Characterization of 1,387 NSCLCs with MET exon 14 (METex14) skipping alterations (SA) and potential acquired resistance (AR) mechanisms.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 9511-9511
Author(s):  
Mark M. Awad ◽  
Jessica Kim Lee ◽  
Russell Madison ◽  
Anthony Classon ◽  
Jamie Kmak ◽  
...  
Keyword(s):  
Donor Site ◽  
Acquired Resistance ◽  
Circulating Tumor Dna ◽  
Acceptor Site ◽  
Polypyrimidine Tract ◽  
Comprehensive Overview ◽  
Functional Sites ◽  
Paired Samples ◽  
Combination Strategies ◽  
Rational Combination

9511 Background: METex14 SA are oncogenic drivers in NSCLC. Due to the numerous sites around ex14 that bind the spliceosome complex, many variations can result in deleterious alterations (alts). We present a comprehensive overview of these ex14 SA across 1,387 NSCLCs and characterized potential AR mechanisms. Methods: Hybrid-capture based comprehensive genomic profiling (CGP) was performed on samples from 60,495 NSCLC patients (pts). A scoring system was applied leveraging our large database of samples with METex14 SA to optimize accurate reporting of these variants. Paired samples were collected ≥ 60 days apart (median 462). Results: 1,393 METex14 SA were identified in samples (1,278 tissue, 109 circulating tumor DNA (ctDNA)) from 1,387 NSCLC pts (2.3%) spanning multiple functional sites: donor (42%), acceptor (4.7%), poly-pyrimidine tract (15%), acceptor and polypyrimidine tract (13%), D1010 (23%), Y1003 (2.1%), and whole exon deletions (0.3%). 6 samples (5 tissue, 1 ctDNA) harbored 2 METex14 SA, each including a mutation (mut) at the donor or acceptor site. MDM2 and CDK4 amplifications (amps) co-occurred in 32% and 19% of METex14 samples, respectively, but were more common with polypyrimidine tract (37% and 23%) vs donor site (32%, p = 0.07 and 18%, p = 0.07) alts. MET co-amp was present in 12% of cases and frequency did not significantly differ by functional site. 66 (4.8%) cases (57 tissue, 9 ctDNA) had known NSCLC co-drivers, including KRAS (68%) and EGFR (14%) mut, a subset of which may represent AR. Paired samples with a METex14 SA in the 1st sample were available for 36 pts. The METex14 SA was detected in the 2nd sample for 32 pts, excluding 3 with low ctDNA. 22/36 (61%) had reportable acquired alts detected including 9 with ≥1 acquired MET muts [D1228X (4), Y1230X (3), Y1003F (1), D1228A/E/H + L1195V (1)] and 3 with acquired MET amp. Other acquired alts included ERBB2 amp and mut (1 each), EGFR ex19ins (1), KRAS amp (1), PIK3CA mut (1), AKT2 amp (1) and others with unknown functional significance. Potential AR alts were present with primary METex14 SA spanning all functional sites. Conclusions: In a dataset of > 60,000 advanced NSCLCs, METex14 SA were present in 2.3% of cases, and represented 6 major subtypes. Among paired cases, potential AR mechanisms included secondary MET alts (33%), and acquired alts in EGFR, ERBB2, KRAS, and PI3K pathways. Acquired alts were independent of the type of METex14 SA. Characterizing common co-occuring may be critical for predicting responses to MET inhibitors and informing rational combination strategies.

scholarly journals Characterization of Non–Small-Cell Lung Cancers With MET Exon 14 Skipping Alterations Detected in Tissue or Liquid: Clinicogenomics and Real-World Treatment Patterns

2021 ◽  
pp. 1354-1376
Author(s):  
Jessica K. Lee ◽  
Russell Madison ◽  
Anthony Classon ◽  
Ole Gjoerup ◽  
Mark Rosenzweig ◽  
...  
Keyword(s):  
Real World ◽  
Acquired Resistance ◽  
Resistance Mechanisms ◽  
Pi3k Pathway ◽  
Treatment Patterns ◽  
Small Cell ◽  
Small Cell Lung ◽  
Comprehensive Overview ◽  
Tissue Samples ◽  
Functional Sites

PURPOSE MET exon 14 ( METex14) skipping alterations are oncogenic drivers in non–small-cell lung cancer (NSCLC). We present a comprehensive overview of METex14 samples from 1,592 patients with NSCLC, associated clinicogenomic characteristics, potential mechanisms of acquired resistance, treatment patterns, and outcomes to MET inhibitors. METHODS Hybrid capture–based comprehensive genomic profiling (CGP) was performed on samples from 69,219 patients with NSCLC. For treatment patterns and outcomes analysis, patients with advanced METex14-altered NSCLC were selected from the Flatiron Health-Foundation Medicine clinicogenomic database, a nationwide deidentified electronic health record–derived database linked to Foundation Medicine CGP for patients treated between January 2011 and March 2020. RESULTS A total of 1,592 patients with NSCLC (2.3%) were identified with 1,599 METex14 alterations spanning multiple functional sites (1,458 of 60,244 tissue samples and 134 of 8,975 liquid samples). Low tumor mutational burden and high programmed death ligand 1 expression were enriched in METex14-altered samples. MDM2, CDK4, and MET coamplifications and TP53 mutations were present in 34%, 19%, 11%, and 42% of tissue samples, respectively. Comparing tissue and liquid cohorts, coalteration frequency and acquired resistance mechanisms, including multiple MET mutations, EGFR, ERBB2, KRAS, and PI3K pathway alterations, were generally similar. Positive percent agreement with the tissue was 100% for METex14 pairs collected within 1 year (n = 7). Treatment patterns showed increasing adoption of MET inhibitors in METex14-altered NSCLC after receipt of CGP results; the real-world response rate to MET inhibitors was 45%, and time to treatment discontinuation was 4.4 months. CONCLUSION Diverse METex14 alterations were present in 2%-3% of NSCLC cases. Tissue and liquid comparisons showed high concordance and similar coalteration profiles. Characterizing common co-occurring alterations and immunotherapy biomarkers, including those present before or acquired after treatment, may be critical for predicting responses to MET inhibitors and informing rational combination strategies.

scholarly journals A broad analysis of splicing regulation in yeast using a large library of synthetic introns

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009805
Author(s):  
Dvir Schirman ◽  
Zohar Yakhini ◽  
Yitzhak Pilpel ◽  
Orna Dahan
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Splice Site ◽  
Donor Site ◽  
Regulation Of Gene Expression ◽  
Combinatorial Design ◽  
Acceptor Site ◽  
Functional Sites ◽  
Splicing Efficiency ◽  
Splicing Machinery

RNA splicing is a key process in eukaryotic gene expression, in which an intron is spliced out of a pre-mRNA molecule to eventually produce a mature mRNA. Most intron-containing genes are constitutively spliced, hence efficient splicing of an intron is crucial for efficient regulation of gene expression. Here we use a large synthetic oligo library of ~20,000 variants to explore how different intronic sequence features affect splicing efficiency and mRNA expression levels in S. cerevisiae. Introns are defined by three functional sites, the 5’ donor site, the branch site, and the 3’ acceptor site. Using a combinatorial design of synthetic introns, we demonstrate how non-consensus splice site sequences in each of these sites affect splicing efficiency. We then show that S. cerevisiae splicing machinery tends to select alternative 3’ splice sites downstream of the original site, and we suggest that this tendency created a selective pressure, leading to the avoidance of cryptic splice site motifs near introns’ 3’ ends. We further use natural intronic sequences from other yeast species, whose splicing machineries have diverged to various extents, to show how intron architectures in the various species have been adapted to the organism’s splicing machinery. We suggest that the observed tendency for cryptic splicing is a result of a loss of a specific splicing factor, U2AF1. Lastly, we show that synthetic sequences containing two introns give rise to alternative RNA isoforms in S. cerevisiae, demonstrating that merely a synthetic fusion of two introns might be suffice to facilitate alternative splicing in yeast. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome. In addition, it provides a valuable resource to study the regulation of constitutive and alternative splicing in a model organism.

Genomic analysis of circulating tumor DNA in 3,334 patients with advanced prostate cancer to identify targetable BRCA alterations and AR resistance mechanisms.

2021 ◽  
Vol 39 (6_suppl) ◽  
pp. 25-25
Author(s):  
Hanna Tukachinsky ◽  
Russell Madison ◽  
Jon Chung ◽  
Lucas Dennis ◽  
Bernard Fendler ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Advanced Prostate Cancer ◽  
Acquired Resistance ◽  
Genomic Analysis ◽  
Circulating Tumor Dna ◽  
Resistance Mechanisms ◽  
Cancer Tissue ◽  
Castration Resistant Prostate Cancer ◽  
High Level ◽  
Tumor Dna

25 Background: Comprehensive genomic profiling (CGP) by next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) from plasma provides a minimally invasive method to identify targetable genomic alterations (GAs) and resistance mechanisms in patients with metastatic castration-resistant prostate cancer (mCRPC). The circulating tumor fraction in patients with mCRPC and the clinical validity of GAs detected in plasma remain unknown. We evaluated the landscape of GAs using ctDNA-based CGP and assessed concordance with tissue-based CGP. Methods: Plasma from 3,334 patients with advanced prostate cancer (including 1,674 mCRPC screening samples from the TRITON2/3 trials and 1,660 samples from routine clinical CGP) was analyzed using hybrid-capture-based gene panel NGS assays. Results were compared with CGP of 2,006 metastatic prostate cancer tissue biopsies. Concordance was evaluated in 837 patients with both tissue (archival or contemporaneous) and plasma NGS results. Results: 3,127 patients [94%] had detectable ctDNA. BRCA1/2 were mutated in 295 patients [8.8%]. In concordance analysis, 72/837 [8.6%] patients had BRCA1/2 mutations detected in tissue, 67 [93%] of whom were also identified by ctDNA, and 20 patients were identified using ctDNA but not tissue [23% of all patients identified using ctDNA]. ctDNA detected subclonal BRCA1/2 reversions in 10 of 1,660 [0.6%] routine clinical CGP samples. AR alterations, including amplifications and hotspot mutations, which were detected in 940/2,213 patients [42%]. Rare AR compound mutations, rearrangements, and novel in-frame deletions were identified. Altered pathways included PI3K/AKT/mTOR [14%], WNT/β-catenin [17%], and RAS/RAF/MEK [5%]. Microsatellite instability was detected in 31/2,213 patients [1.4%]. Conclusions: In the largest study of mCRPC plasma samples conducted to date, CGP of ctDNA recapitulated the genomic landscape detected in tissue biopsies, with a high level of agreement in detection of BRCA1/2 alterations. It also identified patients who may have gained somatic BRCA1/2 alterations since archival tissue was collected. ctDNA detected more acquired resistance GAs than tissue, including novel AR-activating variants. The large percentage of patients with rich genomic signal from ctDNA, and the sensitive, specific detection of BRCA1/2 alterations position liquid biopsy as a compelling clinical complement to tissue CGP for patients with mCRPC.

Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells

1993 ◽  
Vol 13 (8) ◽  
pp. 5085-5098
Author(s):  
A M Carothers ◽  
G Urlaub ◽  
D Grunberger ◽  
L A Chasin
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Consensus Sequence ◽  
Donor Site ◽  
Chinese Hamster ◽  
Base Substitution ◽  
Acceptor Site ◽  
Rnase Protection ◽  
Single Base

Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence.

BRCA Reversion Mutations in Circulating Tumor DNA Predict Primary and Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma

2018 ◽  
Vol 9 (2) ◽  
pp. 210-219 ◽  
Author(s):  
Kevin K. Lin ◽  
Maria I. Harrell ◽  
Amit M. Oza ◽  
Ana Oaknin ◽  
Isabelle Ray-Coquard ◽  
...  
Keyword(s):  
Ovarian Carcinoma ◽  
Parp Inhibitor ◽  
Acquired Resistance ◽  
Circulating Tumor Dna ◽  
High Grade ◽  
Tumor Dna

scholarly journals Efficacy of Sym004 in Patients With Metastatic Colorectal Cancer With Acquired Resistance to Anti-EGFR Therapy and Molecularly Selected by Circulating Tumor DNA Analyses

JAMA Oncology ◽  
2018 ◽  
Vol 4 (4) ◽  
pp. e175245 ◽  
Author(s):  
Clara Montagut ◽  
Guillem Argilés ◽  
Fortunato Ciardiello ◽  
Thomas T. Poulsen ◽  
Rodrigo Dienstmann ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Metastatic Colorectal Cancer ◽  
Acquired Resistance ◽  
Circulating Tumor Dna ◽  
Dna Analyses ◽  
Tumor Dna

scholarly journals Overactive IGF1/Insulin Receptors and NRASQ61R Mutation Drive Mechanisms of Resistance to Pazopanib and Define Rational Combination Strategies to Treat Synovial Sarcoma

Cancers ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 408 ◽  
Author(s):  
Cinzia Lanzi ◽  
Laura Dal Bo ◽  
Enrica Favini ◽  
Monica Tortoreto ◽  
Giovanni Beretta ◽  
...  
Keyword(s):  
Cell Line ◽  
Synovial Sarcoma ◽  
Molecular Mechanisms ◽  
Soft Tissue Sarcomas ◽  
Insulin Receptors ◽  
Mek Inhibitor ◽  
Combination Strategies ◽  
Rational Drug ◽  
Rational Combination ◽  
Induced Apoptosis

Pazopanib is approved for treatment of advanced soft tissue sarcomas, but primary and secondary drug resistance limits its clinical utility. We investigated the molecular mechanisms mediating pazopanib resistance in human synovial sarcoma (SS) models. We found reduced cell sensitivity to pazopanib associated with inefficient inhibition of the two critical signaling nodes, AKT and ERKs, despite strong inhibition of the main drug target, PDGFRα. In the CME-1 cell line, overactivation of IGF1 and Insulin receptors (IGF1R/InsR) sustained AKT activation and pazopanib resistance, which was overcome by a combination treatment with the double IGF1R/InsR inhibitor BMS754807. In the highly pazopanib resistant MoJo cell line, NRASQ61R mutation sustained constitutive ERK activation. Transfection of the NRAS mutant in the pazopanib sensitive SYO-1 cell line increased the drug IC50. MoJo cells treatment with pazopanib in combination with the MEK inhibitor trametinib restored ERK inhibition, synergistically inhibited cell growth, and induced apoptosis. The combination significantly enhanced the antitumor efficacy against MoJo orthotopic xenograft abrogating growth in 38% of mice. These findings identified two different mechanisms of intrinsic pazopanib resistance in SS cells, supporting molecular/immunohistochemical profiling of tumor specimens as a valuable approach to selecting patients who may benefit from rational drug combinations.

scholarly journals Untangling the sequence of events during the S2→ S3transition in photosystem II and implications for the water oxidation mechanism

2020 ◽  
Vol 117 (23) ◽  
pp. 12624-12635 ◽  
Author(s):  
Mohamed Ibrahim ◽  
Thomas Fransson ◽  
Ruchira Chatterjee ◽  
Mun Hon Cheah ◽  
Rana Hussein ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Water Oxidation ◽  
Donor Site ◽  
Oxidation Mechanism ◽  
Oxygenic Photosynthesis ◽  
Acceptor Site ◽  
Oxygen Evolving Complex ◽  
Ps Ii ◽  
Sequence Of Events ◽  
Large Channel

In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2→ S3transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2→ S3transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QAand QB, are observed. At the donor site, tyrosine YZand His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a “water wheel”-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2→ S3transition mirrors the appearance of OXelectron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.

scholarly journals Mechanisms of Acquired Resistance to Savolitinib, a Selective MET Inhibitor in MET-Amplified Gastric Cancer

2020 ◽  
pp. 222-232 ◽  
Author(s):  
Melanie M. Frigault ◽  
Aleksandra Markovets ◽  
Barrett Nuttall ◽  
Kyoung-Mee Kim ◽  
Se Hoon Park ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Progression ◽  
Tumor Tissue ◽  
Treatment Options ◽  
Acquired Resistance ◽  
Circulating Tumor Dna ◽  
Resistance Mechanisms ◽  
Met Inhibitor ◽  
Tumor Dna ◽  
Generation Sequencing

PURPOSE Some gastric cancers harbor MET gene amplifications that can be targeted by selective MET inhibitors to achieve tumor responses, but resistance eventually develops. Savolitinib, a selective MET inhibitor, is beneficial for treating patients with MET-driven gastric cancer. Understanding the resistance mechanisms is important for optimizing postfailure treatment options. PATIENTS AND METHODS Here, we identified the mechanisms of acquired resistance to savolitinib in 3 patients with gastric cancer and MET-amplified tumors who showed a clinical response and then cancer progression. Longitudinal circulating tumor DNA (ctDNA) is useful for monitoring resistance during treatment and progression when rebiopsy cannot be performed. RESULTS Using a next-generation sequencing 100-gene panel, we identified the target mechanisms of resistance MET D1228V/N/H and Y1230C mutations or high copy number MET gene amplifications that emerge when resistance to savolitinib develops in patients with MET-amplified gastric cancer. CONCLUSION We demonstrated the utility of ctDNA in gastric cancer and confirmed this approach using baseline tumor tissue or rebiopsy.

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