• A CRISPR test for rapidly and sensitively detecting circulating EGFR mutations

      Tsou, J.-H.; Leng, Q.; Jiang, F. (MDPI AG, 2020)
      The detection of EGFR mutations in circulating cell-free DNA can enable personalized therapy for cancer. The current techniques for detecting circulating EGFR mutations are expensive and time-consuming with moderate sensitivity. Emerging CRISPR is revolutionizing medical diagnostics and showing a great promise for nucleic acid detection. This study aims to develop CRISPR-Cas12a as a simple test to sensitively detect circulating EGFR mutations in plasma. Serially diluted samples of DNA containing heterozygous EGFR mutations (L858R and T790M) in wild-type genomic DNA are concurrently tested for the mutations by a CRISPR-Cas12a system and droplet digital PCR (ddPCR). The CRISPR-Cas12a system can detect both L858R and T790M with a limit of detection of 0.005% in less than three hours. ddPCR detects the mutations with a limit of detection of 0.05% for more than five hours. Plasma samples of 28 lung cancer patients and 20 cancer-free individuals are tested for the EGFR mutations by CRISPR-Cas12a system and ddPCR. The CRISPR-Cas12a system could detect L858R in plasma of two lung cancer patients whose tissue biopsies are positive for L858R, and one plasma sample of three lung cancer patients whose tissue biopsies are positive for T790M. ddPCR detects L858R in the same two plasm samples, however, does not detect T790M in any of the plasma samples. This proof of principle study demonstrates that the CRISPR-Cas12a system could rapidly and sensitively detect circulating EGFR mutations, and thus, has potential prognostic or therapeutic implications. Copyright 2020 by the authors.
    • Plasma EGFR mutation testing in non-small cell lung cancer: A value proposition

      Oellerich, M.; Christenson, R.H.; Beck, J. (Elsevier B.V., 2019)
      Genomics-driven precision medicine using targeted therapies requires advanced molecular diagnostic tests. Decisions about the use and reimbursement for such tests are increasingly being made on the basis of more outcome-based and value-based approaches. The value proposition concept is a tool to assess the benefits of laboratory testing to each stakeholder of the care pathway with respect to outcomes. This concept was applied to the use of noninvasive plasma epidermal growth factor receptor (EGFR)mutation testing in patients with advanced or metastatic non-small cell lung cancer (NSCLC)to guide treatment with EGFR tyrosine kinase inhibitors (TKIs). Using the value proposition framework, we evaluated published key evidence regarding clinical validity, economic implications, and limitations of this approach. It has been shown that plasma EGFR mutation testing is essential for guiding clinical decisions regarding prediction of eligibility of individual patients for TKI treatment, real-time monitoring, or adjustment of treatment regimens and tracking resistance. The appropriate use of plasma EGFR mutation testing has been shown to deliver both clinical and economic benefits to stakeholders across the entire care pathway; especially in clinical situations where biopsy material is inadequate or unavailable and where it leads to fewer tissue biopsies.
    • Post-surgery circulating tumor cells and AXL overexpression as new poor prognostic biomarkers in resected lung adenocarcinoma

      Bayarri-Lara, C.I.; de Miguel Pérez, Diego; Ortega, F.G. (MDPI AG, 2019)
      Background: The prognosis of early stage non-small cell lung cancer (NSCLC) is quite disappointing and the benefits of adjuvant therapy are relatively small. Thus, there is an urgent need to identify novel prognostic and predictive biomarkers. Lung adenocarcinoma has distinct clinical-pathological characteristics and novel therapeutic strategies are under active evaluation in the adjuvant setting. Here, we investigated the prognostic impact of circulating tumor cells (CTCs) and gene and miRNA tissue expression in resectable NSCLC. Patients and methods: We assessed the association between CTC subpopulations and the outcome of resected early stage lung adenocarcinoma (ADC) patients at three different time-points (CTC1-3) (before surgery, after one month, and after six months) in comparison to squamous cell carcinoma (SCC). Furthermore, gene and miRNA tissue expression, immunoprofiling, and epithelial-to-mesenchymal transition (EMT) markers were correlated with outcome. Results: ADC (n = 47) and SCC (n = 50) revealed different tissue expression profiles, resulting in the presence of different CTC subpopulations. In ADC, miR- 155 correlated with AXL and IL6R expression, which were related to the presence of EMT CTC1 (p = 0.014 and p = 0.004). In the multivariate analysis, CTC2 was an independent prognostic factor for relapse-free survival, and CTC3 and AXL were independent prognostic for overall survival only in ADC. Neither the surgery nor the adjuvant treatment influenced the prognosis of these patients. Conclusions: Our study elucidate the prognostic impact of tissue AXL expression and the presence of CTCs after surgery in adenocarcinoma patients. Tissue AXL expression and CTC EMT activation could potentially represent biomarkers for the stratification of ADC patients that might benefit from new adjuvant therapies. Copyright 2019 by the authors.
    • The triad hsp60-mirnas-extracellular vesicles in brain tumors: Assessing its components for understanding tumorigenesis and monitoring patients

      Graziano, Francesca; Iacopino, Domenico Gerardo; Cammarata, Giacomo; Scalia, Gianluca; Campanella, Claudia; Giannone, Antonino Giulio; Porcasi, Rossana; Florena, Ada Maria; De Macario, Everly Conway; Macario, Alberto J.L.; et al. (MDPI AG, 2021-03-23)
      Brain tumors have a poor prognosis and progress must be made for developing efficacious treatments, but for this to occur their biology and interaction with the host must be elucidated beyond current knowledge. What has been learned from other tumors may be applied to study brain tumors, for example, the role of Hsp60, miRNAs, and extracellular vesicles (EVs) in the mechanisms of cell proliferation and dissemination, and resistance to immune attack and anticancer drugs. It has been established that Hsp60 increases in cancer cells, in which it occurs not only in the mitochondria but also in the cytosol and plasma-cell membrane and it is released in EVs into the extracellular space and in circulation. There is evidence suggesting that these EVs interact with cells near and far from their original cell and that this interaction has an impact on the functions of the target cell. It is assumed that this crosstalk between cancer and host cells favors carcinogenesis in various ways. We, therefore, propose to study the triad Hsp60-related miRNAs-EVs in brain tumors and have standardized methods for the purpose. These revealed that EVs with Hsp60 and related miRNAs increase in patients’ blood in a manner that reflects disease status. The means are now available to monitor brain tumor patients by measuring the triad and to dissect its effects on target cells in vitro, and in experimental models in vivo. © 2021 by the authors.