Droplet digital PCR is a sensitive method for detecting refractory acute myeloid leukemia (AML) clones in peripheral blood and saliva ddPCR for detecting AML clones

Presented at AACR 2018 Annual Meeting

  • Session Category: Prevention Research
  • Session Title: Prevention, Interception, and Early Detection Research
  • Session Date and Time: Tuesday, April 17, 2018 1:00 PM - 5:00 PM
  • Location: McCormick Place South, Exhibit Hall A, Poster Section 11
  • Poster Board Number: 7
  • Permanent Abstract Number: 3253

ABSTRACT: Droplet digital PCR (ddPCR) is a highly sensitive and rapid method for detecting mutant allele frequency (MAF). In preliminary work, our lower limit of detection for common myeloid gene mutations was 0.001% in peripheral blood and bone marrow compared to 0.1% with flow cytometry and 0.01% with real-time quantitative PCR, and turn-around-time is 1 day. Furthermore, we detected leukemic mutant alleles in peripheral blood (PB), introducing the possibility of sparing painful bone marrow biopsy procedures to determine treatment response. Thus, we hypothesized that ddPCR is a feasible and accurate method for monitoring leukemic disease burden in PB for the prospective care of patients (pts) with AML. Eighteen patients (pts) with de novo, relapse/refractory, and secondary AML were recruited to an IRB approved study (NCT02435550) and bone marrow (BM), peripheral blood (PB), and saliva were collected at standard clinical visits. Gene mutations were identified by whole exome sequencing (WES) of BM specimens at study entry. For ddPCR interrogation, genomic DNA was isolated (Qiagen), and select primers and probes (Bio-Rad/IDT) were developed based on variants identified in WES data. Case-specific primers and probes were validated on archived specimens obtained at study entry. 12/18 pt mutanomes met criteria for primer/probe design. 8 pts are in the primer/probe design and validation stage and 4 have completed validation and serial analyses. WES identified, and ddPCR confirmed, at least 1 mutation per patient at the study entry timepoint. The mutations included NRAS G13R, NRAS G12A, CSF3R T618I, and IDH2 R172K. In 2 cases, we observed a reduction in both PB and saliva MAF that were consistent with the reduction in both BM and PB blasts after treatment, resulting in complete remissions. Although PB blasts were reduced in a third pt receiving ruxolitinib, the persistence of their CSF3R MAF in PB indicated a resistant AML clone. WES revealed the presence of NRAS G13R variant in a secondary AML pt, however, WES did not detect this NRAS G13R variant in a cryopreserved BM specimen obtained at the pts MDS diagnosis. Interestingly, ddPCR was able to detect NRAS G13R variant at 0.1% MAF in a PB sample obtained at MDS diagnosis, demonstrating the ultra-sensitive detection of rare variants within a sample, and highlighting the subclonal evolution of this pt’s malignancy. Rapid detection of myeloid-related somatic mutations in a variety of tissue sources (i.e. saliva, PB) will allow for non-invasive monitoring of AML tumor burden. ddPCR may be used to observe molecular response to treatment and to detect relapse prior to clinically indicated BM biopsies.


ResearchMatthew Matassa