D’Assoro, Antonio B.

Introduction:Deep Vein Thrombosis (DVT) of both lower and upper limbs is increasingly being recognized as a clinical entity in patients with chronic diseases hospitalized for several comorbidities. The occurrence of DVT in sites other than lower limbs is an emerging clinical presentation in a setting of a Department of Medicine. The relationship between score models, based on risk factors, symptoms and clinical signs of lower limbs DVT, and circulating biomarkers is not well studied. Hypothesis:We hypothesized to give an additional value to score models using circulating biomarkers in the prediction of DVT in patients hospitalized in Internal Medicine Department. Methods:We prospectively recruited 178 consecutive patients who presented a D-dimer value >500 ng/ml. In each patient Wells modified score for DVT, Hamilton score, Kahn score and St. André Hospital score were applied. We measured serum levels of albumin (%) and protein S (%). Diagnosis of DVT was made by Extended Compression Ultra-Sonography (ECUS) standardized procedure. All patients underwent also an angiographic-CT-scan Results:In 85 (48%) patients the diagnosis of DVT was confirmed. Sites of DVT were as follows: Lower limbs venous thrombosis n=71 (84%); Upper limb venous thrombosis n=8 (9%); portal vein thrombosis n=6 (7%). Patients with DVT had significantly lower albumin levels as compared with controls (mean ± SD 47.6 ± 7.9 vs 55.4 ± 4.6 %, respectively, P<0.001) and lower Protein S (60.7 ± 26.3 vs. 87.6 ± 11.9 %, P<0.001). In this setting, the use of clinical risk scores based on symptoms and clinical signs showed low accuracy (AUC 0.690). We gave an additional value to these scores by testing in combination biomarkers and two of the best items of all clinical score models used to predict DVT: we evaluated a ROC curve analysis including value of Albumin (%), Protein S (%), previous DVT and cancer obtaining a higher accuracy (AUC 0.901, Positive Likelihood Ratio (LR+) 12.97 and Negative Likelihood Ratio (LR-) 0.17). Conclusions:This study demonstrates that biomarkers improve the prediction of clinical scores in the diagnosis of DVT of all sites in hospitalized patients.

Abstract Background: Aurora A (AURKA) is a mitotic kinase responsible for centrosome segregation and mitotic spindle formation. In normal cells, expression of AURKA is highly regulated and is predominantly restricted to G2/M phases of the cell cycle. Unlike healthy cells, cancer cells overexpress AURKA through all phases of the cell cycle resulting in the acquisition of alternate non-mitotic functions. Little is known about cellular functions regulated by AURKA and its interaction with other signaling molecules. Here, we report a novel interaction between AURKA and the mitogen-activated protein kinase (MAPK) pathway in wild type BRAF breast cancer cells as well as demonstrate an additive cytotoxic effect of AURKA- and MEK1/2-specific inhibitors against estrogen positive (ER+) and triple negative (ER-, PR-, HER2-) breast cancer cells. Results: We show that treatment of ER+ HER2- MCF-7, ER- HER2+ SKBR3 and ER- HER2- BT549 cells with AURKA specific inhibitors alisertib, MK8745 and Aurora A Inhibitor I resulted in over 2-fold increase in the levels of both pMEK1/2 and pERK1/2 compared to the untreated controls. The activation of the MAPK pathway was rapid with changes seen within 5 min after treatment with AURKA inhibitors and was sustained for at least 48 hours. No differences in phosphorylation of MEK1/2 or ERK1/2 were observed in BRAFG464V triple negative MDA-MB-231 cells. Treatment with AURKA inhibitors resulted in downregulation of pAURKA and a significant increase in levels of total AURKA protein. The pull-down assay with Ras-binding domain coated agarose beads followed by western blot analysis with anti-pan-RAS Ab revealed no changes in active GTP-bound RAS in alisertib-treated MCF-7 cells compared to the untreated control. Consistently, no significant changes were observed in RAS-inducible phosphorylation of RAF1 activation site at Ser338 as demonstrated by western blot. Treatment with the pan RAF inhibitor TAK-632 did not diminish alisertib-induced pERK and pMEK1/2. Alternatively, treatment with the MEK1/2 specific inhibitor PD0325901 completely abrogated alisertib-induced phosphorylation of MEK1/2 and ERK1/2. Furthermore, combined treatment of alisertib and PD0325901 in vitro revealed significant additive cytotoxic effect in MCF-7 and BT549 cells when compared to either agent used alone (p&lt; 0.008 and p&lt;0.011; p &lt;0.04 and p&lt;0.028). Conclusions: Our data suggests that AURKA is a RAF1-independent negative regulator of MAPK activity in breast cancer cells. The in-depth analysis of the AURKA-MEK1/2 interaction is currently under investigation. The results reveal a promising new strategy for the treatment of wild type BRAF, TNBC patients using a combination of AURKA and MEK1/2 inhibitors. Citation Format: Malgorzata Gil, Archana Chidambaram, Thaer Khoury, Kazuaki Takabe, Igor Puzanov, Irwin Gelman, Antonio D’Assoro, Mateusz Opyrchal. Inhibition of AURKA induces Raf1-independent activation of MAPK pathway in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2123. doi:10.1158/1538-7445.AM2017-2123