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

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 730-730 ◽  
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.

scholarly journals Towards Identifying the Target of Autoimmunity in Aplastic Anemia

Blood ◽  
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.

scholarly journals Identification of an HLA class I allele closely involved in the autoantigen presentation in acquired aplastic anemia

Blood ◽  
2017 ◽  
Vol 129 (21) ◽  
pp. 2908-2916 ◽  
Author(s):  
Yoshitaka Zaimoku ◽  
Hiroyuki Takamatsu ◽  
Kazuyoshi Hosomichi ◽  
Tatsuhiko Ozawa ◽  
Noriharu Nakagawa ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Antigen Presentation ◽  
Somatic Mutations ◽  
Critical Role ◽  
Hla Class I ◽  
Class I ◽  
Key Points

Key Points Somatic mutations of HLA-B*40:02 are very frequently detected in granulocyte of patients with acquired aplastic anemia. Antigen presentation via HLA-B4002 may play a critical role in the pathophysiology of acquired aplastic anemia.

scholarly journals Loss-of-Function Mutations in HLA-Class I Alleles in Acquire Aplastic Anemia: Evidence for the Involvement of Limited Class I Alleles in the Auto-Antigen Presentation of Aplastic Anemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2584-2584
Author(s):  
Hiroki Mizumaki ◽  
Kazuyoshi Hosomichi ◽  
Tanabe Mikoto ◽  
Takeshi Yoroidaka ◽  
Tatsuya Imi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Aplastic Anemia ◽  
Antigen Presentation ◽  
Deep Sequencing ◽  
Research Funding ◽  
Hla Class I ◽  
Class I ◽  
Loss Of Function ◽  
Targeted Deep Sequencing

Abstract [Background] Acquired aplastic anemia (AA) is a rare syndrome characterized by pancytopenia and bone marrow hypoplasia. The cytotoxic T lymphocyte (CTL) attack against autologous hematopoietic stem progenitor cells (HSPCs) is thought to be responsible for bone marrow failure in the majority of AA cases; however, little is known about the target antigens of the CTLs. HLA class I-allele lacking leukocytes (HLA-LL) due to copy-number neutral loss of heterozygosity in the short arm of chromosome 6 (6pLOH) or somatic loss-of-function mutations in HLA class I genes are detected in approximately 20% of patients with newly diagnosed AA, and the presence of HLA-LL represents compelling evidence to support that CTLs specific to HSPCs are involved in the development of AA. Our recent studies using single nucleotide polymorphism array (SNP-A) genotyping and droplet digital polymerase chain reaction (ddPCR) revealed that HLA-B*40:02 is the most frequently lost among all class I alleles that are lost as a result of 6pLOH (Zaimoku, et al. Blood 2017). Various somatic loss-of-function mutations in B*40:02 revealed by deep sequencing in the study substantiated the important role of HLA-B4002 in the autoantigen presentation of AA. However, in the other 6pLOH(+) AA patients who did not possess HLA-B4002, which accounted for 20% of the total AA cases involving patients possessing HLA-LL, the allele in the missing haplotype that was responsible for the autoantigen presentation was largely unknown because the lost fragment of chromosome 6p usually contained 2 or more HLA class I alleles. [Objectives/Methods] To identify class I alleles other than HLA-B*40:02 that are critically involved in the auto-antigen presentation of AA, we screened a total of 624 patients for the presence of HLA-LL using monoclonal antibodies specific to class I HLA alleles, SNP-A, and ddPCR, and performed targeted deep sequencing of HLA class I genes by using SeqCap EZ Choice pobes (Roche) and MiSeq sequencer (Illumina). The paired fractions, including granulocytes that lacked an HLA-A allele and granulocytes that retained the HLA-A allele, as well as CD3+ T cells, were sorted using monoclonal antibodies specific to HLA-A alleles with a BD FACSAria Fusion system (BD Biosciences), and were subjected to DNA extraction. All DNA samples of granulocytes and control cells (CD3+ T cells or buccal mucosa cells) were prepared for targeted deep sequencing. [Results] One hundred and fourteen patients were found to be positive for HLA-LL and 62 (54.4%) of the 114 HLA-LL(+) patients did not carry B*40:02 (severe, n=30; non-severe, n=32; male, n=38; female, n=24; median age, 62 [range, 6-93] years). Apart from B*40:02 (45.6%), A*02:06 (24.6%) was the second-most frequent HLA class I allele in the lost haplotype. The targeted deep sequencing of 20 patients with HLA-LL revealed 6pLOH alone in 11 patients, and somatic loss-of-function mutations plus 6pLOH in 9 patients; none of the patients were positive for somatic loss-of-function mutations alone. Of note, somatic loss-of-function mutations were found in only 5 alleles (A*02:06 in four, B*40:01 in two, B*40:03, A*31:01, and B*54:01 in one each) out of 27 different alleles contained in the lost haplotype. Among the 9 patients with somatic loss-of-function mutations, the median number of mutations per patient was 1 (range, 1-2); these included a missense mutation (n=1), frameshift deletions (n=3) and nonsense mutations (n=7) (Figure). Four patients had a breakpoint of 6pLOH in between the HLA-A and C loci; their lost alleles were A*02:06 (n=2) and A*31:01 (n=2), and the occurrence of 6pLOH in the four patients was therefore attributed to the two HLA-A alleles. Sixty-six percent of the HLA-LL(+) B*40:02(-) patients had at least one of the five alleles in the lost haplotypes. The frequencies of each "high risk" allele found in patients possessing HLA-LL are summarized in Table. [Conclusions] In addition to B*40:02, five class I alleles including HLA-A*02:06, A*31:01, B*54:01, B*40:03 and B*40:01 are thought to play an essential role in the auto-antigen presentation by the HSPCs of Japanese AA patients. The frequencies of the six class I alleles in general Japanese population are much higher than those in the general Caucasian populations but similar to the frequencies in East Asian populations. The higher frequencies of the six alleles in comparison to Caucasian countries may account for the higher incidence of AA in East Asia. Disclosures Takamatsu: Ono: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen: Honoraria; Celgene: Honoraria, Research Funding. Nakao:Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria.

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

Hematology ◽  
2018 ◽  
Vol 2018 (1) ◽  
pp. 457-466 ◽  
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.

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

Blood ◽  
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.

Human-Leukocyte-Histocompatibility Antigens Predict Response to Rituximab and Donor Lymphocyte Infusion (DLI) After Non-Myeloablative Allogeneic Stem Transplantation (NST) for Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2548-2548
Author(s):  
Issa F. Khouri ◽  
Roland Bassett ◽  
Pedro Cano ◽  
Susan O'Brien ◽  
Yvonne Hsu ◽  
...  
Keyword(s):  
T Cell ◽  
Conflicts Of Interest ◽  
Chronic Gvhd ◽  
Hla Class I ◽  
Median Number ◽  
Class I ◽  
Hematopoietic Stem ◽  
Hla Alleles ◽  
Cell Counts ◽  
Significant Difference

Abstract Abstract 2548 Background: The effectiveness of allogeneic NST in CLL has been attributed to a graft-versus-leukemia effect due to elimination of tumor cells by alloimmune effector lymphocytes. In a previous study (Khouri et al, Exp Hematol 2004), we found that when patients with CLL develop relapse after NST, immunomanipulation (IMM) via withdrawal of immunosuppression and DLI with rituximab can induce sustained remissions in some patients. The underlying mechanism of this GVL effect is unknown. The simultaneous presence of the killer-immunoglobulin-like receptor (KIR) 3DL1 and its corresponding human leucocyte antigen (HLA) class I ligands bearing the Bw4 epitope has been described in CLL (Verheyden S, Leukemia 2006). Purpose: Considering that the HLA class I molecules may act as restriction elements for GVL targets after NST in CLL, we investigated the potential association of certain common class I HLA alleles and response to IMM. Methods: We studied all 43 CLL pts who required IMM after NST in sequential phase II protocols at the U.T. MD Anderson Cancer Center from February 1996 to August 2007 because of persistent disease or because of progression. In our analysis, we examined the most common alleles and serotypes expressed in the patients studied. This included HLA-A1, A2, A3, A24, B7, B8, B35, B44, B60, B62, BW4, BW6, CW7. These allele groups are known to be common in most world populations. Rituximab was given at a dose of 375 mg/m2 intravenously followed by 3 weekly doses of 1000 mg/m2. A DLI of 1 × 107 CD3-positive T cells/kg was given after the first two doses of rituximab if no GVHD occurred. An escalated DLI dose was given at 6-week intervals if there was persistent active disease and no GVHD. Results: Median age (range) was 55 years (39-73) and the median Hematopoietic Stem Cell Comorbidity Index was 3 (range, 0–8). The median number of prior chemotherapies was 3. At their transplant, 48% of pts had refractory disease, 90% had Binet stage B/C, and 60% had a beta-2 microglobulin of =/> 3. P53 deletion was detected in 11 of 35 pts (31%) tested. Mixed T-cell chimerism was observed in 60 % of pts at day 90. The median number of DLI infused was 2 (range, 1–6); their median maximal dose was 43.6 (range, 1–200) × 106 CD3+/Kg. In these 43 pts, 20 (47%) experienced complete remission (CR), by CT scans, marrow and flow analysis. Pts characteristics at time of study entry (described above) for NST, and at the time of initiation of IMM (this included white blood cell counts, LDH, % lymphocyte in marrow, % CD5-CD19, lymph node size by computed tomography, maximum dose DLI, number of DLIs, GVHD prior IMM and grade, T-cell chimerism), as well as HLA subtypes were assessed. The major determinants to achieve CR following IMM included receipt of a PBSC graft and achievement of a good (> 90%) donor T-cell chimerism at day 90 (p = 0.035), and having a combination of HLA-A1-positive, HLA-A2-negative, and HLA-B44-negative (p= 0.0009). The rate of CR to IMM was 9%, 36%, 50%, 91% respectively in patients who had none of the HLA factors described, I, 2, and all 3 respectively. There was no statistically significant difference in pts and disease characteristics between HLA-A1-positive or negative, HLA-A2 positive or negative nor between HLA-B44-positive or negative pts. In addition the risks of acute II-IV [Cumulative incidence (CI)=23%) and chronic GVHD (CI=69%) were not different between the respective subtypes. With a median follow-up in surviving pts of 37.2 months (range, 11.4–131.1), the progression-free survival rates at 5-year for patients with HLA-A1+/A2-/B44- vs those who had none of those HLA types were 68% vs 15%, respectively, (p<0.0001). Conclusions: Our results represent the first report showing certain HLA alleles might be predictive for response to GVL and achieving long-term remission in CLL. Verification of our findings in a larger cohort of pts is highly warranted for better selecting pts for IMM for the treatment of recurrent malignancy in CLL. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2021 ◽  
Author(s):  
Yoshitaka Zaimoku ◽  
Bhavisha A Patel ◽  
Sharon D Adams ◽  
Ruba N Shalhoub ◽  
Emma M Groarke ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Bone Marrow Cells ◽  
Late Onset ◽  
Clonal Evolution ◽  
Gene Mutations ◽  
Hla Class I ◽  
Class I ◽  
Hematopoietic Stem ◽  
Monosomy 7 ◽  
Hla Loss

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.

Anti-Leukemia Activity of Alloreactive NK Cells in Haploidentical HSCT in Pediatric Patients: Re-Defining the Role of Activating and Inhibitory KIR

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3002-3002 ◽  
Author(s):  
Daniela Pende ◽  
Stefania Marcenaro ◽  
Michela Falco ◽  
Stefania Martini ◽  
Maria Ester Bernardo ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Subset ◽  
Hla Class I ◽  
Class I ◽  
Leukemia Cells ◽  
Hla Alleles ◽  
Time Points ◽  
Selection Of

Abstract T-cell depleted hematopoietic stem cell transplantation from haploidentical donors (haplo-HSCT) has been reported to benefit from the graft-versus-leukemia effect mediated by natural killer (NK) cells when donor displays NK alloreactivity versus the recipient. NK alloreactivity is mediated by NK receptors, namely Killer Ig-like receptors (KIR) which are specific for allotypic determinants that are shared by different HLA-class I alleles (referred to as KIR ligands). It is known that KIR2DL1 recognizes HLA-C alleles characterized by Lys at position 80 (C2 group), KIR2DL2/3 recognize HLA-C alleles characterized by Asn at position 80 (C1 group), KIR3DL1 recognizes HLA-B alleles sharing the Bw4 supertypic specificity (Bw4 group) and KIR3DL2 recognizes HLA-A3 and –A11 alleles. KIR2D/3DL are inhibitory receptors that, upon engagement with the cognate ligand, inhibit lysis. Activating KIRs, highly homologous in the extracellular domain to the inhibitory counterparts, are KIR2DS1, KIR2DS2 and KIR3DS1, but only KIR2DS1 has been shown to specifically recognize C2 group of alleles expressed on B-EBV cells. We analyzed 21 children with leukemia receiving haplo-HSCT from a relative after a myeloablative conditioning regimen; in all pairs, the expression of a given KIR ligand (HLA class I allele) of the donor was missing in the patient (i.e. KIR ligand-mismatched haplo-HSCT). T-cell depletion was performed through positive selection of CD34+ cells; no pharmacological immune suppression was employed after HSCT. KIR genotype of all donors was evaluated to detect the presence of the various inhibitory and activating KIR genes. Phenotypic analyses were performed on NK cells derived from the donor and the patient at different time points after HSCT. Thanks to the availability of new mAbs able to discriminate between the inhibitory and the activating forms of a certain KIR, we could identify the alloreactive NK cell subset at the population level. These alloreactive NK cells express the KIR specific for the KIR ligand-mismatch (permissive inhibitory KIR) and the activating KIR (if present), while they do not express all inhibitory KIR specific for the patient HLA alleles and NKG2A. Thus, in most instances, we could precisely identify the size of the alloreactive NK cell subset in the donor and in the reconstituted repertoire of the recipient. Functional assays were performed to assess alloreactivity, using appropriate B-EBV cell lines and, if available, patient’s leukemia blasts. In some cases, also NK cell clones were extensively studied, for phenotype and receptor involvement in killing activity. We found that, in most transplanted patients, variable proportions of donor-derived alloreactive NK cells displaying anti-leukemia activity were generated and maintained even at late time-points after transplantation. Donor-derived KIR2DL1+ NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells, including patient leukemia blasts. Differently, KIR2DL2/3+ NK cells displayed poor alloreactivity against leukemia cells carrying HLA alleles belonging to the C2 specificity. Unexpectedly, this was due to recognition of C2 by KIR2DL2/3, as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably, however, C2/C2 leukemia blasts were killed by KIR2DL2/3+ (or by NKG2A+) NK cells that co-expressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role for the KIR2DS2 activating receptor in leukemia cell lysis could not be established. Taken together, these findings provide new information on NK alloreactivity in haplo-HSCT that may greatly impact on the selection of the optimal donor.

HLA Class I Allele-Lacking Hematopoietic Stem/Progenitor Cells Support Long-Term Clonal Hematopoiesis without Oncogenic Driver Mutations in Acquired Aplastic Anemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3894-3894
Author(s):  
Tatsuya Imi ◽  
Takamasa Katagiri ◽  
Kazuyoshi Hosomichi ◽  
Noriharu Nakagawa ◽  
Yoshitaka Zaimoku ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Progenitor Cells ◽  
Somatic Mutations ◽  
Hla Class I ◽  
Driver Mutations ◽  
Hematopoietic Stem ◽  
Target Sequencing ◽  
Clonal Hematopoiesis ◽  
Oncogenic Driver

Abstract [Background] Clonal hematopoiesis is currently known to be common in patients with acquired aplastic anemia (AA). One of the most common abnormalities underlying clonal hematopoiesis in AA patients is copy-number neutral loss of heterozygosity (LOH) in the short of 6 chromosome (6pLOH) caused by acquired uniparental disomy. Hematopoietic stem/progenitor cells (HSPCs) having undergone 6pLOH are thought to evade attack by cytotoxic T lymphocytes (CTLs) specific to auto-antigens by lacking particular HLA-A alleles. These HSPCs then produce HLA class I allele-lacking [HLA(-)] leukocytes to support hematopoiesis in patients with AA patients in remission. Our recent study showed that HLA(-) granulocytes are detected in about 24% of newly-diagnosed AA patients, and the aberrant granulocytes often account for more than 95% of the total granulocytes and persist for many years. The sustainability of 6pLOH(+) HSPC clones suggests that these HSPCs may suffer from secondary somatic mutations that confer a proliferative advantage on them over normal HSPCs. Alternatively, 6pLOH(+) HSPCs may persist and continue to support hematopoiesis according to their inherent sustainability, just like the PIGA mutant HSPCs we previously described (Katagiri et al. Stem Cells, 2013). To test these hypotheses, we determined the sequences of genes associated with the clonal expansion of HSPCs in HLA(-) granulocytes. [Patients and Methods] Eleven AA patients whose percentages of HLA(-) granulocytes ranged 6.4%-99.8% (median 94.2%) of the total granulocyte population were chosen for this study. The patients (male/female, 5/6 and age 27-79 [median 53] years) had been diagnosed with severe (n=5) or non-severe (n=6) AA 2-25 [median 12.5] years earlier, and 7 and 4 patients achieved complete response and partial response, respectively after treatments with cyclosporine (CsA) alone (n=4), CsA+antithymocyte globulin (ATG, n=3), CsA+anabolic steroids (AS, n=2), AS+romiplostim (n=1), and AS alone (n=1). The lineage combinations of HLA(-) cells were granulocyte, monocytes, B cells and T cells (GMBT) in 6, GMB in 4 and GM in 1. HLA(-) and normal [HLA(+)] granulocytes were sorted from the blood leukocytes of the 11 patients and the DNA of each cell population as well as that of buccal mucosa cells was subjected to target sequencing of 61 myelodysplastic syndrome (MDS)-related genes with MiSeq. DNA samples from 5 patients including 4 patients whose HLA(-) cell percentages were greater than 95% were further analyzed by whole-exome sequencing (WES) using HiSeq. The percentage of 6pLOH(+) cells in the total granulocytes or sorted HLA(-) granulocytes were estimated using digital droplet PCR or deep sequencing of HLA alleles. [Results] Target sequencing of 8 of the 11 patients revealed somatic mutations in the HLA(-) granulocytes of 3 patients. HLA(-) granulocytes-specific mutations were found in DNMT3A, PRR5L, SMC3A, and LRCH1 (Table). The variant allele frequencies (VAF) of these mutations were far lower (5.1%-20%) than those of HLA(-) granulocytes that accounted for 95% of sorted cells. WES revealed 22 non-synonymous and 9 synonymous mutations in the HLA(-) granulocytes from 4 of the 5 patientsthat included 3 new patients and 2 patients whose samples were negative for mutations revealed by the target sequencing. The VAF of these mutations ranged from 20.7-52.5% (median 44.1%, Table). Very-high VAFs of several mutant genes suggested that these mutations occurred simultaneously with or soon after the occurrence of 6pLOH. A patient who achieved remission after romiplostim therapy without ATG showed various gene mutations that were thought to have occurred after 6pLOH. Despite of their highly biased hematopoiesis supported by single or few clones, recurrent or MDS-related oncogenic mutations were not detected in any of the 11 patients. Of note, the percentages of 6pLOH(+) cells in the sorted HLA(-) granulocytes were ≤75% (36.7%, 46%, 74%, and 75%) in 4 patients, indicating the presence of granulocytes lacking HLA-A alleles through mechanisms other than 6pLOH. [Conclusions] HLA(-) HSPCs caused by 6pLOH or other unknown mechanisms support long-term hematopoiesis without the development of oncogenic driver mutations that are associated with clonal hematopoiesis of MDS; as such, clonal hematopoiesis by 6pLOH(+) HSPCs may not portend a poor prognosis. Disclosures Nakao: Alexion Pharmaceuticals: Honoraria, Research Funding.

scholarly journals Distinct Escape Mechanisms of HLA Class I Allele-Lacking Hematopoietic Stem Progenitor Cells (HSPCs) from GPI-Deficient HSPCs in Acquired Aplastic Anemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1216-1216
Author(s):  
Noriaki Tsuji ◽  
Kohei Hosokawa ◽  
Takeshi Yoroidaka ◽  
Tanabe Mikoto ◽  
Hiroki Mizumaki ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Anabolic Steroids ◽  
Hla Class I ◽  
Class I ◽  
Immune Selection ◽  
Hematopoietic Stem ◽  
Clonal Hematopoiesis ◽  
Immune Pressure ◽  
Company Limited ◽  
Selection Of

[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<0.05). In 4 of the 9 patients on treatment and in 4 patients who had been free of therapy, the percentages of HLA(-) cells in HSPCs were similar to those of Gs (1%/1%, 1%/1%, 14%/15% and 100%/98% in the 4 treated patients; 16%/11% in the untreated patient; 13%/13%, 95%/95%, 99%/99% and 99%/99% in the 4 patients in remission after therapy). BM subsets including HSCs of 2 patients whose Gs and PB HSPCs lacked HLA-A2 almost completely, were 99% negative for HLA-A2. In contrast, in the remaining 6 patients, the percentages of HLA(-) cells in HSPCs were markedly lower than those in Gs (0%/25%, 2%/7%, 4%/65%, 6%/77% [Case 1], 9%/75% and 10%/39% [Case 2]), suggesting the persistence of CTL attack against HPCs or more differentiated myeloid cells (Figure 1). In contrast to HLA(-) PB HSPCs, there was no discordance in the percentage of GPI(-) cells between PB Gs and HSPCs (4%/4%, 49%/50%, 94%/100%, 96%/96%, 96%/99% and 99%/92%) in all 6 AA/PNH patients including 3 responding to CsA. Lastly, serial changes in the percentage of PB HLA(-) and GPI(-) Gs during CsA therapy were compared in seven patients who possessed both aberrant cells. In 4 patients who were responding to CsA, the HLA(-) G percentage gradually decreased in 3 and remained stable in the remaining one, while the GPI(-) G percentage reciprocally increased in 3 and remained stable in one. In contrast, in 3 patients who obtained sustained remission after CsA therapy, the HLA(-) G percentage increased after cessation of CsA. The GPI(-) G percentages decreased in 1 and remained unchanged in 2 (Figure 2). [Conclusions] Immune selection that favors the survival of HLA(-) HSPCs or HPCs takes place even in AA patients who have been in remission for many years after successful IST and may contribute to clonal hematopoiesis by HLA(-) HSPCs. Given no signs of selection at the HSPC stage and the reciprocal increases in the percentage of GPI(-) Gs in AA/PNH patients responding to CsA therapy, the proliferation of GPI(-) HSPCs may not be affected by immune pressure in convalescent patients. Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Ono Pharmaceutical: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Novartis Pharma K.K: Honoraria; SynBio Pharmaceuticals: Consultancy; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Bristol-Myers Squibb: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Alaxion Pharmaceuticals: Honoraria; Kyowa Kirin: Honoraria.

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