210P - Ultra-sensitive monitoring of leukemia patients using superRCA mutation detection assays

Background

Rare tumor-specific mutations in patient samples serve as excellent markers to monitor the course of malignant disease and responses to therapy in clinical routine, and improved assay techniques are needed for broad adoption. We describe herein - superRCA assays - which provides for rapid and highly specific detection of DNA sequence variants present at very low frequencies in DNA samples. Using a standard flow cytometer we demonstrate precise, ultra-sensitive detection of single-nucleotide mutant sequences from malignant cells against a 100,000-fold excess of DNA, to follow the course of patients treated for acute myeloid leukemia (AML).

Methods

Sequence of interest are first enriched by targeted PCR amplification from a patient sample The amplified sequences are next converted to DNA circles that are subjected to rolling-circle amplification (RCA). Padlock probes specific for mutant or wild-type sequences are then used to probe the repeated sequences of the RCA products with exquisite specificity, followed by RCA of the circularized probes. The large DNA clusters that result from each starting DNA circle are referred to as superRCA products.

Results

With the spike-in samples, we have succcessfully demonstrated that superRCA assay can reliably detect IDH mutation at 1 in 100,000 levels, as comparison, the ddPCR assays sensitivity was limited at 1 in 1000 levels due to high false positive event rate and high standard deviations. In 3 longitudinal AML patient studies, these patients were initially high with IDH mutations at the diagnosis of AML, then after the stem cell transplantation treatment, the mutation levels was decreased to undetectable levels both with superRCA and ddPCR assay. But with superRCA assay, we were able to detect IDH mutation at 1 in 10,000 before the patient were fall complete remission, while with ddPCR assay it was impossible to detect such intermediate conditions for the patient due to the limited sensitivity of ddPCR assay.

Conclusions

The superRCA assay procedure is suitable for routine use by virtue of its high sensitivity and simplicity. The 3-hr protocol only requires a sequence of five additions to a DNA sample, separated by incubations, before reaction products are analyzed using a standard flow cytometer.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

The research was funded by The Swedish Research Council, the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement n. 294409 (ProteinSeq), IngaBritt och Arne Lundbergs Forskningsstiftelse, Vinnova Medtech4Healthmedtech4health-collaborative projects for improved health (2019-01464), The Swedish Foundation for Strategic Research (SB16-0046), Torsten Söderbergs Stiftelse (M130/16) and The Swedish Cancer Society (19 0384 Pj).

Disclosure

L. Chen: Financial Interests, Institutional, Officer: Rarity Bioscience AB. U. Landegren: Financial Interests, Institutional, Ownership Interest: Navinci AB. All other authors have declared no conflicts of interest.