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•We identified 64 unique fusion partners. Rare fusion partners were identified more often in adults than children and included additional members of the ETS and CREB TF families. The remaining RFPs were classified as other transcription factors and non-transcription factors.•Five genes were identified to be altered at a higher prevalence in rare fusion partner tumors including CCNE1, KRAS, MYC, PIK3CA, and RAD21.Pathway enrichment associated alterations in cell cycle control and DNA damage response genes as driving the differences between fusion partners.•Potentially clinically actionable genomic variants were more prevalent in tumors harboring rare fusion partners than common fusions. Commonly altered genes associated with targetability included PIK4CA, CHEK2, ATM, EZH2, and KRAS.

Comprehensive genomic profiling of urothelial carcinoma (n = 1287) reveals a subset (∼0.3%) of urinary tract tumors driven by complete loss of function of the SMARCB1(switch/sucrose nonfermentable-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1) gene. The tumors are genomically and clinically distinct from conventional urothelial carcinoma, with limited additional genomic drivers, a low tumor mutational burden, an earlier presentation in life, and a female preponderance. Optimum benefit might be derived from nonconventional therapy.

IMPORTANCE: Molecular aberrations in the phosphatidylinositol-3-kinase (PI3K) pathway drive tumorigenesis. Frequently co-occurring alterations in hormone receptors and/or human epidermal growth factor receptor 2 (HER2) may be relevant to mechanisms of response and resistance. OBJECTIVE: To identify patterns of aberration in the PI3K and interactive pathways that might lead to targeted therapy opportunities in clinical practice. DESIGN, SETTING, AND PARTICIPANTS: From January 2013 through December 2014, 19 784 consecutive tumor samples (>40 cancer types) were sent from thousands of clinicians in 60 countries to a single commercial laboratory for molecular profiling, including next generation sequencing, protein expression (immunohistochemical analysis [IHC]), and gene amplification (fluorescent in situ hybridization or chromogenic in situ hybridization). MAIN OUTCOMES AND MEASURES: Patterns in targetable genomic and proteomic alterations in the PI3K pathway and coincidence with hormone receptor and HER2 alterations. EXPOSURES: Molecular profiling across solid tumors. RESULTS: Overall, 38% of patients had an alteration in 1 or more PI3K pathway components, most commonly phosphatase and tensin homologue (PTEN) loss (by IHC) (30% of all patients), followed by mutations in PIK3CA (13%), PTEN (6%), or AKT1 (1%). Seventy percent of patients with endometrial cancer and more than 50% of patients with breast, prostate, anal, hepatocellular, colorectal, and cervical cancer exhibited alterations in at least 1 PI3K pathway gene and/or gene product. Examples of frequent aberrations included PTEN loss in hepatocellular (57% of patients), colorectal (48%), gastric (36%), prostate (52%), and endometrial cancer (49%); PIK3CA mutations in endometrial (37%), breast (31%), cervical (29%), and anal cancer (27%). PIK3CA, PTEN, and AKT1 mutations occurred more frequently in the presence of hormone receptor overexpression (androgen, progesterone, or estrogen receptor). PIK3CA mutations were also more common in the HER2-positive than in the HER2-negative group; the opposite pattern was seen for PTEN mutation or PTEN loss. CONCLUSIONS AND RELEVANCE: PI3K pathway aberrations are among the most common in cancer. They do not segregate by classic cancer histologic characteristics. Patterns of biomarker coalterations involving HER2 and hormone receptors may be important for optimizing combination treatments across cancer types.

The HER2(official name ERBB2) gene encodes a membrane receptor in the epidermal growth factor receptor family amplified and overexpressed in adenocarcinoma. Activating mutations also occur in several cancers. We report mutation analyses of the HER2 kinase domain in 7497 histologically diverse cancers. Forty-five genes, including the kinase domain of HER2 with HER2 IHC and dual in situhybridization, were analyzed in tumors from 7497 patients with cancer, including 850 breast, 770 colorectal, 910 non–small cell lung, 823 uterine or cervical, 1372 ovarian, and 297 pancreatic cancers, as well as 323 melanomas and 2152 other solid tumors. Sixty-nine HER2 kinase domain mutations were identified in tumors from 68 patients (approximately 1% of all cases, ranging from absent in sarcomas to 4% in urothelial cancers), which included previously published activating mutations and 13 novel mutations. Fourteen cases with coexisting HER2mutation and amplification and/or overexpression were identified. Fifty-two of 68 patients had additional mutations in other analyzed genes, whereas 16 patients (23%) had HER2mutations identified as the sole driver mutation. HER2mutations coexisted with HER2gene amplification and overexpression and with mutations in other functionally important genes. HER2mutations were identified as the only driver mutation in a significant proportion of solid cancers. Evaluation of anti-HER2 therapies in nonamplified, HER2-mutated cancers is warranted.

Advanced urothelial carcinoma (UC) has a poor prognosis and new treatments are needed. In our study, multiplatform molecular profiling of UC identified unconventional treatment options in most cases. Different molecular profiles are exhibited between bladder and nonbladder UCs.

IQ® Technology, a homogeneous, universal-detection platform, originally designed for high-throughput screening (HTS) of kinases and phosphatases, has now been applied to protease screening. Representative enzymes from the major classes of proteases have been assayed in the IQ® format. Enzyme activity and compound inhibition data are presented for such enzymes as Trypsin, Matrix Metalloproteinase 3 (MMP-3) and Calpain 1. The technology has been tested in 96- to 384- to 1536-well microplate formats and is universally suited for automated screening. IQ® Technology is a direct, noncompetitive assay that does not require antibodies or radioisotopes. Fluorophore-labeled peptides are used as enzyme substrates. Kinase or phosphatase activity is quantified by direct measurement of the phosphorylation state of the substrate. For protease activity, cleavage is quantified with a peptide substrate containing a phospho-residue distal to the fluorphore. Cleavage of the substrate liberates the fluorphore-labeled terminus from the terminus containing the phospho-residue. Protease activity is measured by the change in fluorescence intensity that occurs when a proprietary compound binds specifically to phosphoryl groups on peptides and quenches the fluorescence. Iq® Technology can be used with any peptide sequence and is insensitive to high concentrations of ATP and substrate. The IQ® Technology has been validated against a large number of detergents, organics, and other reagents found in reaction mixtures and has been optimized for HTS applications exhibiting representative Z' values of 0.7. (JALA 2004;9:171-6)

IQ® Technology, a homogeneous, universal-detection platform, originally designed for high-throughput screening (HTS) of kinases and phosphatases, has now been applied to protease screening. Representative enzymes from the major classes of proteases have been assayed in the IQ® format. Enzyme activity and compound inhibition data are presented for such enzymes as Trypsin, Matrix Metalloproteinase 3 (MMP-3) and Calpain 1. The technology has been tested in 96- to 384- to 1536-well microplate formats and is universally suited for automated screening. IQ® Technology is a direct, noncompetitive assay that does not require antibodies or radioisotopes. Fluorophore-labeled peptides are used as enzyme substrates. Kinase or phosphatase activity is quantified by direct measurement of the phosphorylation state of the substrate. For protease activity, cleavage is quantified with a peptide substrate containing a phospho-residue distal to the fluorphore. Cleavage of the substrate liberates the fluorphore-labeled terminus from the terminus containing the phospho-residue. Protease activity is measured by the change in fluorescence intensity that occurs when a proprietary compound binds specifically to phosphoryl groups on peptides and quenches the fluorescence. Iq® Technology can be used with any peptide sequence and is insensitive to high concentrations of ATP and substrate. The IQ® Technology has been validated against a large number of detergents, organics, and other reagents found in reaction mixtures and has been optimized for HTS applications exhibiting representative Z’ values of 0.7.

The IQ®Technology has been developed to serve as a homogeneous, universal detection platform for HTS of kinases and phosphatases. The technology is a direct, noncompetitive assay format that does not require antibodies or radioactive reagents to measure phosphorylation state. Fluorophore-labeled peptides are used as enzyme substrates, and kinase or phosphatase activity is quantitated by direct measurement of the phosphorylation state of the substrate. Phosphorylation is measured by the change in fluorescence intensity that occurs when a proprietary iron-containing compound binds specifically to phosphoryl groups on peptides. This change in observed fluorescence is proportional to the extent of phosphorylation of the fluorophore-labeled peptide. The technology provides a universal method that can be used with any peptide sequence and is insensitive to high concentrations of ATP. Inhibition at the ATP-binding site versus the phosphorylation site can be differentiated and compound selectivity identified using the same detection method as in the primary screen. The technology has been tested against a large number of detergents, organics, and other reagents found in reaction mixtures, and the detection method eliminates common issues associated with fluorescent and chromogenic compounds. The technology has been formatted for 96-, 384-, and 1,536-well microplate formats, and a representative Z′ value of 0.7 was obtained. IC50values generated using this platform correlate with previously reported values, and screening of a small compound library was performed to evaluate the assay further.

The N-terminal pseudosubstrate site within the protein kinase Cα (PKCα)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCα-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940–8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCα-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Rα39–177: this protein contained the full regulatory domain of human PKCα fused to glutathione S-transferase (GST), but lacked amino acids 1–38 (including the pseudosubstrate sequence) and amino acids 178–270 (including the C2 region)]. GST-Rα39–177 significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCα holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33–86 within GST-Rα39–177 dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159–242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of multiple regions within the PKCα-regulatory domain that play a direct role in the inhibition of catalytic domain activity.

IQ™ Technology is a homogeneous, universal detection platform designed specifically for high-throughput screening of kinases and phosphatases. The technology has been tested in 96- to 384- to 1536-well microplate formats and is ideally suited for automated drug discovery screening systems. IQ™ Technology is a direct, non-competitive assay format that does not require antibodies or radioactive reagents to measure phosphorylation state. Kinase or phosphatase activity is quantitated by direct measurement of the phosphorylation state of fluorophore-labeled peptide substrates. Phosphorylation is measured by the change in fluorescence intensity that occurs when a proprietary iron-containing compound binds specifically to phosphoryl groups on peptides. This change in observed fluorescence is proportional to the extent of phosphorylation of the fluorophore-labeled peptide, because the iron-containing compound quenches the fluorescent emission exclusively on the phosphorylated form of the fluorophore-labeled peptide. The IQ™ Technology provides a universal method that can be used with any peptide sequence.

IQ™ Technology is a homogeneous, universal detection platform designed specifically for high-throughput screening of kinases and phosphatases. The technology has been tested in 96- to 384- to 1536-well microplate formats and is ideally suited for automated drug discovery screening systems. IQ™ Technology is a direct, non-competitive assay format that does not require antibodies or radioactive reagents to measure phosphorylation state. Kinase or phosphatase activity is quantitated by direct measurement of the phosphorylation state of fluorophore-labeled peptide substrates. Phosphorylation is measured by the change in fluorescence intensity that occurs when a proprietary iron-containing compound binds specifically to phosphoryl groups on peptides. This change in observed fluorescence is proportional to the extent of phosphorylation of the fluorophore-labeled peptide, because the iron-containing compound quenches the fluorescent emission exclusively on the phosphorylated form of the fluorophore-labeled peptide. The IQ™ Technology provides a universal method that can be used with any peptide sequence.

Homologous recombination DNA damage repair (HR-DDR) deficiency has been used to treat patients with various solid tumors with PARP inhibitors. However, the clinical characteristics of patients with melanoma who have HR-DDR gene mutations and the consequences of PARP inhibition are poorly understood. We compared the commercially available next-generation sequencing data from 84 patients with melanomas from our institution with a dataset of 1,986 patients as well as 1,088 patients profiled in cBioportal. In total, 21.4% of patients had ≥1 functional HR-DDR mutation, most commonly involving BRCA1, ARID1A, ATM, ATR,and FANCA. Concurrent NF1, BRAF,and NRASmutations were found in 39%, 39%, and 22% of cases, respectively. HR-DDR gene mutation was associated with high tumor mutational burden and clinical response to checkpoint blockade. A higher prevalence of HR-DDR mutations was observed in the datasets from Foundation Medicine (Cambridge, CA) and those from the Cancer Genome Atlas. Treatment of HR-DDR‒mutated patient-derived xenograft models of melanoma with PARP inhibitor produced significant antitumor activity in vivo and was associated with an increased apoptotic activity. RNA sequencing analysis of PARP inhibitor–treated tumors indicated alterations in the pathways involving extracellular matrix remodeling, cell adhesion, and cell-cycle progression. Melanomas with HR-DDR mutations represent a unique subset, which is more likely to benefit from checkpoint blockade and may be targeted with PARP inhibitor.

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