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 Abstract 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. study registered


Introduction
Immune aplastic anemia (AA) is caused by T cells that destroy hematopoietic stem cells, and marrow failure is successfully treated with hematopoietic cell transplantation or immunosuppressive therapy (IST). 1 Eltrombopag (EPAG) combined with IST yielded higher hematologic responses and survival compared to IST alone, 2 but long-term outcomes such as relapse and clonal evolution remain clinically problematic and biologically not well understood.
The immune pathophysiology of AA has been, in part, inferred from frequent somatic loss of HLA class I alleles. Increased frequency of some HLA alleles has been reported in AA patients of various ethnicities, [3][4][5][6][7][8][9][10][11][12][13][14] and has been confirmed in a recent genome-wide association study. 15 Somatic loss of HLA class I alleles may result from copy-neutral chromosome 6p loss of heterozygosity (6p LOH) 11,16,17 or acquired inactivating HLA gene mutations. 18,19 A limited set of HLA-A and HLA-B alleles are more likely to acquire somatic mutations; 18,19 a T cell line specific for a missing HLA class I allele has been isolated from an AA patient. 20 Loss in a recurrently mutated HLA allele may characterize a specific immune pathogenesis and associated clinical manifestations. Previous studies suggested better IST responses and survival in patients with HLA loss 11,18,21 and poor outcomes, including frequent clonal evolution, in patients who harbored HLA alleles related to somatic mutations, irrespective of somatic loss of an HLA allele. 19 Clonality is common in AA. 22 Paroxysmal nocturnal hemoglobinuria (PNH)-clones, cells deficient in glycosylphosphatidylinositol (GPI)-anchored proteins due to acquired PIGA mutations, are most frequent. PNH is closely related to immune marrow failure, and the GPI anchor itself may be a target of immune attack. 23,24 Somatic mutations are also present in genes recurrently mutated in myelodysplastic syndromes or acute myeloid leukemia, especially DNMT3A, ASXL1, and BCOR. 17 However, the allelic burden of these clones in AA are usually small, clones remain stable for years, and affected cells infrequently drive evolution to myeloid neoplasms, which are usually characterized by complete or partial loss of chromosome 7. 1,25,26 Of all clonal associations, the mechanism of HLA loss is most clearly related to escape from immune cell destruction.
Our aim was to clarify and enlarge on the clinical significance of HLA class I allele loss and recurrently mutated genotypes in a large cohort of patients with immune AA.

Study design and participants
A total of 544 patients with AA, aged two years or older, treated at the National Institutes of Health Clinical Center, Bethesda, MD, between May 1999 and August 2019 were enrolled in this retrospective study. Patients were enrolled in various IST protocols approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute (clinicaltrials.gov: NCT00001964, NCT00061360, NCT00195624, NCT00260689, NCT00944749, NCT01193283, and NCT01623167) which entailed informed consent. All horse anti-thymocyte globulin (hATG)-based IST protocols have been published 2,27-29 and briefly described in supplemental Methods.

Definitions
All definitions have been consistent across protocols. Severe AA was diagnosed when at least two of the following three criteria were met: a neutrophil count < 0.5 × 10 9 /L, a reticulocyte count < 60 × 10 9 /L, and a platelet count < 20 × 10 9 /L. Hematologic responses were assessed six months after institution of hATG: an overall response was defined as blood counts no longer meeting the above stated criteria for severe AA; a complete response required a neutrophil count of at least 1.0 × 10 9 cells/L, a hemoglobin level of at least 10 g/dL, and a platelet count of at least 100 × 10 9 /L. High-risk clonal evolution was defined as acquisition of either chromosome 7 abnormalities, complex cytogenetics, myelodysplastic syndrome, or acute myeloid leukemia, and low-risk clonal evolution was the other cytogenetic changes, as previously published. 30

Detection of HLA class I loss
Detailed methods for detection of HLA loss are described in the supplemental Methods. In brief, HLA loss was assessed by either HLA flow cytometry plus deep nucleotide sequencing or deep sequencing alone. HLA flow cytometry was performed using cryopreserved peripheral blood mononuclear cells stained with HLA-A and HLA-B allele specific monoclonal antibodies, and assessed in CD33 hi monocytes. Deep sequencing of HLA class I genes was performed using sorted peripheral blood cell subpopulations, including HLA allele-lacking and -expressing monocytes; loss was detected by flow cytometry or using Downloaded from http://ashpublications.org/blood/article-pdf/doi/10.1182/blood.2021012895/1832235/blood.2021012895.pdf by guest on 01 November 2021 whole blood for the deep sequencing-only samples. HLA class I genes were enriched by locus specific long-range PCR, as previously described. 31 To precisely detect low-frequency mutations, we used hla-mapper (version 2.3), 32 and utilized thresholds defined by baseposition error rates in T cell samples, as previously published for non-HLA genes. 33,34 We inferred 6p LOH using read counts of allele-specific single-nucleotide polymorphisms in HLA genes.

Statistics
Statistical analyses were performed using R ( Americans, Hispanics, and Asians, 36 and the results were combined by fixed-effect metaanalysis to elucidate consistent impacts across ethnic groups.

Data sharing statement
All somatic HLA gene mutation information are available in supplemental data.  Our sequencing method allowed us to identify variant allele frequency as low as 0.3% in more than 50% of base positions of single nucleoide variants and 90% of indels (Supplemental Figure 1). The accuracy of the method was supported by the following observations and experiments: HLA class I gene mutations were not detected from additional 16 control T cell samples nor from monocytes of patients expressing respective HLA alleles as determined by flow cytometry (except for missense mutations and one intronic mutation); somatic mutations detected from patient samples were generally predicted to inactivate affected HLA alleles, irrespective of variant allele frequencies and samples (Supplemental  Figure 11C). 18 Eleven missense mutations and two synonymous mutations were predicted to create alternative splice sites and generate truncated proteins (Supplemental Table 1). Of note, 12 of 24 functionally confirmed intronic mutations and seven unconfirmed mutations did not involve donor 5'-GT nor acceptor AG-3' canonical splice sites (Supplemental Table 2): eight of these mutations proximate to the splice sites have been predicted to reduce splicing efficiency; six mutations at 19 to 21 nucleotides from the 3' splice site were found to impair the consensus branchpoint sequences, CTGAC, CTCAC, or CTCAG, also critical for splicing; 39 the remaining five mutations in the middle of HLA-B intron 3 (c.619+123A>G and c.619+133C>G) have been reported to create alternative 5' splice sites. 18 One mutation in intron 7 of HLA-A and two mutations in cytoplasmic tail (exon 7) of HLA-C (S357A and E359del) were assumed to be passenger mutations, as these clones were small (1.0%, 3.0%, and 0.6%) and multiple clones with HLA-B mutations (14, seven, and six) were also detected from the three patients.

Patients
The proportions of HLA allele-lacking cells in total monocytes were reduced in response to IST plus EPAG in all 18 patients sequentially tested, irrespective of hematological responses, and in two patients, HLA allele-lacking cells had disappeared at six months; they were thereafter stable over many years in 13 patients (Supplemental Figure 12), as has been reported. 18 HLA loss and other hematopoietic clones     Correlations of HLA loss and HLA genotypes with pretreatment blood values and hematologic responses to IST were weak (Supplemental Table 4). However, loss of HLA-B*40:02 and certain minor alleles was related to higher blood counts and all patients who lost these alleles responded to IST (Supplemental Figure 16; Supplemental Table 5). These relationships are consistent with the predictive value of reticulocytes for IST response, 40,41 and the generally high response rate to IST in Japanese patients with HLA loss, in whom HLA-B*40:02 was most frequently inactivated, 18,42 but HLA-B*14:02 and HLA-B*08:01 genotypes are virtually absent. 43 High-risk clonal evolution was correlated with the most frequently inactivated allele HLA-B*14:02 (P = .0092; Figure 3A), as previously suggested by Babushok et al, 19 and with HLA loss (P = .00040; Figure 3B), especially with loss of HLA-A*02:01 and HLA-B*14:02 (Supplemental Figure 17A; Supplemental Table 5). In particular, HLA-B*14:02 genotype was strongly correlated among patients without HLA loss (P = .0072), while none of 43 patients who did not carry any of the mutation-associated HLA genotypes exhibited high-risk clonal evolution (Supplemental Figure 17B, C); this suggested that the presence of specific HLA class I-mediated immunity, rather than HLA loss itself, predisposed to high-risk clonal evolution. A combined HLA risk, defined as the presence of HLA loss or HLA-B*14:02 genotype (irrespective of loss), yielded a better prediction factor for high-risk clonal evolution than did either HLA loss alone or HLA-B*14:02 alone (Supplemental Figure 17D).
The HLA associations with high-risk clonal evolution were independent of patient age (Table   3; Supplemental Table 6), a known predictor of secondary myeloid malignancies ( Figure   3C). 44,45 A prediction model for high-risk clonal evolution incorporating the combined HLA risk and age 40 years or older clearly divided patients into three risk groups ( Figure 3D; Supplemental Figure 17E), which was validated in various subgroups of patients (Supplemental Figure 18).

Origin of monosomy 7
We further studied somatic HLA class I gene mutations in monosomy 7 clones evolved in 11 patients with immune AA, using cryopreserved peripheral blood mononuclear cells; four of the patients had HLA allele lacking-leukocytes present before clonal evolution and three did not, and in the other four cases, samples prior to the evolution event were not available. In We confirmed the high prevalence of HLA class I alleles recurrently inactivated by somatic mutations across four major ethnic groups in the United States, which included previously reported HLA-B*14:02 and HLA-B*40:02, 18,19,46 and an unreported allele HLA-B*07:02. Further, correlations of HLA loss with patient age at disease onset, peripheral blood counts, IST-response, and high-risk clonal evolution suggest specific pathogeneses related to certain HLA class I alleles.
In cancer generally, HLA allele loss is frequent and represents escape from immune surveillance, 47,48 which clinically correlate with poor outcomes after treatment with immune checkpoint inhibitors 49 and relapse of acute myeloid leukemia after hematopoietic cell transplantation. 50 While HLA loss in immune AA is protective of the stem cell under selfdestructive immune attack, it also should allow escape from immune surveillance and therefore of malignant clonal evolution. Indeed, we observed this association and tracked it in two patients. Nevertheless, high-risk clonal evolution is more likely consequent to HLA class I-mediated destruction of bone marrow cells than due to immune escape, as HLA-B*14:02, Downloaded from http://ashpublications.org/blood/article-pdf/doi/10.1182/blood.2021012895/1832235/blood.2021012895.pdf by guest on 01 November 2021 the most frequently mutated and overrepresented class I allele in AA, was associated with high-risk clonal evolution irrespective of HLA mutation status, as previously suggested. 19 Most (but not all) monosomy 7 clonal evolution arose from cells with intact HLA genes; high-risk evolution was not observed in any patient without an HLA allele prone to loss. A simple prediction model incorporating HLA loss and HLA-B*14:02, as a convincing indicator of class I-mediated pathogenesis, in addition to age, stratified the incidence of high-risk clonal evolution, even within subgroups. Monitoring for clonal evolution after IST is desirable in patients with HLA allele loss or HLA-B*14:02; early hematopoietic cell transplantation could be considered for the high-risk group patients aged 40 years or older, rather than IST as generally recommended for the aged individuals. 51 The underlying mechanisms responsible for the predominant development of monosomy 7 in immune AA (and other marrow failure syndromes) remain elusive. 52 We detected a small monosomy 7 clone, not visible by conventional bone marrow cytogenetics, at least two years before the clinical diagnosis of clonal evolution in one patient; extended time periods may be necessary for aneuploid clones (and somatically mutated clones 53 ) to expand and become clinically evident, either by accumulating additional genetic aberrations or altered environmental selection. Relatively infrequent mutations in non-HLA genes among patients with HLA loss argues against HLA gene mutations arising due to to an intrinsically higher somatic mutation rate or from genetic drift in a paucicellular stem cell pool. 24 More sensitive methods would benefit both the monitoring and deeper understanding of secondary myeloid malignancies in AA.
PNH clones and HLA allele lacking clones are both assumed to represent immune escape of hematopoietic stem cells from immune cell attack and destruction, manifested by the recurrent presence of multiple clones with somatic mutations in PIGA 54 or HLA class I genes. 18 PNH and HLA absent clones were always mutually exclusive, as in the 27 patients who had both types of clones. Loss of surface GPI-anchored proteins may confer no additional survival advantage on HLA allele loss, and the reverse. A remarkable difference between the two types of escape clones was their relationship to clonal evolution, which occurred regardless of the presence of PNH clones but much more frequently in patients with HLA allele loss. Malignant clones always evolved from non-PNH phenotype cells as previously reported, 25 but they did arise from clones with HLA allele loss.
Our deep sequencing method for HLA class I genes allowed detection of somatic mutations at an allele frequency as low as 0.3%. Many coexisting small clones with somatic mutations in HLA-A and HLA-B alleles were identified, most of which functionally confirmed by flow cytometry. Even intronic mutations, not involving canonical splice sites, and a synonymous mutation were found to reduce cell surface expression of HLA class I alleles; such mutations likely have been underreported in studies using exome or RNA sequencing data and in those lacking functional validation. A limitation of our sequencing method was the lower sensitivity for 6p LOH detection, the result of unbalanced amplification of heterozygous HLA alleles and use of whole blood for some patients. Underestimation of 6p LOH would not be predicted to affect the overall detection of HLA loss, as most 6p LOH clones coexist with clones with inactivating mutations in an HLA allele, but detection of such small clones may be of value if they predicted secondary myeloid malignancies.
In summary, our study suggested the presence of distinct pathogenic pathways related to HLA genotype and loss in immune AA. Most important in the clinic was the relationship between high-risk malignant clonal evolution and class I-mediated immunity, as for the most frequently inactivated HLA-B*14:02 genotype and HLA loss in general, which provide a simple prediction tool for this serious complication.