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In this issue of Blood, Ahci and colleagues describe a simple and robust method for identifying the phenomenon of leukemic HLA loss often associated with treatment failure of hematopoietic stem cell transplantation from haploidentical donors (haplo-HSCT).1

Comprehensive knockout of HLA class II (HLA-II) β-chain genes is complicated by their high polymorphism. In this study, we developed CRISPR/Cas9 genome editing to simultaneously target HLA-DRB, -DQB1, and -DPB1 through a single guide RNA recognizing a conserved region in exon 2. Abrogation of HLA-II surface expression was achieved in five different HLA-typed, human EBV-transformed B lymphoblastoid cell lines (BLCLs). Next-generation sequencing–based detection confirmed specific genomic insertion/deletion mutations with 99.5% penetrance in sorted cells for all three loci. No alterations were observed in HLA-I genes, the HLA-II peptide editor HLA-DMB, or its antagonist HLA-DOB, showing high on-target specificity. Transfection of full-length HLA-DPB1 mRNA into knockout BLCLs fully restored HLA-DP surface expression and recognition by alloreactive human CD4 T cells. The possibility to generate single HLA-II–expressing BLCLs by one-shot genome editing opens unprecedented opportunities for mechanistically dissecting the interaction of individual HLA variants with the immune system.

Although quantitative PCR (qPCR) has been explored for chimerism monitoring after allogeneic stem cell transplantation (SCT), evidence regarding its clinical utility compared with standard short tandem repeat (STR) is still limited. We retrospectively studied commercial qPCR and STR chimerism with respective positivity thresholds of .1% and 1% in 359 peripheral blood (PB) and 95 bone marrow (BM) samples from 30 adult patients after first HLA-matched SCT for myeloid malignancies or acute lymphatic leukemia. Concordance between the 2 methods was 79.5%, with all discordant samples positive in qPCR but negative in STR. Of the latter, sporadic qPCR positivity without clinical correlates was seen mostly in BM samples early post-transplant. In 7 of 21 patients with available follow-up samples in the first months after transplantation, qPCR but not STR revealed low levels (1%) of sustained host chimerism in PB, reflecting delayed engraftment or persistent mixed chimerism (PMC). These conditions were associated with donor-recipient cytomegalovirus (CMV) serostatus and early CMV reactivation but not with immunosuppressive regimens or clinical outcome. qPCR predicted all 8/8 relapses with samples in the 6 months before onset by sustained positivity in both PB and BM compared with 1/8 relapses predicted by STR mainly in BM. The response kinetics to donor lymphocyte infusions for the treatment of PMC or relapse was shown by qPCR but not STR to be protracted over several months in 3 patients. Our results demonstrate the superior clinical utility of qPCR compared with STR for monitoring subtle changes of host chimerism associated with different clinical conditions, making a case for its use in the clinical follow-up of transplant patients.

Aim Next-generation sequencing (NGS) provides a cost and time efficient approach for routine HLA typing. Analysis software typically presumes a diploid genotype with counts of sequencing reads from homozygous or heterozygous genotypes. However, chimeric samples with more than two alleles per locus and imbalanced read counts may occur, for instance, after partially HLA-mismatched hematopoietic stem cell transplantation. Detection of alleles at even very low allele frequencies can then become diagnostically relevant in the context of post-transplant relapse. Here, we describe and validate a novel algorithm for rare HLA allele detection using NGS data. Methods Sequencing reads are mapped against expected HLA-alleles in a chimeric DNA-sample. Only polymorphic nucleotide positions differentiating the expected allele sequences are considered for further analysis. Reads are classified based on the frequency of discriminatory di-nucleotides. The classification algorithm was validated using in vitro admixtures of DNA samples down to allelic frequencies of 0.5 percent based on NGS data targeting exons 2 and 3 for the HLA genes A, B, C, DQB1, DRB1, and DPB1. Results We found that sensitivity and specificity are allele and locus-specific and depend on the number of sequence differences between the alleles. In each of the analyzed admixture samples a subset of the 12 amplicons harbored sufficient sequence differences to detect genotypes at dilutions as low as 0.5 percent. Fig. 1 shows an example for 3 amplicons. Conclusions The proposed algorithm may support the application of cost effective NGS for sensitive detection of HLA microchimerism in different settings including transplantation and pregnancy. Download : Download high-res image (200KB) Download : Download full-size image