Introduction MET gene copy number gain (CNG) may be a predictive biomarker for MET inhibition in lung cancer, but the most appropriate method and criteria for defining MET positivity are uncertain. Methods MET copy number was assessed by fluorescence in situ hybridization (FISH) in lung adenocarcinoma. Positivity criteria included mean MET/cell ≥5 (low ≥5 – <6, intermediate ≥6 –<7, high ≥7) and MET/CEP7 ratio ≥1.8 (low ≥1.8 – ≤2.2, intermediate >2.2 – < 5, high ≥5). Associated clinical and molecular characteristics were captured. Results 99/686 cases (14%) had mean MET/cell ≥ 5, 52/1164 (4.5%) had MET/CEP7 ≥1.8. Other oncogenic drivers (in EGFR, KRAS, ALK, ERBB2, BRAF, NRAS, ROS1 or RET) were detectable in 56% of the mean MET/cell ≥ 5 group and 47% of the MET/CEP 7 ratio ≥1.8 group, suggesting many MET ‘positive’ cases are not truly MET-addicted. Concomitant drivers in low, indeterminate and high categories of mean MET/cell were 32/52 (62%), 12/19 (63%) and 11/27 (41%) (p=0.2) and in MET/CEP7: 15/29 (52%), 9/18 (50%) and 0/4 (0%) respectively (p=0.04). MET/CEP7 ≥1.8, in the absence of other oncogenes, was associated with a higher rate of adrenal metastases (p=0.03), but not with never smoking status. Conclusions FISH MET/CEP7 ≥ 5 defined a MET ‘positive’ group with no oncogenic overlap. As this method and criteria are also associated with the highest response rate to MET inhibition it represents the clearest definition of a MET CNG-addicted state. However, a MET-associated phenotype may also exist across MET/CEP7 ≥ 1.8 cases when no other oncogene overlap occurs.
ROS1 rearrangement/fusion detection in the clinical setting is complex and all methodologies have inherent limitations of which users must be aware to correctly interpret results.
Introduction: MNNG HOS Transforming gene (MET) amplification and MET exon 14 (METex14) alterations in lung cancers affect sensitivity to MET proto-oncogene, receptor tyrosine kinase (MET [also known by the alias hepatocyte growth factor receptor]) inhibitors. Fluorescence in situ hybridization (FISH), next-generation sequencing (NGS), and immunohistochemistry (IHC) have been used to evaluate MET dependency. Here, we have determined the association of MET IHC with METex14 mutations and MET amplification. Methods: We collected data on a tri-institutional cohort from the Lung Cancer Mutation Consortium. All patients had metastatic lung adenocarcinomas and no prior targeted therapies. MET IHC positivity was defined by an H-score of 200 or higher using SP44 antibody. MET amplification was defined by copy number fold change of 1.8x or more with use of NGS or a MET-to-centromere of chromosome 7 ratio greater than 2.2 with use of FISH. Results: We tested tissue from 181 patients for MET IHC, MET amplification, and METex14 mutations. Overall, 71 of 181 patients (39%) were MET IHC-positive, three of 181 (2%) were MET-amplified, and two of 181 (1%) harbored METex14 mutations. Of the MET-amplified cases, two were FISH positive with MET-to-centromere of chromosome 7
Mammalian topoisomerase II␣ (Topo II) is a highly regulated enzyme essential for many cellular processes including the G 2 cell cycle checkpoint. Because Topo II gene expression is regulated posttranscriptionally during the cell cycle, we investigated the possible role of the 3-untranslated region (3-UTR) in controlling Topo II mRNA accumulation. Reporter assays in stably transfected cells demonstrated that, similar to endogenous Topo II mRNA levels, the mRNA levels of reporter genes containing the Topo II 3-UTR varied during the cell cycle and were maximal in S and G 2 /M relative to G 1 . Topo II 3-UTR sequence analysis and RNA-protein binding assays identified a 177-nucleotide (base pairs 4772-4949) region containing an AUUUUUA motif sufficient for protein binding. Multiple proteins (84, 70, 44, and 37 kDa) bound this region, and the binding of 84-and 37-kDa (tentatively identified as the adenosine-or uridinerich element-binding factor AUF1) proteins was enhanced in G 1 , correlating with decreased Topo II mRNA levels. The binding activity was enhanced in cellular extracts or cells treated with thiol-reducing agents, and increased binding correlated with decreased Topo II mRNA levels. These results support the hypothesis that cell cycle-coupled Topo II gene expression is regulated by interaction of the 3-UTR with redox-sensitive protein complexes. Topo II) 1 is a multifunctional protein involved in many cellular processes including replication, repair, transcription, recombination, chromosome condensation and segregation, and the G 2 cell cycle checkpoint pathway (1-5). Topo II levels (mRNA, protein, and activity) increase in late S phase, peak in G 2 /M, and rapidly decrease following mitosis (4, 6 -8). Consistent with these observations, inhibitors of Topo II have been shown to arrest mammalian cells before mitosis in the G 2 phase of the cell cycle (6, 9 -12), and deregulated expression of Topo II results in cell death (13). In HeLa cells synchronized by mitotic shake-off, Topo II mRNA levels increased 16-fold in late S to G 2 /M phases (14 -18 h after plating) relative to G 1 (6). This increase in Topo II mRNA level was associated with a significant increase in Topo II mRNA stability, but with only marginal changes in the transcription rate. Mammalian topoisomerase II␣ (The regulation of mRNA stability has emerged as an important control mechanism of gene expression. Although the mechanisms that alter mRNA stability of different genes have unique features, in general, sequences located in the 3Ј-untranslated region (UTR) and their interactions with specific proteins regulate mRNA stability (14 -17). The most common 3Ј-UTR stability determinants are adenosine-or uridine-rich elements (AREs), which include AUUUA, AUUUUA, and AUUUUUA motifs (18 -22). Furthermore, a family of proteins, the AU-binding proteins, including AUF1 (23), Hel-N1 (24), AUH (25), HuR (26), and AUBF (27), have the capacity to bind with high affinity to mRNA containing ARE. Binding of AUbinding proteins (e.g. to AREs correlates with eit...
The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool in a number of genetic engineering processes. The Cre recombinase has been shown to act on DNA sequences that vary considerably from that of its bacteriophage recognition sequence, loxP. However, little is known about the sequence requirements for functional lox-like sequences. In this study, we have implemented a randomized library approach to identify the sequence characteristics of functional lox site domains. We created a randomized spacer library and a randomized arm library, and then tested them for recombination in vivo and in vitro. Results from the spacer library show that, while there is great plasticity, identity between spacer pairs is the most important factor influencing function, especially in in vitro reactions. The presence of one completely randomized arm in a functional loxP recombination reaction revealed that only three wild-type loxP arms are necessary for successful recombination in Cre-expressing bacteria, and that there are nucleotide preferences at the first three and last three positions of the randomized arm for the most efficiently recombined sequences. Finally, we found that in vitro Cre recombination reactions are much more stringent for evaluating which sequences can support efficient recombination compared to the 294-CRE system.
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