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1.  Discovery of 4-Aryl-2-benzoyl-imidazoles As Tubulin Polymerization Inhibitor with Potent Antiproliferative Properties 
Journal of medicinal chemistry  2013;56(8):3318-3329.
A series of 4-aryl-2-benzoyl-imidazoles were designed and synthesized based on our previously reported 2-aryl-4-benzoyl-imidazole (ABI) derivatives. The new structures reversed the aryl group and the benzoyl group of previous ABI structures and were named as reverse ABI (RABI) analogs. RABIs were evaluated for biological activity against 8 cancer cell lines including multidrug-resistant cancer cell lines. In vitro assays indicated that several RABI compounds had excellent antiproliferative properties with IC50 values in the low nanomolar range. The average IC50 of the most active compound 12a is 14 nM. In addition, the mechanism of action of these new analogs was investigated by cell cycle analysis, tubulin polymerization assay, competitive mass spectrometry binding assay and molecular docking studies. These studies confirmed that these new RABI analogs maintain their mechanisms of action by disrupting tubulin polymerization, similar to their parental ABI analogs.
doi:10.1021/jm4001117
PMCID: PMC3668676  PMID: 23547728
2.  Estrogen regulates histone deacetylases to prevent cardiac hypertrophy 
Molecular Biology of the Cell  2013;24(24):3805-3818.
Angiotensin II stimulation of HDAC2 production, phosphorylation by CK2, and resulting modulation of target genes, which promote cardiac hypertrophy, are opposed by estrogen/ERβ. Angiotensin II also represses class II HDAC4 and 5 production and stimulates their phosphorylation, which expels them from the nucleus, and estrogen prevents this.
The development and progression of cardiac hypertrophy often leads to heart failure and death, and important modulators of hypertrophy include the histone deacetylase proteins (HDACs). Estrogen inhibits cardiac hypertrophy and progression in animal models and humans. We therefore investigated the influence of 17-β-estradiol on the production, localization, and functions of prohypertrophic (class I) and antihypertrophic (class II) HDACs in cultured neonatal rat cardiomyocytes. 17-β-Estradiol or estrogen receptor β agonists dipropylnitrile and β-LGND2 comparably suppressed angiotensin II–induced HDAC2 (class I) production, HDAC-activating phosphorylation, and the resulting prohypertrophic mRNA expression. In contrast, estrogenic compounds derepressed the opposite effects of angiotensin II on the same parameters for HDAC4 and 5 (class II), resulting in retention of these deacetylases in the nucleus to inhibit hypertrophic gene expression. Key aspects were confirmed in vivo from the hearts of wild-type but not estrogen receptor β (ERβ) gene–deleted mice administered angiotensin II and estrogenic compounds. Our results identify a novel dual regulation of cardiomyocyte HDACs, shown here for the antihypertrophic sex steroid acting at ERβ. This mechanism potentially supports using ERβ agonists as HDAC modulators to treat cardiac disease.
doi:10.1091/mbc.E13-08-0444
PMCID: PMC3861078  PMID: 24152730
3.  Selective androgen receptor modulators for the treatment of late onset male hypogonadism 
Asian Journal of Andrology  2014;16(2):256-261.
Several testosterone preparations are used in the treatment of hypogonadism in the ageing male. These therapies differ in their convenience, flexibility, regional availability and expense but share their pharmacokinetic basis of approval and dearth of long-term safety data. The brevity and relatively reduced cost of pharmacokinetic based registration trials provides little commercial incentive to develop improved novel therapies for the treatment of late onset male hypogonadism. Selective androgen receptor modulators (SARMs) have been shown to provide anabolic benefit in the absence of androgenic effects on prostate, hair and skin. Current clinical development for SARMs is focused on acute muscle wasting conditions with defined clinical endpoints of physical function and lean body mass. Similar regulatory clarity concerning clinical deficits in men with hypogonadism is required before the beneficial pharmacology and desirable pharmacokinetics of SARMs can be employed in the treatment of late onset male hypogonadism.
doi:10.4103/1008-682X.122339
PMCID: PMC3955335  PMID: 24407183
hypogonadism; late onset hypogonadism; selective androgen receptor modulators; testosterone
4.  Posterior fossa and spinal gangliogliomas form two distinct clinicopathologic and molecular subgroups 
Background
Gangliogliomas are low-grade glioneuronal tumors of the central nervous system and the commonest cause of chronic intractable epilepsy. Most gangliogliomas (>70%) arise in the temporal lobe, and infratentorial tumors account for less than 10%. Posterior fossa gangliogliomas can have the features of a classic supratentorial tumor or a pilocytic astrocytoma with focal gangliocytic differentiation, and this observation led to the hypothesis tested in this study - gangliogliomas of the posterior fossa and spinal cord consist of two morphologic types that can be distinguished by specific genetic alterations.
Results
Histological review of 27 pediatric gangliogliomas from the posterior fossa and spinal cord indicated that they could be readily placed into two groups: classic gangliogliomas (group I; n = 16) and tumors that appeared largely as a pilocytic astrocytoma, but with foci of gangliocytic differentiation (group II; n = 11). Detailed radiological review, which was blind to morphologic assignment, identified a triad of features, hemorrhage, midline location, and the presence of cysts or necrosis, that distinguished the two morphological groups with a sensitivity of 91% and specificity of 100%. Molecular genetic analysis revealed BRAF duplication and a KIAA1549-BRAF fusion gene in 82% of group II tumors, but in none of the group I tumors, and a BRAF:p.V600E mutation in 43% of group I tumors, but in none of the group II tumors.
Conclusions
Our study provides support for a classification that would divide infratentorial gangliogliomas into two categories, (classic) gangliogliomas and pilocytic astrocytomas with gangliocytic differentiation, which have distinct morphological, radiological, and molecular characteristics.
doi:10.1186/2051-5960-2-18
PMCID: PMC3931494  PMID: 24529209
Ganglioglioma; Pilocytic astrocytoma; Glioneuronal; BRAF; Mutation
5.  Guidelines from the Canadian Association of Pathologists for establishing a telepathology service for anatomic pathology using whole-slide imaging 
The use of telepathology for clinical applications in Canada has steadily become more attractive over the last 10 years, driven largely by its potential to provide rapid pathology consulting services throughout the country regardless of the location of a particular institution. Based on this trend, the president of the Canadian Association of Pathologists asked a working group consisting of pathologists, technologists, and healthcare administrators from across Canada to oversee the development of guidelines to provide Canadian pathologists with basic information on how to implement and use this technology. The guidelines were systematically developed, based on available medical literature and the clinical experience of early adopters of telepathology in Canada. While there are many different modalities and applications of telepathology, this document focuses specifically on whole-slide imaging as applied to intraoperative pathology consultation (frozen section), primary diagnosis, expert or second opinions and quality assurance activities. Applications such as hematopathology, microbiology, tumour boards, education, research and technical and/or standard-related issues are not covered.
doi:10.4103/2153-3539.129455
PMCID: PMC4023030  PMID: 24843826
Anatomical pathology; telepathology; whole slide imaging
6.  Discovery and Preclinical Characterization of Novel Small Molecule TRK and ROS1 Tyrosine Kinase Inhibitors for the Treatment of Cancer and Inflammation 
PLoS ONE  2013;8(12):e83380.
Receptor tyrosine kinases (RTKs), in response to their growth factor ligands, phosphorylate and activate downstream signals important for physiological development and pathological transformation. Increased expression, activating mutations and rearrangement fusions of RTKs lead to cancer, inflammation, pain, neurodegenerative diseases, and other disorders. Activation or over-expression of ALK, ROS1, TRK (A, B, and C), and RET are associated with oncogenic phenotypes of their respective tissues, making them attractive therapeutic targets. Cancer cDNA array studies demonstrated over-expression of TRK-A and ROS1 in a variety of cancers, compared to their respective normal tissue controls. We synthesized a library of small molecules that inhibit the above indicated RTKs with picomolar to nanomolar potency. The lead molecule GTx-186 inhibited RTK-dependent cancer cell and tumor growth. In vitro and in vivo growth of TRK-A-dependent IMR-32 neuroblastoma cells and ROS1-overexpressing NIH3T3 cells were inhibited by GTx-186. GTx-186 also inhibited inflammatory signals mediated by NFκB, AP-1, and TRK-A and potently reduced atopic dermatitis and air-pouch inflammation in mice and rats. Moreover, GTx-186 effectively inhibited ALK phosphorylation and ALK-dependent cancer cell growth. Collectively, the RTK inhibitor GTx-186 has a unique kinase profile with potential to treat cancer, inflammation, and neuropathic pain.
doi:10.1371/journal.pone.0083380
PMCID: PMC3873281  PMID: 24386191
7.  Whole-genome sequencing identifies genetic alterations in pediatric low-grade gliomas 
Nature genetics  2013;45(6):602-612.
The commonest pediatric brain tumors are low-grade gliomas (LGGs). We utilized whole genome sequencing to discover multiple novel genetic alterations involving BRAF, RAF1, FGFR1, MYB, MYBL1 and genes with histone-related functions, including H3F3A and ATRX, in 39 LGGs and low-grade glioneuronal tumors (LGGNTs). Only a single non-silent somatic alteration was detected in 24/39 (62%) tumors. Intragenic duplications of the FGFR1 tyrosine kinase domain (TKD) and rearrangements of MYB were recurrent and mutually exclusive in 53% of grade II diffuse LGGs. Transplantation of Trp53-null neonatal astrocytes containing TKD-duplicated FGFR1 into brains of nude mice generated high-grade astrocytomas with short latency and 100% penetrance. TKD-duplicated FGFR1 induced FGFR1 autophosphorylation and upregulation of the MAPK/ERK and PI3K pathways, which could be blocked by specific inhibitors. Focusing on the therapeutically challenging diffuse LGGs, our study of 151 tumors has discovered genetic alterations and potential therapeutic targets across the entire range of pediatric LGGs/LGGNTs.
doi:10.1038/ng.2611
PMCID: PMC3727232  PMID: 23583981
8.  Orally Bioavailable Tubulin Antagonists for Paclitaxel-Refractory Cancer 
Pharmaceutical research  2012;29(11):3053-3063.
Purpose
To evaluate the efficacy and oral activity of two promising indoles, (2-(1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone [compound II] and (2-(1H-indol-5-ylamino)-thiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone [compound IAT], in paclitaxel- and docetaxel-resistant tumor models in vitro and in vivo.
Methods
The in vitro drug-like properties, including potency, solubility, metabolic stability, and drug-drug interactions were examined for our two active compounds. An in vivo pharmacokinetic study and antitumor efficacy study were also completed to compare their efficacy with docetaxel.
Results
Both compounds bound to the colchicine-binding site on tubulin, and inhibited tubulin polymerization, resulting in highly potent cytotoxic activity in vitro. While the potency of paclitaxel and docetaxel was compromised in a multidrug-resistant cell line that overexpresses P-glycoprotein, the potency of compounds II and IATwas maintained. Both compounds had favorable drug-like properties, and acceptable oral bioavailability (21–50%) in mice, rats, and dogs. Tumor growth inhibition of greater than 100% was achieved when immunodeficient mice with rapidly growing paclitaxel-resistant prostate cancer cells were treated orally at doses of 3–30 mg/kg of II or IAT.
Conclusions
These studies highlight the potent and broad anticancer activity of two orally bioavailable compounds, offering significant pharmacologic advantage over existing drugs of this class for multidrug resistant or taxane-refractory cancers.
doi:10.1007/s11095-012-0814-5
PMCID: PMC3646298  PMID: 22760659
paclitaxel resistant cancer; P-glycoprotein; pharmacokinetics; tubulin; xenograft
9.  Novel Tubulin Polymerization Inhibitors Overcome Multidrug Resistance and Reduce Melanoma Lung Metastasis 
Pharmaceutical research  2012;29(11):3040-3052.
Purpose
To evaluate abilities of 2-aryl-4-benzoyl-imidazoles (ABI) to overcome multidrug resistance (MDR), define their cellular target, and assess in vivo antimelanoma efficacy.
Methods
MDR cell lines that overexpressed P-glycoprotein, MDR-associated proteins, and breast cancer resistance protein were used to evaluate ABI ability to overcome MDR. Cell cycle analysis, molecular modeling, and microtubule imaging were used to define ABI cellular target. SHO mice bearing A375 human melanoma xenograft were used to evaluate ABI in vivo antitumor activity. B16-F10/C57BL mouse melanoma lung metastasis model was used to test ABI efficacy to inhibit tumor lung metastasis.
Results
ABIs showed similar potency to MDR cells compared to matching parent cells. ABIs were identified to target tubulin on the colchicine binding site. After 31 days of treatment, ABI-288 dosed at 25 mg/kg inhibited melanoma tumor growth by 69%; dacarbazine at 60 mg/kg inhibited growth by 52%. ABI-274 dosed at 25 mg/kg showed better lung metastasis inhibition than dacarbazine at 60 mg/kg.
Conclusions
This new class of antimitotic compounds can overcome several clinically important drug resistant mechanisms in vitro and are effective in inhibiting melanoma lung metastasis in vivo, supporting their further development.
doi:10.1007/s11095-012-0726-4
PMCID: PMC3659804  PMID: 22410804
2-aryl-4-benzoyl-imidazoles (ABI); antimelanoma; melanoma lung metastasis; multidrug resistance; tubulin polymerization inhibitor
10.  Development and validation of a rapid and sensitive high-performance liquid chromatography-mass spectroscopy assay for determination of 17-(allylamino)-17-demethoxygeldanamycin and 17-(amino)-17-demethoxygeldanamycin in human plasma 
A sensitive method was developed and validated for the measurement of 17-(allylamino)-17-demethoxygeldanamycin (17AAG) and its active metabolite 17-amino-17-demethoxygeldanamycin (17AG) in human plasma using 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG) as an internal standard. After the addition of internal standard, 200 µL of plasma was extracted using ice cold acetonitrile followed by analysis on a Thermo Finnigan triple-quadruple mass spectrometer coupled to an Agilent 1100 HPLC system. Chromatography was carried out on a 50 × 2.1 mm Agilent Zorbax SB-phenyl 5 µm column coupled to a 3mm Varian metaguard diphenyl pre-column using glacial acetic acid 0.1% and a gradient of acetonitrile and water at a flow rate of 500 µL/min. Atmospheric pressure chemical ionization and detection of 17AAG, 17AG and 17DMAG were accomplished using selected reaction monitoring of m/z 584.3 > 541.3, 544.2 > 501.2, and 615.3 >572.3 respectively in negative ion mode. Retention times for 17AAG, 17AG, and 17DMAG were 4.1, 3.5, and 2.9 minutes, respectively, with a total run time of 7 minutes. The assay was linear over the range 0.5–3000 ng/mL for 17AAG and 17AG. Replicate sample analysis indicated within- and between-run accuracy and precision within 15%. The recovery of 17AAG and 17AG from 200 µL of plasma containing 1, 25, 300, and 2500 ng/mL was 93% or greater. This high performance liquid chromatographic tandem mass spectroscopy (HPLC/MS/MS) method is superior to previous methods. It is the first analytical method reported to date for the quantitation of both 17AAG and its metabolite 17AG and can reliably quantitate concentrations of both compounds as low as 0.5 ng/mL.
doi:10.1016/j.jchromb.2008.06.029
PMCID: PMC3782378  PMID: 18635408
11.  Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI–III) analogues targeting tubulin polymerization as antiproliferative agents 
Journal of medicinal chemistry  2012;55(16):7285-7289.
Novel ABI–III compounds were designed and synthesized based on our previously reported ABI-I and ABI–II analogs. ABI–III compounds are highly potent against a panel of melanoma and prostate cancer cell lines, with the best compound having an average IC50 value of 3.8 nM. They are not substrate of Pgp and thus may effectively overcome Pgp mediated multidrug resistance. ABI–III analogs maintain their mechanisms of action by inhibition of tubulin polymerization.
doi:10.1021/jm300564b
PMCID: PMC3426659  PMID: 22783954
Melanoma; prostate cancer; multidrug resistance; tubulin polymerization inhibitor; antiproliferative activity
12.  Development and Validation of a Highly Sensitive Liquid Chromatography/Mass Spectrometry Method for Simultaneous Quantification of Lenalidomide and Flavopiridol in Human Plasma 
Therapeutic drug monitoring  2008;30(5):620-627.
Lenalidomide, an immunomodulatory agent, and flavopiridol, a broad cyclin-dependent kinase inhibitor, are both active therapies for clinical use in genomic high risk chronic lymphocytic leukemia (CLL). A high-performance liquid chromatographic assay with tandem mass spectrometric detection has been developed to simultaneously quantify lenalidomide and flavopiridol in human and mouse plasma to facilitate their combined clinical development. Samples were prepared by liquid-liquid extraction with acetonitrile- (ACN) containing internal standard (IS), genistein, followed by evaporation of solvent and reconstitution in 95/5 H2O/ACN. Lenalidomide and IS were separated by reverse phase liquid chromatography on a C-18 column using a gradient of H2O and ACN, each with 0.1% formic acid. Atmospheric pressure chemical ionization (APCI) in positive-ion mode with single reaction monitoring on a triple quadrupole mass spectrometer were applied to detect transitions of lenalidomide (260.06 > 149.10) and flavopiridol (402.09 > 341.02). Lower limits of quantification (LLOQ) of lenalidomide and flavopiridol were 1nM and 0.3nM respectively. Recoveries of lenalidomide and flavopiridol from human plasma ranged from 99% to 116% throughout their linear ranges. Within and between-run precision and accuracy of replicate samples were all less than 15%. This is the most sensitive analytical method reported to date for both lenalidomide and flavopiridol. This sensitivity will enable late terminal phase concentration measurements and accurate pharmacokinetic parameter estimation in a planned clinical trial with lenalidomide and flavopiridol in CLL patients.
doi:10.1097/FTD.0b013e318185813d
PMCID: PMC3740534  PMID: 18708993
Lenalidomide; Pharmacokinetics; Flavopiridol; LCMS
13.  Distinct disease-risk groups in pediatric supratentorial and posterior fossa ependymomas 
Acta neuropathologica  2012;124(2):247-257.
Purpose
No reliable classification exists for the therapeutic stratification of children with ependymoma, such that disease-risk might be identified and patients treated to ensure a combination of maximal cure rates and minimal adverse therapeutic effects. This study examined associations between clinicopathological and cytogenetic variables and outcome in a trial cohort of children with ependymoma, with the aim of defining a practical scheme for grading this heterogeneous tumor.
Methods
Intracranial ependymomas (n=146) from children treated on the RT1 trial at St. Jude Children’s Research Hospital were evaluated for the status of multiple pathological features. Interphase FISH (iFISH) defined the status of chromosomes 1q, 6q (LATS1), and 9p21 (CDKN2A). Data relating to these variables were compared with survival data in order to model disease-risk groups.
Results
Extent of surgical resection was a significant determinant of outcome. Tumor cell density and mitotic count were associated with outcome among children with posterior fossa ependymomas (n=119). Among pathologic factors, only brain invasion was associated with outcome in children with supratentorial ependymomas (n=27). Gain of 1q was independently associated with outcome and in combination with clinicopathological variables defined a three-tier system of disease-risk for posterior fossa tumors.
Conclusions
Among children developing posterior fossa ependymomas treated with maximal surgical resection and conformal radiotherapy, key clinicopathological variables and chromosome 1q status can be used to define tiers of disease-risk. In contrast, risk factors for pediatric supratentorial tumors are limited to subtotal resection and brain invasion.
doi:10.1007/s00401-012-0981-9
PMCID: PMC3554251  PMID: 22526017
14.  Inhibitors of Tubulin Assembly Identified through Screening a Compound Library 
Chemical biology & drug design  2008;72(6):513-524.
Tubulin is the proposed target for drugs against cancer and helminths and is also a validated target in kinetoplastid parasites. With the aim of identifying new lead compounds against Leishmania sp., tubulin isolated from L. tarentolae was used to screen a 10 000 compound library. One compound, Chembridge No. 7992831 (5), displayed an IC50 of 13 μm against Leishmania tubulin in an in vitro assembly assay and showed a greater than threefold selectivity over mammalian tubulin. Another compound, Chembridge No. 9067250 (8), exhibited good activity against mammalian tubulin (IC50 = 5.0 μm). This compound was also toxic to several cancer cell lines with IC50 values in the region of 1 μM. Subsequent testing of analogues of 8 contained within the library identified two compounds with greater potency against mammalian tubulin (IC50 values of 1.1 and 2.8 μM). The more potent antitubulin agent also showed promising activity against cancer cell lines in vitro, with IC50 values ranging from 0.18 to 0.73 μM.
doi:10.1111/j.1747-0285.2008.00729.x
PMCID: PMC3677961  PMID: 19090918
Tubulin; screen; library; Leishmania
15.  PLAG1 Alteration in Carcinoma Ex Pleomorphic Adenoma: Immunohistochemical and Fluorescence In Situ Hybridization Studies of 22 Cases 
Head and Neck Pathology  2012;6(3):328-335.
Carcinoma ex pleomorphic adenoma (CA-ex-PA) may arise with nearly any histologic subtype of carcinoma of the salivary gland. In the absence of recognizable residual pleomorphic adenoma (PA) or a prior history of PA, distinction of CA-ex-PA from morphologically similar de novo carcinomas may be difficult. Oncogenic rearrangement of PLAG1 (pleomorphic adenoma gene 1) has been established in PA; however, it has not yet been proven that PLAG1 alteration persists in carcinomas developed from preceding PA. We evaluated 22 histologically diverse CA-ex-PA by immunohistochemistry for PLAG1, and/or by FISH targeting PLAG1. Of these, 17 cases were immunoreactive (1+ to 3+) and 5 were immunonegative/rare positive for PLAG1. For comparison, 39 various salivary gland neoplasms were immunostained for PLAG1, of which all scored negative/rare positive. Twelve of 19 CA-ex-PA analyzed by PLAG1 FISH (63 %) were positive for gene rearrangement, 2 showed only a trisomy/polysomy profile, and 5 had a normal pattern. One FISH-positive tumor showed amplification of PLAG1. One of 3 cases analyzed for HMGA2 FISH was positive for gene rearrangement. In our series, the majority of CA-ex-PA harbored altered PLAG1 or HMGA2 genes detectable by FISH. While PLAG1 immunostain was specific for CA-ex-PA against other carcinomas, its application as a standalone discriminatory test was limited by variable expression. We conclude that most CA-ex-PA, regardless of morphologic subtype, carry altered PLAG1 or HMGA2 genes, and that FISH for PLAG1, along with immunohistochemistry for PLAG1, may help discriminate CA-ex-PA from its de novo carcinoma counterpart.
doi:10.1007/s12105-012-0353-8
PMCID: PMC3422590  PMID: 22485045
Carcinoma ex pleomorphic adenoma; PLAG1; Fluorescence in situ hybridization; Immunohistochemistry; Salivary gland
16.  Disseminated Carcinoma Ex Pleomorphic Adenoma in an Adolescent Confirmed by Application of PLAG1 Immunohistochemistry and FISH for PLAG1 Rearrangement 
Head and Neck Pathology  2012;6(3):377-383.
A 16-year-old previously asymptomatic boy presented with complaints of fatigue, weight loss, and back pain for several months. Imaging studies revealed a large superior mediastinal mass, numerous bilateral pulmonary nodules, and multiple lytic bone lesions. A needle biopsy from a sternal lesion showed a poorly differentiated carcinoma, immunoreactive for cytokeratins and EMA and immunonegative for various organ/tissue-specific markers. His past medical history was significant for excision of a parotid gland tumor 5 years earlier. Histologic review of the salivary gland tumor revealed a pleomorphic adenoma containing a microscopic focus of invasive carcinoma (carcinoma ex pleomorphic adenoma). By immunohistochemistry, both the salivary gland tumor and the disseminated carcinoma expressed PLAG1 with a strong nuclear pattern. Fluorescence in situ hybridization (FISH), using dual-color, break-apart probes for PLAG1, showed rearrangement of the gene in both the salivary gland and the disseminated tumors. FISH demonstrated additional cytogenetic aberrations in the carcinoma, including polysomy for chromosome 8 (in both the primary salivary gland and the metastatic tumors) and PLAG1 amplification (in the metastatic tumor). We conclude that in the proper clinicopathologic setting, application of PLAG1 immunohistochemistry and FISH for PLAG1 gene rearrangement may be valuable in establishing the diagnosis of carcinoma ex pleomorphic adenoma as the source of a cancer of unknown primary site.
doi:10.1007/s12105-012-0330-2
PMCID: PMC3422588  PMID: 22297681
Carcinoma ex pleomorphic adenoma; Carcinoma of unknown primary; PLAG1; Salivary gland
17.  A Subset of Cutaneous and Soft Tissue Mixed Tumors Are Genetically Linked to Their Salivary Gland Counterpart 
Genes, chromosomes & cancer  2011;51(2):140-148.
Neoplasms morphologically similar to mixed tumors and myoepitheliomas of the salivary glands, under the broad concept of myoepithelial neoplasia, have recently been defined in the skin and soft tissue; however, to date, no data have supported a shared genetic background with their salivary gland counterpart. From a large body of research, it has been well established that rearrangement of pleomorphic adenoma gene 1 (PLAG1) leads to aberrant expression of its protein and is pathogenically relevant in the development of salivary mixed tumors. On the other hand, in soft tissue lesions, compelling evidence suggests that EWSR1 is involved in a significant subset. To examine the hypothesis that there is a genetic link between these histologically similar tumors at different sites, we randomly selected 20 benign myoepitheliomas/mixed tumors of skin and soft tissue (10 cases each). Nineteen cases could be immunostained for PLAG1, of which 11 cases showed distinct nuclear staining with moderate or strong intensity in a significant number of cells. Interphase fluorescence in situ hybridization (FISH) for PLAG1 was successfully performed in 11 cases (7 in skin, 4 in soft tissue) and was positive for gene rearrangement in 8 cases (5 in skin, 3 in soft tissue). All PLAG1-rearranged tumors, except one, had clear-cut ductal structures and were immunoreactive for PLAG1. In our series, tumors with PLAG1 alteration shared a common morphologic phenotype characterized by prominent tubuloductal differentiation, suggesting that myoepithelial neoplasms with genuine salivary gland-like morphology, so-called soft tissue/cutaneous mixed tumors, are genetically related to their salivary gland counterpart.
doi:10.1002/gcc.20938
PMCID: PMC3233652  PMID: 22038920
18.  Association of Age at Diagnosis and Genetic Mutations in Patients with Neuroblastoma 
Context
Neuroblastoma is diagnosed over a wide age range from birth through young adulthood, and older age at diagnosis is associated with a decline in survivability.
Objective
To identify genetic mutations that are associated with age at diagnosis in patients with metastatic neuroblastoma.
Design, Setting and Patients
We performed whole genome sequencing of DNA from diagnostic tumors and their matched germlines from 40 patients with metastatic neuroblastoma obtained between 1987 and 2009. Age groups at diagnosis included infants (0-<18 months), children (18 months-<12 years), and adolescents and young adults (≥12 years). To confirm the findings from this discovery cohort, validation testing using tumors from an additional 64 patients obtained between 1985 and 2009 was also performed. Formalin-fixed paraffin-embedded tumor tissue was used for immunohistochemistry and fluorescent in situ hybridization. Telomere lengths were analyzed using the whole genome sequencing data, quantitative polymerase chain reaction and fluorescent in situ hybridization.
Main Outcome Measure
Somatic recurrent mutations in tumors from patients with neuroblastoma correlated with the age at diagnosis and telomere length.
Results
We identified mutations in the ATRX gene in 100% (5/5) (95% CI, 50% – 100%) of tumors from patients in the adolescent and young adult group, 17% (5/29) (95% CI, 7% – 36%) of tumors from children, and 0% (0/6) (95% CI, 0% – 40%) of tumors from infants in the discovery cohort (n=40). In the validation cohort (n=64), we identified mutations in the ATRX gene in 33% (9/27) (95% CI, 17% – 54%) of tumors from patients in the adolescent and young adult group, 16% (4/25) (95% CI, 6% – 35%) of tumors from children, and 0% (0/12) (95% CI, 0% – 24%) of tumors from infants. We identified mutations in the ATRX gene in 44% (14/32) (95% CI, 28% – 62%) of tumors from patients in the adolescent and young adult group, 17% (9/54) (95% CI, 9% – 29%) of tumors from children, and 0% (0/18) (95% CI, 0% – 17%) of tumors from infants in the combined cohort (n=104). ATRX mutations were associated with an absence of ATRX protein in the nucleus and with long telomeres.
Conclusions
ATRX mutations were associated with age at diagnosis in children and young adults with stage 4 neuroblastoma.
Clinical Protocol
“Molecular Characterization of Neuroblastic Tumor: Correlation with Clinical Outcome” (clinical trials.gov: NCT00588068).
doi:10.1001/jama.2012.228
PMCID: PMC3527076  PMID: 22416102
19.  Medulloblastoma: clinicopathological correlates of SHH, WNT, and non-SHH/WNT molecular subgroups 
Acta neuropathologica  2011;121(3):381-396.
Medulloblastoma is heterogeneous, being characterized by molecular subgroups that demonstrate distinct gene expression profiles. Activation of the WNT or SHH signaling pathway characterizes two of these molecular subgroups, the former associated with low-risk disease and the latter potentially targeted by novel SHH pathway inhibitors. This manuscript reports the validation of a novel diagnostic immunohistochemical method to distinguish SHH, WNT, and non-SHH/WNT tumors and details their associations with clinical, pathological and cytogenetic variables. A cohort (n = 235) of medulloblastomas from patients aged 0.4–52 years was studied for expression of four immunohistochemical markers: GAB1, β-catenin, filamin A, and YAP1. Immunoreactivity (IR) for GAB1 characterizes only SHH tumors and nuclear IR for β-catenin only WNT tumors. IRs for filamin A and YAP1 identify SHH and WNT tumors. SHH, WNT, and non-SHH/WNT tumors contributed 31, 14, and 55% to the series. All desmoplastic/nodular (D/N) medulloblastomas were SHH tumors, while most WNT tumors (94%) had a classic phenotype. Monosomy 6 was strongly associated with WNT tumors, while PTCH1 loss occurred almost exclusively among SHH tumors. MYC or MYCN amplification and chromosome 17 imbalance occurred predominantly among non-SHH/WNT tumors. Among patients aged 3–16 years and entered onto the SIOP PNET3 trial, outcome was significantly better for children with WNT tumors, when compared to SHH or non-SHH/WNT tumors, which showed similar survival curves. However, high-risk factors (M+ disease, LC/A pathology, MYC amplification) significantly influenced survival in both SHH and non-SHH/WNT groups. We describe a robust method for detecting SHH, WNT, and non-SHH/WNT molecular subgroups in formalin-fixed medulloblastoma samples. In corroborating other studies that indicate the value of combining clinical, pathological, and molecular variables in therapeutic stratification schemes for medulloblastoma, we also provide the first outcome data based on a clinical trial cohort and novel data on how molecular subgroups are distributed across the range of disease.
doi:10.1007/s00401-011-0800-8
PMCID: PMC3519926  PMID: 21267586
20.  Assessing telomeric DNA content in pediatric cancers using whole-genome sequencing data 
Genome Biology  2012;13(12):R113.
Background
Telomeres are the protective arrays of tandem TTAGGG sequence and associated proteins at the termini of chromosomes. Telomeres shorten at each cell division due to the end-replication problem and are maintained above a critical threshold in malignant cancer cells to prevent cellular senescence or apoptosis. With the recent advances in massive parallel sequencing, assessing telomere content in the context of other cancer genomic aberrations becomes an attractive possibility. We present the first comprehensive analysis of telomeric DNA content change in tumors using whole-genome sequencing data from 235 pediatric cancers.
Results
To measure telomeric DNA content, we counted telomeric reads containing TTAGGGx4 or CCCTAAx4 and normalized to the average genomic coverage. Changes in telomeric DNA content in tumor genomes were clustered using a Bayesian Information Criterion to determine loss, no change, or gain. Using this approach, we found that the pattern of telomeric DNA alteration varies dramatically across the landscape of pediatric malignancies: telomere gain was found in 32% of solid tumors, 4% of brain tumors and 0% of hematopoietic malignancies. The results were validated by three independent experimental approaches and reveal significant association of telomere gain with the frequency of somatic sequence mutations and structural variations.
Conclusions
Telomere DNA content measurement using whole-genome sequencing data is a reliable approach that can generate useful insights into the landscape of the cancer genome. Measuring the change in telomeric DNA during malignant progression is likely to be a useful metric when considering telomeres in the context of the whole genome.
doi:10.1186/gb-2012-13-12-r113
PMCID: PMC3580411  PMID: 23232254
21.  Synthesis and antiproliferative activity of novel 2-aryl-4-benzoyl-imidazole derivatives targeting tubulin polymerization 
Bioorganic & medicinal chemistry  2011;19(16):4782-4795.
We previously reported the discovery of 2-aryl-4-benzoyl-imidazoles (ABI-I) as potent antiproliferative agents for melanoma. To further understand the structural requirements for the potency of ABI analogs, gain insight in the structure-activity relationships (SAR), and investigate metabolic stability for these compounds, we report extensive SAR studies on the ABI-I scaffold. Compared with the previous set of ABI-I analogs, the newly synthesized ABI-II analogs have lower potency in general, but some of the new analogs have comparable potency to the most active compounds in the previous set when tested in two melanoma and four prostate cancer cell lines. These SAR studies indicated that the antiproliferative activity was very sensitive to subtle changes in the ligand. Tested compounds 3ab and 8a are equally active against highly paclitaxel resistant cancer cell lines and their parental cell lines, indicating that drugs developed based on ABI-I analogs may have therapeutic advantages over paclitaxel in treating resistant tumors. Metabolic stability studies of compound 3ab revealed that N-methyl imidazole failed to extend stability as literature reported because de-methylation was found as the major metabolic pathway in rat and mouse liver microsomes. However, this sheds light on the possibility for many modifications on imidazole ring for further lead optimization since the modification on imidazole, such as compound 3ab, did not impact the potency.
doi:10.1016/j.bmc.2011.06.084
PMCID: PMC3152597  PMID: 21775150
22.  Definition of Disease-Risk Stratification Groups in Childhood Medulloblastoma Using Combined Clinical, Pathologic, and Molecular Variables 
Journal of Clinical Oncology  2010;29(11):1400-1407.
Purpose
Medulloblastomas are heterogeneous and include relatively good-prognosis tumors characterized by Wnt pathway activation, as well as those that cannot be successfully treated with conventional therapy. Developing a practical therapeutic stratification that allows accurate identification of disease risk offers the potential to individualize adjuvant therapy and to minimize long-term adverse effects in a subgroup of survivors.
Methods
Using formalin-fixed paraffin-embedded (FFPE) tissue for immunohistochemistry, fluorescent in situ hybridization, and direct sequencing to identify tumors with a Wnt pathway signature and those harboring copy number abnormalities (CNAs) of potential prognostic significance (MYC/MYCN amplification, CNAs of chromosome 6 and 17), we evaluated clinical, pathologic, and molecular outcome indicators and stratification models in a cohort (n = 207) of patients with medulloblastoma 3 to 16 years of age from the International Society of Pediatric Oncology CNS9102 (PNET3) trial.
Results
Metastatic disease and large-cell/anaplastic (LC/A) phenotype were the clinicopathologic variables associated with poor progression-free survival (PFS). Nuclear immunoreactivity for β-catenin, CTNNB1 mutation, and monosomy 6 all identified a group of good-prognosis patients. MYC amplification was associated with poor outcome, but other CNAs were not. Low-risk medulloblastomas were defined as β-catenin nucleopositive tumors without metastasis at presentation, LC/A phenotype, or MYC amplification. High-risk medulloblastomas were defined as tumors with metastatic disease, LC/A phenotype, or MYC amplification. Low-risk, standard-risk, and high-risk categories of medulloblastoma had significantly (P < .0001) different outcomes.
Conclusion
Integrating assays of molecular biomarkers undertaken on routinely collected diagnostic FFPE tissue into stratification schemes for medulloblastoma alongside clinical and pathologic outcome indicators can refine current definition of disease risk and guide adjuvant therapy.
doi:10.1200/JCO.2010.30.2810
PMCID: PMC3525837  PMID: 20921458
23.  The Origins of Oxidant Stress in Parkinson's Disease and Therapeutic Strategies 
Antioxidants & Redox Signaling  2011;14(7):1289-1301.
Abstract
Parkinson's disease (PD) is a major world-wide health problem afflicting millions of the aged population. Factors that act on most or all cell types (pan-cellular factors), particularly genetic mutations and environmental toxins, have dominated public discussions of disease etiology. Although there is compelling evidence supporting an association between disease risk and these factors, the pattern of neuronal pathology and cell loss is difficult to explain without cell-specific factors. This article focuses on recent studies showing that the neurons at greatest risk in PD—substantia nigra pars compacta dopamine neurons—have a distinctive physiological phenotype that could contribute to their vulnerability. The opening of L-type calcium channels during autonomous pacemaking results in sustained calcium entry into the cytoplasm of substantia nigra pars compacta dopamine neurons, resulting in elevated mitochondrial oxidant stress and susceptibility to toxins used to create animal models of PD. This cell-specific stress could increase the negative consequences of pan-cellular factors that broadly challenge either mitochondrial or proteostatic competence. The availability of well-tolerated, orally deliverable antagonists for L-type calcium channels points to a novel neuroprotective strategy that could complement current attempts to boost mitochondrial function in the early stages of the disease. Antioxid. Redox Signal. 14, 1289–1301.
doi:10.1089/ars.2010.3521
PMCID: PMC3048813  PMID: 20712409
24.  Biological Activity of 4-Substituted Methoxybenzoyl-Aryl-Thiazole (SMART): An Active Microtubule Inhibitor 
Cancer research  2010;71(1):216-224.
Formation of microtubules is a dynamic process that involves polymerization and depolymerization of αβ-tubulin heterodimers. Drugs that enhance or inhibit tubulin polymerization can destroy this dynamic process, arresting cells in the G2/M phase of the cell cycle. Although drugs that target tubulin generally demonstrate cytotoxic potency in the sub-nanomolar range, resistance due to drug efflux is a common phenomenon among the antitubulin agents. We recently reported a class of 4-Substituted Methoxybenzoyl-Aryl-Thiazoles (SMART) that exhibited great in vitro potency and broad spectrum cellular cytotoxicity. Evaluation of the in vitro and in vivo anti-cancer activities of three SMART compounds, SMART-H (H), SMART-F (F) and SMART-OH (OH) with varying substituents at the 4-position of aryl ring, demonstrated that they bind potently to the colchicine binding site in tubulin, inhibit tubulin polymerization, arrest cancer cells in G2/M phase of the cell cycle, and induce their apoptosis.
The SMART compounds also equi-potently inhibit the growth of parental and MDR-over-expressing cells in vitro, indicating that they can overcome multidrug resistance. In vivo anti-tumor efficacy studies in human prostate (PC-3) and melanoma (A375) cancer xenograft models demonstrated that SMART-H and SMART-F treatments resulted in %T/C values ranging from 4–30%. In addition, in vivo SMART-H treatment for 21 days at the higher dose (15 mg/kg) failed to produce any apparent neurotoxicity. These studies provide the first in vivo evidence and proof-of-concept that SMART compounds are similarly efficacious to currently FDA approved antitubulin drugs for cancer treatment, but they can circumvent P-glycoprotein-mediated drug resistance.
doi:10.1158/0008-5472.CAN-10-1725
PMCID: PMC3039446  PMID: 21084278
tubulin; P-glycoprotein; pharmacokinetics; xenograft
25.  MYB upregulation and genetic aberrations in a subset of pediatric low-grade gliomas 
Acta neuropathologica  2010;120(6):731-743.
Recent studies of genetic abnormalities in pediatric low-grade gliomas (LGGs) have focused on activation of the ERK/MAPK pathway by KIAA1549-BRAF gene fusions in the majority of pilocytic astrocytomas (PAs) and by rare mutations in elements of the pathway across histopathologically diverse LGGs. This study reports that MYB, an oncogene not previously implicated in gliomagenesis, is activated in a diverse subset of pediatric LGGs. The study cohort comprised 57 pediatric LGGs and a comparative cohort of 59 pediatric high-grade gliomas (HGGs). The LGG cohort included 34 PAs and 23 diffuse gliomas; fibrillary astrocytomas (n=14), oligodendroglial tumors (n=7), and angiocentric gliomas (n=2). MYB copy number abnormalities were disclosed using Affymetrix 6.0 SNP arrays and confirmed using interphase fluorescence in situ hybridization. Novel MYB amplifications that upregulate MYB RNA and protein expression were demonstrated in 2/14 diffuse astrocytomas. In addition, focal deletion of the terminal region of MYB was seen in 1 of 2 angiocentric gliomas (AGs). Increased expression of MYB was demonstrated by quantitative RT-PCR and immunohistochemistry. MYB upregulation at the protein level was demonstrated in a proportion of diffuse LGGs (60%), pilocytic astrocytomas (41%), and HGGs (19%), but abnormalities at the genomic level were only a feature of diffuse gliomas. Our data suggest that MYB may have a role in a subset of pediatric gliomas, through a variety of mechanisms in addition to MYB amplification and deletion.
doi:10.1007/s00401-010-0763-1
PMCID: PMC3066475  PMID: 21046410
MYB; glioma; pediatric; amplification

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