RESUMO
Two hundred twenty-four breast cancer patients with paired tissue and plasma samples were enrolled from 3 clinical centers to evaluate sensitivity and specificity of a digital PCR HER2 amplification assay. All patients were histologically confirmed diagnosis of locally advanced and recurrent or metastatic breast cancer with stage III/IV and had tissue HER2 status determinations using IHC/FISH. For the whole 224 advanced breast cancer patients, the sensitivity between dPCR in plasma and IHC/FISH in tissue samples is 43.75% (42/96), the specificity is 84.38% (108/128) and the overall concordance is 66.96% (150/224). Interestingly, when we looked at stage III, stage IV and recurrent or metastatic breast cancer separately, compared with IHC/FISH in tissue samples, the sensitivity of dPCR in plasma increases from 37.93% (11/29) for stage III to 41.67% (15/36) for stage IV cancer. Recurrent breast cancer patient had an increased sensitivity of 51.61% (16/31). This is consistent with our expectation sensitivity would increase concordantly as tumor burden goes up. On the other hand, specificity decreased from 92.68% (38/41) for stage III to 86.44% (51/59) for stage IV cancer. Recurrent breast cancer patient had a specificity of only 67.86% (19/28). This is, in part, due to inter- and intra-tumor heterogeneity. Many patients determined to be negative for HER2 amplification in tissue biopsy could have HER2 positive tumors at other sites, which was detected by the liquid biopsy. This study suggested the necessity of liquid biopsy for HER2 amplification detection and demonstrated digital PCR can be used as a companion diagnostic tool to determine HER2 amplification status. It also suggested that a liquid biopsy should follow a negative result from tissue biopsy to avoid false negative results especially for late-stage breast cancer patients and ones who experienced relapse or became resistant to current therapy. Future studies should focus on therapeutic effects on patients determined to be HER2 positive through liquid biopsy and collecting additional tissue biopsies to identify HER2 positive tumor when the original tissue biopsy and liquid biopsy don't agree.
RESUMO
BACKGROUND: Developing liquid biopsy technology with higher sensitivity and specificity especially for low-frequency mutations remains crucial. This study demonstrated superior performance of the newly developed digital PCR (dPCR) kit for ctDNA-based EGFR p.T790M detection in metastatic non-small-cell lung cancer (NSCLC) against ARMS-PCR. METHODS: This large-scale, multi-centered diagnostic study recruited 1,045 patients including 1,029 patients diagnosed with advanced NSCLC and 16 patients with specific samples between April 1st 2018 and November 30th 2019. EGFR p.T790M in plasma samples from mNSCLC patients were tested using dPCR with ADx-ARMS PCR and Cobas® EGFR Mutation Test V2 as comparator assays to confirm cut-off value for dPCR and evaluate its performance against ARMS-PCR-based assays. Efficacy was evaluated for patients with EGFR p.T790M detected by dPCR or ARMS-PCR, who underwent Osimertinib treatment. RESULTS: The sensitivity, specificity, and concordance of dPCR against ADx-ARMS PCR was 98.15%, 88.66% and 90.16%, respectively for 1,026 plasma samples. Additional 9.26% patients were detected positive by dPCR. The majority of those samples had a mutation allele frequency between 0.1% and 1%. In 45 paired tissue and plasma samples, the sensitivity improved from 30.77% to 53.85% by dPCR with the specificity over 90%. The response of Osimertinib in 74 EGFR p.T790M-positive patients detected by dPCR, including 26 determined as negative by ARMS-PCR, were evaluated to have an ORR of 44.59% and a DCR of 90.54%. CONCLUSIONS: dPCR is a sensitive and accurate tool for ctDNA-based EGFR p.T790M detection due to its significantly improved sensitivity without compromising specificity, and dPCR is equivalent to ARMS-PCR as a companion diagnostic tool while benefiting more patients under Osimertinib treatment. TRIAL REGISTRATION: Chinese Clinical Trial Registry identifier: ChiCTR2100043147.
RESUMO
A real-time dPCR system was developed to improve the sensitivity, specificity and quantification accuracy of end point dPCR. We compared three technologies - real-time qPCR, end point dPCR and real-time dPCR - in the context of SARS-CoV-2. Some improvement in limit of detection was obtained with end point dPCR compared with real-time qPCR, and the limit of detection was further improved with the newly developed real-time dPCR technology through removal of false-positive signals. Real-time dPCR showed increased linear dynamic range compared with end point dPCR based on quantitation from amplification curves. Real-time dPCR can improve the performance of TaqMan assays beyond real-time qPCR and end point dPCR with better sensitivity and specificity, absolute quantification and a wider linear range of detection.
