Rare Pre-Existing MDS Subclones Contribute to Secondary AML Progression

Background: MDS is composed of a dominant founding clone and typically one or more subclones that descend from the founding clone. Using whole genome sequencing (WGS) of secondary AML patients who were initially diagnosed with MDS, we previously showed that AML progression always coincided with expansion of at least one subclone. However, this emergent subclone was not always detectable in the antecedent MDS samples using high-coverage sequencing. It is currently unclear whether these emerging secondary AML-specific clones represent new mutations acquired in cells during treatment or selection of a rare subclone that was below the level of detection in the diagnostic MDS sample. Identification of such pre-existing subclones at MDS diagnosis could provide important prognostic information. Using an ultra-sensitive, error-corrected sequencing approach coupled with high coverage depths, we sought to determine whether secondary AML-specific mutations could be detected in antecedent MDS samples.

Methods: We studied 4 MDS patients who eventually progressed to secondary AML (mean 601 days to transformation; range 131-955) that had DNA from initial MDS banking, secondary AML, and skin (as a normal control). DNA from the secondary AML sample was whole genome (3 cases) or exome sequenced (1 case). Custom enrichment panels were designed to target somatic mutations identified in the secondary AML samples and used to sequence the prior MDS samples. All 4 MDS cases had at least one subclone present in the secondary AML sample that was not detected by standard panel-based re-sequencing of the initial MDS sample (539x average coverage). We designed custom molecular barcode-based reagents to perform high-sensitivity, error-corrected sequencing targeting mutations that were detected only at progression to secondary AML. These mutation sites were then sequenced to ultra-deep coverage in prior MDS samples. To determine the sensitivity and specificity of the assay, we first sequenced known dilutions of cell line DNA and showed a sensitivity of 75% to detect variant allele fractions (VAFs) of 0.06% (one mutant cell in 800 cells) while maintaining a specificity of >99.9%. Only reads with unique molecular barcodes that were seen at least three times and had >90% identical reads within a read family were considered. Samples were sequenced to an average total coverage depth of 187,000x (range 107k-474k fold) corresponding to 10,398 unique molecular barcodes of which 3,549 passed filtering (at least two unique read families were required to call a variant), resulting in a mean predicted maximum VAF sensitivity of 0.05% (one mutant cell in 1,000 cells).

Results: Using error-corrected barcode sequencing all 4 MDS cases had a subset of mutations detected that were originally only detected in the secondary AML; the mean VAF of detected mutations was 0.25% (range 0.02% to 3.9%) in the antecedent MDS samples, corresponding to 1 mutant cell in 200 cells. However, not all of the 'secondary AML-specific' mutations were detected in MDS samples. On average, only 17% (range 10-39%) of the 'secondary AML-specific' subclone mutations were detected in each MDS sample. This suggests that while some mutations pre-exist in an ancestral subclone in the MDS sample, others are acquired during the intervening months to years between MDS and progression to secondary AML. As an example, in two patients who were given decitabine prior to secondary AML progression, mutations detected in the secondary AML subclone that were not detected in the antecedent MDS sample using error-corrected barcode sequencing were more frequently C->G transversions compared to commonly acquired C->T transitions seen in MDS samples (p=.005; Mantel-Haenszel test). Increased C->G transversions are associated with DNA damage induced by decitabine, suggesting that in these two patients an ancestral subclone pre-existed in MDS that acquired additional decitabine-induced mutations during treatment (i.e., C->G transversions).

Conclusion: Collectively, the data indicate that very rare subclones pre-exist at MDS diagnosis. These pre-existing subclones can subsequently give rise to the dominant clone at progression to secondary AML. Additional mutations -- some induced by chemotherapy treatment --- are also acquired in ancestral subclones that pre-exist in MDS samples. The combination of subclonal mutations may ultimately contribute to disease progression.