Monitoring therapy responses at the leukemic subclone level by ultra-deep amplicon resequencing in acute myeloid leukemia

Abstract

In our individualized systems medicine program, personalized treatment options are identified and administered to chemorefractory acute myeloid leukemia (AML) patients based on exome sequencing and ex vivo drug sensitivity and resistance testing data. Here, we analyzed how clonal heterogeneity affects the responses of 13 AML patients to chemotherapy or targeted treatments using ultra-deep (average 68 000 × coverage) amplicon resequencing. Using amplicon resequencing, we identified 16 variants from 4 patients (frequency 0.54-2%) that were not detected previously by exome sequencing. A correlation-based method was developed to detect mutation-specific responses in serial samples across multiple time points. Significant subclone-specific responses were observed for both chemotherapy and targeted therapy. We detected subclonal responses in patients where clinical European LeukemiaNet (ELN) criteria showed no response. Subclonal responses also helped to identify putative mechanisms underlying drug sensitivities, such as sensitivity to azacitidine in DNMT3A mutated cell clones and resistance to cytarabine in a subclone with loss of NF1 gene. In summary, ultra-deep amplicon resequencing method enables sensitive quantification of subclonal variants and their responses to therapies. This approach provides new opportunities for designing combinatorial therapies blocking multiple subclones as well as for real-time assessment of such treatments.

The evolutionary landscape of chronic lymphocytic leukemia treated with ibrutinib targeted therapy

“…We hypothesized that subclones within each patient may be differentially affected by ibrutinib treatment resulting in clonal shifts, and that these changes may be apparent early during therapy 18 , 19 . We therefore performed whole-exome sequencing (WES) on a median of 3 (range 3–5) pre-relapse peripheral blood CLL samples with median depth of coverage of ×107 (interquartile range [IQR] of 97–119X, Supplementary Data 1 ).…”

“…However, this therapy also exerts strong selective pressure, which can promote the eventual outgrowth of resistant subclones. We therefore undertook an effort to perform unbiased sequencing of serially collected samples in order to define the evolutionary dynamics of ibrutinib-treated CLL, as we and others have shown that closely timed temporal sampling of tumor samples is a powerful approach to characterize such changes 16 , 18 , 19 .…”

The contribution of evolutionary game theory to understanding and treating cancer

“…Furthermore, often only a fraction of the tumor is isolated, which does not represent the complete genomic and phenotypic landscape, and the detection of small lesions and deriving biopsies from them is a major challenge [91,123]. [36,115,116,121]. To be able to use the genotypic information obtained from ctDNA, we need to know the relationship between mutations and their phenotypic impact, i.e., the genotype-phenotype map [4,120].…”

Harnessing Tumor Evolution to Circumvent Resistance

“…The authors showed that the kinetics of resistance depended on the pre-treatment size and relative fitness of the resistant subclone [13]. In AML, ultra-deep amplicon resequencing of serial blood samples similarly highlighted the impact of treatment on clonal heterogeneity [70]. Thus, liquid biopsies can capture the selective pressures imposed by treatment, track the outgrowth of resistant subclones, and may also inform adaptive therapeutic strategies that target the tumor’s changing composition.…”