Genetic Aberrations in Adult B-Precursor Acute Lymphoblastic Leukemia---Characterization of Clinically Relevance Subtypes and Functional Study on Novel Mutants Identified

Background: B-cell precursor acute lymphoblastic leukemia (B-ALL) accounts for 75%~80% of adult ALL. It has long been recognized that cytogenetic alterations have great impact on the treatment outcomes.Adult ALL is characterized by a high occurrence of Philadelphia (Ph) chromosome, a subset of very high-risk ALL. Recently, genome-wide approaches have identified a new subgroup of BCR-ABL1 negative childhood ALL with gene expression profiles similar to Ph (+) patients, called Ph-like ALL which conferred a poor outcome. Furthermore, novel genetic alterations with therapeutic relevance have been identified in Ph-like ALL and lead to the use of the approved and investigational targeted therapy in childhood ALL. In addition, novel CREBBP mutations are found to be associated with inferior survival and drug resistance in pediatric ALL. The treatment outcomes of adult ALL patients are worse than children. The occurrence and prognostic relevance of these newly identified genetic aberrations in adult ALL are largely unknown. Purpose: Our aims of this study are (1) to set up the methodology for identifying the Ph-like subgroup and to characterize the genetic subtypes of CRLF2 deregulation and kinase fusions involved in adult Ph-like ALL; (2) to detect the common gene deletions involving B-lymphocyte development, differentiation, and cell cycle control in adult B-ALL; (3) to detect the recurrent mutations in key cellular pathways with prognostic and/or therapeutic relevance in adult B-ALL; (4) to study the in vitro biological functions and drug response to CREBBP mutants identified in this project; (5) to correlate the genetic aberrations and their co-existed alterations with clinical features and outcomes in adult B-ALL. Materials and Methods: RNA and gDNA of bone marrow samples at diagnosis from 150 adult patients with B-ALL (Ph-negative N=100 and Ph-positive N=50) will be extracted. The Ph-like B-ALL will be identified by using prediction analysis of microarrays with a low density microarray card. Multiplex Ligase Probe Amplification methodology will be used to detect the deletions of IKZF1, PAX5, CDKN2A/B and CRLF2. We will use RT-PCR to detect ABL1, JAK2 and PDGFRB translocations, and FISH analysis for IGH-CRLF2. Gene mutational analyses will be performed by next generation sequencing with Ion Torrent PGMTM. Wild-type and new CREBBP mutants will be constructed and expressed in B-ALL cell lines to study the cell growth and survival. Cell cycle, apoptosis and cell differentiation will be analyzed by flow cytometry. Apoptosis-related proteins due to CREBBP mutations will be examined by Western blot analysis and other associated proteins by coimmunoprecipitation analysis. Acetylation at histone H3 and H4 will be measured by Western blot analysis. Histone deacetylase inhibitor responsiveness in B-ALL cell lines with expressing wild-type and CREBBP mutants will be analyzed for cell viability using MTT cell viability assay. Significance: The results of this study will give insight into the genetic basis underlying the worse outcome of adult B-ALL as compared to pediatric cases. The identification of genetic alterations with prognostic and therapeutic relevance will provide an opportunity to incorporate tyrosine kinase or JAK inhibitors to the current chemotherapy regimens, which are expected to improve the treatment outcomes of adult patients with B-ALL.

Project ID:PC10309-0117
External Project ID:MOST103-2314-B182-052