N Engl J Med 2009;361: R2 Jung Kook Wi / prof. Kyung Sam Cho Loss of Mismatched HLA in Leukemia after Stem-Cell Transplantation

Background Transplantation of hematopoietic stem cells –from a –from a haploidentical family donor –Curative option for patients with high-risk hematologic cancers The major limitation of this strategy –the risk of severe GVHD –alloreactions mediated by donor T cells –against the recipient’s unshared HLA haplotype The feasibility & efficacy of infusions of haploidentical donor T cells haploidentical donor T cells

Background Relapse still ??? Mechanism ??? The infusion of donor T cellsRapid reconstitution of the immune systemThe graft-versus-leukemia Genomic loss Of the recipient’s mismatched HLA haplotype tumor escape from the selective pressure Mechanism of tumor escape from the selective pressure of a patient-specific graft-versus-leukemia reaction

MethodsPatientsRetrospectively –Hematologic cancers –One or more haploidentical hematopoietic stem-cell transplantations –At the San Raffaele Hospital in Milan ( ) All (43 patient) : high-risk hematologic myeloid cancers –36 with AML & 7 with high-risk MDS –26 with transplantation & 17 more than one

Methods Chimerism Analyses Monthly in samples of bone marrow aspirate –short-tandem-repeat amplification (STR) –genomic HLA typing Loss of Heterozygosity –The use of polymerase-chainreaction amplification of 12 highly polymorphic short-tandem-repeat markers spanning the entire length of chromosome 6 –The Illumina Human CNV370-Quad Bead Array –The Affymetrix Human SNP Array 6.0 single-nucleotide- polymorphism (SNP) array

Methods In Vitro Evaluation of Graft-versus-Leukemia Effect Separated peripheral-blood mononuclear cells –from the stem-cell donor for Patient 16 –from Patient days after the first hematopoietic stem-cell transplantation85 days after the first hematopoietic stem-cell transplantation 96 days after the second transplantation96 days after the second transplantation –from a healthy HLA-mismatched subject Cells plated with irradiated mononuclear cells The function of responder cells from the mixed lymphocyte culture –tested after each stimulation –the use of 51Cr-release, enzyme-linked immunospot (ELISpot) –[3H] thymidine-incorporation assays

Results Clinical Observations Studies of donor–host hematopoietic chimerism –carried out monthly after transplantation in all 43 patients –short-tandem-repeat amplification and HLA typing –looking for a reappearance of the host hematopoiesis in the BM –often predicts relapse In all 17 patients, relapse was confirmed –host origin on the basis of short-tandem-repeat chimerism five Surprisingly, in five of these patients, not genomic HLA typing of BM cells did not detect h ost-specific HLA alleles.

Figure 1 Identification of Mutant Variants of Leukemic Cells Not Detected by Chimerism Analysis with HLA Typing.

Figure 2. Genomic Loss of the Patient-Specific HLA Haplotype by Leukemic Cells after Transplantation in Patient 16.

Figure 3. Loss of the Patient-Specific HLA Haplotype through Extensive Rearrangements in Chromosome 6, Leading to Acquired Uniparental Disomy.

Figure 3. Loss of the Patient-Specific HLA Haplotype through Extensive Rearrangements in Chromosome 6, Leading to Acquired Uniparental Disomy.

Functional Study of the Graft-versus- Leukemia Response

Conclusions After transplantation of haploidentical hematopoietic stem cells and infusion of donor T cells, leukemic cells can escape from the donor’s anti - leukemic T cells through. the loss of the mismatched HLA haplotype. This event leads to relapse.