Dasatinib, paclitaxel, and carboplatin in women with advanced-stage or recurrent endometrial cancer: A pilot clinical and translational study.

PURPOSE: To evaluate the effect of dasatinib therapy on EphA2 signaling in cancers of women with measurable (biopsy amenable) advanced-stage, chemo-naïve primary or recurrent endometrial cancer. Preliminary efficacy was also assessed. PATIENTS AND METHODS: We performed a pilot study of single-agent dasatinib lead-in, followed by triplet dasatinib, paclitaxel, and carboplatin. We measured the downstream effectors of EphA2 signaling in pre- and post-dasatinib treatment biopsy tissue samples; we also determined the severity of adverse events and patients' progression-free survival and overall survival durations. RESULTS: Eighteen patients were recruited and given dasatinib (150 mg orally daily for 14 days), followed by paclitaxel, carboplatin and dasatinib (daily) for six cycles (21-day cycles). Seventeen patients were evaluable for toxicity and 11 patients for response. A reverse phase protein array and proximity ligation assay revealed that CRAF/BRAF dimerization, caveolin-1 level, and Notch pathway signaling were predictive of response and resistance to dasatinib. Overall, the objective response rate was 45% (95% CI: 17%-77%), with median progression-free survival duration of 10.5 months and median overall survival duration of 30.4 months. The most common grade 3 or 4 adverse events were neutropenia (76%), thrombocytopenia (53%), anemia (53%), and fatigue (12%). CONCLUSIONS: Caveolin-1 expression, in combination with CRAF/BRAF heterodimerization, is associated with resistance to EphA2 targeting by dasatinib. The triplet combination showed interesting clinical activity in endometrial cancer with acceptable toxicity. Pretreatment with dasatinib may accentuate combination therapy toxicity.

A prospective phase II study of chemoradiation followed by adjuvant chemotherapy for FIGO stage I-IIIA (1988) uterine papillary serous carcinoma of the endometrium.

OBJECTIVE: To prospectively evaluate tumor control, survival, and toxic effects in patients with International Federation of Gynecology and Obstetrics (1988) stage I-IIIA papillary serous carcinoma of the endometrium treated with concurrent chemoradiation and adjuvant chemotherapy. METHODS: Thirty-two patients were enrolled from October 2001 through July 2009. Patients underwent full surgical disease staging and postoperative concurrent weekly paclitaxel (50 mg/m2) and pelvic RT to 45 Gy plus a vaginal cuff boost followed by 4 cycles of adjuvant paclitaxel (135 mg/m2). RESULTS: Thirty patients (94%) were evaluable (3 with stage IA disease, 11 IB, 3 IC, 1 IIB, and 12 IIIA). Eighteen patients (60%) received all 5 planned courses of concurrent chemotherapy, 10 (33%) received 4 courses, and 2 (7%) received 3 courses. All 30 patients received RT; 27 (90%) received the full dose, 2 received 43.2 Gy, and 1 received 39.6 Gy owing to toxic effects. Twenty-three patients (77%) completed all 4 cycles of adjuvant paclitaxel, 3 (10%) completed 3 cycles, 2 (7%) completed 2 cycles, and 2 received no adjuvant therapy. Overall survival (OS), progression-free survival (PFS), and local control rates for all patients were 93%, 87%, and 87%, respectively, at 2 years and 85%, 83%, and 87%, respectively, at 5years. Six patients developed (20%) grade 3/4 toxicities from the treatment. Four patients (13%) had grade 3 or more severe bowel complications and two patients developed symptomatic pelvic fractures. CONCLUSIONS: Treatment with concurrent paclitaxel and pelvic RT followed by 4 courses of systemic paclitaxel produced favorable results in patients with surgically staged I-III UPSC.

Phase 1-2 study of docetaxel plus aflibercept in patients with recurrent ovarian, primary peritoneal, or fallopian tube cancer.

BACKGROUND: Biologically targeted therapies have been postulated as a viable strategy to improve outcomes for women with ovarian cancer. We assessed the safety, tolerance, pharmacokinetics, relevant circulating and image-derived biomarkers, and clinical activity of combination aflibercept and docetaxel in this population. METHODS: For the phase 1 (pharmacokinetic) study, eligible patients had measurable, recurrent or persistent epithelial ovarian, primary peritoneal, or fallopian tube carcinoma with a maximum of two prior chemotherapy regimens. Aflibercept was administered intravenously over three dose levels (2, 4, or 6 mg/kg; one dose every 21 days) to identify the maximum tolerated dose for the phase 2 study. Pharmacokinetics were assessed and dynamic imaging was done during a lead-in phase with single-agent aflibercept (cycle 0) and during combination therapy with intravenous docetaxel (75 mg/m(2)). Eligibility for the phase 2 study was the same as for phase 1. Patients were enrolled in a two-stage design and given aflibercept 6 mg/kg intravenously and docetaxel 75 mg/m(2) intravenously, every 3 weeks. The primary endpoint was objective response rate (ORR) as assessed by Response Evaluation Criteria in Solid Tumors version 1.0. The trial has completed enrolment and all patients are now off study. The trial is registered at ClinicalTrials.gov, number NCT00436501. FINDINGS: From the phase 1 study, the recommended phase 2 doses of aflibercept and docetaxel were found to be 6 mg/kg and 75 mg/m(2), respectively. Log-linear pharmacokinetics (for unbound aflibercept) were observed for the three dose levels. No dose-limiting toxicities were noted. 46 evaluable patients were enrolled in the phase 2 trial; 33 were platinum resistant (15 refractory) and 13 were platinum sensitive. The confirmed ORR was 54% (25 of 46; 11 patients had a complete response and 14 had a partial response). Grade 3-4 toxicities observed in more than two patients (5%) were: neutropenia in 37 patients (80%); leucopenia in 25 patients (54%); fatigue in 23 patients (50%); dyspnoea in ten patients (22%); and stomatitis in three patients (7%). Adverse events specifically associated with aflibercept were grade 1-2 hypertension in five patients (11%), and grade 2 proteinuria in one patient (2%). INTERPRETATION: Combination aflibercept plus docetaxel can be safely administered at the dose and schedule reported here, and is associated with substantial antitumour activity. These findings suggest that further clinical development of this combination in ovarian cancer is warranted. FUNDING: US National Cancer Institute, US Department of Defense, Sanofi-Aventis, Gynecologic Cancer Foundation, Marcus Foundation, and the Commonwealth Foundation.

Phase II study of intraperitoneal recombinant interleukin-12 (rhIL-12) in patients with peritoneal carcinomatosis (residual disease < 1 cm) associated with ovarian cancer or primary peritoneal carcinoma.

BACKGROUND: Pharmacokinetic advantages of intraperitoneal (IP) rhIL-12, tumor response to IP delivery of other cytokines as well as its potential anti-angiogenic effect provided the rationale for further evaluation of IPrhIL-12 in patients with persistent ovarian or peritoneal carcinoma. METHODS: A phase 2 multi-institutional trial (NCI Study #2251) of IP rIL-12 (300 nanogram/Kg weekly) was conducted in patients with ovarian carcinoma or primary peritoneal carcinoma. Patients treated with primary therapy for ovarian cancer who had no extraabdominal/parenchymal disease or bulky peritoneal disease were eligible. Four to 8 weeks from last chemotherapy, eligible patients underwent a laparotomy/laparoscopy. Patients with residual disease < or = 1 cm were registered for the treatment phase 2-5 weeks post surgery. The effect of IP rIL-12 on the expression of TNFalpha , INFalpha , IL-10, IP-10, IL-8, FGF, VEGF was also studied. RESULTS: Thirty-four patients were registered for the first screening phase of the study. Median age was 56.6 years (range: 31-71); 12 completed the second phase and were evaluable for response/toxicity. Performance scores of IL-12 treated patients were 0 (11 pts) and 1 (1 pt). There were no treatment related deaths, peritonitis or significant catheter related complications. Toxicities included grade 4 neutropenia (1), grade 3 fatigue (4), headache (2), myalgia (2), non-neutropenic fever (1), drug fever (1), back pain (1), and dizziness (1). The best response observed was SD. Two patients had SD and 9 had PD, and 1 was evaluable for toxicity only. Peritoneal fluid cytokine measurements demonstrated a > or = 3 fold relative increase post-rhIL-12: IFN-gamma, 5/5 pts; TNF-alpha , 1/5; IL-10, 4/5; IL-8, 5/5; and VEGF, 3/5. IP10 levels were increased in 5/5 patients. Cytokine response profiles suggest either NK or T-cell mediated effects of IP rhIL-12. Cytokine/chemokine results also suggest a pleiotropic response since proteins with potential for either anti-tumor (IFN-gamma , IP-10) or pro-tumor growth effects (VEGF, IL-8) were detected. CONCLUSION: IP IL-12 can safely be administered at this dose and schedule to patients after first line chemotherapy for ovarian/peritoneal carcinoma. The maximum response was stable disease. Future IP therapies with rhIL-12 will require better understanding and control of pleiotropic effects of IL-12.

Cytokines, GM-CSF and IFNgamma administered by priming and post-chemotherapy cycling in recurrent ovarian cancer patients receiving carboplatin.

BACKGROUND: Monocyte/macrophages (MO/MA), a polymorphic population of innate immune cells, have the potential to mediate antitumor effects, and may also contribute to protumor effects. A priming and post-chemotherapy schedule of the myeloid cell mobilizing and immune stimulatory growth factor, granulocyte monocyte stimulating factor (GM-CSF, Leukine) and the MO/MA activating cytokine recombinant interferon gamma 1b (rIFN-gamma1b, Actimmune) has been developed. The pre- and post-chemotherapy design is based upon known in vivo kinetics and immune modulatory effects of these molecules. Carboplatin (Paraplatin) was selected as the cornerstone of treatment of epithelial ovarian cancer (EOC). METHODS: We studied hematopoietic and immunologic effects of GM-CSF and rIFN-gamma1b before and after carboplatin in patients with recurrent EOC. Potentially chemotherapy-sensitive patients with recurrent measurable tumors received subcutaneous GM-CSF (starting at 400 mug/day) for 7 days plus subcutaneous rIFN-gamma1b (100 mug) on days 5 and 7, before and after intravenous carboplatin (area under the curve of 5). We performed standard hematologic assessment and monitored monocyte (MO), dendritic cell, major cell subset counts, and antibody-dependent cell-mediated cytotoxicity (ADCC) against a Her2neu+ tumor cell line, as well as selected plasma inflammatory cytokine, chemokine and growth factor levels. RESULTS: Our analysis comprised only the first 3 months of treatment in the initial 25 patients. Relative to pretreatment baseline values, white blood cell, neutrophil, MO, and eosinophil counts increased (P

Pilot study of Flt3 ligand comparing intraperitoneal with subcutaneous routes on hematologic and immunologic responses in patients with peritoneal carcinomatosis and mesotheliomas.

PURPOSE: This study compared the clinical toxicity and hematological effects of i.p. and s.c. administration of fms-like tyrosine kinase-3-ligand (Flt3-L; Amgen, Thousand Oaks, CA), a truncated glycoprotein that increases dendritic cells (DCs) and monocytes. EXPERIMENTAL DESIGN: Patients with peritoneal carcinomatosis or mesothelioma were randomly assigned to treatment with Flt3-L (25 micro g/kg, maximum 1500 micro g), i.p. or s.c., days 1-5 and 8-12, then changed to the alternative route at 4 weeks. Treatment was continued s.c. or i.p. at 8 weeks. RESULTS: Fifteen patients (14 evaluable) were randomized to receive i.p. (n = 8) or s.c. (n = 7) injections. Their median age was 55 years (range, 40-68 years). Primary tumors were as follows: ovarian/peritoneal cancer (n = 9); gastrointestinal cancer (n = 2); and mesothelioma (n = 4). Treatment was well tolerated without serious toxicity (24 i.p. cycles; 32 s.c. cycles). Treatment (i.p. or s.c.) resulted in significant increases in WBCs (WBC, monocytes, and Lin(-)DR(+) DCs), and platelets (during washout). Both interleukin (IL)-12(p70) and IL-10 were secreted by monocyte-derived DCs after in vitro exposure to maturation factors. Increased IL-12 versus IL-10 secretion responses and higher proportions of the CD11c(+) DC subset in post-Flt3-L specimens suggested a maturational shift toward the monocyte-derived DC phenotype had occurred. Three patients (2 with mesothelioma and 1 with gastrointestinal cancer) had stable disease for 8, 8, and 12+ months, respectively. CONCLUSIONS: Flt3-L, administered either i.p. or s.c., is well tolerated and produced similar increases in monocytes, DCs, and platelets. DCs from peripheral blood and peritoneal fluids showed cell surface phenotypic and cytokine maturational responses to activation stimuli. These findings suggested that Flt3-L, in combination with suitable activating agents, could be developed further in patients with epithelial ovarian cancer.

Phase I study of intraperitoneal recombinant human interleukin 12 in patients with Müllerian carcinoma, gastrointestinal primary malignancies, and mesothelioma.

PURPOSE: The purpose is to determine dose-limiting toxicity, pharmacokinetics,pharmacodynamics, and immunobiology after i.p. injections of recombinant human IL-12 (rhIL-12). EXPERIMENTAL DESIGN: rhIL-12 was administered to 29 previously treated patients with peritoneal carcinomatosis from Müllerian carcinomas, gastrointestinal tract carcinomas and peritoneal mesothelioma in a Phase I trial. rhIL-12 doses were increased from 3 to 600 ng/kg. Three or more patients at each level received weekly i.p. injections of rhIL-12. RESULTS: Dose-limiting toxicity (elevated transaminase) occurred in 2 of 4 patients at the 600 ng/kg dose. More frequent toxicities included fever, fatigue, abdominal pain, nausea, and catheter-related infections. Ten patients received 300 ng/kg with acceptable frequency and severity of side effects. Two patients (one with ovarian cancer and one with mesothelioma) had no remaining disease at laparoscopy. Eight patients had stable disease and 19 progressive disease. At 300 ng/kg i.p., IL-12 was cleared from peritoneal fluid in a biphasic manner with a terminal-phase half-life of 18.7 h; peritoneal fluid levels of IL-12 5 min after i.p. injection were 100-200 pg/ml, and serum levels reached approximately 10 pg/ml between 24 and 36 h. IL-1-alpha, IL-2, IL-10, tumor necrosis factor alpha, and IFN-gamma were determined in serum and peritoneal fluid. IFN-gamma, IL-10, and tumor necrosis factor alpha were detected most frequently. Immunobiological effects included peritoneal tumor cell apoptosis, decreased tumor cell expression of basic fibroblast growth factor and vascular endothelial growth factor, elevated IFN-gamma and IFN-inducible protein 10 transcripts in peritoneal exudate cells, and increased proportions of peritoneal CD3(+) relative to CD14(+) cells. CONCLUSIONS: rhIL-12 at 300 ng/kg by weekly i.p. injection is biologically active and adequately tolerated for Phase II studies.

HTS-Compatible Patient-Derived Cell-Based Assay to Identify Small Molecule Modulators of Aberrant Splicing in Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a genetic disorder characterized by muscle wasting, myotonia, cataracts, cardiac arrhythmia, hyperinsulinism and intellectual deficits, and is caused by expansion of a CTG repeat in the 3'UTR of the Dystrophia Myotonica-Protein Kinase (DMPK) gene. The DMPK transcripts containing expanded CUG repeats accumulate in nuclear foci and ultimately cause mis-splicing of secondary genes through the dysregulation of RNA-binding proteins including Muscleblind 1 (MBNL1) and CUG binding protein 1 (CUGBP1). Correction of mis-splicing of genes such as the Skeletal muscle-specific chloride channel 1 (CLCN1), Cardiac troponin T (TNNT2), Insulin receptor (INSR) and Sarcoplasmic/endoplasmic reticulum Ca(2+)ATPase 1 (SERCA1) may alleviate some of the symptoms of DM1; hence identification of small molecule modulators is an important step towards a therapy for DM1 patients. Here we describe the generation of immortalized myoblast cell lines derived from healthy (DMPK CTG(5)) and DM1 patient (DMPK CTG(1000)) fibroblasts by constitutive overexpression of human telomerase reverse transcriptase (hTERT) and inducible overexpression of the Myoblast determination factor (MYOD). MBNL1-containing nuclear foci, mis-splicing events and defective myotube differentiation defects characteristic of DM1 were observed in these cells. A CLCN1 luciferase minigene construct (CLCN1-luc) was stably introduced to monitor intron 2 retention in the DM1 cellular context (a reported splicing defect in DM1). The assay was validated by performing a high-throughput screen (HTS) of ~13,000 low molecular weight compounds against the CLCN1-luc DM1 myoblast cell line, providing an ideal system for conducting HTS to better understand and treat DM1.

An efficient rapid system for profiling the cellular activities of molecular libraries.

Rapid quantitative methods for characterizing small molecules, peptides, proteins, or RNAs in a broad array of cellular assays would allow one to discover new biological activities associated with these molecules and also provide a more comprehensive profile of drug candidates early in the drug development process. Here we describe a robotic system, termed the automated compound profiler, capable of both propagating a large number of cell lines in parallel and assaying large collections of molecules simultaneously against a matrix of cellular assays in a highly reproducible manner. To illustrate its utility, we have characterized a set of 1,400 kinase inhibitors in a panel of 35 activated tyrosine-kinase-dependent cellular assays in dose-response format in a single experiment. Analysis of the resulting multidimensional dataset revealed subclusters of both inhibitors and kinases with closely correlated activities. The approach also identified activities for the p38 inhibitor BIRB796 and the dual src/abl inhibitor BMS-354825 and exposed the expected side activities for Glivec/STI571, including cellular inhibition of c-kit and platelet-derived growth factor receptor. This methodology provides a powerful tool for unraveling the cellular biology and molecular pharmacology of both naturally occurring and synthetic chemical diversity.

Intraperitoneal fluid neopterin, nitrate, and tryptophan after regional administration of interleukin-12.

Activated monocytes-macrophages may be associated with antitumor activity, and activation of these cells by certain cytokines, primarily interferon gamma (IFN-gamma), can be indicated by alterations in the concentrations of neopterin, nitrate, or tryptophan. Specimens of peritoneal fluid were obtained from patients with intra-abdominal neoplasia who were undergoing treatment in a phase I trial of weekly intraperitoneal recombinant interleukin-12 (rhIL-12), an inducer of IFN-gamma. Concentrations of neopterin, nitrate, tryptophan, IFN-gamma, and tumor necrosis factor alpha (TNF-alpha) were determined at various times during the first 48 hours in 11 patients who received intraperitoneal rhIL-12 in doses ranging from 100 to 1,500 ng/kg. An increase in peritoneal fluid nitrate concentrations was observed in nine of these patients. Increased concentrations of TNF-alpha and IFN-gamma were detected in 3 of 9 and 8 of 11 patients, respectively. Increased peritoneal fluid neopterin concentrations were detected by 24 hours after the injection in all patients studied. A significant increase in the ascitic fluid neopterin level could still be detected after 1 or 2 weeks of treatment (mean +/- standard error, 7.8 +/- 1.5 nM vs. 4.6 +/- 0.3 nM; Wilcoxon test, p = 0.0019), which is consistent with monocyte-macrophage activation. In contrast, the tryptophan concentration was lower (4.7 +/- 1.1 microM vs. 6.1 +/- 1.2 microM; p = 0.0428) after 1 or 2 weeks of treatment. There was a significant correlation between the dose of rhIL-12 and posttreatment neopterin concentrations (r(s) = 0.559, p = 0.0102). The intraperitoneal delivery of rhIL-12 appears to be associated with an immediate, sustained, and dose-dependent increase in peritoneal fluid neopterin, associated in most patients by an increase in IFN-gamma and in certain patients by an increase in nitrate and a decrease in tryptophan concentrations.