Molecularly targeted therapies have been exceptionally successful in the treatment of some malignancies, most notably chronic myeloid leukemia (CML). However in other cancers, e.g. Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL), drug resistance is a frequent occurrence. Resistance mutations may pre-exist as a consequence of intra-tumor heterogeneity; thus the extent of heterogeneity may determine the likelihood of treatment failure. Intra-tumor heterogeneity is challenging to quantitate, as heterogeneous mutations are likely to occur below the background error rate of conventional approaches for mutation detection. To overcome this limitation and explore the relationship between intra-tumor heterogeneity and drug resistance in Ph+ leukemias, we utilized single-molecule Duplex Sequencing (DS) of the ABL1 gene. DS eliminates sequencing errors by independently tagging the two strands of individual DNA molecules; true mutations are present at the same position in both strands, while PCR and sequencing errors are only present in one strand. DS has an error rate below 0.00001%, and thus enables quantitation of heterogeneous mutations with unprecedented resolution. We find that CP-CML, a disease that is often well controlled with targeted therapy, has an extremely low burden of intra-tumor heterogeneity at the time of diagnosis, with a sub-clonal mutation burden similar to that of normal individuals. Simulations which take into account the low cancer stem cell fraction of CP-CML and the sub-clonal mutation burden we measured reveal that only a minority of CP-CML patients will harbor pre-existing drug resistance, which may explain the unusual success of targeted therapy in this setting. Next, we studied patients with advanced Ph+ leukemias (refractory BP-CML and Ph+ ALL), which have a high rate of treatment failure and poor outcomes. We find that patients with BP-CML and Ph+ALL have a significant elevation in sub-clonal heterogeneity relative to those with CP-CML. We demonstrate that the higher extent of intra-tumor heterogeneity correlates with a significantly higher rate of drug resistance mutations and compound resistance (i.e. two concurrent resistance mutations) in patients. Unlike CP-CML, pre-existing mutations conferring resistance to targeted therapy are nearly certain in refractory BP-CML and Ph+ ALL as a consequence of the load of intra-tumor heterogeneity and high cancer stem cell fraction. We support this concept by three distinct approaches: (i) detection of pre-existing sub-clones that drive treatment failure; (ii) modeling the growth kinetics of resistant clones; and (iii) extrapolation of the mutation burden measured by DS. Our results suggest that intra-tumor heterogeneity influences the likelihood of resistance to molecularly targeted therapy. Quantitation of sub-clonal mutation load may be broadly applicable in other malignancies for predicting the likelihood of response to targeted therapy and monitoring disease progression.

Citation Format: Michael W. Schmitt, Justin R. Pritchard, Lan Beppu, J. Graeme Hodgson, Victor M. Rivera, Lawrence A. Loeb, Jerald P. Radich. Sub-clonal heterogeneity and risk of treatment failure in Philadelphia-positive leukemias [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2071. doi:10.1158/1538-7445.AM2017-2071

©2017 American Association for Cancer Research.
2017
American Association for Cancer Research.