ALBERT

Description: Introduction Although steady progress of effective chemotherapy in childhood acute lymphoblastic leukemia (ALL) carried with exceeding 80% of individuals now cured, the majority of adult patients with ALL are not cured by chemotherapy, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative option. However, relapse remains the most leading cause of death after allo-HSCT. Adverse genetic alterations are generally accepted to be responsible for treatment failure and relapse. Several structural chromosomal alterations including rearrangement of the myeloid-lymphoid or mixed-lineage leukemia gene (MLL) and Philadelphia chromosome (Ph), have been mostly found in relapsed ALL. However, many Ph-negative (Ph-) ALL patients with normal karyotype , lacking known risk factors, also experienced relapse. The underlying pathologic determinants leading to relapse and prognostic markers in these cases remain poorly understood. More importantly, allo-HSCT is a distinct treatment option from tradtional chemotherapy and has 2 important forms to eliminate and select on malignant cells. The malignant cells that go on to causing relapse must initially survive ablation of chemotherapy before allo-HSCT and conditioning regimen in allo-HSCT. Then, after allo-HSCT, they must survive the effect of graft-versus- leukemia (GVL) reaction. Following this rationale, we hypothesized that there may be pivotal genetic causes confer leukemic cells a fitness advantage to undergo huge selective pressures and expand after allo-HSCT. To elucidate the genomic basis underlying relapse after allo-HSCT to aid to discover novel predictive biomarkers and identify therapeutic targets, we carried out the first whole-exome sequencing analysis in longitudinal matched samples from diagnosis to relapse after allo-HSCT in adult patients with the most common subtype of ALL, Ph- B-cell ALL (B-ALL). Methods Whole-exome sequencing was conducted for 9 genomic DNA samples from 3 relapsed cases with Ph- B-ALL (discovery cohort) at 3 specific time points including: diagnosis, complete remission (CR) after induction chemotherapy before allo-HSCT, relapse after allo-HSCT to discover candidate relapse-associated mutated genes. We identified putative somatic mutations by comparing each tumor ( diagnostic samples or relapsed samples) to normal (CR samples) from the same patient. To confirm candidate somatic gene mutations, screen relapse-associated gene mutations and define the frequency of somatic mutations identified by whole-exome sequencing analysis, we further carried out target genes whole coding regions sequencing in an ALL extended validation cohort including 58 adult Ph- B-ALL cases, where 27 patients experienced relapse at a median time of 6.5 (range 2-33) months after allo-HSCT and 31 patients did not relapse after allo-HSCT at a median follow-up for 34 (range 12–56) months. Results (1) We discovered novel associations of recurrently mutated genes (CREBBP, KRAS, PTPN21) with the pathogenesis of adult Ph- B-ALL relapse after allo-HSCT, which were mutated in at least two relapsed cases, but were not mutated in non- relapsed patients. (2) The generation of high-depth whole-exome sequencing data in longitudinal matched samples from diagnosis to relapse after allo-HSCT in initial 3 patients allowed us to directly assessed the evolution of somatic mutations. Our data suggested that in the progression of leukemia relapse after allo-HSCT, the relapse clone had a clear relationship to the diagnosis clone, either arising from a subclone already exsiting in the diagnostic tumor, or originating from a common preleukemic progenitor with the diagnosis clone. In the latter pattern, the relapse clone acquires new genetic alterations while retaining some but not all of the alterations found in the diagnostic tumor. In contrast, in some cases, leukemia recurrences afer allo-HSCT may be composed of second malignancies with completely distinct sets of mutations from the primary tumor. Conclusions Our study is the first to explore genetic basis of adult Ph- B-ALL from diagnosis to relapse after allo-HSCT over time, which will provide novel genetic biomarkers on risk “index” to improve individualized treatment intensification and intervention strategies, and potential therapeutic targets for Ph--ALL relapse after allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction CEBPA gene encodes CCAAT/enhancer-binding protein-alpha (C/EBPα), a crucial granulocytic differentiation factor and tumor suppressor in hematologic and non-hematologic malignancies. CEBPA gene is mutated in approximately 5–14% of acute myeloid leukemia (AML) patients and exhibited 3 different types of mutations, germ-line N-terminal mutation, N-terminal frameshift mutation and C-terminal mutation. Although murine models provide the functional consequences of CEBPA gene mutations in human hematopoiesis, the impacts of different mutations on abrogating C/EBPα functions and those of germ-line N-terminal mutations on bone marrow microenvironment have been received less attention. Methods In our previous research, we have provided the first report of multiple mutations of CEBPA contributing to the transformation of donor cells to the leukemic phenotype after allogeneic hematopoietic stem cell transplantation and identified 3 different CEBPA gene mutant forms which disrupted 3 major functional domains of C/EBPα respectively (H Xiao, Blood 2011; 117: 5257-5260). The patient and his donor-sister both harbored the N-terminal germ-line mutation (584_589dup disrupting the TAD2 domain of protein). Susceptible donor hematopoietic cells evolved to overt AML by developing two somatic CEBPA mutations, the N-terminal frameshift mutation (247dupC causing overproduction of truncated 30-KDa isoform (C/EBPα- p30) lacking the TAD1 domain) and the C-terminal mutation (914_916dup disrupting the bZIP domain), in the patient's microenvironment. We used these 3 mutant forms, as well as CEBPA gene wild type to subclone into pLenti6.3-MSC vector. Human leukemia cell lines (NB4, Kasumi-1, HL60, K562) and mouse myeloid progenitor cell line (32Dcl3) were transduced by different mutant forms to assess the impacts on blockage the major functions of C/EBPα including antileukemia effect and inducing granulocyte differentiation. To further discover previously unkown tumor suppressor genes dysregulated by C/EBPα mutant forms, the gene expression profiles of NB4 cells stably transfected with CEBPA gene different mutant forms were compared with that of CEBPA gene wild type vector by using Affymetrix PrimeView human gene expression microarray analysis. On the other hand, human normal bone marrow mesenchymal stromal cell ( MSC ) was transduced by N-terminal germ-line mutation to assess the impacts on the capacity of MSC to differentiate towards the osteogenic/adipogenic lineages, migrate and protect to leukemia cells. Results (1) The N-terminal germ-line mutation retains the functions of inducing apoptosis in leukemic cells and granulocyte differentiation of C/EBPα.The truncated C/EBPα-p30 protein mutant, produced from the N-terminal frameshift mutation, abrogates the effect of inducing apoptosis in leukemia cells. The C-terminal mutation (914_916 dup) abrogates both the effects of inducing apoptosis in leukemic cells and of promoting G-CSF-induced differentiation of 32Dcl3 cells into mature neutrophile granulocyte. (2) Gene expression microarray profiling analysis showed that compared with CEBPA gene wild type, leukemia-associated CEBPA somatic mutations, the N-terminal frameshift mutation and the C-terminal mutation, significantly inhibited the expression of ULBP2, an innate surface ligand of the natural killer (NK) cell receptor NKG2D, ultimately contributed to resistance to NK cell-mediated cytotoxicity. (3) MSCs harboring CEBPA N-terminal germ-line mutation showed impaired differentiation potential to osteogenic lineage by downregulation the expression of osteogenic genes (BSP1 and Runx2) , however similar in differentiation potential to adipogenic lineage, migration and the protection to leukemia cells, compared with MSCs with CEBPA wild type. Conclusions Our data provide clues to support that the N-terminal frameshift mutation of CEBPA works as a class I mutation, while the C-terminal mutation works as both of class I and class II mutations in inducing leukemia. Furthermore, those leukemia-associated CEBPA somatic mutations abrogate the tumor suppressor function of C/EBPα by inhibiting NKG2D-mediated NK cell cytotoxicity. The N-terminal germ-line mutation retains the major functions of C/EBPα, however impaired the osteogenic differentiation potential of bone marrow stromal cells, which is important to support hematopoiesis. Disclosures: No relevant conflicts of interest to declare.