Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of childhood associated with a poor prognosis. Recently, massively parallel sequencing has identified recurrent mutations in the SKI domain of SETBP1 in a variety of myeloid disorders. These lesions were detected in nearly 10% of patients with JMML and have been characterized as secondary events. We hypothesized that rare subclones with SETBP1 mutations are present at diagnosis in a large portion of patients who relapse, but are below the limits of detection for conventional deep sequencing platforms. Using droplet digital polymerase chain reaction, we identified SETBP1 mutations in 17/56 (30%) of patients who were treated in the Children's Oncology Group sponsored clinical trial, AAML0122. Five-year event-free survival in patients with SETBP1 mutations was 18% ± 9% compared with 51% ± 8% for those without mutations (P = .006).

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Molecular tests for genetic mutations play an important role in the diagnosis of cancer. Somatic mutations that drive the pathological features of most tumors have increasing promise as biomarkers for cancer prognosis and therapeutic efficacy. The detection of somatic mutations poses an analytical challenge due to the heterogeneous nature of most samples, where a gene carrying a mutation may differ from the highly abundant wild type sequence by only a single nucleotide. Although a variety of methods exist for mutation analysis, many have poor selectivity and fail to detect mutant sequence below 1 in 100 wildtype sequences. Methods that provide better discrimination and quantitation of somatic mutations are desirable. Here we present a simple strategy using droplet digital™ PCR (ddPCR™) for the detection of somatic mutations with high selectivity and sensitivity. Based on the simple principle of sample partitioning into water-in-oil microdroplets, this ddPCR method increases the abundance of a mutant DNA sequence up to 20,000 times compared to an equivalent bulk PCR reaction. Using conventional TaqMan chemistries and workflow, selectivities of up to 1/100,000 can readily be achieved in any laboratory. Here we present results on the use of ddPCR for the detection and quantitation of several clinically important mutations, including KRAS, c-KIT D816V and JAK2 from clinical samples such as bone marrow aspirates and FFPE. Results from ddPCR are compared to those of conventional approaches including allele specific real-time PCR and sequencing. This ddPCR method may play an important role in the earlier detection of cancer, monitoring the progress of disease and response to therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4859. doi:1538-7445.AM2012-4859

Juvenile Myelomonocytic Leukemia (JMML) is an aggressive myeloproliferative neoplasm of childhood with a 5-year event free survival of 52% after hematopoietic stem cell transplantation (HSCT). A hallmark of JMML is aberrant Ras pathway activation due to mutations in NF1, NRAS, KRAS, PTPN11 and CBL. However, robust predictors of response are lacking, as individual mutations are not reliably associated with outcome, and relapse remains the most common reason for treatment failure. Recently, massively parallel sequencing has identified recurrent mutations in the SKI domain of SETBP1 in a variety of myeloid disorders, including JMML (Piazza et al Nat Genet 2012, Makishima et al Nat Genet 2013, Sakaguchi et al Nat Genet, 2013). These mutations had a lower allelic frequency compared to Ras pathway mutations, but were associated with poor prognosis. These and other data suggested that SETBP1 mutations contribute to disease progression rather than initiation. We identified several patients with JMML who had clonal SETBP1 mutations detected at relapse. Analysis of mononuclear cell extracted DNA from serial samples of two patients who relapsed revealed an increase in the SETBP1 mutant allele frequency over time (Figure 1). Similarly, analysis of colonies plated in methylcellulose from serial time points indicated that the percentage of individual myeloid progenitor colonies that were heterozygous or homozygous for the SETBP1 mutation increased with each sequential sample despite intensive treatment. Based on these data, we tested the hypothesis that rare SETBP1 mutant clones exist at diagnosis in many patients who relapse, and that these rare cells undergo positive selection during treatment. Using a droplet digital PCR (ddPCR) technology with a detection threshold as low as 0.001% of mutant DNA, we identified SETBP1 mutations in 16/53 (30%) of diagnostic JMML specimens from children treated on Children's Oncology Group trial AAML0122. Of these mutations, 12 were subclonal and 4 were clonal. Event free survival (EFS) at 4 years in patients with SETBP1 mutations was 19% ± 10% compared to 51% ± 8% in those with wild type SETBP1 (p=0.006). While samples of patients who relapsed on the AAML0122 trial were not available for analysis, one patient recently undergoing treatment who had a subclonal SETBP1 mutation (0.45% allelic fraction) detected at diagnosis by ddPCR, demonstrated an overt SETBP1 mutation at relapse. Finally, we isolated and analyzed hematopoietic stem (HSC), multipotent progenitor (MPP), common myeloid progenitor (CMP), and granulocyte-monocyte progenitor (GMP) populations from a relapsed sample with a SETBP1 mutation. Sanger sequencing demonstrated that all four progenitor compartments were affected by the mutation. Analysis of additional samples is underway. We conclude that the presence of a subclonal mutation in SETBP1 is a novel biomarker of adverse outcome in JMML. Understanding the mechanisms underpinning SETBP1-mediated resistance and relapse, and further identifying therapeutic vulnerabilities of HSCs expressing these mutant proteins will be critical to improve outcomes for patients with JMML and other myeloid malignancies. Furthermore, the presence of a subclonal SETBP1 mutation at diagnosis might identify JMML patients who will benefit from more intensive conditioning prior to HSCT or from novel therapeutic strategies. Figure 1 Figure 1. Disclosures Troup: Bio-Rad Laboratories: Employment.