High-Accuracy Determination of Microsatellite Instability Compatible with Liquid Biopsies

Abstract

Background Microsatellite instability (MSI) has recently emerged as a predictive pan-tumor biomarker of immunotherapy efficacy, stimulating the development of diagnostic tools compatible with large-scale screening of patients. In this context, noninvasive detection of MSI from circulating tumor DNA stands as a promising diagnostic and posttreatment monitoring tool. Methods We developed drop-off droplet-digital PCR (ddPCR) assays targeting BAT-26, activin A receptor type 2A (ACVR2A), and defensin beta 105A/B (DEFB105A/B) microsatellite markers. Performances of the assays were measured on reconstitution experiments of various mutant allelic fractions, on 185 tumor samples with known MSI status, and on 72 blood samples collected from 42 patients with advanced colorectal or endometrial cancers before and/or during therapy. Results The 3 ddPCR assays reached analytical sensitivity <0.1% variant allelic frequency and could reliably detect and quantify MSI in both tumor and body fluid samples. High concordance between MSI status determination by the three-marker ddPCR test and the reference pentaplex method were observed (100% for colorectal tumors and 93% for other tumor types). Moreover, the 3 assays showed correlations with r ≥ 0.99 with other circulating tumor DNA markers and their dynamic during treatment correlated well with clinical response. Conclusions This innovative approach for MSI detection provides a noninvasive, cost-effective, and fast diagnostic tool, well suited for large-scale screening of patients that may benefit from immunotherapy agents, as well as for monitoring treatment responses.

Personalized tumor-specific DNA junctions to detect circulating tumor in patients with endometrial cancer

“…To our knowledge, only a few studies have reported data on ctDNA in endometrial cancer [ 11 – 16 , 24 ]. CtDNA was detected preoperatively in 15% (10/68 patients) to 41% (21/51 patients) of patients with endometrial cancer, using tumor-associated mutations [ 12 – 14 ].…”

“…Total plasma cell-free DNA (cfDNA) levels in endometrial cancer patients were reported to be increased compared to healthy controls [ 9 , 10 ], but lacks specificity to robustly discriminate pathological levels versus benign controls. A few studies have evaluated the feasibility of using tumor-specific mutations to detect ctDNA in the plasma of endometrial cancer patients [ 11 – 16 ]. In particular, ctDNA screening for point mutations present in primary gynecological tumors using digital droplet PCR reported promise for detecting recurrence preceding anatomic findings on standard computed tomography scans by an average of 7 months [ 11 ].…”

Evaluation of 3 molecular-based assays for microsatellite instability detection in formalin-fixed tissues of patients with endometrial and colorectal cancers

“…Immunohistochemistry (IHC) (evaluation of MMR protein expression including MLH1, MSH2, MSH6 and PMS2) and pentaplex PCR-based assays (directed against 5 microsatellite regions including at least BAT-25 and BAT-26 mononucleotide markers such as Promega MSI Analysis System) are currently the reference assays recommended for dMMR/MSI testing 9 , 27 – 29 . However, novel molecular-based methods emerge for the detection of MSI, including different real-time PCR and droplet digital PCR (ddPCR)-based methods 30 – 33 as well as various custom Next-Generation Sequencing (NGS) approaches 34 – 43 . In this study, we aimed to evaluate the performance of 3 molecular assays, including a custom capture-based NGS approach, Idylla MSI and Bio-Rad ddPCR MSI assays, for the determination of MSI status for theranostic purposes in patients with endometrial and colorectal cancers.…”

“…With a 100% sensitivity and specificity, the Bio-Rad ddPCR MSI assay consists in a fast and cost-effective multiplex assay compatible with large-scale testing of patients, though requiring a specialized laboratory to interpret the data. In a recent study, a custom implementation of ddPCR using specialized targets allowed to reach an unmatched sensitivity for MSI screening compared to previously used approaches, making this methodology applicable for MSI analysis in liquid biopsies 30 .…”

Liquid Biopsy for Prognosis and Treatment in Metastatic Colorectal Cancer: Circulating Tumor Cells vs Circulating Tumor DNA

“…Immunotherapy is a well-established therapeutic option for MMR-deficient mCRC [ 3 , 4 ]. While few studies reported >98% accuracy of ctDNA MMR-deficient noninvasive identification, an ongoing prospective trial (NCT03594448) is investigating serial LB as a detection method for MMR-deficient mCRC [ 107 , 108 ].…”