ALBERT

Description: Introduction Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the best curative option for many patients with Acute Myeloid Leukemia and related malignancies, mostly due to the antileukemic activity of immune cells contained in the graft. Although in most of the cases HSCT can accomplish apparent disease eradication, frequently residual leukemic cells are able to evade elimination, eventually outgrowing and resulting in clinical relapse. Apparently indistinguishable from disease at diagnosis, relapses commonly display a more aggressive behavior, and most of the available therapeutic options are ineffective. Next-generation sequencing provides the opportunity to track specific alterations and disease subclones during the clinical history of patients, and from this information to identify mechanisms by which leukemic cells evade elimination. Methods Serial disease samples collected longitudinally during the complex clinical history of a patient with high-risk myelodysplastic syndrome were analyzed by genomic HLA typing and by high-depth exome sequencing (minimum depth of coverage 70x). Patient fibroblasts and mononucleated peripheral blood cells harvested at remission served as reference for germline HLA typing and exome sequence. Whenever possible, leukemic blast were FACS-purified. Sequencing data were aligned to the reference genome using the BWA aligner and analyzed using the tools developed from the Cancer Genome Analysis group at the Broad institute to identify newly acquired mutations, clonal segregation of novel and pre-existing mutations and quantitative clonal dynamics. Results A 54 year old patient with high-risk refractory anemia with excess blasts (pancytopenia, blasts 19%, adverse cytogenetics) underwent unmanipulated T cell-repleted HLA-haploidentical HSCT in the presence of active disease. After one year of remission a first relapse occurred. HLA typing of the leukemic blasts harvested at relapse demonstrated the selective genomic loss of the mismatched HLA haplotype targeted by donor T cells, a frequent mechanism of leukemia immune evasion after haploidentical HSCT, first described by our group (Vago et al, N Engl J Med, 2009). The patient was re-transplanted from a different haploidentical donor mismatched for the HLA alleles retained by the mutated variants; accordingly, T cells from this donor were expectedly alloreactive against the relapsed leukemia. The patient re-obtained complete remission, which was maintained for five years, before the occurrence of a second relapse. Unexpectedly, at this relapse leukemic cells had lost the HLA molecules mismatched with the second donor, and expressed those that had gone amiss at first relapse. Since at both relapses HLA haplotype loss was due to a stable genomic alteration, chromosome 6p acquired uniparental disomy (aUPD), any linear relation between the first and second relapse could be ruled out (Figure 1). Leukemic samples at diagnosis, first relapse and second relapse were further characterized by high-depth exome sequencing, revealing that most of the mutations present in each of the three samples were not found in the other two: only a minute fraction of the mutations could be identified in all three disease presentations, possibly comprising the “driver mutations” harbored by the original leukemic stem cell clone. Amongst these shared mutations we are currently validating the functional and epidemiological relevance of missense alterations in the TP53 tumor suppressor gene, in the NHEJ1 gene (related to chromosomal instability), and in the BTNL8 gene, encoding the costimulatory receptor B7-H5, which may putatively play a role in leukemia recognition by T cells. Conclusions Our results demonstrate that antileukemic immunity has a profound impact on leukemia clonal evolution, driving the selection of immuno-privileged subclones. Importantly, the genetic alterations shared between the leukemic variants that emerged at years of distance during the clinical history of our patient suggest the long-term persistence of a reservoir of leukemic stem cells which are resistant to, or in stable equilibrium with, the immune pressure that sculpted their progeny. Finally, the longitudinal approach adopted in this study holds promise for the identification of leukemia “driver” mutations, to provide new insights into leukemogenesis and new rationales for targeted eradicating therapies. Disclosures: Bordignon: MolMed SpA: Employment. Bonini:MolMed SpA: Consultancy.

Description: Introduction Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the best curative option for many patients with hematologic malignancies. Still, disease relapse remains a major clinical issue, and therapeutic options available to date are largely unsatisfactory. Thus interest has risen in how to anticipate the detection of residual malignant cells by more sensitive techniques, such as those based on quantitative PCR (qPCR), to prompt preemptive treatments. Adding further complexity to the issue of leukemia recurrence, our group described in the context of HSCT from partially incompatible donors novel variants of post-transplantation leukemia relapse, characterized by genomic loss of the mismatched HLA (Vago et al, N Engl J Med, 2009). These leukemia variants are not detected by routine diagnostic assays and are expected to be resistant to donor lymphocyte infusions, warranting the development of novel specific diagnostic assays and therapeutic algorithms. Methods Here we prospectively validated a commercial hematopoietic chimerism assay based on qPCR detection of 34 insertion/deletion (indel) polymorphisms mapping on 19 different chromosomes, all outside the HLA complex (AlleleSEQR® Chimerism Assay, Celera Genomics). The assay was tested on 403 bone marrow samples harvested from 115 transplanted patients during 18 months of follow-up. Results obtained from qPCR-based chimersim were compared with standard Short Tandem Repeat (STR) chimerism analysis performed in parallel on the same samples. To discriminate between “classical” and “HLA loss” relapses, we developed chimerism assays targeted to specific HLA-A allele groups, based on the same qPCR technology of the commercial “outside HLA” assay and targeted to display its same sensitivity. Results qPCR chimerism displayed high informativity, providing at least a patient-specific indel marker in all the patients analyzed and at least two markers in 109/115 patients (94.7%). When two polymorphic indel markers were analyzed in parallel on the same sample concordance was extremely high (r2=0.97). As expected, qPCR chimerism demonstrated a higher sensitivity in detection of residual host cells as compared to STR: 131/403 samples exhibit a host chimerism above 0.5% (32.5%) and 91/403 above 1% (22.6%), whereas 79/395 (20%) were positive in STR. qPCR chimerism could predict impending relapse with a sensitivity of 37.5% and a specificity of 72.5% for a 0.5% host threshold, and with a sensitivity of 29.2% and a specificity of 83.5% for a 1% host threshold, comparing favourably in terms of sensitivity with STR (sensitivity 20.8%, specificity 84.8%). We designed and validated 5 qPCR reactions able to specifically detect 9 different HLA-A allele groups, displaying a combined frequency of 81% in Caucasians, and providing an informative patient-specific HLA-A allele as chimerism marker to more than 50% of the patients transplanted from mismatched related donors in our centre. Technical validation of the newly developed reactions with artificial mixes of HLA-typed DNA demonstrated for each reaction the sensitivity to detect at least 0.2% target cells and efficiency above 80%. Clinical utility was tested on 80 bone marrow samples harvested from transplanted patients, using the new “inside HLA” reactions in combination with the commercial “outside HLA” assay: combination of the results obtained from the two assays allowed us to unequivocally discriminate HLA loss relapses (n=5) from classical relapses (n=15). Conclusion Hematopoietic chimerism detection by qPCR appears promising, displaying a higher sensitivity and a superimposable specificity as compared to techniques currently in use, and allowing by the novel assay we developed the simultaneous detection of leukemic variants characterized by genomic HLA loss. Disclosures: Bonini: MolMed SpA: Consultancy. Bost:Celera Genomics: Former Employee, Former Employee Other; KimerDx: Employment. Fleischhauer:Celera Genomics: Research Funding.

Description: INTRODUCTION: Although allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) can accomplish apparent leukemia eradication in most patients, residual tumor cells can persist over time and eventually outgrow, resulting in clinical relapse. The genetic landscape of relapsing leukemia is often markedly different from its counterpart at diagnosis, due to clonal evolution (Ding et al, Nature, 2012) and selection of treatment-resistant variants (Vago et al, N Engl J Med, 2009). A potential solution to treat, or even prevent, relapse is to identify and specifically target mutations occurring very early in the leukemic transformation process, and thus putative hallmarks of cancer progenitors. Next-Generation Sequencing (NGS) provides the opportunity to track disease subclones during the clinical history of patients, pinpoint founder alterations and identify the mechanisms by which leukemia evades elimination. METHODS: In the present study, we combined immunogenetic analyses and NGS to detail the complex clinical history of a patient with therapy-related myelodysplasia (t-MDS), diagnosed years after sequential intensive chemotherapy for B cell Acute Lymphoblastic Leukemia (B-ALL). Leukemic cells collected at serial time-points during the patient disease history (initial diagnosis of B-ALL, first presentation of t-MDS, relapse after first allo-HSCT, relapse after second allo-HSCT) were characterized by means of genomic HLA typing, HLA allele-specific quantitative PCR and whole exome sequencing, with a minimum depth of coverage of 70x. Patient fibroblasts and peripheral blood mononuclear cells (PBMCs) collected before the occurrence of t-MDS, as well as PBMCs from the two allogeneic HSC donors, served as controls and reference exomes. Targeted resequencing of mutations of interest was performed using the Fluidigm Access Array system. Gene segments encompassing newly-identified mutations in TP53, NHEJ1 and BTNL8and their respective wild-type counterparts were cloned into plasmids, and used to design and validate specific droplet digital PCR assays, using the Bio-Rad QX100 instrument. RESULTS: A 54-year-old patient with high-risk t-MDS received two subsequent allo-HSCTs from partially-incompatible family donors, and after each transplant experienced disease recurrences. Genomic HLA typing and HLA allele-specific qPCR demonstrated that both relapses were due to mutant variants of the original leukemia that had evaded immune pressure through genomic loss of the HLA haplotype targeted by the respective donor’s T cells. Immunogenetic studies ruled out any linear relationship between the two relapses, suggesting that both might have derived from a common HLA-heterozygous progenitor. Accordingly, whole exome sequencing demonstrated direct clonal evolution from the initial presentation of t-MDS to first relapse, whereas the large majority of mutations present at second relapse were unique to the sample. Only five non-synonymous coding mutations were present in all three t-MDS samples, comprising disrupting mutations in TP53, in NHEJ1 (a key factor in DNA double strand-break repair), and in the T cell costimulatory receptor BTNL8. Of notice secondary mutations, detected only in one or two of the three disease presentations, were predicted to result in partially overlapping functional effects, and could be modeled in a conjoint DNA damage repair network, centered around the putative founder mutation in TP53. By ultra-sensitive droplet digital PCR, the same TP53 mutation was backtracked throughout the whole nine years of patient clinical history, including the phases of apparent complete remission, up to a preleukemic progenitor present in the patient bone marrow at the time of B-ALL initial diagnosis. CONCLUSIONS: Our results demonstrate that therapies, and the immune pressure of allo-HSCT in particular, can have a dramatic effect in shaping leukemia clonal evolution. Importantly, by identifying leukemia founder mutations, we might further our insights into leukemogenesis, and identify novel targets for post-transplantation diagnostics and leukemia-eradicating therapies. Disclosures Bonini: MolMed S.p.A.: Consultancy.