Leukemia specific loss of heterozygosity of MHC in a CLL patient: Disease state impacts timing of confirmatory typing
A 63 year old white male with refractory B-CLL presented for allogeneic HSCT evaluation; HLA typing was performed on PBL at time of WBC= 53K, ALC= 47K and revealed homozygosity at Class I locus and heterozygosity at Class II locus. Two siblings were full mismatches with the recipient and an unrelated search initiated. The patient was treated with Fludaribine and Rituxan complicated by aplastic anemia and bacteremia. Prior to transplant, confirmatory typing performed on PB revealed two full haplotypes at Class I and II. Sample identification error and the presence of third party lymphocyte engraftment as a result of prior red cell or granulocyte transfusion(s) were ruled out by STR analysis of 8 loci of all samples, T and B cells from cryopreserved PB at blast crisis were HLA typed independently. T cell typing yielded both complete haplotypes (genotype verified by offspring HLA typing); B cells typed for homozygous haplotype indicating loss of heterozygosity of MHC locus. Microarray based comparative genomic hybridization of tumor cells confirmed LOH at 6p.
- "...nancies may result in the identification of polymorphisms due solely to somatic mutations . For this reason, our laboratory's policy is to verify all putative novel alleles discovered in hem..."Therefore, theoretically , after identification of a novel allele during routine clinical testing, all that would be needed to complete the IMGT/ HLA submission requirements would be to sequence a second DNA sample from the patient. It should be pointed out that characterizing peripheral blood specimens from patients with hematologic malignancies may result in the identification of polymorphisms due solely to somatic mutations . For this reason, our laboratory's policy is to verify all putative novel alleles discovered in hematopoietic cell transplant patients by testing buccal cell-derived DNA.
[Show abstract] [Hide abstract] ABSTRACT: Loss of heterozygosity (LOH) is a common event in malignant cells. In this work we introduce a new approach to identify patients with loss of heterozygosity in the HLA region either at first diagnosis or after HLA mismatched allogeneic HSCT. Diagnosis of LOH requires a high purity of recipient target cells. FACS is time consuming and also frequently prevented by rather nonspecific or unknown immune phenotype. The approach for recipient cell enrichment is based on HLA targeted complement-dependent cytotoxicity (CDC). Relative fluorescent quantification (RFQ) analysis of HLA intron length polymorphisms then allows analysis of HLA heterozygosity. The approach is exemplified in recent clinical cases illustrating the detection of an acquired allele loss. As illustrated in one case with DPB1, distinct HLA loci in donor and patient were sufficient for both proof of donor cell removal and evaluation of allele loss in the patient's leukemic cells. Results were confirmed using HLA-B RFQ analysis and leukemia-associated aberrant immunophenotype (LAIP) based cell sort. Both results confirmed suspected loss of HLA heterozygosity. Our approach complements or substitutes for FACS-based cell enrichment; hence it may be further developed as novel routine diagnostic tool. This allows rapid recipient cell purification and testing for loss of HLA heterozygosity before and after allogeneic HSCT in easily accessible peripheral blood samples.
- "...In addition, loss of HLA heterozygosity was also described prior to any treatment [4, 6–10] in malignant cells, which might be a serious confounder in a patient's HLA typing. Despite its vital importan..."In addition, loss of HLA heterozygosity was also described prior to any treatment [4, 6–10] in malignant cells, which might be a serious confounder in a patient's HLA typing. Despite its vital importance, copy number neutral loss of heterozygosity is easily missed by standard methods.
- [Show abstract] [Hide abstract] ABSTRACT: Genomic loss of the mismatched HLA is a recently described mechanism of leukemia immune escape and relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Here we first evaluated its incidence, risk factors, and outcome in 233 consecutive transplants from partially HLA-mismatched related and unrelated donors (MMRD and MMUD, respectively). We documented 84 relapses, 23 of which with HLA loss. All the HLA loss relapses occurred after MMRD HSCT, and 20/23 in patients with acute myeloid leukemia. Upon MMRD HSCT, HLA loss variants accounted for 33% of the relapses (23/69), occurring later than their 'classical' counterparts (median: 307 vs 88 days, P<0.0001). Active disease at HSCT increased the risk of HLA loss (HR:10.16, CI:2.65-38.92, P=0.001), whereas older patient age had a protective role (HR:0.16, CI:0.05-0.46, P=0.001). A weaker association with HLA loss was observed for graft T cell dose and occurrence of chronic graft-versus-host disease. Outcome after 'classical' and HLA loss relapses was similarly poor, and second transplantation from a different donor appeared to provide a slight advantage for survival. In conclusion, HLA loss is a frequent mechanism of evasion from T cell alloreactivity and relapse in patients with myeloid malignancies transplanted from MMRDs, warranting routine screening in this transplantation setting.Leukemia accepted article preview online, 05 November 2014. doi:10.1038/leu.2014.314.0Comments 5Citations
- 0Comments 0Citations
- [Show abstract] [Hide abstract] ABSTRACT: The HLA genes are the most polymorphic of the human genome, and novel HLA alleles are continuously identified, often by clinical Sanger sequencing-based typing (SBT) assays. Introduction of next-generation sequencing (NGS) technologies for clinical HLA typing may significantly improve this process. Here we compare four cases of novel HLA alleles identified and characterized by both SBT and NGS. The tested NGS system sequenced broader regions of the HLA loci, and identified novel polymorphisms undetected by SBT. Subsequent characterization of the novel alleles in isolation of coencoded alleles by SBT required custom-designed primers, while the NGS system was able to sequence both alleles in phase. However, the tested assay was unable to amplify buccal cell DNA for subsequent NGS sequencing, presumably due to the lower quality of these samples. While NGS assays will undoubtedly increase novel allele identification, more stringent DNA sample requirements may be necessary for this new technology.0Comments 0Citations