Relapse of Leukemia with Loss of Mismatched HLA Due to Uniparentaldisomy Follwing Haploidentical Hematopoietic Transplantation.
Authors: Takahashi Yoshiyuki , Bustos Itzel , Akatsuka Yoshiki , Muramatsu Hideki , Nishio Nobuhiro , Hama Asahito , Yagasaki Hiroshi , Saji Hiroh , Kato Motohiro , Ogawa Seishi , Kojima Seiji 
Published: October 2019

Abstract
Abstract 2457
Poster Board II-434

Introduction:
Down-regulation or loss of human leukocyte antigen (HLA) expression can lead to impaired T-cell recognition and a blunted immune response to malignant tumors. We investigated HLA expression on leukemic cells derived from patients at the time of diagnosis and relapse after HLA haploidentical hematopoietic stem cell transplantation (HSCT) using flow cytometry with locus-specific antibodies. We hypothesized that the loss of HLA haplotype caused leukemic cells to escape immunosurveillance and consequently led to relapse of the disease.

Materials and methods:
The CD13+/34+ leukemic blasts were sorted by flow cytometry from bone marrow cells at the time of diagnosis and at the time of relapse. Genomic DNA was extracted from leukemic cells by fluorescence-activated cell-sorter as well as phytohemagglutinin-stimulated patient-derived T cells and subjected to single-nucleotide polymorphism (SNP) array analysis using GeneChip NspI arrays (Affymetrix, Tokyo, Japan). Allele-specific copy number was detected using Copy Number Analyser for GeneChip® software. The frequencies of cytotoxic T lymphocyte precursor (CTLp) specific for the recipient mismatched HLA molecules were analyzed using a standard limiting dilution assay. Allo-HLA-restricted CTL clones were isolated by standard limiting dilution and expanded for cytotoxicity assay against mismatched HLA transduced HLA class I-deficient 721.221 B-LCLs.

Results:
Two of three relapsed patients after HLA haploidentical HSCT demonstrated loss of HLA alleles on leukemic cells at the time of relapse and this loss was limited to the mismatched alleles in both patients. However, none of seven relapsed patients experienced haplotype loss following HLA matched HSCT. SNP array analyses of sorted leukemic cells at the time of diagnosis and at the time of relapse further revealed the copy number-neutral loss of heterozygosity, namely acquired uniparental disomy (UPD) on the short arm of chromosome 6, resulting in the total loss of the mismatched HLA haplotype. Recipient alloantigen-specific cytotoxic T-cell clones were generated from the donor that did not recognize the leukemic cells at the time of relapse, whereas those cells taken at diagnosis were recognized and efficiently killed. In one of the patients, we sought to determine if the number of CTLp had changed during the post-transplant course. A limiting dilution analysis with a split-well 51Cr-release assay was carried out to compare the CTLp frequencies specific for the mismatched antigens between the recipient and donor. Surprisingly, the CTLp frequencies reactive with recipient T cell blasts in CD8+ T cells obtained around Days 100, 180, and 300 (4 months before relapse) were undetectable, while the CTLp frequency obtained at Day 520 (1 month after the third DLI or 2 weeks after remission confirmed by bone marrow aspirate) was 8.6 × 10-5 [95% confidence interval (CI), 1.49 × 10-6 – 5.0 × 10-5]. The CTLp frequency in the donor CD8+ cells was 4.3 × 10-5 (95%CI, 7.2 × 10-5 – 2.5 × 10-5), which was close to that obtained after DLI in the recipient.

Conclusions:
These results suggest that the cytotoxic T lymphocyte response to mismatched HLA alleles can eradicate leukemic cells; however, escape from immunosurveillance by the loss of mismatched HLA alleles using UPD may be involved in relapse after haploidentical HSCT.

Disclosures:
No relevant conflicts of interest to declare.