funding ◽

Somatic Mutations ◽

Risk Allele ◽

Hla Class I ◽

Class I ◽

Risk Alleles ◽

Total Cohort ◽

B 41 ◽

Hla Loss

Acquired aplastic anemia (AA) is an autoimmune bone marrow aplasia caused by T cell-mediated destruction of hematopoietic stem and progenitor cells. The antigenic target in AA remains unknown. Recently, we and others identified frequent somatic loss of function of several human leukocyte antigen (HLA) class I alleles in hematopoietic cells that survive the AA immune attack, suggesting these alleles ("risk alleles") present autoantigen in the affected patients. We hypothesize that risk alleles share structural features and peptide-binding characteristics that may inform our understanding of the immune mechanism of AA. To identify additional AA HLA class I risk alleles, we have partnered with two multi-institutional consortia, the North American Pediatric Aplastic Anemia Consortium (NAPAAC) and the Center for International Blood and Marrow Transplant Research (CIBMTR), to evaluate 507 AA patients for somatic HLA loss. Using a combination of targeted massively parallel sequencing of HLA class I genes and single nucleotide polymorphism array genotyping, we identified HLA loss in 19% of the NAPAAC cohort (30 of 156 unselected AA patients) and 13% of the CIBMTR cohort (46 of 351 patients selected to maximize discovery of less common risk alleles). Copy number-neutral loss of heterozygosity of chromosome arm 6p (6p CN-LOH) was the most common cause of HLA loss, occurring in 50 patients (10% of total cohort, 12% NAAPAC and 9% CIBMTR). Somatic mutations were present in 45 patients (9% of total cohort, 12% of NAPAAC and 7% CIBMTR). Patients with HLA loss had a median of 1 mutant clone per patient (range 1-7). Of the somatic mutations, 80% were predicted to disrupt expression of the affected allele (through loss of start, nonsense, or frameshift mutations). The other 20% were missense mutations affecting residues in the peptide binding groove (6 mutations) and the α3 domain (9 mutations) of the HLA class I protein. A total of 19 distinct AA HLA risk alleles were identified, of which 13 were newly identified in this study. Several risk alleles (HLA-B*14:02, HLA-B*40:02, HLA-A*02:01) were enriched in AA patients compared to ethnicity matched controls. Among the 507 patients in the study, at least one risk allele was present in 436/493 (88%) of AA patients and in 34/39 (87%) of the 6p CN-LOH events. Mutations clustered in several groups of alleles (supertypes) known to bind overlapping peptide repertoires, with alleles in B27 and B44 supertypes being most commonly affected. There were no mutations in A01, A01A03, A01A24, A24, B58, B62, or in any of the HLA-C alleles analyzed. Using several metrics to estimate the strength of the autoimmune selection on a given allele, including the frequency of somatic loss and similarity to other HLA risk alleles, we developed an AA HLA risk allele pathogenicity index ranging from very high (B*14:02 and B*40:02) and high pathogenicity (A*33:03, B*08:01, B*13:02, B*14:01, B*27:03, B*27:05, B*38:02, B*41:02 and B*49:01) to those unlikely to be pathogenic. Interestingly, even within high risk supertypes such as B44, there was a wide variation in predicted pathogenicity, ranging from high (for B*40:02 and B*41:02) to low pathogenicity (for B*44:02 and B*44:03), suggesting differences in autopeptide binding. Our study provides a comprehensive analysis of AA HLA risk alleles in a large diverse cohort of AA patients. Our results suggest that HLA risk alleles have shared autoantigen binding specificities that define their pathogenicity in AA, which can be used to identify candidate AA autoantigens. HLA risk allele pathogenicity may have future clinical utility as an adjunctive diagnostic test, as well as for prognostic assessment and haploidentical donor selection. Disclosures Lee: AstraZeneca: Research Funding; Kadmon: Research Funding; Takeda: Research Funding; Novartis: Research Funding; Amgen: Research Funding; Syndax: Research Funding; Pfizer: Consultancy, Research Funding; Incyte: Consultancy, Research Funding. Monos:Omixon: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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Somatic Loss of HLA Class I Alleles Is a Common Genetic Alteration in Acquired Aplastic Anemia and Reveals Aplastic Anemia Risk Alleles

Blood ◽

10.1182/blood.v128.22.730.730 ◽

2016 ◽

Vol 128 (22) ◽

pp. 730-730 ◽

Cited By ~ 1

Author(s):

Daria V. Babushok ◽

Jamie Duke ◽

Hongbo M. Xie ◽

Deborah Ferriola ◽

Natasha Stanley ◽

...

Keyword(s):

Aplastic Anemia ◽

Somatic Mutations ◽

Hla Class I ◽

Median Number ◽

Class I ◽

Loss Of Function ◽

Immune Selection ◽

Hla Alleles ◽

Hla Loss ◽

Pediatric Onset

Abstract Acquired aplastic anemia (AA) is a rare bone marrow failure syndrome. AA is believed to be immune-mediated, supported by in vitro studies and the success of empiric immunosuppressive therapy. Recently, a chromosomal alteration-copy number-neutral loss of heterozygosity of chromosome arm 6p, the site of the Major Histocompatibility Complex and the Human Leukocyte Antigen (HLA) genes-has been identified as a recurrent somatic change in AA. Clonal hematopoiesis marked by 6p CN-LOH is hypothesized to emerge by immune escape of hematopoietic cells lacking certain HLA alleles. However, because of the large size of the genomic region involved by 6p CN-LOH and the strong linkage disequilibrium among other genes in the region, specific alleles targeted by the immune selection in AA are unknown. In a previous study, we reported two patients with somatic loss-of-function mutations in HLA class I genes, leading us to hypothesize that loss of HLA alleles in AA may be common and likely defines a subset of patients with unique characteristics and disease course. To characterize the prevalence of HLA allele loss in AA, we performed targeted next generation sequencing of HLA-A, B, and C genes, in conjunction with single nucleotide polymorphism array genotyping of bone marrow (BM) or peripheral blood DNA in 74 patients with AA. 52 patients had pediatric-onset AA, and 22 had adult-onset AA. Somatic status of mutations was confirmed by sequencing paired constitutional DNA. Eleven patients (15%) were found to have somatic loss of HLA alleles: 5 patients had 6p CN-LOH, 3 patients had loss-of-function mutations (frameshift, nonsense, or start codon loss) of the HLA class I alleles, and 3 patients were found to have both 6p CN-LOH as well as loss-of-function HLA mutations. HLA loss was more frequent in pediatric-onset AA (9 of 52 patients, 17%) as compared to adults (2 of 22 patients, 9%), although the difference did not reach statistical significance. No HLA mutations were identified in 19 patients with classical Paroxysmal Nocturnal Hemoglobinuria, nor in 20 healthy relatives (p=0.06). Among the 11 patients with somatic HLA loss, 8 patients had evidence of oligoclonal hematopoiesis with several independent clones carrying different alterations of the same HLA allele. Among the 6 patients with loss-of-of function HLA mutations, the median number of HLA mutations per patient was 1.5 (range 1-3). Of the 8 patients with acquired 6p CN-LOH, the median number of distinct 6p CN-LOH events per patient was 2 (range 1-4). In the 3 patients harboring both 6p CN-LOH as well as the loss-of-function HLA mutations, both mechanisms led to the recurrent loss of the same allele. Strikingly, only a few distinct HLA class I alleles were targeted by mutations. The most frequently affected were HLA-B*40:02:01 (5 independent mutations in 2 patients) and HLA-B*14:02:01 (3 mutations in 2 patients, and as well as loss through polyclonal 6p CN-LOH in 2 patients). Additionally, one patient each had loss of HLA-A*68:01:01 and HLA-A*33:03:01 through mutational inactivation as well as through 6p CN-LOH. To investigate whether HLA mutations are sufficient to cause clonal expansion or whether other somatic mutations are required, we performed comparative whole exome sequencing (WES) of paired BM and skin DNA in five patients carrying inactivating HLA mutations. Four of the five patients had no other mutations affecting protein sequence or untranslated regulatory regions. One patient had additional somatic mutations, which were subclonal to and co-segregated with the three independent inactivating mutations in the HLA-B*40:02:01 allele. Serial follow-up confirmed that HLA mutations persisted overtime, with a relative expansion of one of the HLA-B*40:02:01 mutant clones bearing a protein-altering mutation in the BCL9 gene. Our results show that loss of HLA class I alleles is common in AA, second only to PIGA gene mutations. The affected alleles are non-random, with immune selection most commonly targeting HLA-B* 40:02:01 and HLA-B*14:02:01 alleles, providing the first evidence of specificity of immune attack in AA. The resultant hematopoiesis caused by selection of cells with HLA allele loss is typically oligoclonal and commonly occurs in the absence of other somatic mutations. Acquisition of additional mutations can lead to clonal dominance overtime. Further studies are underway to better understand the role of HLA loss in patient outcomes and AA pathogenesis. Disclosures No relevant conflicts of interest to declare.

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Paroxysmal Nocturnal Hemoglobinuria Clones Are Infrequent in Hepatitis-Associated Aplastic Anemia at the Time of Diagnosis

Blood ◽

10.1182/blood-2019-125620 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5016-5016

Author(s):

Wenrui Yang ◽

Xin Zhao ◽

Guangxin Peng ◽

Li Zhang ◽

Liping Jing ◽

...

Keyword(s):

Aplastic Anemia ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Clonal Evolution ◽

Extrinsic Factors ◽

Clone Size ◽

Hematopoietic Stem ◽

Over Time ◽

Pnh Clone

Aplastic anemia (AA) is an immune-mediated bone marrow failure, resulting in reduced number of hematopoietic stem and progenitor cells and pancytopenia. The presence of paroxysmal nocturnal hemoglobinuria (PNH) clone in AA usually suggests an immunopathogenesis in patients. However, when and how PNH clone emerge in AA is still unclear. Hepatitis associated aplastic anemia (HAAA) is a special variant of AA with a clear disease course and relatively explicit immune pathogenesis, thus serves as a good model to explore the emergence and expansion of PNH clone. To evaluate the frequency and clonal evolution of PNH clones in AA, we retrospectively analyzed the clinical data of 90 HAAA patients that were consecutively diagnosed between August 2006 and March 2018 in Blood Diseases Hospital, and we included 403 idiopathic AA (IAA) patients as control. PNH clones were detected in 8 HAAA patients (8.9%,8/90) at the time of diagnosis, compared to 18.1% (73/403) in IAA. Eight HAAA patients had PNH clone in granulocytes with a median clone size of 3.90% (1.09-12.33%), and 3 patients had PNH clone in erythrocytes (median 4.29%, range 2.99-10.8%). Only one HAAA patients (1/8, 12.5%) had a PNH clone larger than 10%, while 24 out of 73 IAA patients (32.9%) had larger PNH clones. Taken together, we observed a less frequent PNH clone with smaller clone size in HAAA patients, compared to that in IAAs. We next attempted to find out factors that associated with PNH clones. We first split patients with HAAA into two groups based on the length of disease history (≥3 mo and < 3mo). There were more patients carried PNH clone in HAAA with longer history (21.4%, 3/14) than patients with shorter history (6.6%, 5/76), in line with higher incidence of PNH clone in IAA patients who had longer disease history. Then we compared the PNH clone incidence between HAAA patients with higher absolute neutrophil counts (ANC, ≥0.2*109/L) and lower ANC (< 0.2*109/L). Interestingly, very few VSAA patients developed PNH clone (5%, 3/60), while 16.7% (5/30) of non-VSAA patients had PNH clone at diagnosis. We monitored the evolution of PNH clones after immunosuppressive therapy, and found increased incidence of PNH clone over time. The overall frequency of PNH clone in HAAA was 20.8% (15/72), which was comparable to that in IAA (27.8%, 112/403). Two thirds of those new PNH clones occurred in non-responders in HAAA. In conclusion, PNH clones are infrequent in HAAA compared to IAA at the time of diagnosis, but the overall frequency over time are comparable between the two groups of patients. In SAA/VSAA patients who are under the activated abnormal immunity, longer clinical course and relatively adequate residual hematopoietic cells serve as two important extrinsic factors for HSCs with PIGA-mutation to escape from immune attack and to expand. Disclosures No relevant conflicts of interest to declare.

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