Proteomics has the potential to provide answers in cancer pathogenesis and to direct targeted therapy through the comprehensive analysis of protein expression levels and activation status. The realization of this potential requires the development of new, rapid, high-throughput technologies for performing protein arrays on patient samples, as well as novel analytic techniques to interpret them. Herein, we describe the validation and robustness of using reverse phase protein arrays (RPPA) for the analysis of primary acute myelogenous leukemia samples as well as leukemic and normal stem cells. In this report, we show that array printing, detection, amplification, and staining precision are very high, reproducible, and that they correlate with traditional Western blotting. Using replicates of the same sample on the same and/or separate arrays, or using separate protein samples prepared from the same starting sample, the intra-and interarray reproducibility was extremely high. No statistically significant difference in protein signal intensities could be detected within the array setups. The activation status (phosphorylation) was maintained in experiments testing delayed processing and preparation from multiple freezethawed samples. Differences in protein expression could reliably be detected in as few as three cell protein equivalents. RPPA prepared from rare populations of normal and leukemic stem cells were successfully done and showed differences from bulk populations of cells. Examples show how RPPAs are ideally suited for the largescale analysis of target identification, validation, and drug discovery. In summary, RPPA is a highly reliable, reproducible, high-throughput system that allows for the rapid large-scale proteomic analysis of protein expression and phosphorylation state in primary acute myelogenous leukemia cells, cell lines, and in human stem cells. [Mol Cancer Ther 2006;5(10):2512 -21]

GDC-0449, an orally active small molecule that targets the hedgehog pathway, appears to have antitumor activity in locally advanced or metastatic basal-cell carcinoma. (ClinicalTrials.gov number, NCT00607724.)

Purpose The hedgehog (Hh) signaling pathway, a key regulator of cell growth and differentiation during development is implicated in pathogenesis of certain cancers. Vismodegib (GDC-0449) is a small-molecule inhibitor of smoothened, a key component of Hh signaling. This phase I trial assessed GDC-0449 treatment in patients with solid tumors refractory to current therapies or for which no standard therapy existed. Experimental Design Sixty-eight patients received GDC-0449 at 150 mg/d (n = 41), 270 mg/d (n = 23), or 540 mg/d (n = 4). Adverse events, tumor responses, pharmacokinetics, and pharmacodynamic down-modulation of GLI1 expression in noninvolved skin were assessed. Results Thirty-three of 68 patients had advanced basal cell carcinoma (BCC), 8 had pancreatic cancer, 1 had medulloblastoma; 17 other types of cancer were also represented. GDC-0449 was generally well-tolerated. Six patients (8.8%) experienced 7 grade 4 events (hyponatremia, fatigue, pyelonephritis, presyncope, resectable pancreatic adenocarcinoma, and paranoia with hyperglycemia), and 27.9% of patients experienced a grade 3 event [most commonly hyponatremia (10.3%), abdominal pain (7.4%), and fatigue (5.9%)]. No maximum tolerated dose was reached. The recommended phase II dose was 150 mg/d, based on achievement of maximal plasma concentration and pharmacodynamic response at this dose. Tumor responses were observed in 20 patients (19 with BCC and 1 unconfirmed response in medulloblastoma), 14 patients had stable disease as best response, and 28 had progressive disease. Evidence of GLI1 down-modulation was observed in noninvolved skin. Conclusions GDC-0449 has an acceptable safety profile and encouraging anti-tumor activity in advanced BCC and medulloblastoma. Further study in these and other cancer types is warranted.

Background Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations are common in acute myeloid leukemia (AML) and are associated with rapid relapse and short survival. In relapsed/refractory (R/R) AML, the clinical benefit of FLT3 inhibitors has been limited by rapid generation of resistance mutations, especially FLT3-D835. Gilteritinib is a potent, highly selective oral FLT3/AXL inhibitor with preclinical activity against FLT3-ITD and FLT3-D835 mutations. The aim of this Phase 1/2 study was to assess the safety, tolerability, and pharmacokinetic (PK) effects of gilteritinib in FLT3 mutation-positive (FLT3mut+) R/R AML. Methods This ongoing pharmacodynamic-driven Phase 1/2 trial (NCT02014558) enrolled subjects from October 2013 to August 2015 who were aged ≥18 years and were either refractory to induction therapy or had relapsed after achieving remission with prior therapy. Subjects were enrolled in one of seven dose-escalation or dose-expansion cohorts that were assigned to receive once-daily doses of oral gilteritinib (20, 40, 80, 120, 200, 300, or 450 mg). Cohort expansion was based on safety/tolerability, FLT3 inhibition in correlative assays, and antileukemic activity; the 120 and 200 mg dose cohorts were further expanded to include FLT3mut+ patients only. Safety and tolerability, and PK effects were the primary endpoints; antileukemic response was the main secondary endpoint. Safety and tolerability were assessed by monitoring dose-limiting toxicities and treatment-emergent adverse events, and safety assessments (eg, clinical laboratory evaluations, electrocardiograms) in the Safety Analysis Set. Findings A total of 252 adults with R/R AML, including 58 with wild-type FLT3 and 194 with FLT3 mutations (FLT3-ITD, n=162; FLT3-D835, n=16; FLT3-ITD and -D835, n=13; other, n=3), received oral gilteritinib (20–450 mg) once daily in one of seven dose-escalation (n=23) or dose-expansion (n=229) cohorts. Gilteritinib was well tolerated in this heavily pretreated population; Grade 3 diarrhea and hepatic transaminase elevation limited dosing above 300 mg/d. The most common Grade 3/4 adverse events were febrile neutropenia (39%; n=97/252), anemia (24%; n=61/252), thromobocytopenia (13%; n=33/252), sepsis (11%; n=28/252), and pneumonia (11%; n=27/252). Serious adverse events in ≥5% of patients were febrile neutropenia (31%; n=78/252), progressive disease (17%; n=43/252), sepsis (14%; n=36/252), pneumonia (11%; n=27/252), and acute renal failure (10%; n=25/252), pyrexia (8%; n=21/252), bacteremia (6%; n=14/252), and respiratory failure (6%; n=14/252). Gilteritinib demonstrated consistent, potent inhibition of FLT3 phosphorylation at doses ≥80 mg/d in correlative assays. While responses were observed across all dose levels regardless of FLT3 mutation status (overall response rate [ORR]=40%), response rate was improved in FLT3mut+ patients at doses ≥80 mg/d (ORR=52%). Among patients with FLT3-ITD, the additional presence of FLT3-D835 did not alter response rate; patients with only FLT3-D835 respond...