RESUMEN
BACKGROUND: Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands). METHODS: Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18-21, and KRAS exon 2-3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance. RESULTS: A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752_I759del) (69%), EGFR p.(N771_H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately. CONCLUSIONS: Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine.
Asunto(s)
ADN Tumoral Circulante , Humanos , ADN Tumoral Circulante/sangre , ADN Tumoral Circulante/genética , Mutación , Neoplasias/genética , Neoplasias/sangre , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores ErbB/genética , Receptores ErbB/sangre , Proteínas Proto-Oncogénicas B-raf/genética , Países BajosRESUMEN
BACKGROUND: Activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene have been detected often in solid tumors. Targeted therapy for mutant PIK3CA is now available in the clinic, making molecular diagnostics pivotal. Our aim was to design a multiplex digital PCR (dPCR) assay to evaluate the 4 most common PIK3CA hotspot mutations simultaneously to characterize and quantify these in liquid biopsies. METHODS: A multiplex assay was developed to detect exon 9 p.E542K and p.E545K mutations, and exon 20 p.H1047L and p.H1047R mutations using the Stilla 3-color dPCR Naica system. The assay was evaluated on stock and pre-amplified DNA from cell lines with the above mutations as single and pooled samples, and on cell-free DNA (cfDNA) from healthy blood donors (HBDs) and breast cancer patients, to determine detection thresholds and diagnostic accuracy. RESULTS: The assay distinguished all 4 PIK3CA mutations in (cf)DNA, and also when dual mutations were present. Detection thresholds of stock and pre-amplified cfDNA samples were 0.11 and 0.40 copies/uL (cp/uL) for mutant copies concentration, and 0.003% and 0.68% for variant allele frequencies (VAFs), respectively. The assay confirmed the PIK3CA (mutation) status as defined by targeted next-generation sequencing (NGS) in 82 out of 96 patients that were mutant for PIK3CA, and in 11 out of 12 patients with wild-type PIK3CA. CONCLUSIONS: Our designed multiplex dPCR assay detected PIK3CA mutations with high accuracy in stock and pre-amplified cfDNA. Furthermore, it is affordable and demands less cfDNA input when compared to available uniplex dPCR assays and NGS analyses.
RESUMEN
Phenotypic plasticity, defined as the ability of individual cells with stable genotypes to exert different phenotypes upon exposure to specific environmental cues, represent the quintessential hallmark of the cancer cell en route from the primary lesion to distant organ sites where metastatic colonization will occur. Phenotypic plasticity is driven by a broad spectrum of epigenetic mechanisms that allow for the reversibility of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions (EMT/MET). By taking advantage of the co-existence of epithelial and quasi-mesenchymal cells within immortalized cancer cell lines, we have analyzed the role of EMT-related gene isoforms in the regulation of epithelial mesenchymal plasticity (EMP) in high grade serous ovarian cancer. When compared with colon cancer, a distinct spectrum of downstream targets characterizes quasi-mesenchymal ovarian cancer cells, likely to reflect the different modalities of metastasis formation between these two types of malignancy, i.e. hematogenous in colon and transcoelomic in ovarian cancer. Moreover, upstream RNA-binding proteins differentially expressed between epithelial and quasi-mesenchymal subpopulations of ovarian cancer cells were identified that underlie differential regulation of EMT-related isoforms. In particular, the up- and down-regulation of RBM24 and ESRP1, respectively, represent a main regulator of EMT in ovarian cancer cells. To validate the functional and clinical relevance of our approach, we selected and functionally analyzed the Tropomyosin 1 gene (TPM1), encoding for a protein that specifies the functional characteristics of individual actin filaments in contractile cells, among the ovarian-specific downstream AS targets. The low-molecular weight Tpm1.8/9 isoforms are specifically expressed in patient-derived ascites and promote invasion through activation of EMT and Wnt signaling, together with a broad spectrum of inflammation-related pathways. Moreover, Tpm1.8/9 expression confers resistance to taxane- and platinum-based chemotherapy. Small molecule inhibitors that target the Tpm1 isoforms support targeting Tpm1.8/9 as therapeutic targets for the development of future tailor-made clinical interventions.