Liquid Biopsy and Tissue Biopsy Comparison with Digital PCR and IHC/FISH for HER2 Amplification Detection in Breast Cancer Patients.

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.

A large-scale, multicentered trial evaluating the sensitivity and specificity of digital PCR versus ARMS-PCR for detecting ctDNA-based EGFR p.T790M in non-small-cell lung cancer patients.

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.

Perinatal exposure to antibiotics reduces affiliative behavior after post-weaning in zebra finches (Taeniopygia guttata).

Social behavior is influenced by a host of factors, including the immune system; for example, song quality in male starlings predicts immunocompetence suggesting the development of the immune system is interconnected with aspects social development (Duffy and Ball, 2002). Treating birds with antibiotics during the perinatal period may alter this development, and thereby, social behaviors beyond song. We asked if antibiotic exposure during the perinatal period effected parenting and offspring social behavior (e.g. aggressive and affiliative behaviors) in zebra finches? We treated the drinking water of zebra finch parents and hatchlings from post-hatch day 5-14 with azithromycin or a vehicle control and monitored parenting/social behavior. After weaning, we transferred offspring from the breeding cage to group housing and monitored social behavior and integration into the colony by measuring aggressive and affiliative behaviors. For all treatments we saw a reduction in the number of songs performed by fathers, however, specifically for antibiotic treated offspring there was a reduction in affiliative behaviors relative to vehicle treated controls suggesting the immune system, perhaps via the guts microbiome, influences certain aspects of social behaviors in birds.

Real-time digital polymerase chain reaction (PCR) as a novel technology improves limit of detection for rare allele assays.

BACKGROUND: Tumor heterogeneity may lead to false negative test results for tissue biopsy-based companion diagnostic tests. Real-time polymerase chain reaction (PCR) and digital PCR assays are used to detect rare alleles in cell-free circulating DNA for liquid biopsies; however, those tests lack strong sensitivity at low allele frequencies. We show here a novel real-time digital PCR instrument that utilizes cycle-based amplification curves to further improve the sensitivity and quantification accuracy of digital PCR. METHODS: The novel real-time digital PCR instrument was compared to an endpoint digital PCR system to determine the sensitivity and quantification accuracy of both instruments. Samples were all thermal cycled on the real-time digital PCR instrument but were analyzed on both endpoint and real-time digital PCR instruments to compare the performance without introducing other variables. Contrived samples for epidermal growth factor receptor (EGFR) exon 19 deletion, T790M, and L858R point mutations as well as human epidermal growth factor receptor 2 (HER2) amplification were tested. Different mutant allele frequencies and wildtype to mutant gene copy number ratios were tested for EGFR and HER2, respectively. RESULTS: By removing false positive datapoints using real-time amplification curves, real-time digital PCR improved sensitivity by lowering the baseline for wildtype samples. For EGFR 19del assay, samples with 2 or more fluorescein amidite (FAM) labeled positive wells are determined positive by real-time digital PCR, while a minimum of 5 FAM positive datapoints is needed by endpoint digital PCR. Improved limit of detection for EGFR 19del mutation was also observed. Real-time digital PCR also had better quantification accuracy and sensitivity, resulting in the mutant allele frequencies being closer to the expected values for all EGFR mutations, especially at very low allele frequencies. However, at high allele frequencies or for gene amplification assays, real-time digital PCR is comparable with endpoint digital PCR. CONCLUSIONS: This novel technology with improved sensitivity is important and needed because it addresses current issues with liquid biopsy tests. Due to limited amounts of circulating tumor DNA (ctDNA) obtained for liquid biopsy tests, few copies of mutant alleles are expected. With the lower baseline of real-time digital PCR, false negative test results from tissue biopsy would be more effectively reduced, leading to more patients receiving the targeted therapy they need for better survival.