Molecular mechanisms of acquired resistance to MET tyrosine kinase inhibitors (TKI) are poorly understood. We aimed to characterize the genomic mechanisms of resistance to type I and type II MET TKIs ...and their impact on sequential MET TKI therapy outcomes in patients with metastatic
exon 14-mutant NSCLC.
Genomic alterations occurring at the time of progression on MET TKIs were studied using plasma and tissue next-generation sequencing (NGS).
A total of 20 patients had tissue or plasma available for analysis at the time of acquired resistance to a MET TKI. Genomic alterations known or suspected to be mechanisms of resistance were detected in 15 patients (75%). On-target acquired mechanisms of resistance, including single and polyclonal
kinase domain mutations in codons H1094, G1163, L1195, D1228, Y1230, and high levels of amplification of the
exon 14-mutant allele, were observed in 7 patients (35%). A number of off-target mechanisms of resistance were detected in 9 patients (45%), including
mutations and amplifications in
, and
; one case displayed both on- and off-target mechanisms of resistance. In 2 patients with on-target resistant mutations, switching between type I and type II MET TKIs resulted in second partial responses.
On-target secondary mutations and activation of bypass signaling drive resistance to MET TKIs. A deeper understanding of these molecular mechanisms can support the development of sequential or combinatorial therapeutic strategies to overcome resistance.
•Next-generation sequencing (NGS) of cell-free DNA could expand access to precision medicine.•Technical differences between plasma NGS assays are poorly characterized.•We compare complex variants ...detection between two plasma NGS assays.•Using tumor as a reference, we identify biochemical and bioinformatic differences.
Plasma genotyping represents an opportunity for convenient detection of clinically actionable mutations in advanced cancer patients, such has been well-documented in non-small cell lung cancer (NSCLC). Oncogenic gene fusions are complex variants that may be more challenging to detect by next-generation sequencing (NGS) of plasma cell-free DNA (cfDNA). Rigorous evaluation of plasma NGS assays in the detection of fusions is needed to maximize clinical utility.
: Additional plasma was collected from patients with advanced NSCLC and ALK, ROS1, or RET gene fusions in tissue who had undergone clinical plasma NGS using Guardant360™(G360, Guardant Health). We then sequenced extracted cfDNA with a plasma NGS kit focused on known driver mutations in NSCLC (ctDx-Lung, Resolution Bioscience) with cloud-based bioinformatic analysis and blinded variant calling.
Of 16 patients assayed known to harbor anALK, ROS1, or RET in tumor, G360 detected fusions in 7 cases, ctDx-Lung detected fusions in 13 cases, and 3 cases were detected by neither. Of the 7 fusions detected by both assays, G360 reported lower mutant allelic fractions (AF). In cases missed by G360, tumor derived TP53 mutations were often detected confirming presence of tumor DNA. Raw sequencing data showed that inverted or out-of-frame variants were overrepresented in cases detected using ctDx-Lung but not by G360.
Focusing on complex, clinically actionable mutations using tumor as a reference standard allows for evaluation of technical differences in plasma NGS assays that may impact clinical performance. Noting the heterogeneity of fusion sequences observed in NSCLC, we hypothesize that differences in hybrid capture techniques and bioinformatic calling may be sources of variations in sensitivity among these assays.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The
proto-oncogene encodes a receptor tyrosine kinase that is activated by gene fusion in 1%-2% of non-small cell lung cancers (NSCLC) and rarely in other cancer types. Selpercatinib is a highly ...selective RET kinase inhibitor that has recently been approved by the FDA in lung and thyroid cancers with activating
gene fusions and mutations. Molecular mechanisms of acquired resistance to selpercatinib are poorly understood.
We studied patients treated on the first-in-human clinical trial of selpercatinib (NCT03157129) who were found to have
amplification associated with resistance to selpercatinib. We validated
activation as a targetable mediator of resistance to RET-directed therapy, and combined selpercatinib with the MET/ALK/ROS1 inhibitor crizotinib in a series of single patient protocols (SPP).
amplification was identified in posttreatment biopsies in 4 patients with
fusion-positive NSCLC treated with selpercatinib. In at least one case,
amplification was clearly evident prior to therapy with selpercatinib. We demonstrate that increased MET expression in
fusion-positive tumor cells causes resistance to selpercatinib, and this can be overcome by combining selpercatinib with crizotinib. Using SPPs, selpercatinib with crizotinib were given together generating anecdotal evidence of clinical activity and tolerability, with one response lasting 10 months.
Through the use of SPPs, we were able to offer combination therapy targeting
-amplified resistance identified on the first-in-human study of selpercatinib. These data suggest that MET dependence is a recurring and potentially targetable mechanism of resistance to selective RET inhibition in advanced NSCLC.
Plasma circulating tumor DNA (ctDNA) analysis is routine for genotyping of advanced non-small-cell lung cancer (NSCLC); however, early response assessment using plasma ctDNA has yet to be well ...characterized.
Patients with advanced
-mutant NSCLC across three phase I NCI osimertinib combination trials were analyzed in this study, and an institutional cohort of patients with
,
, and
-mutant advanced NSCLC receiving systemic treatment was used for validation. Plasma was collected before treatment initiation and serially before each cycle of therapy, and key driver mutations in ctDNA were characterized by droplet digital polymerase chain reaction. Timing of plasma versus imaging response was compared in a separate cohort of patients with
-mutant NSCLC treated with osimertinib. Across cohorts, we also studied ctDNA variability before treatment start.
In the NCI cohort, 14/16 (87.5%) patients exhibited ≥ 90% decrease in mutation abundance by the first on-treatment timepoint (20-28 days from treatment start) with minimal subsequent change. Similarly, 47/56 (83.9%) patients with any decrease in the institutional cohort demonstrated ≥ 90% decrease in mutation abundance by the first follow-up draw (7-30 days from treatment start). All 16 patients in the imaging cohort with radiographic partial response showed best plasma response within one cycle, preceding best radiographic response by a median of 24 weeks (range: 3-147 weeks). Variability in ctDNA levels before treatment start was observed.
Plasma ctDNA response is an early phenomenon, with the majority of change detectable within the first cycle of therapy. These kinetics may offer an opportunity for early insight into treatment effect before standard imaging timepoints.
Next-generation sequencing (NGS) is an important component of first-line treatment selection for metastatic non-small-cell lung cancer (NSCLC) and is typically ordered by medical oncologists in the ...outpatient setting after the pathologic diagnosis has been established. Time to treatment initiation is an important clinical challenge, especially for patients with rapidly progressive disease.
Plasma cell-free DNA (cfDNA) NGS was performed on 20 patients with suspected metastatic NSCLC hospitalized at an academic cancer center, before pathologic diagnosis. Clinicopathologic and treatment data were analyzed. Time from pathologic diagnosis to genotyping result was compared with standard care groups who underwent plasma or tumor NGS in routine clinical care.
The median time from pathologic diagnosis to the plasma cfDNA NGS result was 3 days in the study cohort, versus 18 days and 35.5 days in the standard care plasma and tumor NGS groups, respectively. 68.4% of evaluable patients had metastatic NSCLC, and 21.1% had another advanced solid tumor. Forty-five percent of plasma cfDNA results demonstrated actionable or informative genomic variants, and 20% of patients received standard or investigational first-line targeted therapy as a direct result of the plasma cfDNA NGS.
Plasma cfDNA NGS before pathologic diagnosis in hospitalized patients with suspected metastatic NSCLC results in substantially shorter time to genotyping result compared with standard outpatient workflows. This provides important initial evidence for the use of plasma-based genotyping earlier in the diagnostic journey, especially for patients with clinically aggressive disease. Additional studies and innovative approaches toward regulatory and reimbursement considerations are needed.
Plasma cell-free DNA (cfDNA) sequencing is a compelling diagnostic tool in solid tumors and has been shown to have high positive predictive value. However, limited assay sensitivity means that ...negative plasma genotyping, or the absence of detection of mutation of interest, still requires reflex tumor biopsy.
We analyzed two independent cohorts of patients with advanced non-small-cell lung cancer (NSCLC) with known canonical driver and resistance mutations who underwent plasma cfDNA genotyping. We measured quantitative features, such as maximum allelic frequency (mAF), as clinically available measures of cfDNA tumor content, and studied their relationship with assay sensitivity.
In patients with
-mutant NSCLC harboring
T790M, detection of driver mutation at > 1% AF conferred a sensitivity of 97% (368/380) for detection of T790M across three cfDNA genotyping platforms. Similarly, in a second cohort of patients with
or
driver mutations, when the mAF of nontarget mutations was > 1%, sensitivity for driver mutation detection was 100% (43/43). Combining the two NSCLC patient cohorts, the presence of nontarget mutations at mAF > 1% predicts for high sensitivity (> 95%) for identifying the presence of the known driver mutation, whereas mAF of ≤ 1% confers sensitivity of only 26%-54% across platforms. Focusing on 21 false-negative cases where the driver mutation was not detected on plasma next-generation sequencing, other mutations (presumably clonal hematopoiesis) were detected at ≤ 1% AF in 14 (67%).
Plasma cfDNA genotyping is highly sensitive when adequate tumor DNA content is present. The likelihood of a false-negative cfDNA genotyping result is low in a sample with evidence of > 1% tumor content. Bioinformatic approaches are needed to further optimize the assessment of cfDNA tumor content in plasma genotyping assays.
This study deals with total phenolic content, antiproliferative and proapoptotic activity of methanolic extracts from different Teucrium species and the effect on the prooxidant/antioxidant status in ...HCT-116 cells. The total phenolic content of the extracts was measured spectrophotometricaly and the obtained results ranged from 56.62 mg/g to 172.50 mg GA/g. The antiproliferative activity of methanolic extracts from different Teucrium species was determined using MTT cell viability assay, where IC(50) value was used as a parameter for cytotoxicity. The type of cell death was explored by fluorescence microscopy using the acridin orange/ethidium bromide method. MTT assay showed that all extracts significantly reduced cell viability in a dose-dependent manner, with very low IC(50) values. The highest content of phenolic compounds and the best cytotoxic activity on HCT-116 cells after 24 h of exposure was in T. chamaedrys extract, with IC(50) values of 5.48 × 10(-9) μg/mL. After 72 h, methanolic extract of T. arduini appeared to have the best cytotoxic activity on HCT-116, with IC(50) values of 0.37 μg/mL. Treatments caused typical apoptotic morphological changes in HCT-116 cells and showed a high percentage of apoptotic cells. The results of the presented research indicate that some Teucrium extracts are a very rich source of phenols, which may directly contribute to high antiproliferative and proapoptotic activity.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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