Abstract 3686: Single cell assays of heterogeneity and clonality in leukemia

Abstract

Abstract A range of hypotheses have emerged as to why imatinib (a targeted inhibitor of bcr-abl) seems unable to eliminate the most primitive chronic myeloid leukemia (CML) cells, such as bcr-abl amplification and clonal evolution. The biology of the CML imatinib-resistant cell is of critical importance to understanding why despite high percentages of initial clinical responses to imatinib (>80% of patients in chronic phase at some point obtaining complete cytogenetic remission), resistance, relapse and/or progression occurs in 20-30% of cases and most patients in remission still have residual disease detected by sensitive PCR monitoring of the CML specific bcr-abl transcripts. In acute myeloid leukemia (AML) as well, patients are typically treated with a chemotherapeutic regimen that leads to clinical responses in 80-95% of patients. However, over half of these patients will relapse and eventually die of disease suggesting that a subpopulation of leukemic cells are capable of both surviving treatment and causing regrowth of the tumor. In both diseases, it has not been definitively established what the identity of the cells responsible for resistance and relapse is, nor what combination of mutations, gene expression changes, and alternative splicing events are required for these cells’ resistant phenotype. Assessing changes in gene expression in the bulk samples provides a valuable overall picture of the phenotype of the predominant cell type is or if there are large changes in expression in less frequent cells, but will completely mask changes exhibited by rare cells. One limiting factor in our ability to study more levels of heterogeneity in leukemia subpopulations is the lack of appropriately powerful and sensitive techniques to assay more complex facets of cellular behavior in such small populations. We have developed several multiplexed, high sensitivity analysis techniques and applied them to study multiple facets of cell behavior and identity in rare cell samples, down to the single cell level. By analyzing expression and genotype in smaller samples, direct from cells, we can analyze heterogeneity in expression and alternative splicing, combined with both cell surface markers and the presence of multiple mutations. These innovative techniques have allowed us to study clonality in AML samples in detail via a robust assay that can identify multiple mutations occurring concurrently in single cells. We have also optimized a protocol to simultaneously assay gene expression of 24 in 100-500 cell samples from flow sorted leukemia cell populations. Finally multiplexed assays for alternative splice variant detection in single cells has enabled us to study whether alternative splice variant expression of several genes occurs concurrently in the same cells, or in different cells of the population. With these techniques in hand we have the unique opportunity to begin studying the biology of these diseases from a previously unrealistic angle. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3686. doi:1538-7445.AM2012-3686