Development of multiplex digital PCR assays for the detection of PIK3CA mutations in the plasma of metastatic breast cancer patients

Abstract

With the approval of new therapies targeting the PI3K pathway, the detection of PIK3CA mutations has become a key factor in treatment management for HR+/HER2− metastatic breast cancer (MBC). We developed multiplex digital PCR (dPCR) assays to detect and quantify PIK3CA mutations. A first screening assay allows the detection of 21 mutations, with a drop-off system targeting the 542–546 hotspot mutations combined with the simultaneous detection of N345K, C420R, H1047L and H1047R mutations. In the case of a positive result, a sequential strategy based on other assays that we have developped allows for precise mutation identification. Clinical validity was determined by analyzing plasma circulating free DNA (cfDNA) from 213 HR+/HER2− MBC samples, as well as DNA extracted from 97 available matched tumors from 89 patients. Our assays have shown reliable specificity, accuracy and reproducibility, with limits of blank of three and four droplets for the screening assay. Sixty-eight patients (32%) had at least one PIK3CA mutation detectable in their plasma, and we obtained 83.1% agreement between the cfDNA analysis and the corresponding tumors. The high sensitivity and robustness of these new dPCR assays make them well-suited for rapid and cost-effective detection of PIK3CA mutations in the plasma of MBC patients.

Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA

“…Comprehensive target panels are necessary to monitor treatment response on a patient-individual level [ 7 , 8 ]. Ultrasensitive mutation detection in a high background of wild type DNA is typically achieved by using digital PCR (dPCR) [ 3 , 9 , 10 , 11 ]. The risk of false negative results due to the low amounts of DNA caused by splitting samples for multiple singleplex assays can be minimized by multiplex assays [ 12 ].…”

Skin Cancer Research Goes Digital: Looking for Biomarkers within the Droplets

“…In the early 2000s, advances in microfluidics and informatics, coupled with novel water-in-oil emulsion systems for sample partitioning, have allowed the development of more sophisticated equipment capable of subdividing PCR reactions into smaller reaction volumes [ 51 ]. Since then, different digital PCR platforms have been released, and are nowadays available, including the microfluidic chamber-based Biomark ® system from Fluidigm [ 52 ], micro-well chip-based Quantstudio 12k/3D ® from Thermo Fisher Scientific [ 53 ], droplet-based QX100 and QX200 Droplet Digital PCR ® from Bio-Rad [ 30 ], the RainDrop dPCR ® from RainDance technologies [ 54 ], the Crystal digital PCR ® from Stilla Technologies [ 55 ], the BEAMing ® technology from Sysmex Inostics [ 56 ], the Lab On An Array (LOAA) Digital PCR ® system from Optolane [ 57 ] and the QIAcuity Digital PCR ® system from Qiagen [ 58 ]. These systems have proven suitable for cancer research, showing similar results in terms of nucleic acid quantification, specificity and sensitivity.…”

Digital Droplet PCR in Hematologic Malignancies: A New Useful Molecular Tool

“…In the non-invasive prenatal testing field, multiplexing ddPCR using universal locked nucleic acid probes correctly identified several fetal aneuploidies [ 17 ], while in oncology this technique detected 4 different PIK3CA mutations on “liquid biopsy”, with a clinical impact in the management of metastatic breast cancer [ 18 ]. Moreover, an Italian group set a multiplexing ddPCR in patients affected by chronic myeloid leukemia (CML) with unusual BCR-ABL1 atypical transcripts, not quantifiable by standardized QT-PCR, with an optimal detection limit level (0.001%).…”