Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip.
ABSTRACT: EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.
Project description:OBJECTIVES:Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition, mostly caused by germline TP53 mutations. Lung adenocarcinoma (ADC) has been identified as the most frequent LFS-related cancer outside the common LFS core spectrum. EGFR-kinase domain duplication (KDD) is rare in lung cancer and the effective therapy for LFS patients with EGFR-KDD mutated ADC is unclear. This study reports the first case of a TP53-mutated LFS patient with confirmed family history, developing advanced lung ADC harboring EGFR-KDD. MATERIALS AND METHODS:The patient's lung tumor, lymph nodes, liquid biopsies and germline control sample at various disease stages were subjected to next-generation sequencing (NGS). The TP53 germline mutation was confirmed using the peripheral blood of the patient's relatives by Sanger sequencing. RESULTS:A rare EGFR-KDD somatic mutation that was missed in the routine EGFR hotspots test, and a TP53-E285K temperature-sensitive germline mutation were identified by NGS. The patient was diagnosed with breast cancer in 2006 and her family cancer history review revealed that seven out of 13 relatives were diagnosed or died from LFS-spectrum cancers before the age of 45 years. Three of the six relatives were positive for the TP53-E285K germline mutation. This patient received multi-line chemotherapy followed by anlotinib, a multi-target tyrosine kinase inhibitor, upon the identification of EGFR-KDD, and achieved an overall survival of 18 months. CONCLUSIONS:Our study highlights the importance of NGS in discovering rare genetic alterations to guide treatment decision-making, and provides meaningful insight into the potential treatment options for LFS patients with EGFR-KDD mutations.
Project description:Circulating tumor cells (CTCs) are a tumor-derived material utilized for liquid-based biopsy; however, capturing rare CTCs for further molecular analysis remains technically challenging, especially in non-small-cell lung cancer. Here, we report the results of a clinical evaluation of On-chip Sort, a disposable microfluidic chip-based cell sorter, for capture and molecular analysis of CTCs from patients with lung adenocarcinoma. Peripheral blood was collected from 30 metastatic lung adenocarcinoma patients to enumerate CTCs using both On-chip Sort and CellSearch in a blind manner. Captured cells by On-chip Sort were subjected to further molecular analysis. Peripheral blood samples were also used for detection of EGFR mutations in plasma using droplet digital PCR. Significantly more CTCs were detected by On-chip Sort (22/30; median 5; range, 0-18 cells/5 mL blood) than by CellSearch (9/30; median, 0; range, 0-12 cells/7.5 mL) (P < 0.01). Thirteen of 30 patients who had a negative CTC count by CellSearch had a positive CTC count by On-chip Sort. EGFR mutations in CTCs captured by On-chip Sort were observed in 40.0% (8/20) of patients with EGFR-mutated primary tumor. EGFR mutations were often observed in 53.3% (8/15) of patients detected in plasma DNA. Expressions of EGFR and vimentin protein on CTCs were also successfully assessed using On-chip Sort. These results suggest that On-chip Sort is an efficient method to detect and capture rare CTCs from patients with lung adenocarcinoma that are undetectable with CellSearch. Mutation detection using isolated CTCs remains to be further tackled (UMIN000012488).
Project description:The majority of non-small cell lung cancer (NSCLC) patients harbor EGFR-activating mutations that can be therapeutically targeted by EGFR tyrosine kinase inhibitors (EGFR-TKI), such as erlotinib and gefitinib. Unfortunately, a subset of patients with EGFR mutations are refractory to EGFR-TKIs. Resistance to EGFR inhibitors reportedly involves SRC activation and induction of epithelial-to-mesenchymal transition (EMT). Here, we have demonstrated that overexpression of CRIPTO1, an EGF-CFC protein family member, renders EGFR-TKI-sensitive and EGFR-mutated NSCLC cells resistant to erlotinib in culture and in murine xenograft models. Furthermore, tumors from NSCLC patients with EGFR-activating mutations that were intrinsically resistant to EGFR-TKIs expressed higher levels of CRIPTO1 compared with tumors from patients that were sensitive to EGFR-TKIs. Primary NSCLC cells derived from a patient with EGFR-mutated NSCLC that was intrinsically erlotinib resistant were CRIPTO1 positive, but gained erlotinib sensitivity upon loss of CRIPTO1 expression during culture. CRIPTO1 activated SRC and ZEB1 to promote EMT via microRNA-205 (miR-205) downregulation. While miR-205 depletion induced erlotinib resistance, miR-205 overexpression inhibited CRIPTO1-dependent ZEB1 and SRC activation, restoring erlotinib sensitivity. CRIPTO1-induced erlotinib resistance was directly mediated through SRC but not ZEB1; therefore, cotargeting EGFR and SRC synergistically attenuated growth of erlotinib-resistant, CRIPTO1-positive, EGFR-mutated NSCLC cells in vitro and in vivo, suggesting that this combination may overcome intrinsic EGFR-inhibitor resistance in patients with CRIPTO1-positive, EGFR-mutated NSCLC.
Project description:Non-small cell lung cancers (NSCLC) that have developed resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here, we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGF? secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGF? was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGF? receptor in HCC4006 cells prevented EMT but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared with cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly developed EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele was lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy.
Project description:Epidermal growth factor receptor (EGFR) was evaluated as a target antigen for cancer cell capture by microfluidic methods based on antigen-antibody association. A polymer CTC-chip microfluidic device was surface-functionalized with three different anti-EGFR antibodies and used to capture EGFR-expressing cancer cells. Capture efficacy depended on the type of antibody used, and cetuximab efficiently captured cancer cell lines that had a wide range of EGFR expression. Capture efficiency was analyzed from the viewpoint of antigen-antibody association in a kinetic process, i.e., cell rolling well-known in leukocyte adhesion, and antibodies with a smaller dissociation constant were shown to result in more efficient capture. Moreover, a lower limit of cellular EGFR expression level for the capture was estimated and methods to decrease the limit were discussed based on densities of anti-EGFR antibody on the device surface.
Project description:Oncogenic KRAS mutations in colorectal cancer (CRC) are associated with lack of benefit from epidermal growth factor receptor (EGFR)-directed antibody (Ab) therapy. However, the mechanisms by which constitutively activated KRAS (KRAS(G12V)) impairs effector mechanisms of EGFR-Abs are incompletely understood. Here, we established isogenic cell line models to systematically investigate the impact of KRAS(G12V) on tumor growth in mouse A431 xenograft models as well as on various modes of action triggered by EGFR-Abs in vitro. KRAS(G12V) impaired EGFR-Ab-mediated growth inhibition by stimulating receptor-independent downstream signaling. KRAS(G12V) also rendered tumor cells less responsive to Fc-mediated effector mechanisms of EGFR-Abs-such as complement-dependent cytotoxicity (CDC) and Ab-dependent cell-mediated cytotoxicity (ADCC). Impaired CDC and ADCC activities could be linked to reduced EGFR expression in KRAS-mutated versus wild-type (wt) cells, which was restored by small interfering RNA (siRNA)-mediated knockdown of KRAS4b. Immunohistochemistry experiments also revealed lower EGFR expression in KRAS-mutated versus KRAS-wt harboring CRC samples. Analyses of potential mechanisms by which KRAS(G12V) downregulated EGFR expression demonstrated significantly decreased activity of six distinct transcription factors. Additional experiments suggested the CCAAT/enhancer-binding protein (C/EBP) family to be implicated in the regulation of EGFR promoter activity in KRAS-mutated tumor cells by suppressing EGFR transcription through up-regulation of the inhibitory family member C/EBP?-LIP. Thus, siRNA-mediated knockdown of C/EBP? led to enhanced EGFR expression and Ab-mediated cytotoxicity against KRAS-mutated cells. Together, these results demonstrate that KRAS(G12V) signaling induced C/EBP?-dependent suppression of EGFR expression, thereby impairing Fc-mediated effector mechanisms of EGFR-Abs and rendering KRAS-mutated tumor cells less sensitive to these therapeutic agents.
Project description:Non-small-cell lung cancer (NSCLC) is commonly caused by a mutation in the epidermal growth factor receptor (EGFR) and subsequent aberrant EGFR signaling with uncontrolled kinase activity. A deletion mutation in EGFR exon 19 is frequently observed in EGFR gene mutations. We designed a DNAzyme to suppress the expression of mutant EGFR by cleaving the mutant EGFR mRNA. The DNAzyme (named Ex19del Dz) specifically cleaved target RNA and decreased cancer cell viability when transfected into gefitinib-resistant lung cancer cells harboring EGFR exon 19 deletions. The DNAzyme decreased EGFR expression and inhibited its downstream signaling pathway. In addition to EGFR downregulation, Ex19del Dz containing CpG sites activated Toll-like receptor 9 (TLR9) and its downstream signaling pathway via p38 kinase, causing an immunostimulatory effect on EGFR-mutated NSCLC cells. Thus, dual effects of this DNAzyme harboring the CpG site, such as TLR9 activation and EGFR downregulation, leads to apoptosis of EGFR-mutated NSCLC cells. [BMB Reports 2018; 51(1): 27-32].
Project description:BACKGROUND:There is a growing appreciation for radio-sensitiser use in multi-modal cancer treatment models. Squamous cell anal carcinoma (SCAC) is a rare gastrointestinal tumour traditionally treated with concurrent chemotherapy and radiation. Cetuximab, an epidermal growth factor receptor (EGFR) inhibitor, has demonstrated significant efficacy when combined with radiation in squamous cell carcinoma of the head and neck (SccH&N). We wanted to assess EGFR and Kirsten-ras (K-ras) status in SCAC to see whether it compares with SccH&N. METHODS:Over 90 SCAC paraffin-embedded biopsies were mounted onto a tissue microarray and were assessed for EGFR expression by immunohistochemistry. These samples were also assessed for the most frequently mutated K-ras and EGFR exons by high-resolution melting analysis. RESULTS:The EGFR was present in over 90% of samples tested. The K-ras and EGFR mutations were absent in all samples tested, although a synonymous single-nucleotide polymorphism was found in 3 out of 89 samples tested for EGFR exon 19. CONCLUSION:The low rate of K-ras and EGFR mutations, coupled with the high surface expression of EGFR, suggests similarity in the EGFR signalling pathway between SCAC and SccH&N, and thus a potential role for EGFR inhibitors in SCAC. To our knowledge this is the largest cohort of invasive SCAC samples investigated for EGFR and K-ras mutations reported to date.
Project description:Objectives: Tumor pathology examination especially epidermal growth factor receptor (EGFR) mutations molecular testing has been integral part of lung cancer clinical practices. However, the EGFR mutations spatial distribution characteristics remains poorly investigated, which is critical to tumor heterogeneity analysis and precision diagnosis. Here, we conducted an exploratory study for label-free lung cancer pathology diagnosis and mapping of EGFR mutation spatial distribution using ambient mass spectrometry imaging (MSI). Materials and Methods: MSI analysis were performed in 55 post-operative non-small cell lung cancer (NSCLC) tumor and paired normal tissues to distinguish tumor from normal and classify pathology. We then compared diagnostic sensitivity of MSI and ADx-amplification refractory mutation system (ARMS) for the detection of EGFR mutation in pathological confirmed lung adenocarcinoma (AC) and explored EGFR mutations associated biomarkers to depict EGFR spatial distribution base on ambient MSI. Results: Of 55 pathological confirmed NSCLC, MSI achieved a diagnostic sensitivity of 85.2% (23/27) and 82.1% (23/28) for AC and squamous cell carcinoma (SCC), respectively. Among 27 AC, there were 17 EGFR-wild-type and 10 EGFR-mutated-positive samples detected by ARMS, and MSI achieved a diagnostic sensitivity of 82.3% (14/17) and 80% (8/10) for these two groups. Several phospholipids were specially enriched in AC compared with SCC tissues, with the higher ions intensity of phospholipids in EGFR-mutated-positive compared with EGFR-wild-type AC tissues. We also found EGFR mutations distribution was heterogeneous in different regions of same tumor by multi-regions ARMS detection, and only the regions with higher ions intensity of phospholipids were EGFR-mutated-positive. Conclusion: MSI method could accurately distinguish tumor pathology and subtypes, and phospholipids were reliable EGFR mutations associated biomarkers, phospholipids imaging could intuitively visualize EGFR mutations spatial distribution, may facilitate our understanding of tumor heterogeneity.