Emergence of Sub-Clonal Drug Resistance Mutations during CML Therapy
Authors: Schmitt Michael W. , Pritchard Justin R. , Hodgson Graeme , Rivera Victor M. , Aminov Bella I. , Beppu Lan , Loeb Lawrence A. , Radich Jerald P. 
Published: October 2019

Abstract

Tyrosine kinase inhibitors (TKIÕs) vastly improve survival in chronic myeloid leukemia (CML). However resistance is a common occurrence, and is most frequently mediated by point mutations in the Abl kinase. There have been conflicting reports regarding the frequency of resistance mutations in the Abl kinase at the time of CML diagnosis, with some studies indicating that a large number of resistance mutations are pre-existing in many patients. However, a high burden of pre-existing resistance mutations is seemingly at odds with the ability of TKIÕs to initially control CML for months to years in the large majority of patients.
Nearly all prior studies have used dozens of cycles of PCR amplification to enrich the Abl gene prior to mutation detection. PCR amplification introduces thousands of errors throughout the gene, which makes accurate identification of mutations challenging. Methods of mutation detection such as next-generation sequencing (NGS) are also error prone, with a background error rate near 1%. These high rates of background mutation render accurate detection of minority variants difficult or impossible.
We have developed an alternate DNA mutation detection method, termed Duplex Sequencing (Schmitt MW et al, Nature Methods 2015;12:423-425), which improves on the accuracy of DNA sequencing by >100,000 fold. In Duplex Sequencing, the two strands of single DNA molecules are individually tagged and sequenced. True mutations are present at the same position in both strands and are complementary, whereas artifacts arising from amplification or sequencing errors are seen in only one strand and are not scored. We have additionally developed methods for >1,000,000 fold enrichment of the Abl gene from human cells. This enrichment protocol allows for unbiased detection of all possible mutations throughout the Abl gene, with an accuracy exceeding 99.9999%.
We applied Duplex Sequencing to 18 patients with newly diagnosed chronic phase CML (CP-CML). We sequenced the active site exons of Abl from tens of thousands of individual CML genomes; no sub-clonal resistance mutations were seen in any patient. We demonstrate that prior reports indicating frequent resistance mutations at the time of CML diagnosis may be attributable to artifactual mutations introduced by reverse transcription and PCR amplification.
We are now studying patients from the PACE trial (Cortes JE et al. N Engl J Med. 2013;369:1783-1796); these patients were heavily pre-treated, with the majority having received 3 or more prior TKIÕs. We identified 29 patients from the trial with a treatment-emergent mutation not detectable at baseline by conventional methods (NGS or Sanger), and have performed Duplex Sequencing on 9 of these patients to date. In contrast to treatment-naïve patients, we find that these heavily pre-treated patients commonly harbor multiple co-existing resistant sub-clones, most of which fall below the detection limit of conventional methods but are readily detected by Duplex Sequencing. In addition, in 44% of patients, mutations that emerged during ponatinib treatment were found as rare sub-clones at baseline (average detection limit of 1 in 11,412 cells), indicating that the mutant sub-clones pre-existed prior to ponatinib therapy. We are now sequencing the remainder of the cohort, and are increasing our detection limit through interrogation of a larger number of cells per patient.
Our results indicate the feasibility of detecting sub-clonal resistance mutations at the time of initiation of next-line TKI therapy in order to guide treatment, and argue that high-sensitivity methods for mutation detection are required to optimally stratify patients to the most appropriate TKI. Moreover, our approach can detect sub-clonal mutations in any gene, and could be broadly applicable for identification of sub-clonal drug resistance mutations relevant to other targeted therapies.
Figure 1. Detection of sub-clonal mutations in the Abl gene. Top panel: Next-generation sequencing of the Abl gene in a CML patient results in thousands of artifactual errors, preventing detection of low-level mutations. Bottom panel: Duplex Sequencing of the same sample eliminates artifactual errors, revealing a single point mutation in Abl. This mutation (E279K) confers resistance to imatinib. Figure 1. Detection of sub-clonal mutations in the Abl gene. Top panel: Next-generation sequencing of the Abl gene in a CML patient results in thousands of artifactual errors, preventing detection of low-level mutations. Bottom panel: Duplex Sequencing of the same sample eliminates artifactual errors, revealing a single point mutation in Abl. This mutation (E279K) confers resistance to imatinib.

Disclosures
Pritchard: ARIAD: Employment, Equity Ownership. Hodgson:ARIAD Pharmaceuticals: Employment, Equity Ownership. Rivera:ARIAD: Employment, Equity Ownership.