Showing papers in "Clinical Cancer Research in 1999"

Phase I Clinical and Pharmacokinetic Study of PK1 [N-(2-Hydroxypropyl)methacrylamide Copolymer Doxorubicin]: First Member of a New Class of Chemotherapeutic Agents—Drug-Polymer Conjugates

Abstract: PK1 comprises doxorubicin covalently bound to N -(2-hydroxypropyl)methacrylamide copolymer by a peptidyl linker. Following cellular uptake via pinocytosis, the linker is cleaved by lysosomal enzymes, allowing intratumoral drug release. Radically altered plasma and tumor pharmacokinetics, compared to free doxorubicin, and significant activity in animal tumors have been demonstrated preclinically. We aimed to determine the maximum tolerated dose, toxicity profile, and pharmacokinetics of PK1 as an i.v. infusion every 3 weeks to patients with refractory or resistant cancers. Altogether, 100 cycles were administered (range, 20–320 mg/m 2 doxorubicin-equivalent) to 36 patients (20 males and 16 females) with a mean age of 58.3 years (age range, 34–72 years). The maximum tolerated dose was 320 mg/m 2 , and the dose-limiting toxicities were febrile neutropenia and mucositis. No congestive cardiac failure was seen despite individual cumulative doses up to 1680 mg/m 2 . Other anthracycline-like toxicities were attenuated. Pharmacokinetically, PK1 has a distribution t 1/2 of 1.8 h and an elimination t 1/2 averaging 93 h. 131 I-labeled PK1 imaging suggests PK1 is taken up by some tumors. Responses (two partial and two minor responses) were seen in four patients with NSCLC, colorectal cancer, and anthracycline-resistant breast cancer. PK1 demonstrated antitumor activity in refractory cancers, no polymer-related toxicity, and proof of principle that polymer-drug conjugation decreases doxorubicin dose-limiting toxicities. The recommended Phase II dose is 280 mg/m 2 every 3 weeks. Studies are planned in colorectal, NSCLC, and breast cancer patients.

The Nuclear Factor-κB RelA Transcription Factor Is Constitutively Activated in Human Pancreatic Adenocarcinoma Cells

Abstract: 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.

Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in nude mice.

Abstract: Epidermal growth factor receptor (EGFR) regulates the growth and progression of human transitional cell carcinoma (TCC) of the bladder. We have shown that therapy targeting EGFR inhibited the growth of human TCC established orthotopically in nude mice. The purpose of this study was to evaluate whether EGFR-directed therapy affects angiogenesis associated with the growth and metastasis of human TCC. We determined the cytostatic effect and the effect on production of angiogenic factors after in vitro treatment of the human TCC cell line 253J B-V with MAb C225, a chimerized monoclonal anti-EGFR antibody. The 253J B-V cells were implanted orthotopically into athymic nude mice, and established tumors (4 weeks) were treated with i.p. MAb C225. Expression of the angiogenic factors vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) was evaluated by immunohistochemistry and in situ mRNA hybridization analyses and correlated with microvessel density evaluated after immunohistochemical staining with anti-CD31. In vitro treatment with MAb C225 inhibited mRNA and protein production of VEGF, IL-8, and bFGF by 253J B-V cells in a dose-dependent manner. MAb C225 therapy of nude mice with established TCCs growing orthotopically resulted in inhibition of growth and metastasis compared with controls (P <0.0005). VEGF, IL-8, and bFGF expression was significantly lower in treated tumors than in controls. The down-regulation of these angiogenic factors preceded the involution of blood vessels. These studies indicate that therapy with anti-EGFR MAb C225 has a significant antitumor effect mediated, in part, by inhibition of angiogenesis.

The proteasome inhibitor PS-341 in cancer therapy.

Abstract: The anticancer activity of the boronic acid dipeptide proteasome inhibitor PS-341 was examined in vitro and in vivo. PS-341 was a potent cytotoxic agent toward MCF-7 human breast carcinoma cells in culture, producing an IC90 of 0.05 microM on 24 h of exposure to the drug. In the EMT-6 tumor cell survival assay, PS-341 was equally cytotoxic administered p.o. or by i.p. injection up to a dose of 2 mg/kg. PS-341 was also toxic to the bone marrow colony-forming unit-granulocyte macrophage. PS-341 increased the tumor cell killing of radiation therapy, cyclophosphamide, and cisplatin in the EMT-6/Parent tumor, but was not able to overcome the in vivo resistance of the EMT-6/CTX and EMT-6/CDDP tumors. In the tumor growth delay assay, PS-341 administered p.o. had antitumor activity against the Lewis lung carcinoma, both primary and metastatic disease. In combination, regimens with 5-fluorouracil, cisplatin, Taxol and adriamycin, PS-341 seemed to produce primarily additive tumor growth delays against the s.c. tumor and was highly effective against disease metastatic to the lungs. The proteasome is an interesting new target for cancer therapy, and the proteasome inhibitor PS-341 warrants continued investigation in cancer therapy.

Expression of Proinflammatory and Proangiogenic Cytokines in Patients with Head and Neck Cancer

Abstract: Altered immune, inflammatory, and angiogenesis responses are observed in patients with head and neck squamous cell carcinoma (HNSCC), and many of these responses have been linked with aggressive malignant behavior and a decrease in prognosis. In this study, we examined the hypothesis that HNSCC cells produce cytokines that regulate immune, inflammatory, and angiogenesis responses. We identified important regulatory cytokines in supernatants of well-defined and freshly cultured HNSCC cell lines by ELISA and determined whether these cytokines are detected in tumor cell lines and tissue specimens by immunohistochemistry. The serum concentration of the cytokines and cytokine-dependent acute phase inflammatory responses (i.e., fibrinogen, C-reactive protein, and erythrocyte sedimentation rate) from patients with HNSCC was determined, and the potential relationship of serum cytokine levels to tumor volume was analyzed. Cytokines interleukin (IL)-1alpha, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor were detected in similar concentration ranges in the supernatants of a panel of established University of Michigan squamous cell carcinoma (UM-SCC) cell lines and supernatants of freshly isolated primary HNSCC cultures. Evidence for the expression of IL-1alpha, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, and VEGF in HNSCC cells within tumor specimens in situ was obtained by immunohistochemistry. In a prospective comparison of the cytokine level and cytokine-inducible acute-phase proteins in serum, we report that cytokines IL-6, IL-8, and VEGF were detected at higher concentrations in the serum of patients with HNSCC compared with patients with laryngeal papilloma or age-matched control subjects (at P < 0.05). The serum concentrations of IL-8 and VEGF were found to be weakly correlated with large primary tumor volume (R2 = 0.2 and 0.4, respectively). Elevated IL-1- and IL-6-inducible acute-phase responses were also detected in cancer patients but not in patients with papilloma or control subjects (at P < 0.05). We therefore conclude that cytokines important in proinflammatory and proangiogenic responses are detectable in cell lines, tissue specimens, and serum from patients with HNSCC. These cytokines may increase the pathogenicity of HNSCC and prove useful as biomarkers or targets for therapy.

A randomized controlled trial comparing intrathecal sustained-release cytarabine (DepoCyt) to intrathecal methotrexate in patients with neoplastic meningitis from solid tumors.

Abstract: Standard treatment for neoplastic meningitis requires frequent intrathecal (IT) injections of chemotherapy and is only modestly effective. DepoCyt is a sustained-release formulation of cytarabine that maintains cytotoxic concentrations of the drug in the cerebrospinal fluid (CSF) for more than 14 days after a single 50-mg injection. We conducted a randomized, controlled trial of DepoCyt versus methotrexate in patients with solid tumor neoplastic meningitis. Sixty-one patients with histologically proven cancer and positive CSF cytologies were randomized to receive IT DepoCyt (31 patients) or IT methotrexate (30 patients). Patients received up to six 50-mg doses of DepoCyt or up to sixteen 10-mg doses of methotrexate over 3 months. Treatment arms were well balanced with respect to demographic and disease-related characteristics. Responses occurred in 26% of DepoCyt-treated and 20% of methotrexate-treated patients (P = 0.76). Median survival was 105 days in the DepoCyt arm and 78 days in the methotrexate arm (log-rank P = 0.15). The DepoCyt group experienced a greater median time to neurological progression (58 versus 30 days; log-rank P = 0.007) and longer neoplastic meningitis-specific survival (log-rank P = 0.074; median meningitis-specific survival, 343 versus 98 days). Factors predictive of longer progression-free survival included absence of visible central nervous system disease on neuroimaging studies (P<0.001), longer pretreatment duration of CSF disease (P<0.001), history of intraparenchymal tumor (P<0.001), and treatment with DepoCyt (P = 0.002). The frequency and grade of adverse events were comparable between treatment arms. In patients with solid tumor neoplastic meningitis, DepoCyt produced a response rate comparable to that of methotrexate and significantly increased the time to neurological progression while offering the benefit of a less demanding dose schedule.

Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function

Abstract: Inadequate function of dendritic cells (DCs) in tumor-bearing hosts is one mechanism of tumor escape from immune system control and may compromise the efficacy of cancer immunotherapy. Vascular endothelial growth factor (VEGF), produced by most tumors, not only plays an important role in tumor angiogenesis but also can inhibit the maturation of DCs from hematopoietic progenitors. Here, we investigate a novel combination of antiangiogenic and immunotherapy based on this dual role of VEGF. Two s.c. mouse tumor models were used: D459 cells, expressing mutant human p53; and MethA sarcoma with point mutations in the endogenous murine p53 gene. Therapy with anti-mouse VEGF antibody (10 microg i.p. twice a week over 4 weeks) was initiated when tumors became palpable. Treatment of established tumors with anti-VEGF antibody alone did not affect the rate of tumor growth. However, anti-VEGF antibody significantly improved the number and function of lymph node and spleen DCs in these tumor-bearing animals. To investigate the possible effects of this antibody on the immunotherapy of established tumors, tumor-bearing mice were immunized with DCs pulsed with the corresponding mutation-specific p53 peptides, together with injections of anti-VEGF antibody. Therapy with peptide-pulsed DCs alone resulted in considerable slowing of tumor growth but only during the period of treatment, and tumor growth resumed after the end of the therapy. Combined treatment with peptide-pulsed DCs and anti-VEGF antibody resulted in a prolonged and much more pronounced antitumor effect. This effect was associated with the induction of significant anti-p53 CTL responses only in this group of mice. These data suggest that inhibition of VEGF may be a valuable adjuvant in the immunotherapy of cancer.

Prostate-specific membrane antigen is produced in tumor-associated neovasculature.

Abstract: Prostate-specific membrane antigen (PSMA), a type II transmembrane protein, was originally thought to be strictly expressed in prostatic tissue, but recent studies have demonstrated PSMA protein expression in nonprostatic tumor neovasculature as well. Using immunohistochemistry, reverse transcription-PCR assays, and in situ hybridization, we have demonstrated PSMA mRNA transcripts and protein expression in the endothelium of tumor-associated neovasculature of multiple nonprostatic solid malignancies. In addition, we found no PSMA mRNA or protein expression in the vascular endothelial cells of corresponding benign tissue examples. Our findings expand the possible therapeutic role of PSMA and establish it as a unique biomarker specifically produced and expressed by tumor-associated neovasculature but not produced or expressed by normal vessels.

Tissue Microarrays for Gene Amplification Surveys in Many Different Tumor Types

Abstract: Gene amplifications are common in many different tumor types and may confer diagnostic, prognostic, or therapeutic information for patient management. Tedious experiments are often required to determine which tumor types have amplifications of a specific oncogene. To facilitate rapid screening for molecular alterations in many different malignancies, a tissue microarray consisting of samples from 17 different tumor types was generated. Altogether, 397 individual tumors were arrayed in a single paraffin block. To determine whether results from the literature can be reproduced on minute tissue samples (diameter, 0.6 mm), amplification of three extensively studied oncogenes (CCND1, CMYC, and ERBB2) was analyzed in three fluorescence in situ hybridization experiments from consecutive sections cut from the tissue microarray. Amplification of CCND1 was found in breast, lung, head and neck, and bladder cancer, as well as in melanoma. ERBB2 was amplified in bladder, breast, colon, stomach, testis, and lung cancer. CMYC was amplified in breast, colon, kidney, lung, ovary, bladder, head and neck, and endometrial cancer. These results confirm and even extend existing data in the literature on such amplifications. In summary, we applied three fluorescence in situ hybridization experiments to analyze amplifications of three oncogenes in three x 397 tumors within a week. This demonstrates the power of using minute arrayed tissue specimens for tumor screening.

Vascular Stroma Formation in Carcinoma in Situ, Invasive Carcinoma, and Metastatic Carcinoma of the Breast

Abstract: The generation of vascular stroma is essential for solid tumor growth and involves stimulatory and inhibiting factors as well as stromal components that regulate functions such as cellular adhesion, migration, and gene expression. In an effort to obtain a more integrated understanding of vascular stroma formation in breast carcinoma, we examined expression of the angiogenic factor vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF); the VPF/VEGF receptors flt-1 and KDR; thrombospondin-1, which has been reported to inhibit angiogenesis; and the stromal components collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin by mRNA in situ hybridization on frozen sections of 113 blocks of breast tissue from 68 patients including 28 sections of breast tissue without malignancy, 18 with in situ carcinomas, 56 with invasive carcinomas, and 8 with metastatic carcinomas. A characteristic expression profile emerged that was remarkably similar in invasive carcinoma, carcinoma in situ, and metastatic carcinoma, with the following characteristics: strong tumor cell expression of VPF/VEGF; strong endothelial cell expression of VPF/VEGF receptors; strong expression of thrombospondin-1 by stromal cells and occasionally by tumor cells; and strong stromal cell expression of collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin. The formation of vascular stroma preceded invasion, raising the possibility that tumor cells invade not into normal breast stroma but rather into a richly vascular stroma that they have induced. Similarly, tumor cells at sites of metastasis appear to induce the vascular stroma in which they grow. We conclude that a distinct pattern of mRNA expression characterizes the generation of vascular stroma in breast cancer and that the formation of vascular stroma may play a role not only in growth of the primary tumor but also in invasion and metastasis.

Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based vaccine.

Abstract: HER-2/neu is a "self" tumor antigen that is overexpressed in 15-30% of human adenocarcinomas. Vaccine strategies directed against HER-2/neu and other self tumor antigens require development of methods to overcome immune tolerance to self-proteins. In rats, rat neu peptide vaccines have been shown to be an effective way of circumventing tolerance to rat neu protein and generating rat neu-specific immunity. The present report validates that a similar peptide-based vaccine formulation is effective for inducing T-cell immunity to HER-2/neu protein in humans with breast and ovarian cancer. The vaccine formulation included groups of peptides derived from the HER-2/neu extracellular domain (ECD) or intracellular domain (ICD) mixed with granulocyte macrophage colony stimulating factor as an adjuvant. These peptides were 15-18 amino acids in length and designed to elicit a CD4 T helper-specific immune response. Patients underwent intradermal immunization once a month for a total of two to six immunizations. To date, all of the patients immunized with HER-2/neu peptides developed HER-2/neu peptide-specific T-cell responses. The majority of patients (six of eight) also developed HER-2/neu protein-specific responses. Responses to HER-2/neu protein occurred with epitope spreading. Immune T cells elicited by vaccination were shown to migrate outside the peripheral circulation by virtue of generating delayed type hypersensitivity responses distant from the vaccine site, which indicated the potential ability to traffic to the site of tumor. The use of peptide-based vaccines may be a simple, yet effective, vaccine strategy for immunizing humans to oncogenic self-proteins.

Detection of Tumor Messenger RNA in the Serum of Patients with Malignant Melanoma

Abstract: Serum RNases are known to be elevated in patients with cancer. Consequently, it is not clear whether human mRNA with sufficient integrity as to permit reverse transcription-PCR (RT-PCR) amplification is detectable in serum. We examined serum from six patients with malignant melanoma for human tyrosinase mRNA using RT-PCR. Serum from 20 normal volunteers served as controls. Tyrosinase mRNA could be demonstrated in serum from four of the six melanoma patients with detection by gel electrophoresis and confirmation by blotting amplified product to a tyrosinase-specific probe. The serum remained tyrosinase mRNA positive, even if passed through a 0.45 microm filter prior to RNA extraction, indicating that the mRNA was extracellular at the time of extraction. Tyrosinase mRNA could not be detected in any control serum (0 of 20 individuals). The presence and integrity of amplifiable RNA was confirmed in all serum specimens (patients and controls) by RT-PCR amplification of c-abl mRNA. Amplifiable RNA could be demonstrated regardless of whether serum was freshly drawn or stored frozen for several years. We conclude that human mRNA can be extracted and amplified from serum. The ability to amplify tumor mRNA from serum may have important utility in cancer diagnostics and monitoring.

Inverse Relationship between Epidermal Growth Factor Receptor Expression and Radiocurability of Murine Carcinomas

Abstract: The study investigated whether a relationship exists between the extent of epidermal growth factor receptor (EGFR) expression and in vivo radiocurability of murine tumors. EGFR expression was determined in nine carcinomas (four mammary carcinomas, designated MCa-4, MCa-29, MCa-35, and MCa-K; two squamous cell carcinomas, designated SCC-IV and SCC-VII; an ovarian adenocarcinoma, OCa-I; a hepatocarcinoma, HCa-I; and an adenosquamous carcinoma, ACa-SG) syngeneic to C3Hf/Kam mice using Western blot analysis. These tumors greatly differed in their radioresponse, assessed by TCD50 assay, and in their susceptibility to radiation-induced apoptosis. Likewise, the expression of EGFR greatly varied, by as much as 21-fold, and the magnitude of the EGFR expression positively correlated with increased tumor radioresistance. The levels of EGFR inversely correlated with radiation-induced apoptosis, suggesting that the lack of sensitivity to apoptosis induction was a major mechanism responsible for radioresistance of tumors with high EGFR. This correlation was highly significant only for wild-type p53 carcinomas. Radiation activated EGFR autophosphorylation and increased the activity of protein tyrosine kinase, but only in tumors with high EGFR expression. Thus, EGFR expression was a major determinant of tumor radioresponse in vivo. The pretreatment assessment of EGFR expression could predict radiotherapy outcome and may assist in selecting an effective treatment modality.

Minichromosome maintenance proteins as biological markers of dysplasia and malignancy.

Abstract: Dysplasia, an intermediate stage in the progression from normal tissue to neoplasia, is defined morphologically by a loss of normal orientation between epithelial cells, with changes in cellular and nuclear shape and size. However, little is known about the functional properties of dysplastic cells, including their replicative state, largely due to a lack of available biological markers. We have used novel antibodies against minichromosome maintenance (MCM) proteins to examine the proliferative status of a range of histological lesions and to characterize dysplastic cells in functional terms. Immunoperoxidase staining was used to localize the MCM proteins, components of the prereplicative complex that is essential for initiating eukaryotic DNA replication. These proteins are down-regulated in cells undergoing differentiation or quiescence and, thus, serve as specific markers for proliferating cells. In normal and some reactive tissues, MCM expression was present only in restricted proliferative compartments, consistent with our published findings in the uterine cervix. In dysplastic and malignant tissues, in contrast, MCM proteins were expressed in the majority of cells, extending to surface layers of dysplastic stratified epithelia. In carcinomas, the frequency of expression of MCM proteins showed an inverse correlation with the degree of tumor differentiation. Thus, we suggest that dysplastic cells may be characterized in functional terms as remaining in cell cycle, due to deregulation of normal controls over cell proliferation. Antibodies against MCM proteins have potential clinical applications, for example, in the assessment of tumor prognosis in histological sections and the identification of proliferating cells in clinical samples using biochemical or cytological assays.

Antitumor Activity of Sequential Treatment with Topotecan and Anti-Epidermal Growth Factor Receptor Monoclonal Antibody C225

Abstract: Epidermal growth factor (EGF)-related proteins such as transforming growth factor α (TGF-α) control cancer cell growth through autocrine and paracrine pathways. Overexpression of TGF-α and/or its receptor (EGFR) has been associated with a more aggressive disease and a poor prognosis. The blockade of EGFR activation has been proposed as a target for anticancer therapy. Monoclonal antibody (MAb) C225 is an anti-EGFR humanized chimeric mouse MAb that is presently in Phase II clinical trials in cancer patients. Previous studies have suggested the potentiation of the antitumor activity of certain cytotoxic drugs, such as cisplatin and doxorubicin, in human cancer cell lines by treatment with anti-EGFR antibodies. We have evaluated in human ovarian, breast, and colon cancer cell lines, which express functional EGFR, the antiproliferative activity of MAb C225 in combination with topotecan, a cytotoxic drug that specifically inhibits topoisomerase I and that has shown antitumor activity in these malignancies. A dose-dependent supraadditive increase of growth inhibition in vitro was observed when cancer cells were treated with topotecan and MAb C225 in a sequential schedule. In this respect, the cooperativity quotient, defined as the ratio between the actual growth inhibition obtained by treatment with topotecan followed by MAb C225 and the sum of the growth inhibition achieved by each agent, ranged from 1.2 to 3, depending on drug concentration and cancer cell line. Treatment with MAb C225 also markedly enhanced apoptotic cell death induced by topotecan. For example, in GEO colon cancer cells, 5 nm topotecan, followed by 0.5 μg/ml MAb C225, induced apoptosis in 45% cells as compared with untreated cells (6%) or to 5 nm topotecan-treated cells (22%). Treatment of mice bearing established human GEO colon cancer xenografts with topotecan or with MAb C225 determined a transient inhibition of tumor growth because GEO tumors resumed the growth rate of untreated tumors at the end of the treatment period. In contrast, an almost complete tumor regression was observed in all mice treated with the two agents in combination. This determined a prolonged life span of the mice that was significantly different as compared with controls ( P P P

Development of difluoromethylornithine (DFMO) as a chemoprevention agent.

Abstract: D,L-alpha-difluoromethylornithine (DFMO) was synthesized over 20 years ago. It was hoped that this enzyme-activated, irreversible inhibitor of ornithine decarboxylase, the first enzyme in polyamine synthesis, would be effective as a chemotherapy for hyperproliferative diseases, including cancer and/or infectious processes. DFMO was generally found to exert cytostatic effects on mammalian cells and tissues, and its effectiveness as a therapeutic agent has been modest. DFMO was also found to cause treatment-limiting (but reversible) ototoxicity at high doses. This side effect, along with its minimal therapeutic activity, contributed to the loss of interest by many clinicians in further developing DFMO as a cancer therapeutic agent. However, DFMO was subsequently shown to inhibit carcinogen-induced cancer development in a number of rodent models, and interest in developing this compound as a preventive agent has increased. The rationale for the inhibition of ornithine decarboxylase as a cancer chemopreventive agent has been strengthened in recent years because this enzyme has been shown to be transactivated by the c-myc oncogene in certain cell/tissue types and to cooperate with the ras oncogene in malignant transformation of epithelial tissues. Recent clinical cancer chemoprevention trials, using dose de-escalation designs, indicate that DFMO can be given over long periods of time at low doses that suppress polyamine contents in gastrointestinal and other epithelial tissues but cause no detectable hearing loss or other side effects. Current clinical chemoprevention trials are investigating the efficacy of DFMO to suppress surrogate end point biomarkers (e.g., colon polyp recurrence) of carcinogenesis in patient populations at elevated risk for the development of specific epithelial cancers, including colon, esophageal, breast, cutaneous, and prostate malignancies.

Macrophage infiltration and heme oxygenase-1 expression correlate with angiogenesis in human gliomas

Abstract: Macrophages are key participants in angiogenesis. In this study on human brain tumors, we first investigated whether macrophage infiltration is associated with angiogenesis and malignant histological appearance. Immunostaining of macrophages and small vessels in resected glioma specimens indicated that numbers of infiltrating macrophages and small vessel density were higher in glioblastomas than in astrocytomas or anaplastic astrocytomas. Macrophage infiltration was closely correlated with vascular density in human gliomas. Heme oxygenase-1 (HO-1), which is the rate-limiting enzyme in heme catabolism, was also associated with activated macrophages. Expression of mRNA encoding HO-1 was correlated with macrophage infiltration and vascular density in human glioma samples. Infiltrating macrophages were positively stained with anti-HO-1 antibody by immunohistochemical analysis, and in situ hybridization for HO-1 indicated that HO-1 was expressed in infiltrating macrophages in gliomas. HO-1 gene may be a useful marker for macrophage infiltration as well as neovascularization in human gliomas.

Role of Vascular Endothelial Growth Factor C Expression in the Development of Lymph Node Metastasis in Gastric Cancer

Abstract: Neogenesis of lymphatic vessel and lymphatic invasion is frequently found in the stroma of cancers, but the mechanisms of this phenomenon remain unclear. Vascular endothelial growth factor C (VEGF-C) is known to be the only growth factor for the lymphatic vascular system, and its receptor has been identified as Flt4. To clarify the mechanism of lymphatic invasion in cancer, we studied the expression of VEGF-C and flt4 genes in gastric cancer tissues. VEGF-C mRNA was mainly expressed in primary tumors (15 of 32; 47%), but the frequency of VEGF-C mRNA expression was low in normal mucosa (4 of 32; 13%). In primary tumors, there was a significant relationship between VEGF-C and flt4 mRNA expression. In contrast, Flt4 was mainly expressed on the lymphatic endothelial cells but not in cancer cells. A strong correlation was found between VEGF-C expression and lymph node status, lymphatic invasion, venous invasion, and tumor infiltrating patterns. Cancer cells in the lymphatic vessels frequently showed intracytoplasmic VEGF-C immunoreactivity. Furthermore, there was a close correlation between VEGF-C tissue status and the grade of lymph node metastasis. Patients with high expression of VEGF-C protein had a significantly poorer prognosis than did those in low VEGF-C expression group. By the Cox regression model, depth of wall invasion, lymph node metastasis, and VEGF-C tissue status emerged as independent prognostic parameters, and the VEGF-C tissue status was ranked third as an independent risk factor for death. These results strongly suggest that cancer cells producing VEGF-C may induce the proliferation and dilation of lymphatic vessels, resulting in the development of invasion of cancer cells into the lymphatic vessel and lymph node metastasis.

Prognostic significance of elevated cyclooxygenase 2 expression in primary, resected lung adenocarcinomas

Abstract: Recently, we demonstrated that elevated expression of cyclooxygenase 2 (COX-2) is frequently seen in a specific type of lung cancer, i.e., adenocarcinoma, and is possibly associated with its invasion and metastasis. Here, the prognostic significance of elevated COX-2 expression was evaluated in a cohort of 130 adenocarcinoma patients who had consecutively undergone potentially curative resections. Immunohistological examination showed the presence of tumor cells with markedly increased COX-2 immunoreactivity in 93 of 130 (72%) cases. No relationship was found between the increase in COX-2 expression and clinical outcomes when the entire cohort was considered (P = 0.099). Reasoning that the influence of the increase in COX-2 expression may have been obscured by the clinical and molecular pathogenetic complexities in cases with an advanced disease, we also separately analyzed the prognostic significance of increased COX-2 expression after stratification according to the disease stage. A significant relationship between elevated COX-2 expression and shortened patient survival was observed only in a cohort of patients with stage I disease (P = 0.034). These findings suggest that an increase in COX-2 expression may be clinically significant for the prognosis of patients undergoing surgical resection of early-stage adenocarcinomas and, thus, warrant further conclusive studies involving a larger cohort.

A Phase I Study of Active Immunotherapy with Carcinoembryonic Antigen Peptide (CAP-1)-pulsed, Autologous Human Cultured Dendritic Cells in Patients with Metastatic Malignancies Expressing Carcinoembryonic Antigen

Abstract: Dendritic cells (DCs), antigen-presenting cells capable of priming naive T cells to specific antigens in an HLA-restricted fashion, have been demonstrated to induce protective T cell-mediated immunity in tumor-bearing animals. We performed this study to test the safety, feasibility, and clinical response of immunizations with in vitro-generated DCs, loaded with an HLA-A2-restricted peptide fragment of the tumor antigen carcinoembryonic antigen (CEA), for the treatment of patients with advanced CEA-expressing malignancies. Cell preparations enriched for autologous DCs were generated from the patients' plastic adherent peripheral blood mononuclear cells in serum-free media supplemented with granulocyte macrophage colony-stimulating factor and interleukin-4. Within the cell preparation, 66% of the cells expressed the phenotype typical for DCs (CD86high, HLA-DRhigh, and CD14low). The DCs were loaded with the CEA peptide CAP-1 and cryopreserved. Groups of three to six patients received four weekly or biweekly i.v. infusions of the CAP-1-loaded DC in escalating dose levels of 1 x 10(7), 3 x 10(7), and 1 x 10(8) cells/dose. A subset of the patients in the last group also received intradermal injections of 1 x 10(6) DCs. There were no toxicities directly referable to the treatments. One patient had a minor response, and one had stable disease. Skin punch biopsy at DC injection sites demonstrated pleomorphic infiltrates in the three patients evaluated. We conclude that it is feasible and safe to generate and administer large numbers of previously cryopreserved DCs loaded with CAP-1 peptide to patients with advanced malignancies.

Interferon-α-mediated Down-Regulation of Angiogenesis-related Genes and Therapy of Bladder Cancer Are Dependent on Optimization of Biological Dose and Schedule

Abstract: The purpose of this study was to identify and optimize the antiangiogenic activity of IFN-alpha against human bladder cancer cells growing in the bladder of nude mice. 253J B-V IFN(R) cells (resistant to antiproliferative effects of IFN-alpha or IFN-beta) were implanted into the bladder wall of nude mice. Three days later, the mice were treated with s.c. injections of IFN-alpha (70,000 units/week) at different dosing schedules (1, 2, 3, or 7 times/week). Daily therapy with IFN-alpha produced the most significant inhibition of tumor growth, tumor vascularization, and down-regulation of basic fibroblast growth factor and matrix metalloprotease-9 mRNA and protein expression. Changing dose and schedule of IFN-alpha administration had minimal effects on the expression of vascular endothelial growth factor or interleukin 8. The daily s.c. administrations of 5,000 or 10,000 units IFN-alpha-2a produced maximal inhibition of bFGF and MMP-9 expression (mRNA and protein), maximal reduction in tumor vessel density, and maximal reduction in serum levels of bFGF. Daily administration of higher doses of IFN-alpha failed to produce significant antiangiogenic effects. These data suggest that the antiangiogenic activity of IFN-alpha is dependent on frequent administration of optimal biological dose and not maximal tolerated dose.

X-Ray Irradiation Induces Thymidine Phosphorylase and Enhances the Efficacy of Capecitabine (Xeloda) in Human Cancer Xenografts

Abstract: Thymidine phosphorylase (dThdPase) is an essential enzyme for the activation of the cytostatics capecitabine (N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) and its intermediate metabolite 5'-deoxy-5-fluorouridine (5'-dFUrd) to 5-fluorouracil (5-FUra) in tumors. We observed previously that several cytokines and cytostatics up-regulated dThdPase expression and consequently enhanced the efficacy of capecitabine and 5'-dFUrd. In the present study, we found that X-ray irradiation also up-regulated dThdPase expression in several human cancer xenografts. A single-dose local irradiation at 5 Gy increased dThdPase levels by up to 13-fold at 9 days after the irradiation. Whole-body irradiation also up-regulated dThdPase in a tumor, but it did not increase the enzyme level in the liver. We also observed that the irradiation increased the levels of human tumor necrosis factor alpha (TNF-alpha), which is an up-regulator of dThdPase, prior to the dThdPase up-regulation. These results indicate that X-ray irradiation might increase dThdPase levels indirectly through the human TNF-alpha in the tumor tissue. In the WiDr colon and MX-1 mammary human cancer xenograft models, the combination of a single local X-ray irradiation with either capecitabine or 5'-dFUrd was much more effective than either radiation or chemotherapy alone. In contrast, treatment with X-ray irradiation and 5-FUra in combination showed no clear additive effects. Combined modality treatment of cancer patients with cape-citabine and X-ray irradiation would have greater potential usefulness than conventional radiochemotherapy with 5-FUra.

Combination of EGFR, HER-2/neu, and HER-3 Is a Stronger Predictor for the Outcome of Oral Squamous Cell Carcinoma Than Any Individual Family Members

Abstract: In a series of 111 patients with squamous cell carcinoma (SCC), we used immunohistochemistry to examine the expression levels of four epidermal growth factor receptor (EGFR) family members (EGFR, HER-2/neu, HER-3, and HER-4). Expression of the EGFR members was not significantly associated with tumor size. However, their expressions (except for HER-4) were significantly associated with the presence of lymph node metastasis, and all of them were significantly associated with distant metastasis. We further examined the association between the expression levels of the EGFR members and the survival rates in 47 oral SCC patients whose detailed clinical follow-ups were available. The expression of all EGFR members was significantly associated with shortened patient survival, and the association was strongest for HER-2/neu. Furthermore, the combination of HER-2, HER-3, and EGFR but not HER-4 significantly improved the predicting power. The expression level of HER-2/neu was significantly correlated with that of EGFR or HER-3. Similar coexpression patterns were also observed in three oral SCC cell lines studied, but not in four other head and neck SCC cell lines. Taken together, these results indicated that expression levels of EGFR, HER-2/ neu, and HER-3 may help predict the outcome of patients with oral SCC.

Therapy of B-cell lymphoma with anti-CD20 antibodies can result in the loss of CD20 antigen expression.

Abstract: Rituximab is a chimeric antibody with human γ-1 and κ constant regions and murine variable regions. It recognizes the CD20 antigen, a pan B-cell marker. Therapeutic trials in patients with B-cell non-Hodgkin’s lymphoma (NHL) have shown significant efficacy with a primary response rate of 50%, and a secondary response rate of 44% after repeat treatments in prior responders. The selection for proliferating tumor cells that no longer express CD20 may compromise repeated treatment. We have identified a patient who developed a transformed NHL that lost CD20 protein expression after two courses of therapy with rituximab. In a pretreatment lymph node biopsy, 83% of B cells (as defined by CD19 and surface immunoglobulin) expressed surface CD20. A biopsy from the recurrent tumor after two courses of rituximab revealed a diffuse large cell NHL where 0% of B cells expressed CD20 with no evidence of bound rituximab. Cytoplasmic staining showed no CD20 protein. Sequencing of immunoglobulin heavy chain cDNA identified identical variable sequences in the initial and recurrent lymphomas, confirming the association between the two tumors. Literature and database review suggests that approximately 98% of diffuse large cell lymphomas express CD20, which suggests that these tumors rarely survive without CD20. This is the first identified case of loss of CD20 expression in a lymphoma that has relapsed after rituximab therapy, although several other cases have since been identified. Considering the significant number of patients treated with anti-CD20 antibodies, this may occur only rarely and is unlikely to preclude recurrent therapy with anti-CD20 antibodies in the majority of patients. However, because many patients have relapsed after anti-CD20 antibody therapy and have not been biopsied to identify clones with down-regulated CD20 antigen, we do not currently know the true frequency of this phenomenon. When possible, patients should undergo evaluation for CD20 expression before repeated courses of anti-CD20 therapy.

Alterations in Expression of Basic Fibroblast Growth Factor (FGF) 2 and Its Receptor FGFR-1 in Human Prostate Cancer

Abstract: Fibroblast growth factors (FGFs) play an important role in the growth and maintenance of the normal prostate. There is increasing evidence from both animal models and analysis of human prostate cancer cell lines that alterations of FGFs and/or FGF receptors (FGFRs) may play an important role in prostate cancer progression. To better define the role of FGF2 and FGF7 in human prostate cancer in vivo, we have quantified these two growth factors in clinically localized human prostate cancers and uninvolved prostate by ELISA and Western blotting and determined their localization by immunohistochemistry. The expression of two of the primary receptors for these growth factors, FGFR-1 and FGFR-2, were also analyzed by immunohistochemistry and Western blotting in these same samples. We have found that FGF2 is significantly increased in prostate cancers when compared with uninvolved prostate and that the FGF2 is present in the stromal fibroblasts and endothelial cells but not the cancer cells. In addition, we have observed overexpression of both FGFR-1 and FGFR-2 in the prostate cancer epithelial cells in a subset of prostate cancers and that such overexpression is correlated with poor differentiation. Thus, there is both an increase in FGF2 concentration in prostate cancers and an increased expression of a receptor capable of responding to this growth factor, establishing a potential paracrine stimulation of prostate cancer cells by the surrounding stromal cells, which may play an important role in prostate cancer progression.

Osteopontin: Possible Role in Prostate Cancer Progression

Abstract: Human prostate cancer has the propensity to metastasize to the bone where reciprocal cellular interactions between prostate cancer and bone cells are known to occur. Osteopontin (OPN), a noncollagenous bone extracellular matrix, is a secreted adhesive glycoprotein with a functional RGD cell-binding domain that interacts with the alpha(v)beta3 cell surface integrin heterodimer. OPN has been associated with malignant transformation as well as being ligand to the CD44 receptor. Polyclonal antibodies to human OPN (hOPN) were prepared, and specificity was shown by preabsorption with recombinant hOPN. The stimulatory effect of hOPN protein and the inhibitory effect of hOPN antibody on human prostate cancer cell lines LNCaP and C4-2 were assessed by induction or inhibition of anchorage-independent growth, respectively. Expression of hOPN mRNA in prostate cancer cell lines and human prostate cancer tissue specimens were measured by mRNA blot analysis. Protein expression was assessed by immunohistochemistry in human prostate cancer specimens and by Western blot analysis in prostate cancer cell lines. hOPN stimulated anchorage-independent growth of the human prostate cancer cell lines LNCaP and C4-2 in vitro. Antibodies to hOPN inhibited the growth-stimulatory effect by endogenous OPN, which can be overcome by the addition of exogenous hOPN. hOPN mRNA and protein are expressed in human prostate cancer cell lines in vitro and in clinical human prostate cancer specimens. These findings taken together suggest that OPN may act as a paracrine and autocrine mediator of prostate cancer growth and progression.

Leukocytes and platelets of patients with cancer contain high levels of vascular endothelial growth factor

Abstract: Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific mitogen and permeability factor. Malignant human tumors have been shown to produce VEGF. Elevated levels of VEGF have been detected in sera of cancer patients, but its origin is unsettled. We analyzed VEGF concentrations in serum, plasma, whole blood, and peripheral blood mononuclear cells (PBMNCs) and platelets in 56 cancer patients and 52 healthy controls using ELISA. The VEGF concentrations in the lysed whole blood samples [blood VEGF (B-VEGF)] were higher in cancer patients than in healthy controls (median, 464 versus 298 pg/ml; P 0.1). The cancer patients regularly had higher B-VEGF concentrations than healthy individuals with comparable leukocyte or platelet counts. The VEGF content of isolated PBMNCs and platelets was severalfold higher in cancer patients than in healthy controls (median, 10.6 versus 0.9 pg per 10(6) PBMNCs, and median, 1.6 versus 0.5 pg per 10(6) platelets; P<0.0001 and P = 0.0008, respectively). Serum VEGF and B-VEGF correlated strongly (P<0.0001). Very little or no VEGF was found in the plasma. The results indicate that VEGF in the bloodstream is transported by blood cells, including leukocytes and platelets. The blood cells of cancer patients contain greatly elevated amounts of this major angiogenic growth factor, and this reservoir of VEGF may have a role in tumor angiogenesis and metastasis formation. VEGF in serum samples originates from blood cells, and the use of VEGF of whole blood or of isolated blood cells may improve the clinical value of VEGF measurements.

Insulin and insulin-like growth factor-I (IGF-I) receptor overexpression in breast cancers leads to insulin/IGF-I hybrid receptor overexpression: evidence for a second mechanism of IGF-I signaling.

Abstract: The insulin receptor (IR) form hybrids with the closely related insulin-like growth factor-I (IGF-I) receptor (IGF-I-R). Because most human breast carcinomas overexpress both the IR and the IGF-I-R, we evaluated whether the insulin/IGF-I hybrid receptor (Hybrid-R) is also overexpressed in these tumors and what role it plays in breast cancer biology. Using specific ELISAs and Western blots, we measured Hybrid-R content and function in 8 human cultured breast cancer cell lines and 39 human breast cancer specimens. Hybrid-R content and function were also compared to the content and function of the IR and the IGF-I-R. Hybrid-R content exceeded the IGF-I-R content in >75% of breast cancer specimens and was directly related to the molar ratio of both the IR and IGF-I-R content, suggesting that Hybrid-R formation occurred by random assembly of IR and IGF-I-R half-receptors. Hybrid-Rs became tyrosine autophosphorylated when breast cancer cells were exposed to IGF-I but not when they were exposed to insulin. In cells with an elevated Hybrid-R content, Hybrid-R autophosphorylation in response to IGF-I exceeded IGF-I-R autophosphorylation, suggesting that most of the IGF-I effect occurred via the Hybrid-R. Furthermore, Hybrid-Rs mediated growth in response to IGF-I, as indicated by experiments with blocking antibodies to the IGF-I-R. These data indicated therefore that: (a) Hybrid-Rs are present and play a major role in mediating the IGF-I signal in breast cancer; (b) their expression is directly related to IR overexpression; and (c) potential therapies designed to block IGF-I actions in breast cancer must take into account the role of these Hybrid-Rs.

Complete Pathological Remission Is Possible with Systemic Combination Chemotherapy for Inoperable Hepatocellular Carcinoma

Abstract: The purpose of this Phase II study was to determine the response rate, the toxicity, and the effect on survival of the combination of cisplatin, doxorubicin, 5-fluorouracil, and alpha-IFN (PIAF) in advanced unresectable hepatocellular carcinoma. Fifty patients with either unresectable or metastatic disease were treated with PIAF: cisplatin (20 mg/m2 i.v., days 1-4), doxorubicin (40 mg/m2 i.v., day 1), 5-fluorouracil (400 mg/m2 i.v., days 1-4), and alpha-IFN (5 MU/m2 s.c., days 1-4). Treatment was repeated every 3 weeks to a maximum of six cycles. All patients were evaluable for response, toxicity, and survival. As assessed by conventional imaging criteria, there were no complete responses, but 13 patients (26%) had a partial response. Among the 36 patients who had an initially high alpha-fetoprotein level (>500 ng/ml), 15 (42%) had a >50% fall after therapy. Nine patients underwent surgical resection after achieving partial response and, in 4 of these patients, histological examination of the resected specimens revealed no viable tumor cells. All these nine patients are alive, and eight patients remain in complete remission at between 7.6 and 25.8 months at the time of analysis. The overall median survival was 8.9 months. Toxicity was mainly myelosuppression and mucositis. There were two treatment-related deaths due to neutropenic sepsis. PIAF is active in hepatocellular carcinoma despite considerable hematological toxicity. Complete pathological remission is possible with this systemic combination. Apparently, persistent radiological lesions may still represent complete pathological resolution of active disease.

Constitutive and inducible interleukin 8 expression by hypoxia and acidosis renders human pancreatic cancer cells more tumorigenic and metastatic.

Abstract: 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.