Search

Purpose: The most common genetic lesions in pancreatic ductal adenocarcinoma (PDAC) have been identified. However, significant gaps still exist in our understanding of how such genetic alterations act in concert to induce PDAC development. In this study, we investigated the mechanism of tumorigenic transformation in the immortalized human pancreatic ductal epithelial (HPDE) cell line by sequentially introducing PDAC signature alterations into this cell line., Experimental Design: The phenotype for stable expression of mutant K-ras, Her2, p16/p14shRNA, and Smad4shRNA in HPDE cells was examined by assays for cell proliferation, migration, invasion, soft agar, and orthotopic tumorigenesis. The mechanisms of tumorigenic transformation were further explored by gene expression profiling and pathway analyses., Results: The transformed cells exhibited enhanced proliferation, migration, and invasion, displayed anchorage-independent growth in soft agar, and grew orthotopic tumors with some histopathologic features of PDAC. We found that Smad4 played key roles in the tumorigenic transformation of HPDE cells. We further found that MDM2 and Bmi-1 were overexpressed in the tumorigenic HPDE cells and that Bmi-1 overexpression was regulated by Smad4. Ingenuity Pathway Analysis software analysis of microarray data revealed that dysregulation of integrin-linked kinase signaling and the cell cycle were the most significant changes involved in tumorigenic transformation. Altogether, this cell culture model closely recapitulated human pancreatic carcinogenesis from gene lesions, activation of specific signaling pathways, and some histopathologic features., Conclusion: The combination of activated K-ras and Her2 with inactivated p16/p14 and Smad4 was sufficient and essential to transform HPDE cells, thus revealing the potential tumorigenic mechanism.

Purpose: Accumulating evidence suggests that metformin has antitumor activity. The aim of this study was to determine whether metformin use has a survival benefit in patients with pancreatic cancer., Experimental Design: We conducted a retrospective study of patients with diabetes and pancreatic cancer treated at The University of Texas MD Anderson Cancer Center (Houston, TX). Information on diabetes history, including treatment modalities and clinical outcome of pancreatic cancer, was collected using personal interviews and medical record review. Survival analysis was carried out using a Kaplan-Meier plot, log-rank test, and Cox proportional hazards regression models., Results: Among the 302 patients identified, there were no significant differences in demographic or major clinical characteristics between the patients who had received metformin (n = 117) and those who had not (n = 185). The 2-year survival rate was 30.1% for the metformin group and 15.4% for the non-metformin group (P = 0.004; χ(2) test). The median overall survival time was 15.2 months for the metformin group, and 11.1 months for the non-metformin group (P = 0.004, log-rank test). Metformin users had a 32% lower risk of death; the HR (95% confidence interval) was 0.68 (0.52-0.89) in a univariate model (P = 0.004), 0.64 (0.48-0.86) after adjusting for other clinical predictors (P = 0.003), and 0.62 (0.44-0.87) after excluding insulin users (P = 0.006). Metformin use was significantly associated with longer survival in patients with nonmetastatic disease only., Conclusions: Our finding that metformin use was associated with improved outcome of patients with diabetes and pancreatic cancer should be confirmed in independent studies. Future research should prospectively evaluate metformin as a supplemental therapy in this population., (©2012 AACR.)

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer death. No effective therapy is currently available for PDAC because of the lack of understanding of the mechanisms leading to its growth and development. Inflammatory cells, particularly mast cells, have been shown to play key roles in some cancers. We carried out this study to test the hypothesis that mast cells in the tumor microenvironment are essential for PDAC tumorigenesis., Experimental Design: The presence of inflammatory cells at various stages of PDAC development was determined in a spontaneous mouse model of PDAC (K-ras(G12V)). The importance of mast cells was determined using orthotopically implanted PDAC cells in mast cell-deficient Kit(w-sh/w-sh) mice and further confirmed by reconstitution of wild-type bone marrow-derived mast cells. Clinical relevance was assessed by correlating the presence of mast cells with clinical outcome in patients with PDAC., Results: In the spontaneous mouse model of PDAC (K-ras(G12V)), there was an early influx of mast cells to the tumor microenvironment. PDAC tumor growth was suppressed in mast cell-deficient Kit(w-sh/w-sh) mice, but aggressive PDAC growth was restored when PDAC cells were injected into mast cell-deficient mice reconstituted with wild-type bone marrow-derived mast cells. Mast cell infiltration into the tumor microenvironment was predictive of poor prognosis in patients with PDAC., Conclusions: Mast cells play an important role in PDAC growth and development in mouse models and are indicative of poor prognosis in humans, which makes them a potential novel therapeutic target.

Purpose: The resistance of tumors to antiangiogenic therapies is becoming increasingly relevant. There are currently no validated predictive biomarkers for selecting which cancer patients will benefit from antiangiogenic therapy. Also lacking are resistance biomarkers that can identify which escape pathways should be targeted after tumors develop resistance to VEGF treatment. Recent studies showed that anti-VEGF treatment can make tumor cells more aggressive and metastatic. However, the mechanisms and mediators of this are unidentified. Therefore, we aimed this study at directly identifying the tumor cell-initiated mechanisms responsible for the resistance of pancreatic cancer to anti-VEGF treatment., Experimental Design: We established and validated two murine models of human pancreatic cancer resistant to the VEGF-specific antibody bevacizumab in vivo. We used a genome-wide analysis to directly identify which tumor-secreted factors were overexpressed by pancreatic cancer cells that were resistant to anti-VEGF treatment., Results: Rather than direct proangiogenic factors, we identified several proinflammatory factors that were expressed at higher levels in cells resistant to anti-VEGF treatment than in treatment-sensitive control cells. These proinflammatory factors acted in a paracrine manner to stimulate the recruitment of CD11b(+) proangiogenic myeloid cells. Also, we found that secreted factors overexpressed by anti-VEGF treatment-resistant pancreatic cancer cells acted in an autocrine manner to induce epithelial-to-mesenchymal transition (EMT) and were thus responsible for increased aggressiveness of bevacizumab-resistant pancreatic tumors., Conclusions: Our results identified proinflammatory factors and EMT markers as potential biomarkers for selecting patients with pancreatic cancer for antiangiogenic therapy., (©2011 AACR.)

Purpose: This study was designed to determine whether DNA methylation biomarkers are associated with recurrence and survival in colon cancer patients., Experimental Design: A retrospective analysis of 82 patients who received curative surgical resection for American Joint Committee on Cancer (AJCC) high-risk stage II or III colon cancer (1999-2007) was conducted. DNA methylation status was quantitatively evaluated by the pyrosequencing method. We preselected three tumor suppressor genes and one locus of interest; CHFR, ID4, RECK, and MINT1. Mean methylation levels of multiple CpG sites in the promoter regions were used for analysis; 15% or more was defined as methylation positive. The association of recurrence-free survival (RFS) and overall survival (OS) with methylation status was analyzed by the log-rank test, Kaplan-Meier method, and Cox proportional hazards model., Results: Methylation levels of ID4, MINT1, and RECK did not correlate with RFS or OS. CHFR was methylation positive in 63% patients. When methylation status was dichotomized (negative or low: <30%, high: ≥30%), patients with CHFR methylation-high (44%) had worse RFS (P = 0.006) and reduced OS (P = 0.069). When stratified by stage, CHFR methylation-high was associated with reduced RFS (P = 0.004) and OS (P = 0.010) in stage III patients. CHFR methylation-high was commonly associated with N2 disease (P = 0.04) and proximal tumors (P = 0.002). Multivariate analysis indicated AJCC T4 disease and CHFR methylation-high (P = 0.001 and P = 0.015, respectively) were independent predictors for recurrence., Conclusions: The extent of CHFR promoter methylation correlates with RFS, indicating it is a promising epigenetic marker for recurrence.

Purpose: Accurate identification of tissue of origin (ToO) for patients with carcinoma of unknown primary (CUP) may help customize therapy to the putative primary and thereby improve the clinical outcome. We prospectively studied the performance of a microRNA-based assay to identify the ToO in CUP patients., Experimental Design: Formalin-fixed paraffin-embedded (FFPE) metastatic tissue from 104 patients was reviewed and 87 of these contained sufficient tumor for testing. The assay quantitates 48 microRNAs and assigns one of 25 tumor diagnoses by using a biologically motivated binary decision tree and a K-nearest neighbors (KNN). The assay predictions were compared with clinicopathologic features and, where suitable, to therapeutic response., Results: Seventy-four of the 87 cases were processed successfully. The assay result was consistent or compatible with the clinicopathologic features in 84% of cases processed successfully (71% of all samples attempted). In 65 patients, pathology and immunohistochemistry (IHC) suggested a diagnosis or (more often) a differential diagnosis. Out of those, the assay was consistent or compatible with the clinicopathologic presentation in 55 (85%) cases. Of the 9 patients with noncontributory IHC, the assay provided a ToO prediction that was compatible with the clinical presentation in 7 cases., Conclusions: In this prospective study, the microRNA diagnosis was compatible with the clinicopathologic picture in the majority of cases. Comparative effectiveness research trials evaluating the added benefit of molecular profiling in appropriate CUP subsets are warranted. MicroRNA profiling may be particularly helpful in patients in whom the IHC profile of the metastasis is nondiagnostic or leaves a large differential diagnosis., (©2011 AACR.)

Pancreatic cancer (PC) is a highly lethal disease with complex etiology involving both environmental and genetic factors. Although cigarette smoking is known to explain 25% of cases, data from recent studies suggest that obesity and long-term type II diabetes are two major modifiable risk factors for PC. Furthermore, obesity and diabetes seem to affect the clinical outcome of patients with PC. Understanding the mechanistic effects of obesity and diabetes on the pancreas may identify new strategies for prevention or therapy. Experimental and epidemiologic evidence suggests that the antidiabetic drug metformin has protective antitumor activity in PC. In addition to insulin resistance and inflammation as mechanisms of carcinogenesis, obesity and diabetes are linked to impairments in endothelial function and coagulation status, which increase the risks of thrombosis and angiogenesis and, in turn, the risk of PC development and progression. The associations of the ABO blood group gene and NR5A2 gene variants with PC discovered by recent genome-wide association studies may link insulin resistance, inflammation, and thrombosis to pancreatic carcinogenesis. These exciting findings open new avenues for understanding the etiology of PC and provide opportunities for developing novel strategies for prevention and treatment of this disease.

Purpose: Nucleolin is a major nucleolar protein that has been shown to be overexpressed in rapidly dividing cells and plays an essential role in cell proliferation and survival. However, the expression and significance of nucleolin in pancreatic ductal adenocarcinoma (PDA) have not been studied., Experimental Design: We used a tissue microarray consisting of 1.0-mm cores of tumor and paired nonneoplastic pancreatic tissue from 69 pancreaticoduodenectomy specimens with stage II PDA. Nucleolin expression was evaluated by immunohistochemistry and scored quantitatively by image analysis. Nucleolin expression was classified as nucleolin-high or nucleolin-low using the median nucleolin labeling index of 3.5% as cutoff. Staining results were correlated with clinicopathologic features and survival., Results: Both PDAs and PDA cell lines showed nucleolar staining for nucleolin. Nucleolin expression was higher in PDAs and PDA cell lines than in nonneoplastic ductal epithelial cells. Among the 69 stage II PDAs, 34 (49%) were nucleolin-high. The median overall survival was 65.2 +/- 16.3 months for patients who had nucleolin-high PDAs compared with 19.5 +/- 3.3 months for patients whose tumors were nucleolin-low (P = 0.03, log-rank method). No significant correlation between nucleolin expression and other clinicopathologic parameters was found. In multivariate analysis, nucleolin expression was a prognostic factor for overall survival in patients with stage II PDA independent of patient's age, gender, tumor size, differentiation, and lymph node status., Conclusions: Nucleolin was overexpressed in PDAs and PDA cell lines. A high level of nucleolar expression of nucleolin was an independent prognostic marker for better survival for patients with stage II PDAs., (Copyright 2010 AACR.)

Purpose: To show whether single nucleotide polymorphisms (SNP) of drug metabolic genes were associated with toxicity of 2',2'-difluoro 2'-deoxycytidine (gemcitabine)-based chemoradiotherapy and overall survival (OS) of patients with pancreatic cancer., Experimental Design: We evaluated 17 SNPs of the CDA, dCK, DCTD, RRM1, hCNT1, hCNT2, hCNT3, and hENT1 genes in 154 patients with potentially resectable pancreatic adenocarcinoma who were enrolled in clinical trials at The University of Texas M.D. Anderson Cancer Center (Houston, TX) from February 1999 to January 2006, with follow-up until April 2009. Patients received neoadjuvant concurrent gemcitabine and radiation therapy with or without gemcitabine-cisplatin induction therapy. The association of genotypes with toxicity or OS was tested, respectively, by logistic regression and Cox regression analysis., Results: None of the 17 SNPs, individually, had a significant association with OS. A combined genotype effect of CDA A-76C, dCK C-1205T, DCTD T-47C, hCNT3 C-69T, hENT1 T-549C, and hENT1 C913T on OS was observed. Patients carrying 0 to 1 (n = 43), 2 to 3 (n = 77), or 4 to 6 (n = 30) variant alleles had median survival time of 31.5, 21.4, and 17.5 months, respectively. The hazard ratio of dying was 1.71 (95% confidence interval, 1.06-2.76) and 3.16 (95% confidence interval, 1.77-5.63) for patients carrying two to three or four to six at-risk genotypes (P = 0.028 and P < 0.001), respectively, after adjusting for clinical predictors. CDA C111T, dCK C-1205T, dCK A9846G, and hCNT3 A25G, individually and jointly, had a significant association with neutropenia toxicity., Conclusions: These observations suggest that polymorphic variations of drug metabolic genes were associated with toxicity of gemcitabine-based therapy and OS of patients with resectable pancreatic cancer.

The identification of high-risk subsets of colon cancers has undergone extensive study over the years, and multidimensional gene signatures have shown promise for colon cancer. However, several hurdles need to be overcome in order to show the benefit of this approach and successfully apply them to the clinic. (Clin Cancer Res 2009;15(24):7451-2).

Purpose: Development of novel agents and drug combinations are urgently needed for treatment of pancreatic cancer. Oxaliplatin belongs to an important class of DNA-damaging organoplatinum agents, useful in pancreatic cancer therapy. However, increased ability of cancer cells to recognize and repair DNA damage enables resistance to these agents. Poly (ADP ribose) polymerase-1 is a sensor of DNA damage with key roles in DNA repair. Here, we report the therapeutic activity of the poly (ADP ribose) polymerase-1 inhibitor BSI-401, as a single agent and in combination with oxaliplatin in orthotopic nude mouse models of pancreatic cancer, and its effect on oxaliplatin-induced acute neurotoxicity., Experimental Design: We determined in vitro the effect of BSI-401 and its synergism with oxaliplatin on the growth of pancreatic cancer cells. Activity of different dosages of parenteral and oral BSI-401, alone and in combination with oxaliplatin, was evaluated in orthotopic nude mouse models with luciferase-expressing pancreatic cancer cells. The effect of BSI-401 in preventing oxaliplatin-induced acute cold allodynia was measured in rats using a temperature-controlled plate., Results: BSI-401 alone and in synergism with oxaliplatin significantly inhibited the growth of pancreatic cancer cells in vitro. In nude mice, i.p. [200 mg/kg once a week (QW) x 4] and oral [400 mg/kg days 1-5 of each week (QD5 + R2) x 4] administration of BSI-401 significantly reduced tumor burden and prolonged survival (46 versus 144 days, P = 0.0018; 73 versus 194 days, P = 0.0017) compared with no treatment. BSI-401 combined with oxaliplatin had potent synergistic antitumor activity (46 versus 132 days, P = 0.0063), and significantly (P = 0.0148) prevented acute oxaliplatin-induced neurotoxicity., Conclusions: BSI-401, alone or in combination with oxaliplatin, is a promising new therapeutic agent that warrants further evaluation for treatment of pancreatic cancer.

Purpose: The current research was undertaken to examine the association between genetic variations in DNA repair and pancreatic cancer risk., Experimental Design: We analyzed 9 single nucleotide polymorphisms of 7 DNA repair genes (LIG3, LIG4, OGG1, ATM, POLB, RAD54L, and RECQL) in 734 patients with pancreatic adenocarcinoma and 780 healthy controls using the Taqman method. Information on cigarette smoking, alcohol consumption, medical history, and other risk factors was collected by personal interview., Results: The homozygous mutant genotype of LIG3 G-39A [odds ratio (OR), 0.23; 95% confidence interval (CI), 0.06-0.82; P = 0.027] and ATM D1853N (OR, 2.55; 95% CI, 1.08-6.00; P = 0.032) was significantly associated with altered risk for pancreatic cancer. A statistically significant interaction of ATM D1853N and LIG4 C54T genotype with diabetes on the risk of pancreatic cancer was also detected. Compared with nondiabetics with the ATM D1853N GG genotype, nondiabetics with the GA/AA, diabetics with the GG, and diabetics with the GA/AA genotypes, respectively, had ORs (95% CI) of 0.96 (0.74-1.24), 1.32 (0.89-1.95), and 3.23 (1.47-7.12; P(interaction) = 0.032, likelihood ratio test). The OR (95% CI) was 0.91 (0.71-1.17), 1.11 (0.73-1.69), and 2.44 (1.34-4.46) for nondiabetics carrying the LIG4 CT/TT genotype, diabetics with the CC genotype, and diabetics carrying the CT/TT genotype, respectively, compared with nondiabetics carrying the CC genotype (P(interaction) = 0.02)., Conclusions: These observations suggest that genetic variations in DNA repair may act alone or in concert with other risk factors on modifying a patient's risk for pancreatic cancer.

Purpose: Mitogen-activated protein 4 kinase 4 (MAP4K4) is a serine/threonine kinase and belongs to the mammalian STE20/MAP4K family. Recent studies have shown that MAP4K4 is overexpressed in many types of human cancer and cancer cell lines. MAP4K4 plays an important role in transformation, invasiveness, adhesion, and cell migration. However, the expression of MAP4K4 and its significance in pancreatic ductal adenocarcinoma (PDA) has not been studied., Experimental Design: We examined the expression of MAP4K4 by immunohistochemistry using tissue microarrays consisting of 66 stage II PDA and their paired benign pancreatic tissue. The staining results were categorized as MAP4K4-H or MAP4K4-L. The results were correlated with clinicopathologic features and patient survival., Results: MAP4K4 was overexpressed (MAP4K4-H) in 30 of 66 (46%) PDAs and was higher than the paired benign pancreatic tissue samples (19%; P=0.002). The median overall and recurrence-free survival for patients with MAP4K4-H PDAs were 19.5 and 9.3 months, respectively, compared with 65.2 and 28.8 months for patients with MAP4K4-L tumor (P=0.02 and 0.0005, log-rank test). MAP4K4 expression was associated with poor overall and recurrence-free survival in univariate analysis (P=0.02 and 0.001). In multivariate analysis, MAP4K4 expression significantly correlated with overall and recurrence-free survival (P=0.025 and 0.004) independent of age, tumor size, differentiation, and stage. MAP4K4 expression was also associated with higher frequency of recurrence/metastasis, larger tumor size, and increased number of positive lymph nodes (P<0.05)., Conclusion: MAP4K4 was overexpressed in about half of PDAs. Overexpression of MAP4K4 was associated with worse prognosis and is a prognostic marker for stage II PDAs.

Purpose: Pancreatic cancer is almost always lethal, and the only U.S. Food and Drug Administration-approved therapies for it, gemcitabine and erlotinib, produce objective responses in <10% of patients. We evaluated the clinical biological effects of curcumin (diferuloylmethane), a plant-derived dietary ingredient with potent nuclear factor-kappaB (NF-kappaB) and tumor inhibitory properties, against advanced pancreatic cancer., Experimental Design: Patients received 8 g curcumin by mouth daily until disease progression, with restaging every 2 months. Serum cytokine levels for interleukin (IL)-6, IL-8, IL-10, and IL-1 receptor antagonists and peripheral blood mononuclear cell expression of NF-kappaB and cyclooxygenase-2 were monitored., Results: Twenty-five patients were enrolled, with 21 evaluable for response. Circulating curcumin was detectable as drug in glucuronide and sulfate conjugate forms, albeit at low steady-state levels, suggesting poor oral bioavailability. Two patients showed clinical biological activity. One had ongoing stable disease for >18 months; interestingly, one additional patient had a brief, but marked, tumor regression (73%) accompanied by significant increases (4- to 35-fold) in serum cytokine levels (IL-6, IL-8, IL-10, and IL-1 receptor antagonists). No toxicities were observed. Curcumin down-regulated expression of NF-kappaB, cyclooxygenase-2, and phosphorylated signal transducer and activator of transcription 3 in peripheral blood mononuclear cells from patients (most of whom had baseline levels considerably higher than those found in healthy volunteers). Whereas there was considerable interpatient variation in plasma curcumin levels, drug levels peaked at 22 to 41 ng/mL and remained relatively constant over the first 4 weeks., Conclusions: Oral curcumin is well tolerated and, despite its limited absorption, has biological activity in some patients with pancreatic cancer.

Purpose: The goals of this study were to determine if single-nucleotide polymorphisms in DNA damage repair genes and cell cycle regulating genes affect clinical response to combined gemcitabine radiation therapy and the overall survival (OS) of patients with pancreatic cancer., Experimental Design: We evaluated six single-nucleotide polymorphisms of the ATM, ATM and Rad3-related (ATR), CHEK1, and CHEK2 genes in 119 patients with potentially resectable pancreatic cancer who were enrolled in clinical trials at The University of Texas M. D. Anderson Cancer Center from February 1999 to January 2006, with follow-up until February 2007. Patients received neoadjuvant concurrent gemcitabine and radiation therapy with or without gemcitabine-cisplatin induction therapy. Genotypes were determined and tested for associations with OS by Kaplan-Meier estimation, the log-rank test, and Cox regression analysis. P values of

Purpose: Aurora-A and p16 play a major role in cell cycle checkpoint regulation. Both of them are important in the maintenance of centrosome duplication. Therefore, we hypothesized that polymorphisms in the two genes may interact or work together to influence the finely tuned mechanisms of cell cycle regulation that these proteins regulate. The purpose of this study was to investigate the association of the Aurora-A (T91A), and p16 (C540G and C580T) polymorphisms with age at diagnosis of pancreatic cancer., Experimental Design: We genotyped 148 Caucasian patients with a diagnosis of pancreatic cancer for the Aurora-A and p16 polymorphisms using pyrosequencing. We tested the association between age at diagnosis and the Aurora-A and p16 genotypes by comparing Kaplan-Meier curves, evaluating the homogeneity of the curves using the log-rank test. We used Cox proportional hazard regression analysis to estimate the association between time to diagnosis and genotype, adjusting for gender., Results: Patients with the Aurora-A polymorphic genotypes had a median age at diagnosis with pancreatic cancer that was 2.8 years earlier than those with the wild-type genotype [log-rank, P=0.015; hazard ratio (HR), 1.55; 95% confidence intervals (95% CI), 1.09-2.20]. There was no significant association between the p16 genotypes and age at diagnosis. However, the Aurora-A and p16 C580T polymorphisms combined had a synergistic effect on age-associated risk for early diagnosis of pancreatic cancer. Compared with patients with wild-type genotypes for both genes, the median age at diagnosis for patients with one or two polymorphic alleles for both genes was 12.6 years earlier (log-rank, P=0.0002; HR, 3.88; 95% CI, 1.94-7.76). No significant associations between the polymorphisms and the cancer metastatic status or survival after diagnosis were found., Conclusions: Our findings suggest that the Aurora-A polymorphism contributes to a significantly earlier age at diagnosis of pancreatic cancer, and that Aurora-A and p16 C580T polymorphisms synergistically contribute to an earlier age at diagnosis of pancreatic cancer.

Purpose: Effective systemic therapy options for carcinoid tumors are lacking. We conducted in vitro studies and a phase II clinical trial to explore the activity of imatinib in carcinoid tumors., Experimental Design: Cells of the human bronchial carcinoid cell line NCI-H727 and the human pancreatic carcinoid cell line BON-1 were treated with increasing concentrations of imatinib using standard procedures to assess in vitro growth-inhibitory activity. A clinical trial using a two-stage phase II design to assess the response rate and safety profile of imatinib at a dose of 400 mg given twice daily in patients with advanced carcinoid tumors was completed., Results: In both cell lines, there was a dose- and time-dependent cytotoxic effect. The clinical trial enrolled 27 evaluable patients. Median duration on trial was 16 weeks. One patient had a partial response, 17 had stable disease, and 9 had progressive disease by the Response Evaluation Criteria in Solid Tumors criteria. Median progression-free survival time was 24 weeks. Median overall survival is 36 months. Seven patients who achieved a biochemical response had a superior progression-free survival time compared with patients without biochemical response (115 weeks compared with 24 weeks; P = 0.003). An increase in plasma basic fibroblast growth factor was associated with a shorter progression-free survival duration (P = 0.02)., Conclusions: Our data suggest that imatinib is active in vitro and has a modest clinical activity in carcinoid patients. Changes in tumor markers may help select patients who are likely to benefit from therapy.

Purpose: To determine whether the synthetic microRNAs (miRNA) could effectively target tumor cells we designed several miRNA complementary to glioma-associated antigen-1 (Gli-1) mRNA and investigated their ability to inhibit tumor cell proliferation. The sonic hedgehog pathway is an early and late mediator of tumorigenesis in epithelial cancers. Activation of sonic hedgehog signaling seems to precede transformation of tissue stem cells to cancerous stem cells, with the Gli-1 transcription factor functioning as a mediator of environmental signals. Inhibiting cancer cell proliferation by targeting the Gli-1 effector pathway is difficult to achieve by chemotherapeutic agents or short interfering RNA., Experimental Design: We hypothesized that targeting the 3'-untranslated region of Gli-1 mRNA would effectively inhibit tumor cell proliferation. To test this hypothesis, we used synthetic miRNAs of our own design and corresponding duplex/small temporal RNAs by introducing three-nucleotide loops in the 3'-untranslated region Gli-1 sequence of high GU content., Results: We found that miRNA (Gli-1-miRNA-3548) and its corresponding duplex (Duplex-3548) significantly inhibited proliferation of Gli-1+ ovarian (SK-OV-3) and pancreatic (MiaPaCa-2) tumor cells. The miRNAs mediated delayed cell division and activation of late apoptosis in MiaPaCa-2 cells. This is the first demonstration of inhibition of pancreatic tumor cell division by designed miRNA., Conclusions: Gli-1 miRNAs should significantly add to the general understanding of the mechanisms of metastasis and contribute toward the design of better treatments for epithelial cancers.

Plasma protein profiling using separations coupled to matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has great potential in translational research; it can be used for biomarker discovery and contribute to disease diagnosis and therapy. Previously reported biomarker searches have been done solely by MS protein profiling followed by bioinformatics analysis of the data. To add to current methods, we tested an alternative strategy for plasma protein profiling using pancreatic cancer as the model. First, offline solid-phase extraction is done with 96-well plates to fractionate and partially purify the proteins. Then, multiple profiling and identification experiments can be conducted on the same protein fractions because only 5% of the fractions are used for MALDI MS profiling. After MALDI MS analysis, the mass spectra are normalized and subjected to a peak detection algorithm. Over three sets of mass spectra acquired using different instrument variables, approximately 400 unique ion signals were detected. Classification schemes employing as many as eight individual peaks were developed using a training set with 123 members (82 cancer patients) and a blinded validation set with 125 members (57 cancer patients). The sensitivity of the study was 88%, but the specificity was significantly lower, 75%. The reason for the low specificity becomes apparent upon protein identification of the ion signals used for the classification. The identifications reveal only common serum proteins and components of the acute phase response, including serum amyloid A, alpha-1-antitrypsin, alpha-1-antichymotrypsin, and inter-alpha-trypsin inhibitor.

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.

Purpose: Pancreatic adenocarcinoma is currently the fourth leading cause of cancer death in the United States, and most pancreatic cancers develop locally advanced disease or metastasis at the time of diagnosis. The mechanisms by which it invades and metastasizes are not known., Experimental Design: To identify the genes involved in pancreatic cancer metastasis, we analyzed the gene expression profiles between highly metastatic Colo357L3.6pl and parental Colo357FG pancreatic cancer cell lines using cDNA microarrays and confirmed differential gene expression by reverse transcription-PCR, Western blotting, and immunologic analysis of 54 samples from pancreatic cancer patients. The correlation with clinical outcome was also examined. The possible signaling pathways involved with tropomyosin-related kinase B (TrkB) were analyzed., Results: Our findings showed that TrkB was overexpressed in the highly metastatic Colo357L3.6pl cells, which correlated with perineural invasion (P = 0.026), positive retroperitoneal margin (P = 0.0005), and shorter latency to development of liver metastasis (Cox proportional hazard ratio, 0.3; 95% confidence interval, 0.1-0.8; P = 0.01) in patient samples. Extracellular signal-regulated kinases 1 and 2 were activated and Elk-1 and AP-1 DNA binding activity was induced in Colo357L3.6pl cells. Furthermore, interleukin 8 and vascular endothelial growth factor were more strongly expressed in Colo357L3.6pl than Colo357FG cells, and these findings were confirmed in Colo357L3.6pl and Colo357FG orthotopic tumors., Conclusion: These results suggest that overexpression of TrkB and activation of mitogen-activated protein kinase and AP-1, which may in turn induce the expression of vascular endothelial growth factor and interleukin 8, may mediate the cardinal clinical features of locally aggressive growth and metastasis of pancreatic cancer. Our results also imply that TrkB receptor may be a novel therapeutic target for pancreatic cancer.

Purpose: In a recent study, we presented preliminary evidence for biological activity in a Phase I dose-finding study (15-600 mg/m(2)) of recombinant human endostatin in patients with refractory solid tumors. Here, we conducted additional biomarker analyses to correlate changes in tumor biology with dose., Experimental Design: Excisional tumor biopsies were obtained at baseline and after 56 days of endostatin therapy. Laser scanning cytometry (LSC) was used to quantify biomarker levels in whole tissue sections. Apoptosis in tumor cells (TCs) and tumor-associated endothelial cells (ECs) was quantified by fluorescent three-color anti-CD31/terminal deoxynucleotidyl transferase-mediated nick end labeling staining. Microvessel densities were measured by LSC-guided vessel contouring. Levels of tumor-associated EC BCL-2 and hypoxia-inducible factor 1alpha were determined by immunofluorescence and LSC quantification. The results, including tumor blood flow measured by positron emission tomography, were analyzed using a quadratic polynomial model., Results: Significant increases in EC death and decreases in tumor microvessel density were observed, with maximal effects of endostatin at a dose of 249 mg/m(2) (95% confidence interval, 159-338) and 257 mg/m(2) (95% confidence interval, 183-331), respectively. In contrast, levels of TC death were uniformly low and did not correlate with endostatin dose. Maximal nuclear hypoxia-inducible factor 1alpha and minimal EC Bcl-2 levels were observed at approximately 250 mg/m(2), although the changes did not reach statistical significance., Conclusions: The data suggest that endostatin had optimal biological activity at doses approximately 250 mg/m(2) in our cohort of patients. Endostatin's failure to induce high levels of TC death may explain its lack of significant clinical activity in this Phase I trial.

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.

Purpose: Multiple chromosome abnormalities, including gain of chromosome20q, have been detected frequently in human pancreatic cancers. Overexpression of the STK15/BTAK/Aurora A gene located on chromosome 20q13, which encodes a centrosome-associated serine/threonine kinase, has been shown to induce chromosomal instability, leading to aneuploidy and cell transformation in multiple in vitro experimental systems. The purpose of this study was to investigate the expression and copy number alteration of STK15 in pancreatic cancer., Experimental Design: STK15 expression at both the mRNA and protein levels together with the copy number of STK15 gene was measured in nine pancreatic carcinoma cell lines: (a) HPAF-II; (b) Aspc-1; (c) Panc-1; (d) Panc-3; (e) Panc-28; (f) Panc-48; (g) HS766T; (h) MIAPaCa-2; and (i) BxPc3. STK15 protein expression was also examined in normal pancreatic tissues and tumors by Western blotting and immunohistochemistry., Results: STK15 was overexpressed in all of the nine cell lines examined, but gene amplification was infrequent. Western Blot analysis of primary tumor tissues revealed 2-10 times overexpression of STK15 protein compared with normal adjacent tissues from pancreatic cancer patients. Concurrent overexpression of cdc20, an STK15-associated protein, and reduced expression of cdc25, a mitosis-activating protein phosphatase, were detected in the same tumor samples. Elevated STK15 protein expression was detected in 22 of 38 tumor sections (58%) from pancreatic cancer patients. The extent of STK15 expression was not significantly correlated with the size, degree of differentiation, and metastasis status of the tumors., Conclusions: These results show that STK15 is overexpressed in pancreatic tumors and carcinoma cell lines and suggest that overexpression of STK15 may play a role in pancreatic carcinogenesis.

Purpose: Localized pancreatic carcinoma is rarely resectable and is resistant to conventional therapies. ONYX-015 (dl1520) is an E1B-55kD gene-deleted replication-selective adenovirus that preferentially replicates in and kills malignant cells. Endoscopic ultrasound (EUS) has the potential to conveniently and accurately deliver local therapy to the pancreas. Therefore, we undertook a trial of the feasibility, tolerability, and efficacy of EUS injection of ONYX-015 into unresectable pancreatic carcinomas., Experimental Design: Twenty-one patients with locally advanced adenocarcinoma of the pancreas or with metastatic disease, but minimal or absent liver metastases, underwent eight sessions of ONYX-015 delivered by EUS injection into the primary pancreatic tumor over 8 weeks. The final four treatments were given in combination with gemcitabine (i.v., 1,000 mg/m(2)). Patients received 2 x 10(10) (n = 3) or 2 x 10(11) (n = 18) virus particles/treatment., Results: After combination therapy, 2 patients had partial regressions of the injected tumor, 2 had minor responses, 6 had stable disease, and 11 had progressive disease or had to go off study because of treatment toxicity. No clinical pancreatitis occurred despite mild, transient elevations in lipase in a minority of patients. Two patients had sepsis before the institution of prophylactic oral antibiotics. Two patients had duodenal perforations from the rigid endoscope tip. No perforations occurred after the protocol was changed to transgastic injections only., Conclusions: This study indicates that ONYX-015 injection via EUS into pancreatic carcinomas by the transgastic route with prophylactic antibiotics is feasible and generally well tolerated either alone or in combination with gemcitabine. Transgastric EUS-guided injection is a new and practical method of delivering biological agents to pancreatic tumors.

Purpose: We seek to elucidate the role of constitutive nuclear factor kappaB (NFkappaB) activity in human pancreatic cancer cells. We have demonstrated that the transcription factor NFkappaB is activated constitutively in human pancreatic adenocarcinoma and human pancreatic cancer cell lines but not in normal pancreatic tissues or in immortalized/nontumorigenic pancreatic epithelial cells, suggesting that NFkappaB plays a critical role in development of pancreatic adenocarcinoma., Experimental Design: By pooling all of the puromycin resistant clones after inhibitor of nuclear factor-kappaB phosphorylation mutant (IkappaBalphaM) retroviral infection, we generated pancreatic tumor cell lines that express a IkappaBalphaM (S32, 36A) that blocks NFkappaB activity. Inhibition of metastatic phenotype was assayed in an orthotopic nude mouse model. NFkappaB activity was determined by electrophoretic mobility shift assay, and the expression of NFkappaB downstream target genes was analyzed by Northern, Western, and immunohistochemical analyses., Results: We showed that inhibiting constitutive NFkappaB activity by expressing IkappaBalphaM suppresses liver metastasis, but not tumorigenesis, from the metastatic human pancreatic tumor cell line AsPc-1 in an orthotopic nude mouse model. Furthermore, inhibiting NFkappaB activation by expressing IkappaBalphaM significantly reduced in vivo expression of a major proangiogenic molecule, vascular endothelial growth factor, and, hence, decreased neoplastic angiogenesis. Inhibiting NFkappaB activation by expressing IkappaBalphaM and using pharmacologic NFkappaB inhibitor PS-341 also significantly reduced cytokine-induced vascular endothelial growth factor and interleukin-8 expression in AsPc-1 pancreatic cancer cells., Conclusion: These results demonstrated that the inhibition of NFkappaB signaling can suppress the angiogenic potential and metastasis of pancreatic cancer, and suggest that the NFkappaB signaling pathway is a potential target for anticancer agents.

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.

Purpose: The tumor suppressor gene Smad4/DPC4, a key transcription factorin transforming growth factor beta (TGF-beta) signaling cascades,is inactivated in 50% of pancreatic adenocarcinomas. We seek to determine the role of Smad4/DPC4 in the suppression of tumor cell growth and in the regulation of TGF-beta-mediated expression of cell-cycle regulatory genes p15(ink4b) and p21(waf1)., Experimental Design: Smad4/DPC4 is overexpressed by adenoviral infection in CFPac-1 pancreatic cancer cells, in which the Smad4/DPC4 is homozygously deleted, and in Capan-1 pancreatic cancer cells, in which Smad4/DPC4 is not expressed. Expression of the TGF-beta downstream target gene p21(waf1), regulation of the p15(ink4b) promoter, anchorage-independent growth, and tumorigenesis were examined., Results: We demonstrate that expression of Smad4/DPC4 in Capan-1 cells reduced anchorage-independent growth by more than 50%, and inhibited xenograft tumor growth. However, overexpression of Smad4/DPC4 did not inhibit CFPac-1 cell growth. Interestingly, Smad4/DPC4 induced expression of p15(ink4b), p21(waf1), and TGF-beta-responsive reporter gene in Capan-1 but not in CFPac-1 cells. Furthermore, we found a previously unidentified Smad4 binding element (SBE) located in the region between -356 and -329 bp of the p15(ink4b) promoter. The p15(ink4b) promoter reporter gene assays revealed that Smad4-dependent transcriptional activation is mediated by this SBE, which indicates that p15(ink4b) is one of the downstream target genes regulated by Smad/DPC4., Conclusion: These results explain the role of Smad4/DPC4 in TGF-beta-mediated inhibition of cell proliferation in vitro and in vivo. Moreover, these results suggest that Smad4/DPC4-mediated tumor suppression and induction of TGF-beta-regulated cell-cycle-inhibitory genes may depend on additional factors that are absent in CFPac-1 cells.

Gemcitabine has modest activity in the treatment of advanced pancreatic cancer and is a potent radiosensitizer. We conducted a Phase I trial to determine the maximum tolerated dose of weekly gemcitabine delivered concurrently with radiation therapy for the treatment of locally advanced adenocarcinoma of the pancreatic head and to assess the treatment-related toxic effects associated with such a regimen. Eighteen patients with pathologically proven, locally advanced adenocarcinoma of the pancreatic head were enrolled in this study. Patients received seven weekly doses of gemcitabine with 3000 cGy of external beam radiation therapy delivered during the first 2 weeks of therapy. Six patients received gemcitabine at 350 mg/m(2)/week, nine at 400 mg/m(2)/week, and three at 500 mg/m(2)/week. Grade 3-4 hematological toxicity was observed in over half the patients treated. Nonhematological toxicities were significant and included fatigue, anorexia, nausea, vomiting, and dehydration. Forty-four % of the patients required admission to the hospital for management of nausea/vomiting and dehydration. The risk of hospitalization appeared to be dose-related; all of the three patients treated at 500 mg/m(2)/week required hospital admission during treatment. Seventeen patients were evaluated for response, and eight patients (47%) had evidence of a local anticancer effect. Four of these eight patients (24%) had a partial response to therapy. The median survival for the entire group was 6 months. The 1-year survival rate for patients with an objective response to therapy was 66%. The clinical responses observed in this group of patients suggest gemcitabine is a clinically relevant radiosensitizer in patients with pancreatic adenocarcinoma. However, the toxic effects are significant, suggesting that until dose and scheduling issues are explored further, concomitant administration of gemcitabine and radiation therapy should still be considered investigational.

CD26 is a M(r) 110,000 surface glycoprotein with diverse functional properties, including having a potentially significant role in tumor development, and antibodies to CD26 mediate pleomorphic cellular functions. In this report, we show that binding of soluble anti-CD26 monoclonal Ab 1F7 inhibits the growth of the human CD30+ anaplastic large cell T-cell lymphoma cell line Karpas 299 in both in vitro and in vivo experiments. In vitro experiments show that 1F7 induces cell cycle arrest at the G1-S checkpoint, associated with enhanced p21 expression that is dependent on de novo protein synthesis. Furthermore, experiments with a severe combined immunodeficient mouse tumor model demonstrate that 1F7 treatment significantly enhances survival of tumor-bearing mice by inhibiting tumor formation. Our data therefore suggest that anti-CD26 treatment may have potential clinical use for CD26+ hematological malignancies.

Both epidermal growth factor receptor (EGF-R) signaling mechanisms and angiogenesis have been evaluated as independent targets for therapy of human pancreatic carcinoma, but a link between the two processes has been identified only recently. This study evaluated whether EGF-R blockade therapy with anti-EGF-R antibody C225 inhibits pancreatic carcinoma growth and metastasis in an orthotopic nude mouse model via tumor-mediated angiogenesis and whether gemcitabine potentiates this effect. In vitro treatment of human pancreatic carcinoma L3.6pl cells with C225 inhibited EGF-R autophosphorylation, producing a maximum of 20% cytostasis. Treatment with C225 plus gemcitabine resulted in additive cytotoxic effects that increased with increasing gemcitabine concentrations. Dose-dependent decreases in expression of the angiogenic factors vascular endothelial growth factor and interleukin 8 (but not basic fibroblast growth factor) were observed in the C225-treated cells (mRNA and protein levels). In L3.6pl tumors established in the pancreas of nude mice, systemic therapy with C225 alone and C225 in combination with gemcitabine resulted in growth inhibition, tumor regression, and abrogation of metastasis; median tumor volume was reduced from 538 to 0.3 and to 0 mm3, respectively. Gemcitabine treatment alone reduced median tumor volume from 538 to 152 mm3. Liver metastases were present in 50% of the controls, 30% of the gemcitabine-treated animals, and 20% of C225-treated animals. No macroscopically visible liver metastases were observed in the combination treatment group. As early as 11 days after C225 treatment, the median percentage of proliferating cell nuclear antigen-positive cells was substantially reduced compared with gemcitabine treatment alone (26% versus 73%, respectively) versus controls (92%), correlating with in vivo blockade of EGF-R activation. Similarly after 11 days treatment, production of vascular endothelial growth factor and interleukin 8 was significantly lower in C225 and C225 plus gemcitabine-treated tumors versus gemcitabine-treated and control tumors. Significant differences in microvessel density were observed 18 days after C225 or combination treatments (but not gemcitabine alone) in direct correlation with the difference in percentage of apoptotic endothelial cells, as visualized by double immunofluorescence microscopy. These experiments indicate that therapeutic strategies targeting EGF-R have a significant antitumor effect on human L3.6pl pancreatic carcinoma growing in nude mice which is mediated in part by inhibition of tumor-induced angiogenesis, leading to tumor cell apoptosis and regression. Furthermore, this effect is potentiated in combination with gemcitabine.

Dolastatin (DOLA)-10 is a pentapeptide isolated from the mollusc Dolabella auricularia with clinically promising antitumor activity documented in various in vitro and in vivo tumor models. The objectives of this Phase I study were to determine the maximum tolerated dose, evaluate toxic effects, and document any antitumor activity of this novel agent. Using an electrospray ionization mass spectroscopy system, we also characterized the clinical pharmacokinetics, pharmacodynamics, and metabolism of DOLA-10. The maximum tolerated dose was reached at 300 microg/m2. Granulocytopenia, the dose-limiting toxicity, was documented in 33% of the patients treated at that dose level. There were no episodes of thrombocytopenia or severe anemia (Hgb < 8), and no major nonhematological toxicity was observed. Stabilization of tumor growth was observed in four patients, but no objective responses were seen. Whereas a two-compartment model described the DOLA-10 plasma concentration-time data reasonably well, a three-compartment model consistently performed better. After a rapid distribution phase, DOLA-10 plasma levels declined with mean beta and gamma half-lives of 0.99 and 18.9 h, respectively. Significant interpatient and intrapatient variability in DOLA-10 plasma clearances was observed. The mean area under the concentration-time curve increased proportionally as the dose was escalated, but there was significant overlap between dose levels. The area under the concentration-time curve and the percentage of decline in neutrophils were correlated. A single DOLA-10 metabolite was detected in five patients. Unlike the in vitro studies of DOLA-10, the principal metabolite detected was an N-demethyl derivative, confirmed by mass spectroscopy. In all five subjects, the concentration of this metabolite never exceeded 2% of the simultaneously measured parent drug concentration. The available preclinical, pharmacological, and clinical data suggest that further study of escalated DOLA-10 dosing with cytokine support is warranted.

The clinical features and survival times of patients with unknown primary carcinoma (UPC) are heterogeneous. Therefore, the goals of this study were to apply a novel analytical method to UPC patients to: (a) identify novel prognostic factors; (b) explore the interactions between clinical variables and their impact on survival; and (c) illustrate explicitly how the covariates interact. The 1000 patients analyzed were referred to the University of Texas M. D. Anderson Cancer Center from January 1, 1987 through November 30, 1994. Clinical data from these patients were entered into a computerized database for storage, retrieval, and analysis. Multivariate analyses of survival were performed using recursive partitioning referred to as classification and regression tree (CART) analysis. The median survival for all 1000 consecutive UPC patients was 11 months. CART was performed with an initial split on liver involvement, and 10 terminal subgroups were formed. Median survival of the 10 subgroups ranged from 40 months (95% confidence interval, 22-66 months) for UPC patients with one or two metastatic organ sites, with nonadenocarcinoma histology, and without liver, bone, adrenal, or pleural metastases to 5 months (95% confidence interval, 4-7 months) in UPC patients with liver metastases, tumor histologies other than neuroendocrine carcinoma, age >61.5 years, and a small subgroup of patients with adrenal metastases. Two additional trees were also explored. These analyses demonstrated that important prognostic variables were consistently applied by the CART program and effectively segregated patients into groups with similar clinical features and survival. CART also identified previously unappreciated patient subsets and is a useful method for dissecting complex clinical situations and identifying homogeneous patient populations for future clinical trials.

The role and regulation of interleukin 8 (IL-8) in the growth and metastasis of SG, FG, and L3.3 variants derived from COLO 357 human pancreatic cancer cells were determined. After orthotopic implantation in the pancreas of nude mice, SG cells produced the smallest tumors, whereas L3.3 cells produced the largest tumors. SG cells produced no liver metastasis, whereas FG cells produced numerous liver metastases, and L3.3 cells produced more and larger liver metastases. In vitro analysis of IL-8 expression indicated that SG cells expressed the lowest level of IL-8 gene expression as determined by both Northern blot analysis and ELISA, whereas L3.3 cells expressed the highest level of IL-8. Immunohistochemical analysis of tumor lesions indicated that IL-8 overexpression was predominant in the regions surrounding necrotic areas, where cells were exposed to low oxygen tension (hypoxia) and acidic pH. In vitro treatment of FG tumor cells with hypoxia or acidosis led to an increased expression of IL-8. To directly determine the role of IL-8 in the growth and metastasis of pancreatic cancer, FG cells were transfected with IL-8 sense or antisense oligonucleotide expression vectors. The neo-resistance gene-transfected FG cells were used as controls. Decreased IL-8 expression after transfection with IL-8 antisense oligonucleotide expression vector retarded the growth of FG cells in mice after intrapancreatic implantation, which correlated with decreased tumor angiogenesis. Our data demonstrated that hypoxia and acidosis contribute to the overexpression of IL-8, which in turn plays an important role in tumor angiogenesis and contributes significantly to the aggressive biology of human pancreatic cancer.

Pancreatic adenocarcinoma is a leading cause of adult cancer mortality in the United States. Recent studies have revealed that point mutation of the K-ras oncogene is a common event in pancreatic cancer, and oncogenesis mediated by Ras may also involve activation of Rel/nuclear factor (NF)-kappa B transcription factors. Furthermore, the c-rel member of Rel/NF-kappa B transcription factor family was first identified as a cellular homologue of the v-rel oncogene, suggesting that other members of the Rel/NF-kappa B family are potentially oncogenes. We therefore investigated the possibility that Rel/NF-kappa B transcription factors are activated in pancreatic cancer. Immunohistochemical analysis, Western blot and Northern blot analysis, electrophoretic mobility shift assays, and chloramphenicol acetyltransferase assays were performed to determine RelA activity in human pancreatic adenocarcinomas and normal tissues and nontumorigenic or tumorigenic cell lines. RelA, the p65 subunit of NF-kappa B, was constitutively activated in approximately 67% (16 of 24) of pancreatic adenocarcinomas but not in normal pancreatic tissues. Constitutive RelA activity was also detected in 9 of 11 human pancreatic tumor cell lines but not in nontumorigenic Syrian golden hamster cell lines. I kappa B alpha, a previously identified NF-kappa B-inducible gene, was overexpressed in human pancreatic tumor tissues and cell lines, and RelA activation could be inhibited by curcumin and dominant-negative mutants of I kappa B alpha, raf, and MEKK1. This is the first report demonstrating constitutive activation of RelA in nonlymphoid human cancer. These data are consistent with the possibility that RelA is constitutively activated by the upstream signaling pathway involving Ras and mitogen-activated protein kinases in pancreatic tumor cells. Constitutive RelA activity may play a key role in pancreatic tumorigenesis through activation of its downstream target genes.

We examined the expression level of several genes that regulate distinct steps of metastasis in formalin-fixed, paraffin-embedded, archival specimens of primary human pancreatic carcinomas from patients undergoing curative surgery. The expression of epidermal growth factor receptor, E-cadherin, type IV collagenase [matrix metalloproteinase (MMP) 2 and MMP-9), basic fibroblast growth factor, vascular endothelial growth factor/vascular permeability factor, and interleukin 8 was examined by a colorimetric in situ mRNA hybridization technique. Down-regulation of E-cadherin and up-regulation of type IV collagenase (MMP-9 and MMP-2) at the periphery of the neoplasms (P = 0.0167, 0.0102, and 0.0349, respectively) had significant prognostic value. The ratio of type IV collagenase expression (mean of the expression of MMP-2 and MMP-9) to E-cadherin expression (MMP:E-cadherin ratio) at the periphery of the tumors was significantly higher in patients with recurrent disease (4.7 +/- 2.1) than in patients who were disease free (2.3 +/- 1.7; P = 0.0008). Death from pancreatic cancer was significantly associated with a high MMP:E-cadherin ratio (>3.0) by overall survival analysis (P < 0.0002), whereas a low MMP:E-cadherin ratio (<3.0) was found in seven of eight patients alive 28-64 months after surgery. Multivariate analysis of overall survival showed that the MMP:E-cadherin ratio was a significant independent prognostic factor, whereas stage, nodal metastasis, and histological type were not. These data show that multiparametric analysis for several metastasis-related genes may allow physicians to assess the metastatic potential and hence predict the clinical outcome of individual patients with resectable pancreatic carcinoma.

Epithelial ovarian cancer is a major cause of cancer-related mortality in women, making the search for new treatment modalities essential. Sodium phenylacetate (NaPa), a phenylalanine derivative, has been shown to induce cytostasis and differentiation by inhibiting protein isoprenylation. Similar effects have been observed with phenylbutyrate, a phenylacetate congener. We examined in parallel the growth inhibitory activity against human ovarian carcinoma cell lines of phenylacetate, phenylbutyric acid (PB), and certain related compounds, and comparisons were made with lovastatin. On a molar basis, hydroxykynurenine and kynurenine showed the highest activity followed by PB and NaPa. Ovarian carcinoma cell lines were also sensitive to lovastatin in micromolar concentrations. Additive effects were observed when PB was combined with cisplatin or when NaPa or PB were combined with lovastatin. NaPa and PB, but not kynurenine, inhibited incorporation of [3H]mevalonate into ovarian carcinoma cells. An immune modulatory role might also be suggested for PB because it resulted in increased ovarian tumor cell expression of human leukocyte antigen class I and the cluster of differentiation molecule CD58, whereas transforming growth factor-beta2 expression was decreased. Phenylbutyrate, which is the ester form of PB, has shown acceptable pharmacological properties and clinical responses in patients with other malignancies, and might be considered for evaluation in ovarian cancer.

Although the radiosensitizing potential of paclitaxel has been investigated extensively in cancer treatment, a sister taxane, docetaxel, has been studied rarely. We investigated the ability of docetaxel to enhance in vivo tumor radioresponse and influence radiation injury to normal tissue. In addition, mitotic arrest and apoptosis in tumors and normal tissues were assessed after docetaxel administration to determine whether these cellular effects underly its radio-modifying action. Mice bearing in their legs 8-mm isotransplants of a murine mammary carcinoma, designated MCA-4, were treated with 33 mg/kg docetaxel i.v., 9-21 Gy single-dose local tumor irradiation, or both (in which case radiation was given 9 or 48 h after docetaxel). Tumor growth delay was the end point of the treatments. Mitotic arrest and apoptosis were assayed 1-72 h after treatment with docetaxel. Normal tissue radioresponse was determined using jejunal crypt cell survival 3.5 days after mice were exposed to 9.2-14.8 Gy single-dose, total-body irradiation; the mice were treated with 33 mg/kg docetaxel i.v. 3, 9, or 48 h before irradiation. Docetaxel was assessed for its ability to induce mitotic arrest and apoptosis in jejunum 1-72 h after treatment. Docetaxel induced both mitotic arrest and apoptosis in both tumor and jejunum. Mitotic arrest preceded apoptosis and peaked in the tumor at 9-12 h after treatment; it peaked at 3 h in jejunum. Docetaxel enhanced tumor radioresponse by a factor of 1.45 when the drug was given 9 h before radiation and 2.33 when it was given 48 h before. In contrast, it only slightly enhanced radiation-induced damage of the jejunum and only when given 3 or 9 h before irradiation. Thus, docetaxel given within 2 days before irradiation acted as a potent enhancer of tumor radioresponse and increased the therapeutic gain of irradiation.

The bcl-2 proto-oncogene and the p53 tumor suppressor gene are important determinants of tumor cell susceptibility to apoptosis. bcl-2 and mutant p53 proteins inhibit apoptosis in vitro and can provide prognostic information in certain tumor types. We analyzed bcl-2 and p53 expression in archival pancreatic (n = 35) and ampullary (n = 6) adenocarcinomas, resected for cure, and their relationship to overall survival. Patients were treated with 5-fluorouracil and irradiation either pre- (n = 21) or postoperatively (n = 15); 5 patients received surgery alone. Using specific monoclonal antibodies, cytoplasmic bcl-2 and nuclear p53 proteins were detected in 22 of 40 (55%) and 20 of 37 (54%) tumors, respectively. No relationship was found between bcl-2 and p53 expression. Neither bcl-2 nor p53 correlated with histological response to preoperative chemoradiation. Lymph node involvement predicted poor overall survival (P = 0.02). A trend toward improved survival was seen in well-differentiated (P = 0.08) tumors and in those with increased bcl-2 expression (P = 0.06). p53 expression was not related to clinical outcome. In a multivariate analysis, nodal status was the single most important predictor of overall survival. Of note, the combined variable of bcl-2 expression and histological grade was a stronger prognostic variable than nodal status alone. Unlike nodal status, these features can potentially be evaluated in preoperative biopsy specimens.

Topotecan, a semisynthetic water-soluble analogue of camptothecin, inhibits human topoisomerase I (topo I). We performed a Phase I clinical and plasma pharmacological study of topotecan administered by 24-h continuous infusion without and with granulocyte colony-stimulating factor (G-CSF). We also measured topo I-DNA complexes in peripheral blood mononuclear cells (PBMCs) in an attempt to correlate formation of topo I-DNA complexes in patients treated with topotecan with toxicity and/or response. One hundred four courses of topotecan at doses of 2.5-15.0 mg/m2 were administered to 44 patients with solid tumors. The maximum tolerated dose without G-CSF was 10.0 mg/m2; granulocytopenia was the dose-limiting toxic effect. The maximum tolerated dose could not be increased with G-CSF because of severe thrombocytopenia. Plasma pharmacology was obtained in 11 patients treated at 12.5 mg/m2 and 15.0 mg/m2. The topotecan lactone end-infusion plasma levels correlated strongly with the area under the curve. Lactone elimination was biexponential with a mean t1/2alpha of 28 min and a t1/2beta of 3.8 h at 12.5 mg/m2. Topo I-DNA complexes were measured before and after treatment in PBMCs from seven patients. Pretopotecan topo I-DNA complexes were available on two additional patients treated at 15 mg/m2. The mean increase in topo I-DNA complexes at the end of the topotecan infusion was 1.25 times the pretreatment value. There was a statistically significant relationship (P = 0.02) between lack of disease progression and the level of topo I-DNA complexes measured in PBMCs before therapy. For Phase II studies of minimally treated adults with solid tumors, the recommended topotecan starting dose administered by 24-h continuous infusion is 10 mg/m2 without G-CSF.