Absolute Q for Precision Oncology Applications
The Absolute Q was designed with precision medicine for oncology in mind, and late last year we demonstrated how it can impact patient care and treatment using three applications, in our publication in Nature Scientific Reports:
- EGFR p.T790M mutation detection down to just a handful of molecules
- Accurate quantification of the BCR-ABL1 gene fusion using a certified reference material
- Longitudinal monitoring of a novel transcript in a pediatric cancer patient
In this publication, we showcased the Absolute Q digital PCR (dPCR) Platform and microfluidic array partitioning (MAP) technology for the first time - describing in detail how our novel MAP plates are designed to provide consistent, high quality digital PCR with an experimental workflow identical to qPCR.
We used two pre-designed oncology assays to demonstrate the Absolute Q’s ability to detect and quantify rare genomic targets with both sensitivity and specificity. These assays target the well known cancer-related genetic alterations EGFR p.T790M and the gene fusion BCR-ABL1.
Finally, testing the Absolute Q in the clinical application of precision oncology, we tracked a juvenile myelomonocytic leukemia (JMML) patient's response to therapy using an assay which was custom-designed for a gene-fusion unique to the patient.
EGFR p.T790M Rare Mutation Allele Quantification
Early detection of resistance mutations can better inform patient treatment and guide appropriate more efficacious drug selection, and in the case of non-small cell lung cancer (NSCLC) the EGFR p.T790M mutation can indicate such resistance to tyrosine kinase inhibitors.
For this first experiment, we performed EGFR p.T790M mutation quantification using precision circulating cell free DNA (ccfDNA) reference material (Seraseq ctDNA). This material is specifically designed to mimic the quality and composition of nucleic acid expected from an extracted patient blood sample - essentially, the mutation molecules are first fragmented and then mixed into a high concentration, well characterized normal genome.
We tested two mock ccfDNA samples designed to have mutation allele frequencies (MAF) of 1% and 0.1%, and accurately quantified these precision controls. In addition to these, we tested both negative (100% wild-type normal) and no template control samples, and found both to be free of positives in the expected channels.
BCR-ABL1 Gene Fusion Detection
Nearly 95% of chronic myeloid leukemia (CML) cases are characterized by the presence of the BCR-ABL1 fusion gene, and treatment with TKIs is the clinical gold standard. Regular monitoring of BCR-ABL1 transcripts is integral to successful TKI treatment, occasionally allowing drug discontinuation upon achievement of deep remissions.
Using published assay sequences (Alikian et.al, 2017) and gBlock control materials, we precisely quantified the BCR-ABL1 fusion target at an ultra-rare MAF of 0.01%1.
Custom tracking of a juvenile patient's journey to remission
In a previous study(Chao et.al, 2019) , a seven-week old infant was diagnosed with JMML and tested negative for all known associated mutations. NGS sequencing identified a new gene-fusion between the CCDC88C and FLT3 genes. This information was used to develop a custom digital PCR assay.
Using bio-banked samples collected from this patient, we retrospectively monitored the patient’s positive response to treatment using the Absolute Q.
Initially the patient did not respond to cytotoxic chemotherapy (Cytarabine), and as expected from the previous study, the quantity of detectable CCDC88C-FLT3 fusion targets remained high in the blood. After treatment with Sorafenib monotherapy, the patient responded and the concentration plummeted. Finally, after the hematopoietic stem cell transplantation, we identified zero partitions positive for the CCDC88C-FLT3 fusion target for all subsequent samples, indicating and confirming the initial findings of complete molecular remission.
The Absolute Q is a fully integrated digital PCR platform - meaning no technical training past preparing a qPCR mastermix is required to perform dPCR. Furthermore, the Absolute Q leverages digital PCR’s unparalleled sensitivity for rare targets while improving upon the challenges which currently exist in the digital PCR technology space. We believe that simplicity will facilitate and accelerate the adoption of digital PCR for precision oncology.
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1. Alikian, M. et al. RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in
Chronic Myeloid Leukemia. Clin Chem. 63, 525–531 (2017).
2. Chao, A. K., et al. Fusion driven JMML: A novel CCDC88C-FLT3 fusion responsive to sorafenib identified by RNA sequencing.
Leukemia. https://doi.org/10.1038/s41375-019-0549-y (2019).