Risk Factors and Prognosis Analysis of Patient-Specific HLA Genomic Loss Relapse Following ATG-Based T-Cell-Replete Haploidentical Hematopoietic Stem Cell Transplantation

Background Developments in related haploidentical hematopoietic stem cell transplantation (haplo-HSCT) have made two immune systems with incompatible human leukocyte antigens (HLA) adaptable, greatly reducing transplantation-related toxicity. In the haploidentical transplantation system, patient-specific HLA antigens are a primary way in which donor immune cells exert their graft-versus-leukemia effects by alloreactivity, and therefore patient-specific HLA genomic loss is often considered a mechanism of leukemia relapse. Although the incidence and characteristics of HLA loss have been investigated in patients with myeloid leukemia who relapsed after transplantation, patient-specific HLA genomic loss in lymphoid conditions and under different transplantation regimes remain enigmatical. We herein provide the data for HLA loss relapse in lymphoid disease, as well as offer data in myeloid disease based on antithymocyte globulin (ATG) T-cell-replete conditioning. Methods Between May 2012 and May 2021, 106 patients were enrolled in this study, including 104 had hematological relapse and 2 had minimal residual disease (MRD) recurrence after haplo-HSCT. All data were collected from centers within the Zhejiang Provincial Workgroup of HSCT. Firstly, the patients were divided into two groups according to the type of disease: myeloid and lymphoid conditions, and secondly, they were divided into the Classic Group and the Loss Group according to whether the patients had unshared HLA genomic loss at the time of relapse or not. A two-tailed P value of 0.05 was considered statistically significant. Univariate analysis is performed using the χ2 test, Fisher exact test, and Student t test, as appropriate. A multivariate model was used to calculate the odds ratio (OR) with a 95% confidence interval (CI). Post relapse overall survival (prOS) was estimated and plotted using the Kaplan-Meier method. Results Of all patients, HLA loss was detected in 50.9% (54/106) patients and the occurrence was not related to disease type (P=0.44). Patients having either myeloid malignancies or lymphoid malignancies with HLA loss experienced relapse later than those with classic relapse. The demographic and baseline characteristics of patients were summarized in Table 1, and the post-relapse parameters were presented in Table 2. We next determined what factors correlating with HLA loss at relapse by multivariate analysis. For patients with myeloid malignancies, a pre-transplant underweight status based on body mass index (BMI) was a factor strongly correlated with classic type when relapse (OR=0.095, 95%CI: 0.015~0.604, P=0.013); patients suffering de novo aGVHD had an OR of 4.844 (95%CI: 1.143~20.530, P=0.032) (Figure 1). As for lymphoid conditions, patients who experiencing MRD-positivity before hematological relapse (OR=22.623, 95% CI: 1.597~320.532, P=0.021), and >180 days from HSCT to relapse (OR=5.695, 95% CI: 1.073~30.211, P=0.041) were more likely to lose unshared HLA at relapse. Yet male patients (OR=0.037, 95% CI: 0.003~0.411, P=0.037) were inclined to have classic one when relapse (Figure 1). For prognosis, in patients with myeloid conditions, the median prOS was 217 days and 215 days in the Loss Group and Classic Group, respectively (P=0.34). In the Loss Group, the white blood cell (WBC) at diagnosis was related to prOS (>50ⅹ10 9/L vs. ≤50ⅹ10 9/L: median: 157 vs. 261 days, P=0.037). For patients having male donors, patient gender was related to prOS: median prOS was 386 days for male patients and 124 days for female patients (P=0.008). For patients with lymphoid malignancies, the median prOS was 285 days and 244 days in the Loss Group and Classic Group, respectively (P=0.24). The results of the univariate analysis from the Loss Group showed that male patients (median: 40 vs. 194 days; P=0.036) and cGVHD fare (median: 114 vs. 468 days; P=0.002) were related to inferior prOS. Conclusion The occurrence of patient-specific HLA genomic relapse after haplo-HSCT appears later than classic relapse. In myeloid malignancies, the patient pre-transplant BMI and aGVHD fare are associated with the relapsed type. In lymphoid conditions, patient sex, presence of MRD-positive results before relapse, and the time from transplantation to relapse correlate with HLA loss at relapse. Yet this study is based on a limited number of patients, further validation in other centers is warranted. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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.

Copy Neutral LOH Affecting the Entire Chromosome 6 is a Frequent Mechanism of HLA Class I Alterations in Cancer

Total or partial loss of HLA class I antigens reduce the recognition of specific tumor peptides by cytotoxic T lymphocytes favoring cancer immune escape during natural tumor evolution. These alterations can be caused by genomic defects, such as loss of heterozygosity at chromosomes 6 and 15 (LOH-6 and LOH-15), where HLA class I genes are located. There is growing evidence indicating that LOH in HLA contributes to the immune selection of HLA loss variants and influences the resistance to immunotherapy. Nevertheless, the incidence and the mechanism of this chromosomal aberration involving HLA genes has not been systematically assessed in different types of tumors and often remains underestimated. Here, we used SNP arrays to investigate the incidence and patterns of LOH-6 and LOH-15 in a number of human cancer cell lines and tissues of different histological types. We observed that LOH in HLA is a common event in cancer samples with a prevalence of a copy neutral type of LOH (CN-LOH) that affects entire chromosome 6 or 15 and involves chromosomal duplications. LOH-6 was observed more often and was associated with homozygous HLA genotype and partial HLA loss of expression. We also discuss the immunologic and clinical implications of LOH in HLA on tumor clonal expansion and association with the cancer recurrence after treatment.

Clinical Utility of the Detection of the Loss of the Mismatched HLA in Relapsed Hematological Patients After Haploidentical Stem Cell Transplantation With High-Dose Cyclophosphamide

Haploidentical hematopoietic stem cell transplantation (Haplo-HSCT) with high-dose cyclophosphamide (PTCy) has resulted in a low incidence of graft-vs.-host disease (GVHD), graft failure, and non-relapse mortality. However, post-transplantation relapse remains a common cause of treatment failure in high-risk patients. Unraveling the mechanisms of relapse is therefore crucial for designing effective relapse treatment strategies. One of these mechanisms is the loss of the mismatched HLA on the recipient's leukemic cells. To study the incidence and clinical relevance of this phenomenon, we analyzed 181 patients treated with Haplo-HSCT with PTCy (2007–2019), of which 37 relapsed patients after transplantation. According to the kit employed for HLA-loss analysis, among 22 relapsed patients, we identified HLA loss at relapse in 6 of the 22 patients (27%) studied. Based on the results obtained, the genomic loss of HLA was more common in females than males (66 vs. 33%) and HLA-loss relapses occurred later than classical relapses (345 vs. 166 days). Moreover, the patients with HLA-loss had a greater presence of active disease at the time of transplantation and had undergone a larger number of treatment lines than the group with classical relapses (66 vs. 43% and 66 vs. 18%, respectively). Four of these relapses were studied retrospectively, while two were studied prospectively, the results of which could be considered for patient management. Additionally, two relapsed patients analyzed retrospectively had myeloid neoplasms. One patient had not undergone any treatment, and three had undergone donor lymphocyte infusions (DLIs) and chemotherapy. All presented severe GVHD and disease progression. In contrast, the two patients studied prospectively had a lymphoid neoplasm and were not treated with DLIs. One of them was treated with chemotherapy but died from disease progression, and the other patient underwent a second Haplo-HSCT from a different donor and is still alive. We can conclude that the detection of HLA-loss at the onset of relapse after Haplo-HSCT with PTCy could help in clinical practice to select appropriate rescue treatment, thereby avoiding the use of DLIs or a second transplantation from the same donor.

HLA loss of heterozygosity-mediated discordant responses to immune checkpoint blockade in squamous cell lung cancer with renal metastasis

Despite the significant success of immune checkpoint blockade therapy in advanced non-small-cell lung cancer compared with chemotherapy, efficacy varies greatly across patients, and acquired resistance frequently occurs. In particular, during immunotherapy, the dynamic changes in molecular events have not been characterized. The authors report a case of squamous cell lung carcinoma with renal metastasis, treated with pembrolizumab, in which the primary tumor and rare renal metastases showed different responses. Using whole-exome sequencing, the authors found loss of heterogeneity in HLA genes in all tumors and high levels of intratumor heterogeneity in metastases. The increased levels of HLA loss led to therapy resistance during tumor evolution. In addition to tumor mutational burden and PD-L1, HLA loss of heterozygosity and intratumor heterogeneity should be taken into consideration during immunotherapy.

Towards Identifying the Target of Autoimmunity in Aplastic Anemia

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.

73 Orthogonally and functionally validated algorithm for detecting HLA loss of heterozygosity

BackgroundHuman leukocyte antigen (HLA) genes facilitate communication between tumor cells and the immune system through the cell surface presentation of a diverse set of peptides. HLA loss of heterozygosity (LOH) has been associated with reduced immune pressure on neoantigens and impaired response to checkpoint blockade immunotherapy. Although HLA LOH is emerging as a key biomarker for response to immunotherapy, few tools exist to detect HLA LOH. Moreover, the accuracy of these tools is not well understood due to lack of orthogonal validation approaches. Here, we briefly describe DASH (Deletion of Allele-Specific HLAs), an algorithm to detect HLA LOH from exome sequencing data, and present a three-pronged validation approach to assess its performance.MethodsIn-silico evaluation of the limit of detection (LOD) of DASH was performed by deeply sequencing a tumor-normal paired cell line with HLA LOH and mixing reads at different proportions to simulate variable tumor purity and clonality. Direct genomic validation was performed using digital PCR (dPCR) with allele-specific primers targeting both predicted kept and lost alleles in ten patient samples and one cell line. Quantitative immunopeptidomics was performed to compare peptides presented by HLA alleles in tumor cells and adjacent normal cells. The relative increase or decrease of peptide presentation per allele was estimated by predicting the binding of each peptide to the patient-specific alleles.ResultsDASH is a machine learning model built upon the HLA-enhanced ImmunoID NeXT Platform®. We validated the performance of DASH using three orthogonal approaches to better understand the factors driving sensitivity and specificity of the algorithm. Evaluation using cell line mixtures that simulate LOH at various dilutions helped establish the LOD of DASH. For fully clonal tumors, DASH had 100% sensitivity at all tumor purity levels above 8% and 100% specificity at tumor purity levels higher than 24%. Patient-specific and allele-specific dPCR assays provided sensitive, direct evidence of HLA LOH. All samples predicted to have HLA LOH by DASH with high confidence were confirmed by dPCR. Finally, a quantitative immunopeptidomics experiment in one patient with HLA LOH revealed a large decrease in the peptides presented by deleted alleles, revealing the functional implications of HLA LOH.ConclusionsHLA LOH detection methods need to be rigorously validated in order to be used as a clinical biomarker. Here, we introduced three methods to assess performance, demonstrated the strong predictive power of DASH, and highlighted the need to consider tumor purity in such assessments.