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  • Probability of positive genetic testing in patients diagnosed with pheochromocytoma and paraganglioma: Criteria beyond a family history. Surgery Alobuia, W. M., Ammar, S., Tyagi, M., Ghosh, C., Gunda, V., Annes, J. P., Kebebew, E. 2020

    Abstract

    BACKGROUND: Genetic testing for germline pheochromocytoma and paraganglioma susceptibility genes is associated with improved patient management. However, data are currently sparse on the probability of a positive testing result based on an individual's clinical presentation. This study evaluates clinical characteristics for association with testing positive for known pheochromocytoma and paraganglioma susceptibility genes.METHODS: This retrospective analysis examined 111 patients with a diagnosis of pheochromocytoma and paraganglioma who underwent genetic testing. Logistic regression and receiver operating characteristic analyses were performed to identify factors associated with a positive genetic testing result. Probabilities were then calculated for combinations of significant factors to determine the likelihood of a positive test result in each group.RESULTS: Of 32 patients with a family history of pheochromocytoma and paraganglioma, 31 (97%) had a germline mutation detected. Of 79 patients without a family history, 24 (30%) had a pathogenic germline mutation detected. In multivariate analysis, a positive family history, aged ?47 years, and tumor size ?2.9 cm were independent factors associated with a positive genetic testing result. Patients meeting all 3 criteria had a 100% probability compared with 13% in those without any of the criteria. In addition to a positive family history, having either aged ?47 years or tumor size ?2.9 cm resulted in a 90% and 100% probability of a positive result, respectively. In the absence of a family history, the probability in patients who were aged ?47 years and had a tumor size ?2.9 cm was 60%.CONCLUSION: In addition to a family history of pheochromocytoma and paraganglioma, aged ?47 years, and tumor size ?2.9 cm are associated with a higher probability of testing positive for a pheochromocytoma and paraganglioma susceptibility gene mutation. Patients meeting all 3 criteria have a 100% probability of a positive genetic testing result.

    View details for DOI 10.1016/j.surg.2020.08.027

    View details for PubMedID 33023754

  • A combinatorial strategy for targeting BRAF V600E mutant cancers with BRAF V600E inhibitor (PLX4720) and tyrosine kinase inhibitor (ponatinib). Clinical cancer research : an official journal of the American Association for Cancer Research Kebebew, E., Ghosh, C., Kumar, S., Kushchayeva, Y., Gaskins, K., Boufraqech, M., Wei, D., Gara, S. K., Zhang, L., Zhang, Y., Shen, M., Mukherjee, S. 2020

    Abstract

    PURPOSE: Most aggressive thyroid cancers are commonly associated with a BRAF V600E mutation. Preclinical and clinical data in BRAF V600E cancers suggest that combined BRAF and MEK inhibitor treatment result in a response, but resistance is common. One mechanism of acquired resistance is through persistent activation of tyrosine kinase (TK) signaling by alternate pathways. We hypothesized that combination therapy with BRAF and multitargeting TK inhibitors (MTKI) might be more effective in BRAF V600E thyroid cancer than single agent or BRAF and MEK inhibitors.EXPERIMENTAL DESIGN: The combined drug activity was analyzed to predict any synergistic effect using high throughput screening (HTS) of active drugs. We performed follow up in vitro and in vivo studies to validate and determine the mechanism of action of synergistic drugs.RESULTS: The MTKI ponatinib and the BRAF inhibitor PLX4720 showed synergistic activity by HTS. This combination significantly inhibited proliferation, colony formation, invasion and migration in BRAF V600E thyroid cancer cell lines and downregulated pERK/MEK and c-JUN signaling pathways, and increased apoptosis. PLX4720 resistant BRAF V600E cells became sensitized to the combination treatment, with decreased proliferation at lower PLX4720 concentrations. In orthotopic thyroid cancer mouse model, combination therapy significantly reduced tumor growth (p < 0.05), lower number of metastases (p < 0.05) and longer survival (p < 0.05) compared to monotherapy and vehicle control.CONCLUSIONS: Combination treatment with ponatinib and PLX4720 exhibited significant synergistic anticancer activity in preclinical models of BRAF V600E thyroid cancer, in addition to overcoming PLX4720 resistance. Our results suggest this combination should be tested in clinical trials.

    View details for DOI 10.1158/1078-0432.CCR-19-1606

    View details for PubMedID 31937621

  • Diphenylbutylpiperidine Antipsychotic Drugs Inhibit Prolactin Receptor Signaling to Reduce Growth of Pancreatic Ductal Adenocarcinoma in Mice. Gastroenterology Dandawate, P., Kaushik, G., Ghosh, C., Standing, D., Ali Sayed, A. A., Choudhury, S., Subramaniam, D., Manzardo, A., Banerjee, T., Santra, S., Ramamoorthy, P., Butler, M., Padhye, S. B., Baranda, J., Kasi, A., Sun, W., Tawfik, O., Coppola, D., Malafa, M., Umar, S., Soares, M. J., Saha, S., Weir, S. J., Dhar, A., Jensen, R. A., Thomas, S. M., Anant, S. 2020; 158 (5): 1433?49.e27

    Abstract

    Prolactin (PRL) signaling is up-regulated in hormone-responsive cancers. The PRL receptor (PRLR) is a class I cytokine receptor that signals via the Janus kinase (JAK)-signal transducer and activator of transcription and mitogen-activated protein kinase pathways to regulate cell proliferation, migration, stem cell features, and apoptosis. Patients with pancreatic ductal adenocarcinoma (PDAC) have high plasma levels of PRL. We investigated whether PRLR signaling contributes to the growth of pancreatic tumors in mice.We used immunohistochemical analyses to compare levels of PRL and PRLR in multitumor tissue microarrays. We used structure-based virtual screening and fragment-based drug discovery to identify compounds likely to bind PRLR and interfere with its signaling. Human pancreatic cell lines (AsPC-1, BxPC-3, Panc-1, and MiaPaCa-2), with or without knockdown of PRLR (clustered regularly interspaced short palindromic repeats or small hairpin RNA), were incubated with PRL or penfluridol and analyzed in proliferation and spheroid formation. C57BL/6 mice were given injections of UNKC-6141 cells, with or without knockdown of PRLR, into pancreas, and tumor development was monitored for 4 weeks, with some mice receiving penfluridol treatment for 21 days. Human pancreatic tumor tissues were implanted into interscapular fat pads of NSG mice, and mice were given injections of penfluridol daily for 28 days. Nude mice were given injections of Panc-1 cells, xenograft tumors were grown for 2 weeks, and mice were then given intraperitoneal penfluridol for 35 days. Tumors were collected from mice and analyzed by histology, immunohistochemistry, and immunoblots.Levels of PRLR were increased in PDAC compared with nontumor pancreatic tissues. Incubation of pancreatic cell lines with PRL activated signaling via JAK2-signal transducer and activator of transcription 3 and extracellular signal-regulated kinase, as well as formation of pancospheres and cell migration; these activities were not observed in cells with PRLR knockdown. Pancreatic cancer cells with PRLR knockdown formed significantly smaller tumors in mice. We identified several diphenylbutylpiperidine-class antipsychotic drugs as agents that decreased PRL-induced JAK2 signaling; incubation of pancreatic cancer cells with these compounds reduced their proliferation and formation of panco spheres. Injections of 1 of these compounds, penfluridol, slowed the growth of xenograft tumors in the different mouse models, reducing proliferation and inducing autophagy of the tumor cells.Levels of PRLR are increased in PDAC, and exposure to PRL increases proliferation and migration of pancreatic cancer cells. Antipsychotic drugs, such as penfluridol, block PRL signaling in pancreatic cancer cells to reduce their proliferation, induce autophagy, and slow the growth of xenograft tumors in mice. These drugs might be tested in patients with PDAC.

    View details for DOI 10.1053/j.gastro.2019.11.279

    View details for PubMedID 31786131

    View details for PubMedCentralID PMC7103550

  • Adrenal Vein Sampling to Distinguish Between Unilateral and Bilateral Primary Hyperaldosteronism: To ACTH Stimulate or Not? Journal of clinical medicine Sung, T. Y., Alobuia, W. M., Tyagi, M. V., Ghosh, C., Kebebew, E. 2020; 9 (5)

    Abstract

    The aim of this study is to determine the accuracy of adrenal vein sampling (AVS) with and without adrenocorticotropic hormone (ACTH) stimulation to distinguish between unilateral and bilateral primary hyperaldosteronism (PA). Retrospective analysis of a prospective database from a referral center between 1984 and 2009, 76 patients had simultaneous cannulation of bilateral adrenal veins and AVS with and without ACTH stimulation. All patients had adrenalectomies. The selectivity index (SI, cut-off value ?2) was used for confirmation of successful cannulation of the adrenal vein. The lateralization index (LI, cut-off value >2 and >4) was used for distinguishing between unilateral and bilateral PA. The SI ratio was higher with ACTH stimulation compared to without for the right adrenal vein (p = 0.027). The LI >2 ratio was higher with ACTH stimulation compared to without (p = 0.007). For the LI >4 ratio, there was no difference between with and without ACTH stimulation (p = 0.239). However, for a LI >4, 7 patients (9.2%) were not lateralized with ACTH stimulation, but they did lateralize without ACTH stimulation. AVS with ACTH stimulation is associated with a higher SI ratio compared to AVS without ACTH stimulation. However, when using LI >4 for AVS, samples without ACTH stimulation should also be included to detect a subset of patients with unilateral disease that are not detected with ACTH stimulation.

    View details for DOI 10.3390/jcm9051447

    View details for PubMedID 32413990

  • Super-enhancers: novel target for pancreatic ductal adenocarcinoma. Oncotarget Ghosh, C., Paul, S., Dandawate, P., Gunewardena, S. S., Subramaniam, D., West, C., Anant, S., Dhar, A. 2019; 10 (16): 1554?71

    Abstract

    Super-enhancers (SEs) are unique areas of the genome which drive high-level of transcription and play a pivotal role in the cell physiology. Previous studies have established several important genes in cancer as SE-driven oncogenes. It is likely that oncogenes may hack the resident tissue regenerative program and interfere with SE-driven repair networks, leading to the specific pancreatic ductal adenocarcinoma (PDAC) phenotype. Here, we used ChIP-Seq to identify the presence of SE in PDAC cell lines. Differential H3K27AC marks were identified at enhancer regions of genes including c-MYC, MED1, OCT-4, NANOG, and SOX2 that can act as SE in non-cancerous, cancerous and metastatic PDAC cell lines. GZ17-6.02 affects acetylation of the genes, reduces transcription of major transcription factors, sonic hedgehog pathway proteins, and stem cell markers. In accordance with the decrease in Oct-4 expression, ChIP-Seq revealed a significant decrease in the occupancy of OCT-4 in the entire genome after GZ17-6.02 treatment suggesting the possible inhibitory effect of GZ17-6.02 on PDAC. Hence, SE genes are associated with PDAC and targeting their regulation with GZ17-6.02 offers a novel approach for treatment.

    View details for DOI 10.18632/oncotarget.26704

    View details for PubMedID 30899425

    View details for PubMedCentralID PMC6422180

  • The Histone Demethylase KDM3A, Increased in Human Pancreatic Tumors, Regulates Expression of DCLK1 and Promotes Tumorigenesis in Mice. Gastroenterology Dandawate, P., Ghosh, C., Palaniyandi, K., Paul, S., Rawal, S., Pradhan, R., Sayed, A. A., Choudhury, S., Standing, D., Subramaniam, D., Padhye, S. B., Gunewardena, S., Thomas, S. M., Neil, M. O., Tawfik, O., Welch, D. R., Jensen, R. A., Maliski, S., Weir, S., Iwakuma, T., Anant, S., Dhar, A. 2019; 157 (6): 1646?59.e11

    Abstract

    The histone lysine demethylase 3A (KDM3A) demethylates H3K9me1 and H3K9Me2 to increase gene transcription and is upregulated in tumors, including pancreatic tumors. We investigated its activities in pancreatic cancer cell lines and its regulation of the gene encoding doublecortin calmodulin-like kinase 1 (DCLK1), a marker of cancer stem cells.We knocked down KDM3A in MiaPaCa-2 and S2-007 pancreatic cancer cell lines and overexpressed KDM3A in HPNE cells (human noncancerous pancreatic ductal cell line); we evaluated cell migration, invasion, and spheroid formation under hypoxic and normoxic conditions. Nude mice were given orthotopic injections of S2-007 cells, with or without (control) knockdown of KDM3A, and HPNE cells, with or without (control) overexpression of KDM3A; tumor growth was assessed. We analyzed pancreatic tumor tissues from mice and pancreatic cancer cell lines by immunohistochemistry and immunoblotting. We performed RNA-sequencing analysis of MiaPaCa-2 and S2-007 cells with knockdown of KDM3A and evaluated localization of DCLK1 and KDM3A by immunofluorescence. We analyzed the cancer genome atlas for levels of KDM3A and DCLK1 messenger RNA in human pancreatic ductal adenocarcinoma (PDAC) tissues and association with patient survival time.Levels of KDM3A were increased in human pancreatic tumor tissues and cell lines, compared with adjacent nontumor pancreatic tissues, such as islet and acinar cells. Knockdown of KDM3A in S2-007 cells significantly reduced colony formation, invasion, migration, and spheroid formation, compared with control cells, and slowed growth of orthotopic tumors in mice. We identified KDM3A-binding sites in the DCLK1 promoter; S2-007 cells with knockdown of KDM3A had reduced levels of DCLK1. HPNE cells that overexpressed KDM3A formed foci and spheres in culture and formed tumors and metastases in mice, whereas control HPNE cells did not. Hypoxia induced sphere formation and increased levels of KDM3A in S2-007 cells and in HPNE cells that overexpressed DCLK1, but not control HPNE cells. Levels of KDM3A and DCLK1 messenger RNA were higher in human PDAC than nontumor pancreatic tissues and correlated with shorter survival times of patients.We found human PDAC samples and pancreatic cancer cell lines to overexpress KDM3A. KDM3A increases expression of DCLK1, and levels of both proteins are increased in human PDAC samples. Knockdown of KDM3A in pancreatic cancer cell lines reduced their invasive and sphere-forming activities in culture and formation of orthotopic tumors in mice. Hypoxia increased expression of KDM3A in pancreatic cancer cells. Strategies to disrupt this pathway might be developed for treatment of pancreatic cancer.

    View details for DOI 10.1053/j.gastro.2019.08.018

    View details for PubMedID 31442435

    View details for PubMedCentralID PMC6878178

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