SUMMARY We report the preclinical evaluation of PF-06463922, a potent and brain penetrant ALK/ROS1 inhibitor. Compared to other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors due to secondary ALK kinase domain mutations and/or due to the failed control of brain metastases.

Dinoflagellate taxonomy is based primarily on morphology and morphometric data that can be difficult to obtain. In contrast, molecular data can be rapidly and cost-effectively acquired, which has led to a rapid accumulation of sequence data in GenBank. Currently there are no systematic criteria for utilizing taxonomically unassigned sequence data to identify putative species that could in turn serve as a basis for testable hypotheses concerning the taxonomy, diversity, distribution, and toxicity of these organisms. The goal of this research was to evaluate whether simple, uncorrected genetic distances (p) calculated using ITS1/5.8S/ITS2 (ITS region) rDNA sequences could be used to develop criteria for recognizing putative species before formal morphological evaluation and classification. The current analysis used sequences from 81 dinoflagellate species belonging to 14 genera. For this diverse assemblage of dinoflagellate species, the within-species genetic distances between ITS region copies (p 5 0.000-0.021 substitutions per site) were consistently less than those observed between species (p 5 0.042-0.580). Our results indicate that a between-species uncorrected genetic distance of p! 0.04 could be used to delineate most free-living dinoflagellate species. Recently evolved species, however, may have ITS p values <0.04 and would require more extensive morphological and genetic analyses to resolve. For most species, the sequence of the dominant ITS region allele has the potential to serve as a unique species-specific ''DNA barcode'' that could be used for the rapid identification of dinoflagellates in field and laboratory studies.

Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1 G2032R mutation and the ROS1 G2026M gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.PF-06463922 | ROS1 | kinase inhibitor R eceptor tyrosine kinases (RTKs) are vital conduits of extracellular signals that direct cell growth and survival pathways. Unregulated RTK activation through chromosomal rearrangements, point mutations, and gene amplification has been shown to be responsible for the initiation and progression of many cancers. The orphan RTK c-ros oncogene1 (ROS1) normally is expressed transiently in various tissues during development with little to no expression in adult tissues (1). Elevated full-length c-ROS1 expression levels have been observed in 20-30% of patients with nonsmall cell lung cancer (NSCLC) by gene expression profiling (2-4) and in 13% of patients with lung adenocarcinoma using immunohistochemistry (IHC) (5). However, its function, both in normal physiology and disease, remains poorly defined mainly because of its still unidentified ligand. Chromosomal rearrangements resulting in oncogenic activation of ROS1 have been observed in a subset of patients with glioblastoma (6-9), NSCLC (10-14), cholangiocarcinoma (15), ovarian cancer (16), angiosarcoma (17), inflammatory myofibroblastic tumors (18), and Spitzoid melanoma (19). To date, interchromosomal translocations or intrachromosomal deletions have resulted in the production of 20 different N-terminal ROS1 fusion genes in a variety of cancers (Table S1).ROS1 is a distinct receptor with a kinase domain that is phylogenetically related to the anaplastic lymphoma kinase/lymphocyte-specific protein tyrosine kinase (ALK/LTK) and insulin receptor (INSR) RTK families (20), suggesting that tyrosine kinase inhibitors for these receptors could have cross-activity against ROS1. A recent phase I/II cl...

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A reverse-transcriptase-polymerase-chain-reaction (RT-PCR) procedure was evaluated for detection of chicken proventricular necrosis virus (CPNV) in transmissible viral proventriculitis (TVP) -affected chickens. The RT-PCR procedure was compared with indirect immunofluorescence (IFA) and virus isolation for detection of CPNV in experimentally infected chickens. Microscopic lesions characteristic of TVP were detected on days 5-35 postexposure (PE) in CPNV-infected chickens; CPNV was detected by RT-PCR on days 3-14 PE in freshly collected proventriculi, and on days 1-14 PE in formalin-fixed paraffin-embedded (FFPE) proventriculi. CPNV was detected in proventriculi of experimentally infected chickens by IFA on days 3-10 PE, and by virus isolation on days 1-14 PE. With IFA used as a reference, sensitivity of the RT-PCR procedure with freshly collected and FFPE proventriculi was 88% and 100%, respectively; specificity was 83% and 86%, respectively. Proventriculi (FFPE) obtained from suspect TVP cases (n=19) were evaluated for presence of CPNV by RT-PCR and microscopic lesions consistentwith TVP. CPNV was detected by RT-PCR in proventriculi from 8/11 TVP (+) cases (24/36 tissue sections). TVP (+) cases were defined by microscopic lesions characteristic of TVP; CPNV was not detected in proventriculi (0/8 cases, 0/32 tissue sections) in the absence of these lesions. The association between presence of TVP-characteristic microscopic lesions and presence of CPNV was highly significant (P = 0.0014). These findings indicate the utility of the RT-PCR procedure for detection of CPNV and provide additional evidence for an etiologic role for this virus in TVP.