The Utility of Preoperative Vascular Grading in Patients Undergoing Surgery First for Pancreatic Cancer: Does Radiologic Arterial or Venous Involvement Predict Pathologic Margin Status?

Controversy exists on accurately grading vascular involvement on preoperative imaging for pancreatic ductal adenocarcinoma. We reviewed the association between preoperative imaging and margin status in 137 patients. Radiologists graded venous involvement based on the Ishikawa classification system and arterial involvement based on preoperative imaging. For patients with both classifications recorded, we categorized vascular involvement as “None,” “Arterial only,” “Venous only,” or “Both” and examined the association of vascular involvement and pathologic margin status. Of 134 patients with Ishikawa classifications, 63%, 17%, 11%, and 9% were graded as I, II, III, and IV, respectively. Of 96 patients with arterial staging, 74%, 16%, and 10% were categorized as stages i, ii, and iii, respectively. Of 93 patients with both stagings, 61% had no vascular involvement, 7% had arterial only, 14% had venous only, and 17% had both involved. Ishikawa classification was strongly associated with a positive SMA and SMV margin (p<0.001). However, for arterial staging, there was no association with SMA or SMV margin. Overall, Ishikawa grading was more predicative of arterial involvement and remained significant on multivariate analysis. The use of diagnostic imaging in predicting positive margins is more accurate when using a venous grading system

Combining A5 and F4 IgGs improves growth inhibition of ERBB3-positive cell lines and synergizes with ERBB-targeted agents.

<p>Individual, pairwise, and triple combinations of A5, F4 and either trastuzumab or erlotinib (as appropriate) were serially diluted (2-fold dilution scheme) and incubated with the ERBB2-positive cell lines, BT-474, SK-BR-3, NCI-N87, and EGFR-positive cell line ACHN, as noted. Concentration plotted on the x-axis refers to anti-ERBB3 IgG concentration. Trastuzumab and erlotinib are at 1∶200 molar ratios with anti-ERBB3 antibodies. The impact of treatment on cell viability was quantified with Cell-Titer Blue. Vehicle-treated controls for each cell line were set at 100%. Values plotted represent the means ± standard error of the means (SEM) for between 2–6 independent experiments, each carried out in triplicate.</p

Anti-ERBB3 mAbs inhibit ligand-mediated ErbB3 signaling.

<p>A) Serum-starved BT-474, NCI-N87 and ACHN cells were treated with appropriate antibodies or PBS for 30 minutes followed by HRGβ stimulation for 10 minutes. B) NCI-N87 cells were treated with either the A5/F4 oligoclonal or PBS and stimulated with HRGβ, as appropriate, over a 60 minute time course. Cell lysates were analyzed by western blot and probed with anti-pAKT(S473) as a marker for ligand-induced signaling. Membranes were probed with anti-beta actin mAb that served as loading control.</p

Ability of single-chain Fv molecules to inhibit cell viability correlates with epitope.

<p>A) Intact (dI-IV) and sub-domains (dI, dI-II, dIII, dIII-IV) of ERBB3 extracellular domain were expressed and subjected to immunoprecipitations with immobilized scFv under conditions of antibody excess. S  =  supernatant (unbound fraction), P  =  pellet (bound fraction).</p

The A5/F4 oligoclonal exhibits in vivo efficacy.

<p>NCR nu/nu mice harboring 120–150 mm³ NCI-N87 subcutaneous tumor xenografts were randomly assigned into treatment groups (n≥6/group) and received i.p injections as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112376#s2" target="_blank">Methods</a>. Means +/−SEM of percentage tumor volume growth is shown for each measurement. Statistical analysis was performed with Wilcoxon Ranked Sum test.</p

Binding activity of A5 and F4 anti-ERBB3 IgGs.

<p>A) Equilibrium binding of A5 and F4 IgGs to purified ERBB3 extracellular domains was quantified in an ELISA format by incubating 4 fold-serial dilutions of IgG (1333–0.02 nM) and 0 nM control with immobilized ERBB3, and bound antibody was detected with HRP-conjugated anti-human Fc secondary antibody. Binding to ERBB2 was used to control for specificity. Values plotted represent the means ± standard deviation of a representative experiment B) Increasing amounts (0, 0.5, 2, 5 µg) of A5 and F4 IgG were incubated with BT-474 cells (250,000 cells/assay) and binding was detected by flow cytometry with a FITC-conjugated anti-human Fc secondary antibody. Cells were incubated with 2 µg of PE-conjugated anti-ERBB3 antibody (SGP1-PE) as a positive control. Equivalent amounts of mouse IgG1-PE (R&D Systems, cat #IC002P) served as a negative control.</p

Effects of anti-ErbB3 mAbs on basal ErbB3 signaling.

<p>BT-474, NCI-N87 and ACHN cells growing in complete media were serum starved for 12–16 hours and treated with 1 µM A5, 1 µM F4, 1.6 nM trastuzumab (T), 100 nM cetuximab (C), PBS or combinations of A5/F4 (AF), A5/F4/trastuzumab (AFT), or A5/F4/Cetuximab (AFC) for a prescribed time. Cell lysates were analyzed by western blot with pERBB3(Y1289) and pAKT(S473) mAbs as markers of ERBB3 signaling, as well as anti-beta actin mAb as a loading control.</p

Identification of Wee1 as a target in combination with avapritinib for gastrointestinal stromal tumor treatment

Management of gastrointestinal stromal tumors (GISTs) has been revolutionized by the identification of activating mutations in KIT and PDGFRA and clinical application of RTK inhibitors in advanced disease. Stratification of GISTs into molecularly defined subsets provides insight into clinical behavior and response to approved targeted therapies. Although these RTK inhibitors are effective in most GISTs, resistance remains a significant clinical problem. Development of effective treatment strategies for refractory GISTs requires identification of novel targets to provide additional therapeutic options. Global kinome profiling has the potential to identify critical signaling networks and reveal protein kinases essential in GISTs. Using multiplexed inhibitor beads and mass spectrometry, we explored the majority of the kinome in GIST specimens from the 3 most common molecular subtypes (KIT mutant, PDGFRA mutant, and succinate dehydrogenase deficient) to identify kinase targets. Kinome profiling with loss-of-function assays identified an important role for G2/M tyrosine kinase, Wee1, in GIST cell survival. In vitro and in vivo studies revealed significant efficacy of MK-1775 (Wee1 inhibitor) in combination with avapritinib in KIT mutant and PDGFRA mutant GIST cell lines as well as notable efficacy of MK-1775 as a monotherapy in the engineered PDGFRA mutant line. These studies provide strong preclinical justification for the use of MK-1775 in GIST