Developing Ultrasensitive Library-Aliquot-based Droplet Digital PCR for detecting T790M

Patients with non-small cell lung cancer (NSCLC) harboring activating mutations in the epidermal growth factor receptor (EGFR) show significant progression-free survival benefit when they are treated with EGFR tyrosine kinase inhibitors (TKIs), have been recommended as the first-line therapy for NSCLC patents with EGFR-sensitizing mutations.¹ Unfortunately, the vast majority acquire resistance after 9-12 months of treatment. However, the occurrence of this new EGFR T790M secondary mutation is the most well-established biomarker for this resistance. For this reason, fast and accurate detection of T790M is required for the monitoring of disease progression and for designing new treatment strategies opportunely for patients.²

Currently, investigating genetic aberrations in tumor tissues is still considered gold standard, but several drawbacks, such as the heterogeneous nature of a tumor, limitation for real-time monitoring of the genetic aberrations, and the formaldehyde-fixation sample preparation, are needed to overcome.

Alternatively, circulating tumor DNA (ctDNA) is available from liquid biopsies in the form of blood draws. Using ctDNA is minimally invasive and feasible in clinic practice.^{2, 3} However, the sensitivity of the T790M ctDNA test is normally lower than tumor rebiopsies because ctDNA test is largely inhibited by the quantity of input material. Thus, overcoming the limitation of input amount is the key point to use ctDNA for biopsies. Droplet digital polymerase chain reaction (ddPCR) is one such digital PCR technology based on compartmentalization of DNA into droplets. Compartmentalized DNA droplets are then amplified in parallel. As a result, sensitivity is highly increased.^{4, 5} However, to optimize the use of ctDNA as T790M detection, still higher sensitivity analyzing method is required. Recently, library-aliquots-based ddPCR (LAB-ddPCR), in which the isolated ctDNA is first processed for next generation sequencing (NGS) library preparation; this is followed by a ddPCR test on the aliquots of the precapture library. Because the ctDNA is amplified after NGS-library preparation and initial input amount is enhanced, LAB-ddPCR largely increases the detection sensitivity of the T790M ctDNA more than just using ddPCR.⁶ As a result, NGS based ddPCR assay showed a comparable T790M-positive rate (58.57%) in the ctDNA samples as in FFPE-tumor rebiopsies.

Reference:

1. Sundaresan, Tilak K., et al. "Detection of T790M, the acquired resistance EGFR mutation, by tumor biopsy versus noninvasive blood-based analyses." Clinical Cancer Research22.5 (2016): 1103-1110.
2. Sheikine, Yuri, et al. "EGFR testing in advanced non–small-cell lung cancer, a mini-review." Clinical lung cancer 17.6 (2016): 483-492.
3. Sundaresan, Tilak K., et al. "Detection of T790M, the acquired resistance EGFR mutation, by tumor biopsy versus noninvasive blood-based analyses." Clinical Cancer Research22.5 (2016): 1103-1110.
4. Watanabe, Masaru, et al. "Ultra-Sensitive Detection of the Pretreatment EGFR T790M Mutation in Non–Small Cell Lung Cancer Patients with an EGFR-Activating Mutation Using Droplet Digital PCR." Clinical Cancer Research (2015).
5. Borsu, Laetitia, et al. "Clinical application of Picodroplet Digital PCR technology for rapid detection of EGFR T790M in Next-Generation sequencing libraries and DNA from limited tumor samples." The Journal of Molecular Diagnostics 18.6 (2016): 903-911.

6. Yang, Ke, et al. "Developing Ultrasensitive Library-Aliquot-Based Droplet Digital PCR for Detecting T790M in Plasma-Circulating Tumor DNA of Non-small-Cell-Lung-Cancer Patients." Analytical chemistry 90.19 (2018): 11203-11209.