Prevalence of class I–III BRAF mutations among 114,662 cancer patients in a large genomic database

BRAF mutations are relatively common in many cancers, particularly melanoma, colorectal cancer, and thyroid cancer and to a lesser extent in lung cancer. These mutations can be targeted by BRAF and MEK inhibitors, which exhibit good clinical activity. There are conflicting reports of the various relative rates of BRAF Class I mutations (V600 locus), defined as those that exhibit extremely strong kinase activity by stimulating monomeric activation of BRAF, Class II, define as non-V600 mutations that activate BRAF to signal as a RAS-independent dimer, and Class III mutations, defined as “kinase-dead” with low kinase activity as compared to wild type BRAF. Prospective studies have largely focused on patients with tumors harboring Class I BRAF mutations (limited to the V600 locus) where response rates up to 70% with BRAF plus MEK inhibition have been demonstrated. We report on the relative prevalence of various types of BRAF mutations across human cancers in a cohort of 114,662 patients that received comprehensive genomic profiling using next-generation sequencing. Of these patients, 4517 (3.9%) a pathogenic or presumed pathogenic BRAF mutation (3.9%). Of these, 1271 were seen in melanoma, representing 39.7% of all melanomas sequenced, representing the highest rate in all tumors. Class I (V600) mutations were seen overall in 2841 patients (62.1% of BRAF mutations, 2.4% of total cancers). Class II mutations were seen in 746 tumors (16.5% of BRAF mutant, 0.7% of total), and Class III mutations were seen in 801 tumors (17.7% of BRAF, 0.7% of total). Knowledge of the relative prevalence of these types of mutations can aid in the development of agents that might better address non-V600 mutations in cancer.Impact statementThese data represent the largest aggregation of BRAF mutations within a single clinical database to our knowledge. The relative proportions of both BRAF V600 mutations and non-V600 mutations are informative in all cancers and by malignancy, and can serve as a definitive gold-standard for BRAF mutation cancer incidence by malignancy. The rate of BRAF mutation in human cancer in a real-world large database is lower than previously reported likely representing testing more broadly across tumor types. The relative percentages of Class II and Class III BRAF mutations are higher than previously reported, representing almost 35% of BRAF mutations in cancer. These findings provide support for the development of effective treatments for non-V600 BRAF mutations in cancer.     

Kinetic studies on the inactivation of 5-lipoxygenase by 5(S)-hydroperoxyeicosatetraenoic acid

The oxygenation of arachidonic acid (AA) by guinea-pig neutrophil 5-lipoxygenase terminates prematurely at a substrate utilization of only 50%. In the presence of dithiothreitol (DTT), reaction progress continues longer but still terminates prematurely, at about 70% substrate turnover. The addition of more substrate during the first 60 seconds of the initial reaction resulted in continued product formation. However, at times after 120 seconds, the addition of more AA could not produce additional product formation. Together, these results indicate a time-dependent (t1/2 = 0.5-1.0 min), irreversible loss of enzyme activity. To determine if the product 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) mediates the inactivation, it was tested for its ability to irreversibly inhibit the enzyme and found to inactivate 5-lipoxygenase with Ki = 0.05 +/- 0.01 microM and ki = 1.4 +/- 0.4 min-1. DTT changed the apparent affinity of 5-HPETE (Ki = 0.33 +/- 0.09 microM) but had no effect on the rate of inactivation (ki = 1.26 +/- 0.62 min-1). In contrast, the hydroxy derivative of 5-HPETE, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), is a reversible, time-independent inhibitor with Ki = 6.3 +/- 0.9 microM regardless of DTT. The ability of thiols to protect 5-lipoxygenase from production inactivation is due, at least in part, to a non-enzymatic reaction between DTT and 5-HPETE that converts the hydroperoxy acid to a material that can no longer inactivate the enzyme.     

Differential Effector Engagement by Oncogenic KRAS

1. Download high-res image (291KB)
2. Download full-size image

     

Identification of plastid genotypes in Oenothera subsect. Munzia by restriction fragment length polymorphisms (RFLPs)

 Five discrete plastid genotypes (plastomes), designated I–V and typified by Oenothera Hookeri, biennis, Lamarckiana, parviflora and argillicola respectively, have been previously characterized within the European subsect. Euoenothera. The evolutionarily more-derived plastome types (I, II and V) are generally less tolerant of new hybridization events than the ancestral types (III and IV), and were first identified based on their incompatibility reactions with standard hybrid nuclei. Restriction maps for all five plastomes are available for the enzymes PvuII, SalI, KpnI and PstI (Gordon et al. 1982). The present study employs PvuII and KpnI restriction digests to compare 28 of the 45 species of subsect. Munzia with Euoenothera plastomes I–V. The results of plastome RFLP fingerprinting show uniform divergence of the South American taxa from their European congeners; all share the previously documented 45-kb inversion in the large single-copy region reported by Hachtel et al. (1991). However, at least six new plastome types have evolved within subsect. Munzia, giving rise to small-fragment size differences of 0.1–0.7 kb. In two of these cases (Oe. featherstonei and Oe. longiflora) unique fragments occurred. For Oe. featherstonei the unique KpnI fragment resulted from a novel 2.2 kb insertion, whereas in Oe. longiflora an additional PvuII restriction site has been created. Received: 2 June 1998 / Accepted: 14 July 1998     

Gene therapy with iNOS provides long-term protection against myocardial infarction without adverse functional consequences

Previous studies have shown that gene therapy with inducible nitric oxide synthase (iNOS) protects against myocardial infarction at 3 days after gene transfer. However, the long-term effects of iNOS gene therapy on myocardial ischemic injury and cardiac function are unknown. To address this issue, we used a recombinant adenovirus 5 (Ad5) vector (Av3) with deletions of the E1, E2a, and E3 regions, which enables long-lasting recombinant gene expression for at least 2 mo due to lack of inflammation. Mice received intramyocardial injections in the left ventricular (LV) anterior wall of Av3/LacZ (LacZ group) or Av3/iNOS (iNOS group); 1 or 2 mo later, they were subjected to myocardial infarction (30-min coronary occlusion followed by 4 h of reperfusion). Cardiac iNOS gene expression was confirmed by immunoblotting and activity assays at 1 and 2 mo after gene transfer. In the iNOS group, infarct size (percentage of risk region) was significantly reduced (P < 0.05) both at 1 mo (24.2 +/- 3.4%, n = 6, vs. 48.0 +/- 3.6%, n = 8, in the LacZ group) and at 2 mo (23.4 +/- 3.1%, n = 8, vs. 36.6 +/- 2.4%, n = 7). The infarct-sparing effects of iNOS gene therapy were as powerful as those observed 24 h after ischemic preconditioning (23.1 +/- 3.4%, n = 10). iNOS gene transfer had no effect on LV function or dimensions up to 8 wk later (echocardiography). These data demonstrate that iNOS gene therapy mediated by the Av3 vector affords long-term (2 mo) cardioprotection without inflammation or adverse functional consequences, a finding that provides a rationale for further preclinical testing of this therapy.