Naima Ismaili

Purpose: The application of pan-cancer next-generation sequencing panels in the clinical setting has facilitated the identification of low frequency somatic mutations and the testing of new therapies in solid tumors using the "basket trial" scheme. However, little consideration has been given to the relevance of nonsynonymous germline variants, which are likely to be uncovered in tumors and germline and which may be relevant to prognostication and prediction of treatment response. Experimental Design: We analyzed matched tumor and normal DNA from 34 melanoma patients using an Ion Torrent cancer-associated gene panel. We elected to study the germline variant Q472H in the kinase insert domain receptor (KDR), which was identified in 35% of melanoma patients in both a pilot and an independent 1,223 patient cohort. Using patient-derived melanoma cell lines and human samples, we assessed proliferation, invasion, VEGF levels, and angiogenesis by analyzing tumor microvessel density (MVD) using anti-CD34 antibody. Results: Serum VEGF levels and tumor MVD were significantly higher in Q472H versus KDR wild-type (WD) patients. Primary cultures derived from melanomas harboring the KDR variant were more proliferative and invasive than KDR wild type. Finally, using a VEGFR2 antibody, we showed that KDR Q472H cells were sensitive to targeted inhibition of VEGFR2, an effect that was not observed in KDR WT cells. Conclusions: Our data support the integration of germline analysis into personalized treatment decision-making and suggest that patients with germline KDR variant might benefit from antiangiogenesis treatment. Clin Cancer Res; 22(10); 2377–85. ©2015 AACR.

Background: Molecularly targeted therapy is improving response rates and overall survival in subsets of melanoma patients. However, targeted therapy for triple negative patients (BRAF, NRAS and c-KIT wild type) is not yet defined. In addition, new evidence suggests that germline variants may have an impact in melanoma progression and response to therapy. In this study, we attempt to define the utility of a recently developed clinical assay that encompasses targeted sequencing of 50 genes with known impact on cancer progression. Methods: We used the AmpliSeq Cancer Panel HotSpot.V2 from Ion Torrent. The panel targets sequencing of Hotspot regions including 2,800 COSMIC mutations within 50 oncogenes and tumor suppressor genes. Tumor and blood germline DNAs were studied. All identified mutations were independently validated by Sanger sequencing. We then linked the molecular profile to extensive clinicopathological information including treatment and prospective clinical follow-up. Results: We examined 35 tumor samples from 35 melanoma patients (2 patients had 2 specimens). 14/35 (40%) had BRAF mutations, 15/35 (34%) had NRAS, KRAS or HRAS mutations and 1 had c-KIT mutant tumor. Eight patients were triple negative and presented with mutations in TP53, ERBB4, PIK3CA, NOTCH, EZH2 and VHL. We detected germline variants in TP53 (49%), KDR (VEGFR2 - 34%), APC (31%), KIT (23%) and PIK3CA (14%) genes. Notably, KDR Q472H has been shown to affect VEGFR2 function. Patients with KDR Q742H had higher microvessels density and higher VEGF production. In addition, melanoma cell lines with KDR Q472H variant proliferate and invade more when compared with WT cell lines. More important, only cell lines with that variant respond to anti-VEGFR2 treatment. Finally, the presence of the KDR Q472H variant is associated with a higher prevalence of brain metastases and with a shorter survival (15.1 months compared to 54.6 months). Conclusions: Sequencing using a validated clinical assay was informative of several targetable mutations in triple negative melanoma. Inhibitors targeting some of these mutations are already FDA approved for non-melanoma and others are currently tested in clinical trials. Our data also revealed a role of germline variant KDR Q472H in melanoma progression, that was not reported before, suggesting that further functional studies are warranted. Citation Format: Amel Salhi, Ines Pires Da Silva, Kevin P. Lui, Naima Ismaili, Chaowei Wu, Eleazar C. Vega-Saenz de Miera, Richard L. Shapiro, Russell S. Berman, Anna C. Pavlick, Judy Zhong, Adriana Heguy, Iman Osman. Integration of melanoma genotyping in clinical care. []. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl): nr A35.

Primary effusion lymphoma (PEL) is a rare form of non-Hodgkin lymphoma that presents with body cavity effusions. It occurs chiefly in immunodeficient HIV-positive patients. The tumor cells generally express gene sequences of human herpesvirus-8 (HHV-8) and Epstein-Barr virus (EBV). Tumor cells of HIV-negative patients usually express HHV-8 gene sequences, but rarely those of EBV. We report a novel case of PEL in an HIV-negative homosexual male whose tumor cells expressed both HHV-8 and EBV gene sequences and who developed evidence of central nervous system involvement.

Regulation of the hypothalamic–pituitary–adrenal (HPA) axis is critical for adaptation to environmental changes. The principle regulator of the HPA axis is corticotrophin-releasing hormone (CRH), which is made in the parventricular nucleus and is an important target of negative feedback by glucocorticoids. However, the molecular mechanisms that regulate CRH are not fully understood. Disruption of normal HPA axis activity is a major risk factor of neuropsychiatric disorders in which decreased expression of the glucocorticoid receptor (GR) has been documented. To investigate the role of the GR in CRH neurons, we have targeted the deletion of the GR, specifically in the parventricular nucleus. Impairment of GR function in the parventricular nucleus resulted in an enhancement of CRH expression and an up-regulation of hypothalamic levels of BDNF and disinhibition of the HPA axis. BDNF is a stress and activity-dependent factor involved in many activities modulated by the HPA axis. Significantly, ectopic expression of BDNF in vivo increased CRH, whereas reduced expression of BDNF, or its receptor TrkB, decreased CRH expression and normal HPA functions. We find the differential regulation of CRH relies upon the cAMP response-element binding protein coactivator CRTC2, which serves as a switch for BDNF and glucocorticoids to direct the expression of CRH.

Dysregulation of liver X receptor alpha (LXRalpha) activity has been linked to cardiovascular and metabolic diseases. Here, we show that LXRalpha target gene selectivity is achieved by modulation of LXRalpha phosphorylation. Under basal conditions, LXRalpha is phosphorylated at S198; phosphorylation is enhanced by LXR ligands and reduced both by casein kinase 2 (CK2) inhibitors and by activation of its heterodimeric partner RXR with 9-cis-retinoic acid (9cRA). Expression of some (AIM and LPL), but not other (ABCA1 or SREBPc1) established LXR target genes is increased in RAW 264.7 cells expressing the LXRalpha S198A phosphorylation-deficient mutant compared to those with WT receptors. Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXRalpha S198A. Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Thus, our findings reveal a previously unrecognized role for phosphorylation in restricting the repertoire of LXRalpha-responsive genes.

Abstract: The glucocorticoid receptor (GR) is phosphorylated at multiple serine residues in a hormone‐dependent manner. It has been suggested that GR phosphorylation affects turnover, subcellular trafficking, or the transcriptional regulatory functions of the receptor, yet the contribution of individual GR phosphorylation sites to the modulation of GR activity remains enigmatic. This review critically evaluates the literature on GR phosphorylation and presents more recent work on the mechanism of GR phosphorylation from studies using antibodies that recognize GR only when it is phosphorylated. In addition, we present support for the notion that GR phosphorylation modifies protein‐protein interactions, which can stabilize the hypophosphorylated form of the receptor in the absence of ligand, as well as facilitate transcriptional activation by the hyperphosphorylation of GR via cofactor recruitment upon ligand binding. Finally, we propose that GR phosphorylation also participates in the nongenomic activation of cytoplasmic signaling pathways evoked by GR. Thus, GR phosphorylation is a versatile mechanism for modulating and integrating multiple receptor functions.