Phase II Randomized Trial of Sequential or Concurrent FOLFOXIRI-Bevacizumab Versus FOLFOX-Bevacizumab for Metastatic Colorectal Cancer (STEAM).

BACKGROUND: First-line treatment for metastatic colorectal cancer (mCRC) typically entails a biologic such as bevacizumab (BEV) with 5-fluorouracil/leucovorin/oxaliplatin (FOLFOX) or 5-fluorouracil/leucovorin/irinotecan (FOLFIRI). STEAM (NCT01765582) assessed the efficacy of BEV plus FOLFOX/FOLFIRI (FOLFOXIRI), administered concurrently (cFOLFOXIRI-BEV) or sequentially (sFOLFOXIRI-BEV, FOLFOX-BEV alternating with FOLFIRI-BEV), versus FOLFOX-BEV for mCRC. PATIENTS AND METHODS: Patients with previously untreated mCRC (n = 280) were randomized 1:1:1 to cFOLFOXIRI-BEV, sFOLFOXIRI-BEV, or FOLFOX-BEV and treated with 4-6-month induction followed by maintenance. Coprimary objectives were overall response rate (ORR; first-line cFOLFOXIRI-BEV vs. FOLFOX-BEV) and progression-free survival (PFS; pooled first-line cFOLFOXIRI-BEV and sFOLFOXIRI-BEV vs. FOLFOX-BEV). Secondary/exploratory objectives included overall survival (OS), liver resection rates, biomarker analyses, and safety. RESULTS: ORR was 72.0%, 72.8%, and 62.1% and median PFS was 11.9, 11.4, and 9.5 months with cFOLFOXIRI-BEV, sFOLFOXIRI-BEV, and FOLFOX-BEV, respectively. OS was similar between arms. ORR between cFOLFOXIRI-BEV and FOLFOX-BEV did not significantly differ (p = .132); thus, the primary ORR endpoint was not met. cFOLFOXIRI-BEV and sFOLFOXIRI-BEV numerically improved ORR and PFS, regardless of RAS status. Median PFS was higher with pooled concurrent and sequential FOLFOXIRI-BEV versus FOLFOX-BEV (11.7 vs. 9.5 months; hazard ratio, 0.7; 90% confidence interval, 0.5-0.9; p < .01). Liver resection rates were 17.2% (cFOLFOXIRI-BEV), 9.8% (sFOLFOXIRI-BEV), and 8.4% (FOLFOX-BEV). Grade ≥ 3 treatment-emergent adverse events (TEAEs) were observed in 91.2% (cFOLFOXIRI-BEV), 86.7% (sFOLFOXIRI-BEV), and 85.6% (FOLFOX-BEV) of patients, with no increase in serious chemotherapy-associated TEAEs. CONCLUSION: cFOLFOXIRI-BEV and sFOLFOXIRI-BEV were well tolerated with numerically improved ORR, PFS, and liver resection rates versus FOLFOX-BEV, supporting triplet chemotherapy plus BEV as a first-line treatment option for mCRC. IMPLICATIONS FOR PRACTICE: The combination of first-line FOLFIRI with FOLFOX and bevacizumab (concurrent FOLFOXIRI-BEV) improves clinical outcomes in patients with metastatic colorectal cancer (mCRC) relative to FOLFIRI-BEV or FOLFOX-BEV, but it is thought to be associated with increased toxicity. Alternating treatment of FOLFOX and FOLFIRI (sequential FOLFOXIRI-BEV) could improve tolerability. In the phase II STEAM trial, which is the largest study of FOLFOXIRI-BEV in patients in the U.S., it was found that both concurrent and sequential FOLFOXIRI-BEV are active and well tolerated in patients with previously untreated mCRC, supporting the use of these regimens as potential first-line treatment options for this population.

Two phase I, pharmacokinetic, and pharmacodynamic studies of DFP-10917, a novel nucleoside analog with 14-day and 7-day continuous infusion schedules.

Purpose DFP-10917 is a novel deoxycytidine analog with a unique mechanism of action. Brief exposure to high concentrations of DFP-10917 inhibits DNA polymerase resulting in S-phase arrest, while prolonged exposure to DFP-10917 at low concentration causes DNA fragmentation, G2/M-phase arrest, and apoptosis. DFP-10917 demonstrated activity in tumor xenografts resistant to other deoxycytidine analogs. Experimental design Two phase I studies assessed the safety, pharmacokinetic, pharmacodynamic and preliminary efficacy of DFP-10917. Patients with refractory solid tumors received DFP-10917 continuous infusion 14-day on/7-day off and 7-day on/7-day off. Enrollment required age > 18 years, ECOG Performance Status 0-2 and adequate organ function. Results 29 patients were dosed in both studies. In 14-day infusion, dose-limiting toxicities (DLT) consisting of febrile neutropenia and thrombocytopenia occurred at 4.0 mg/m2/day. At 3.0 mg/m2/day, 3 patients experienced neutropenia in cycle 2. The dose of 2.0 mg/m2/day was well tolerated in 6 patients. In 7-day infusion, grade 4 neutropenia was DLT at 4.0 mg/m2/day. The maximum tolerated dose was 3 mg/m2/day. Other toxicities included nausea, vomiting, diarrhea, neutropenia, and alopecia. Eight patients had stable disease for >12 weeks. Paired comet assays performed for 7 patients showed an increase in DNA strand breaks at day 8. Pharmacokinetic data showed dose-proportionality for steady-state concentration and AUC of DFP-10917 and its primary metabolite. Conclusion Continuous infusion of DFP-10917 is feasible and well tolerated with myelosuppression as main DLT. The recommended doses are 2.0 mg/m2/day and 3.0 mg/m2/day on the 14-day and 7-day continuous infusion schedules, respectively. Preliminary activity was suggested. Pharmacodynamic data demonstrate biological activity at the tested doses.

Phase II study of DFP-10917, a deoxycytidine analog, given by 14-day continuous intravenous infusion for chemotherapy-refractory advanced colorectal cancer.

Background DFP-10917 is a cytotoxic deoxycytidine analogue that causes DNA fragmentation, G2/M-phase arrest, and apoptosis. This agent has been shown to have antitumor activity against colorectal cancer (CRC) in preclinical studies and to be tolerable in patients. The purpose of our phase II trial was to evaluate the safety, efficacy and pharmacogenomics of DFP-10917 as well as DNA damage studies in patients with advanced CRC refractory to cytotoxic chemotherapy. Methods In this single-arm, Simon two-stage, phase II trial, patients with chemotherapy-refractory advanced CRC received 2.0 mg/m2/day DFP-10917 via 14-day continuous infusion. Enrollment criteria included age ≥ 18 years, Eastern Cooperative Oncology Group status of 0 or 1, and adequate organ function. The primary endpoint was 3-month progression-free survival, defined as the proportion of patients who did not have progressive disease or death within 3 months of starting therapy. All patients who received any amount of DFP-10917 were included in the safety analysis. DNA damage study was assessed by comet assay. Results Of 28 patients initially enrolled, 26 received DFP-10917. Three patients (12%) were progression free at 3 months. The median progression-free survival was 1.3 months (95% confidence interval, 1.3-1.6 months). There were no complete or partial responses. Most patients (n = 20, 77%) had progressive disease, and only six (23%) had stable disease at any time. The trial was terminated according to the pre-planned stopping rule. The most frequent (≥5%) medication-related grade 3 or higher adverse events were neutropenia (n = 10, 38%), fatigue (n = 4, 15%), anemia (n = 3, 12%), and leukopenia (n = 3, 12%). DNA strand-breaks were detected after infusion (medians of % tail intensity were 2.89 and 12.64 on day 1 and day 15, respectively, p < 0.001, sign test). Conclusion Overall, single-agent DFP-10917 did not show meaningful antitumor activity in chemotherapy-refractory advanced CRC. The safety profile of DFP-10917 was tolerable and similar to that observed in earlier clinical studies.

A Randomized, Double-Blinded, Phase II Trial of Gemcitabine and Nab-Paclitaxel Plus Apatorsen or Placebo in Patients with Metastatic Pancreatic Cancer: The RAINIER Trial.

LESSONS LEARNED: The addition of the heat shock protein 27 (Hsp27)-targeting antisense oligonucleotide, apatorsen, to a standard first-line chemotherapy regimen did not result in improved survival in unselected patients with metastatic pancreatic cancer.Findings from this trial hint at the possible prognostic and predictive value of serum Hsp27 that may warrant further investigation. BACKGROUND: This randomized, double-blinded, phase II trial evaluated the efficacy of gemcitabine/nab-paclitaxel plus either apatorsen, an antisense oligonucleotide targeting heat shock protein 27 (Hsp27) mRNA, or placebo in patients with metastatic pancreatic cancer. METHODS: Patients were randomized 1:1 to Arm A (gemcitabine/nab-paclitaxel plus apatorsen) or Arm B (gemcitabine/nab-paclitaxel plus placebo). Treatment was administered in 28-day cycles, with restaging every 2 cycles, until progression or intolerable toxicity. Serum Hsp27 levels were analyzed at baseline and on treatment. The primary endpoint was overall survival (OS). RESULTS: One hundred thirty-two patients were enrolled, 66 per arm. Cytopenias and fatigue were the most frequent grade 3/4 treatment-related adverse events for both arms. Median progression-free survival (PFS) and OS were 2.7 and 5.3 months, respectively, for arm A, and 3.8 and 6.9 months, respectively, for arm B. Objective response rate was 18% for both arms. Patients with high serum level of Hsp27 represented a poor-prognosis subgroup who may have derived modest benefit from addition of apatorsen. CONCLUSION: Addition of apatorsen to chemotherapy does not improve outcomes in unselected patients with metastatic pancreatic cancer in the first-line setting, although a trend toward prolonged PFS and OS in patients with high baseline serum Hsp27 suggests this therapy may warrant further evaluation in this subgroup.

Inhibition of the mammalian target of rapamycin (mTOR) in advanced pancreatic cancer: results of two phase II studies.

BACKGROUND: The phosphoinositide 3-kinase (PI3K)/Akt pathway is constitutively activated in pancreatic cancer and the mammalian target of rapamycin (mTOR) kinase is an important mediator for its signaling. Our recent in vitro studies suggest that prolonged exposure of pancreatic cancer cells to mTOR inhibitors can promote insulin receptor substrate-PI3K interactions and paradoxically increase Akt phosphorylation and cyclin D1 expression in pancreatic cancer cells (negative feedback loop). The addition of erlotinib to rapamycin can down-regulate rapamycin-stimulated Akt and results in synergistic antitumor activity with erlotinib in preclinical tumor models. METHODS: Two studies prospectively enrolled adult patients with advanced pancreatic cancer, Eastern Cooperative Oncology Group performance status 0-1, adequate hematologic, hepatic and renal parameters and measurable disease. In Study A, temsirolimus was administered intravenously at 25 mg weekly. In Study B, everolimus was administered orally at 30 mg weekly and erlotinib was administered at 150 mg daily. The primary endpoint in both studies was overall survival at 6 months. Secondary endpoints included time to progression, progression-free survival, overall survival, response rate, safety and toxicity. Pretreatment tumor biopsies were analyzed by immunofluorescence and laser scanning cytometry for the expression of pmTOR/mTOR, pAkt/Akt, pErk/Erk, pS6, p4EBP-1 and PTEN. RESULTS: Five patients enrolled in Study A; Two patients died within a month (rapid disease progression and hemorrhagic stroke, respectively). One patient developed dehydration and another developed asthenia. Sixteen patients enrolled in Study B.: 12 males, all ECOG PS = 1. Median cycles = 1 (range 1-2). Grade 4 toxicity: hyponatremia (n = 1), Grade 3: diarrhea (n = 1), cholangitis (n = 3), hyperglycemia (n = 1), fatigue (n = 1). Grade 2: pneumonia (n = 2), dehydration (n = 2), nausea (n = 2), neutropenia (n = 1), mucositis (n = 2) & rash (n = 2). Four patients were hospitalized. Progressive disease occurred in 15 and 1 was non-evaluable. Pretreatment biopsies revealed a higher pAkt/Akt ratio in tumor specimens that in nonmalignant pancreatic tissue. No such trends were noted for the other biomarkers. CONCLUSIONS: Neither study with mTOR inhibitors demonstrated objective responses or disease stability. The negative feedback loop resulting from mTOR inhibition may account for the disease progression and toxicity noted in these studies. Future strategies should aim for a broader targeting of the PI3K pathway in pancreatic cancer. STUDY A: NCT 0075647. STUDY B: NCT00640978.

Phase I clinical study of three times a day oral administration of TAS-102 in patients with solid tumors.

TAS-102 is a novel formulation of the fluorinated pyrimidine analogue trifluorothymidine (FTD) with an inhibitor of thymidine phosphorylase. The purpose of this study was to determine the MTD and DLT for TAS-102 administered three times a day on days 1-5 and 8-12 every 4 weeks. Fifteen patients were enrolled with two patients experiencing dose-limiting fatigue and granulocytopenia at the first dose level (80 mg/m2/day). Granulocytopenia was the primary toxicity: 7 patients experienced grade 3 or 4 granulocytopenia with the first course. No responses were noted, but nine patients demonstrated prolonged stable disease in this heavily pretreated 5-FU refractory population.

Transforming growth factor alpha expression drives constitutive epidermal growth factor receptor pathway activation and sensitivity to gefitinib (Iressa) in human pancreatic cancer cell lines.

The epidermal growth factor receptor (EGFR) is considered an important therapeutic target in pancreatic cancer, but it is currently impossible to identify those patients who are most likely to benefit from EGFR-directed therapy. We examined the biological effects of the EGFR tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) in a panel of nine human pancreatic cancer cell lines. The drug strongly inhibited DNA synthesis and induced low levels of apoptosis at clinically relevant concentrations in a subset of three of the lines (L3.6pl, BxPC3, and Cfpac1). Sensitivity to gefitinib correlated directly with ligand [transforming growth factor-alpha (TGF-alpha)] expression (r(2) = 0.71, P = 0.004) but not with surface EGFR expression. The gefitinib-sensitive cells displayed constitutive baseline EGFR phosphorylation, whereas the gefitinib-resistant cells did not. Exposure to gefitinib or a small interfering RNA construct specific for TGF-alpha reversed the constitutive EGFR phosphorylation and downstream target [extracellular signal-regulated kinases (ERK), AKT] phosphorylation in the gefitinib-sensitive cells but had no effects on ERK or AKT phosphorylation in gefitinib-resistant cells. Baseline EGFR phosphorylation was lower in a subclone of L3.6pl selected for low TGF-alpha expression, and these cells were also resistant to gefitinib-mediated growth inhibition. Gefitinib blocked the growth of tumor xenografts derived from L3.6pl cells but had no effect on the growth of tumors derived from EGFR-independent MiaPaCa-2 cells. Together, our data show that TGF-alpha expression identifies a subset of human pancreatic cancer cells that is dependent on EGFR signaling in vitro and in vivo. Quantification of TGF-alpha expression may therefore represent an effective means of identifying EGFR-responsive primary tumors.

Aggresome disruption: a novel strategy to enhance bortezomib-induced apoptosis in pancreatic cancer cells.

The proteasome inhibitor bortezomib (formerly known as PS-341) recently received Food and Drug Administration approval for the treatment of multiple myeloma, and its activity is currently being evaluated in solid tumors. Bortezomib triggers apoptosis in pancreatic cancer cells, but the mechanisms involved have not been fully elucidated. Here, we show that pancreatic cancer cells exposed to bortezomib formed aggregates of ubiquitin-conjugated proteins ("aggresomes") in vitro and in vivo. Bortezomib-induced aggresome formation was determined to be cytoprotective and could be disrupted using histone deacetylase (HDAC) 6 small interfering RNA or chemical HDAC inhibitors, which resulted in endoplasmic reticulum stress and synergistic levels of apoptosis in vitro and in an orthotopic pancreatic cancer xenograft model in vivo. Interestingly, bortezomib did not induce aggresome formation in immortalized normal human pancreatic epithelial cells in vitro or in murine pancreatic epithelial cells in vivo. In addition, these cells did not undergo apoptosis following treatment with bortezomib, suberoylanilide hydroxamic acid, or the combination, showing tumor selectivity. Taken together, our study shows that inhibition of aggresome formation can strongly potentiate the efficacy of bortezomib and provides the foundation for clinical trials of bortezomib in combination with HDAC inhibitors for the treatment of pancreatic cancer.

Patterns of self-reported symptoms in pancreatic cancer patients receiving chemoradiation.

Pancreatic cancer is a lethal disease characterized by multiple disease-related symptoms. Chemoradiation therapy is a standard of treatment for locally advanced pancreatic cancer. Although shown to prolong survival, there is little information about treatment-related symptoms or the palliative benefits of chemoradiation. We assessed symptoms of patients with locally advanced pancreatic cancer receiving chemoradiation to determine the prevalence, and co-occurrence, of symptoms and to identify the extent to which symptoms interfered with function. Forty-eight patients were treated with chemoradiation on a Phase I protocol. Patients received radiotherapy (50.4 Gy in 28 fractions), capecitabine (median dose 825 mg/m(2) twice daily), and bevacizumab (2.5-10 mg/kg). Symptom severity and its interference with function were prospectively assessed (at presentation, during, and after chemoradiation) in 43 consenting patients using the M.D. Anderson Symptom Inventory. Results showed that 95% of patients reported at least one of the 13 symptoms assessed at presentation. The most commonly reported symptoms of moderate to severe (>or=5 on a 0-10 scale) intensity at presentation were lack of appetite (24%), pain (19%), fatigue (19%), and sleep disturbance (10%). We observed an increase in patients reporting moderate to severe fatigue, nausea, and sleep disturbance during chemoradiation. McNemar tests for paired binary observations showed the proportion of patients reporting moderate to severe symptoms significantly (P<0.001) decreased after chemoradiation at 94 days follow-up (lack of appetite=7%, pain=7%, fatigue=13%, sleep disturbance=7%). This study demonstrates the feasibility and usefulness of symptom assessment in chemoradiation protocols. Future studies with larger cohorts are needed to further characterize multiple symptoms associated with chemoradiation.

Cytotoxic chemotherapy for pancreatic cancer: Advances to date and future directions.

Chemotherapy remains the mainstay of treatment for pancreatic cancer as most patients present with advanced disease, which precludes locoregional treatment. However, the efficacy of chemotherapy is limited. Gemcitabine is the only agent that improves symptoms and confers a modest survival advantage. Many combination therapy regimens have been studied in phase II settings. Eleven randomised phase III trials have been conducted to compare gemcitabine-containing regimens with gemcitabine monotherapy since gemcitabine became available clinically. The combination of gemcitabine plus capecitabine has demonstrated a survival advantage over gemcitabine, whereas gemcitabine plus oxaliplatin and gemcitabine plus cisplatin have shown improved progression-free survival or time to tumour progression but failed to demonstrate a survival advantage over gemcitabine. The search for effective therapy for advanced pancreatic cancer continues. Gemcitabine in combination with cytotoxic agents or molecular targeted agents hold promise.

Pharmacodynamic analysis of target inhibition and endothelial cell death in tumors treated with the vascular endothelial growth factor receptor antagonists SU5416 or SU6668.

PURPOSE: To determine the effects of small molecule inhibitors of vascular endothelial growth factor receptor (VEGFR)-2 (SU5416 and SU6668) on receptor phosphorylation in tumor xenografts and in paired tumor biopsies obtained in three clinical trials in patients with advanced solid malignancies. EXPERIMENTAL DESIGN: The dose-dependent effects of SU6668 on angiogenesis and tumor growth were investigated in orthotopic L3.6pl pancreatic tumors. Excisional or 18G core biopsies were obtained from patients before and after therapy with SU5416 or SU6668. Laser scanning cytometry-mediated analysis was used to quantify levels of phosphorylated and total VEGFRs and platelet-derived growth factor receptors (PDGFR), tumor microvessel densities, vessel sizes, and endothelial and tumor cell apoptosis. RESULTS: Significant inhibition of tumor microvessel density and growth and increased apoptosis were observed at SU6668 maximum tolerated dose (100 mg/kg) in L3.6pl xenografts. At 6 hours post therapy, SU6668 reduced VEGFR and PDGFR phosphorylation in the tumors by 50% and 92%, respectively, but levels rebounded beyond the baselines by 24 hours. Levels of phosphorylated VEGFR-2 and PDGFR also decreased significantly ( approximately 50%) 6 hours after therapy in 1 of 6 primary human tumors treated with SU6668, but these effects were not associated with increased apoptosis. A significant increase in endothelial cell apoptosis was observed in one tumor exposed to SU5416 and was associated with an increase in vessel size, but these changes occurred without an increase in tumor cell death. CONCLUSIONS: SU5416 and SU6668 displayed biological activity in xenografts. However, neither drug produced marked biological activity in primary patient tumors.

Celecoxib inhibits vascular endothelial growth factor expression in and reduces angiogenesis and metastasis of human pancreatic cancer via suppression of Sp1 transcription factor activity.

The aggressive biology of human pancreatic adenocarcinoma has been linked with overexpression of vascular endothelial growth factor (VEGF). Constitutive activation of the transcription factor Sp1 plays a critical role in VEGF overexpression. Recent studies indicated that celecoxib, a selective cyclooxygenase-2 inhibitor, exhibits potent antitumor activity. However, the underlying molecular mechanisms of this activity remain unclear. In the present study, we used a pancreatic cancer model to determine the role of Sp1 in the antitumor activity of celecoxib. Treatment of various pancreatic cancer cells with celecoxib suppressed VEGF expression at both the mRNA and protein level in a dose-dependent manner. VEGF promoter deletion and point mutation analyses indicated that a region between nucleotide -109 and -61 and its intact Sp1-binding sites were required for the inhibition of VEGF promoter activity by celecoxib. Also, celecoxib treatment reduced both Sp1 DNA binding activity and transactivating activity. This decreased activity correlated with reduced Sp1 protein and its phosphorylation as determined using Western blot analysis. Furthermore, in an orthotopic pancreatic cancer animal model, celecoxib treatment inhibited tumor growth and metastasis. The antitumor activity was consistent with inhibition of angiogenesis as determined by evaluating tumor microvessel formation, which correlated with decreased Sp1 activity and VEGF expression. Collectively, our data provide a novel molecular mechanism for the antitumor activity of celecoxib and may help further improve its effectiveness in controlling pancreatic cancer growth and metastasis.

Stat3 activation regulates the expression of vascular endothelial growth factor and human pancreatic cancer angiogenesis and metastasis.

Expression of vascular endothelial growth factor (VEGF), a key angiogenic protein, has been linked with pancreatic cancer progression. However, the molecular basis for VEGF overexpression remains unclear. Immunohistochemical studies have indicated that VEGF overexpression coincides with elevated Stat3 activation in human pancreatic cancer specimens. In our study, more than 80% of the human pancreatic cancer cell lines used exhibited constitutively activated Stat3, with Stat3 activation correlated with the VEGF expression level. Blockade of activated Stat3 via ectopic expression of dominant-negative Stat3 significantly suppressed VEGF expression, angiogenesis, tumor growth, and metastasis in vivo. Furthermore, constitutively activated Stat3 directly activated the VEGF promoter, whereas dominant-negative Stat3 inhibited the VEGF promoter. A putative Stat3-responsive element on the VEGF promoter was identified using a protein-DNA binding assay and confirmed using a promoter mutagenesis assay. These results indicate that Stat3 directly regulates VEGF expression and hence angiogenesis, growth, and metastasis of human pancreatic cancer, suggesting that Stat3 signaling may be targeted for treatment of pancreatic cancer.

A novel model system for studying the double-edged roles of nitric oxide production in pancreatic cancer growth and metastasis.

In the present study, a model system for studying the role of nitric oxide (NO) in tumor growth and metastasis was reported. Incubation of Panc02 murine pancreatic adenocarcinoma cells in vitro with cytokines and interferon led to heterogeneous expression of NO synthase II (NOS II) protein. Clonal sublines expressing different levels of NOS II were then established using a limited dilution technique. After orthotopical implantation into the pancreas of syngeneic C57BL/6 mice, clones with a low level of NOS II expression produced tumors in pancreas, metastasized to the liver, and formed ascites, whereas those having a high level of NOS II expression did not. Liver-metastasis variants having low to high metastatic ability were also established using in vivo/in vitro passage. Compared with parental Panc02 cells exhibiting a high level of NOS II expression, these variants had a decreased level of NOS II expression. Furthermore, the heterogeneous Panc02 cells were injected intravenously into a large number of syngeneic mice. Variants that metastasized to the liver, lung, skin, peritoneum, ovary, and lymph nodes were established. All of the metastatic variants exhibited a lower level of NOS II expression than the parental Panc02 cell line did. However, the phenotypes of NOS II induction and metastatic ability were unstable. Multiple in vitro/in vivo selection led to stable low NOS II expression and high metastatic potential. Finally, to further confirm the role of NOS II expression derived from tumor cells in metastasis, poorly metastatic Panc02-H0 and highly metastatic Panc02-H7 cells were injected into the pancreas of syngeneic NOS II(-/-) mice, and groups of mice received i.p. injections of either phosphate-buffered saline or L-N(6)-(1-iminoethyl) lysine. Inhibition of NOS II activity in vivo significantly promoted distant liver metastasis. Collectively, these data show that NOS II expression is highly heterogeneous and dynamically regulated, which can directly influence tumor growth and metastasis.

Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor, in combination with gemcitabine for advanced pancreatic cancer: a multicenter phase II Trial.

PURPOSE: To determine the response rate, time to disease progression, survival duration and rate, and toxicity with the combination of cetuximab and gemcitabine in patients with epidermal growth factor receptor (EGFR)-expressing advanced pancreatic cancer. PATIENTS AND METHODS: Patients with measurable locally advanced or metastatic pancreatic cancer who had never received chemotherapy for their advanced disease and had immunohistochemical evidence of EGFR expression were eligible for the multicenter phase II trial. Patients were treated with cetuximab at an initial dose of 400 mg/m(2), followed by 250 mg/m(2) weekly for 7 weeks. Gemcitabine was administered at 1,000 mg/m(2) for 7 weeks, followed by 1 week of rest. In subsequent cycles, cetuximab was administered weekly, and gemcitabine was administered weekly for 3 weeks every 4 weeks. RESULTS: Sixty-one patients were screened for EGFR expression, 58 patients (95%) had at least 1+ staining, and 41 were enrolled onto the trial. Five patients (12.2%) achieved a partial response, and 26 (63.4%) had stable disease. The median time to disease progression was 3.8 months, and the median overall survival duration was 7.1 months. One-year progression-free survival and overall survival rates were 12% and 31.7%, respectively. The most frequently reported grade 3 or 4 adverse events were neutropenia (39.0%), asthenia (22.0%), abdominal pain (22.0%), and thrombocytopenia (17.1%). CONCLUSION: Cetuximab in combination with gemcitabine showed promising activity against advanced pancreatic cancer. Further clinical investigation is warranted.

Prostaglandin E2 drives cyclooxygenase-2 expression via cyclic AMP response element activation in human pancreatic cancer cells.

Cyclooxygenase-2 (COX-2) is constitutively expressed in most human primary carcinomas and with its synthesized product, prostaglandin E2 (PGE2), appears to play important roles in tumor invasion, angiogenesis, resistance to apoptosis and suppression of host immunity. However, the molecular mechanisms that control COX-2 expression are unclear. The purpose of this study was to clarify the mechanism of basal and PGE2-mediated COX-2 expression in the highly metastatic L3.6pl human pancreatic cancer cell line. Using RNA interference to disrupt the expression of CREB and the NF-kappaB p65 subunit, we found that both are involved in maintaining basal COX-2 expression in L3.6pl cells. We also demonstrated that PGE2 increased the cyclic AMP concentration, thereby activating protein kinase A (PKA), which in turn phosphorylated the cyclic AMP response element binding protein (CREB), leading to interaction with the cyclic AMP response element in the promoter region of the COX-2 gene. Immunocytochemical analysis confirmed that PGE2 stimulated the translocation of PKA to the nucleus and increased the immuno-reactivity of phosphorylated CREB. Pretreatment with the PKA selective inhibitor H 89 and the E-prostanoid receptor 2 inhibitor AH 6809 reduced COX-2 upregulation by PGE2. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay results further suggested a role for CREB in COX-2 transcriptional control. Understanding the pathways that control COX-2 expression may lead to a better understanding of its dysregulation in pancreatic carcinomas and facilitate the development of novel therapeutic approaches.

A pharmacological study of celecoxib and gemcitabine in patients with advanced pancreatic cancer.

PURPOSE: To evaluate whether celecoxib alters the conversion of gemcitabine into its active metabolite, difluorodeoxycytidine triphosphate (dFdCTP), in peripheral blood mononuclear cells (PBMCs). METHODS: Patients with advanced pancreatic cancer who had not received chemotherapy and had acceptable organ function were eligible for the study. The initial dose of gemcitabine was 750 mg/m(2) administered intravenously at a rate of 10 mg/m(2)/min on days 1, 8, and 15 every 4 weeks. Celecoxib was administered orally at 400 mg twice a day starting 2 days after the first dose of gemcitabine. Serial blood samples were taken during the first and second gemcitabine infusions and the cellular dFdCTP levels from PBMCs were analyzed. RESULTS: Five patients received gemcitabine at 750 mg/m(2) and six patients received it at 650 mg/m(2). Severe adverse events included neutropenia, thrombocytopenia, enteritis, and gastric perforation. Two patient died early during treatment. Cellular pharmacology studies showed that the conversion of gemcitabine into dFdCTP was not affected by celecoxib. CONCLUSION: Despite the increased clinical toxicities encountered with the combination, celecoxib did not alter the conversion of gemcitabine into its active metabolites in PBMCs. Gemcitabine 650 mg/m(2) infusion over 65 min on days 1, 8, and 15 every 4 weeks in combination with celecoxib at 400 mg twice a day was the dose recommended for further study.

Phase I and pharmacological study of oral 9-aminocamptothecin colloidal dispersion (NSC 603071) in patients with advanced solid tumors.

PURPOSE: 9-Aminocamptothecin colloidal dispersion (9-ACCD; NSC 603071) is a specific inhibitor of topoisomerase I that can be given p.o. This Phase I trial was conducted to determine the toxicity profile, maximal tolerated dose, and pharmacokinetics profile, including bioavailability, of p.o. 9-ACCD in patients with advanced solid tumors. EXPERIMENTAL DESIGN: After receiving one i.v. dose of 9-ACCD, patients were treated with 9-ACCD p.o., starting with a 2-week schedule, to establish the safety. Once safety was established, patients were treated continuously for 4 weeks followed by a rest period of 2 weeks at dosages of 0.2, 0.3, 0.45, 0.56, 0.7, and 0.63 mg/m(2)/day. Serial blood samples were collected for the pharmacokinetics study on day 1 after the i.v. dose and day 2 after p.o. administration. Lactone and total 9-aminocamptothecin were analyzed by high-pressure liquid chromatographic assay. RESULTS: Thirty-two patients were treated on the study. The dose-limiting toxicity was myelosuppression at the dosage of 0.7 mg/m(2)/day. Other toxic effects included nausea, vomiting, fatigue, and transient elevation of the total bilirubin level. The maximal tolerated dose was 0.63 mg/m(2)/day. There was no objective response. The mean terminal half-life of p.o. total 9-ACCD was 1.2 +/- 1.2 h, and the volume of distribution was 17.7 +/- 20.6 l/m(2). The mean bioavailability of total 9-ACCD was 68.1 +/- 36.4%. CONCLUSIONS: Despite good tolerance of p.o. administration, the lack of clinical activity and variable absorption of 9-ACCD suggested that further development might not be warranted.

Celecoxib inhibits angiogenesis by inducing endothelial cell apoptosis in human pancreatic tumor xenografts.

Previous studies suggest that antagonists of cyclooxygenases 1 and 2 (COX-1, -2) inhibit angiogenesis in tumor xenografts, but the molecular mechanisms involved remain unclear. Here we characterized the effects of non-selective (indomethacin) and selective (NS398, celecoxib) cyclooxygenase inhibitors on parameters of angiogenesis in human pancreatic adenocarcinoma cells. COX-1 expression was constitutive in 9/9 pancreatic cancer cell lines, whereas COX-2 and cytosolic phospholipase A2 (cPLA2) expression were observed in 4/9 cell lines (BxPC3, Capan2, Cfpac1, and L3.6 pl). Production of the COX product, prostaglandin E2, correlated with expression of cPLA2 and COX-2 and was blocked by non-steroidal anti-inflammatory drugs (NSAIDs, indomethacin or NS398). In contrast to the findings of others, neither indomethacin nor NS398 affected tumor cell secretion of angiogenic factors (VEGF, bFGF, IL-8) at concentrations that produced maximal inhibition of PGE2 production, and higher concentrations increased angiogenic factor production. We also studied the effects of celecoxib in orthotopic L3.6 pl xenografts. Immunofluorescence analyses revealed high-level expression of COX-2 in endothelial cells in L3.6 pl xenografts that increased following therapy with celecoxib, whereas the tumor cells expressed uniformly low levels of COX-2. Celecoxib did not decrease tumor-associated VEGF levels in orthotopic human L3.6 pl xenografts, but the drug did decrease tumor microvessel density (MVD) and increase apoptosis in tumor-associated endothelial cells in a dose-dependent fashion. Together, our results demonstrate that the anti-angiogeneic effects of NSAIDs in human pancreatic cancer cells are exerted via direct effects on endothelial cells.

Epidermal growth factor receptor-targeted therapy for pancreatic cancer.

Epidermal growth factor receptor (EGFR) plays an important role in tumor development and maintenance. It is a cell surface molecule that mediates signal transduction from the cell surface to cytoplasm. Elevated expression of EGFR or its ligand correlates with worse prognosis in a variety of human cancers. Therefore, blockade of EGFR activity would provide a novel strategy for the treatment of cancer. Two classes of EGFR inhibitors, monoclonal antibodies and tyrosine kinase inhibitors, have been described. The preclinical activity of these EGFR inhibitors and phase I clinical data are summarized in this article. A phase II trial of the EGFR inhibitor IMC-C225 in combination with gemcitabine for patients with advanced pancreatic cancer is discussed.