Lipid messengers as targets for antiangiogenic therapy.

Cancer, only second to heart disease, is a leading cause of death in the United States. Despite many years of cancer research little progress has been made in the treatment of many types of cancer. With the advent of molecular biology and advanced biochemical techniques, we have begun to elucidate the various signaling pathways that account for the transformation of normal cells to malignant cells. Our understanding of cancer cell signaling and cell cycle deregulation has paved the way for the rational design of specific inhibitors. Alas, attempts to specifically and exclusively target treatment to the cancer cell have fallen short of expectations for cure and often result in unfortunate drug side effects. More recently, Folkman proposed neovascularization requirements for tumor expansion and metastasis, and this sparked great interest in both the molecular mechanism of tumor-induced angiogenesis and its potential target for anticancer treatment. In this review, we first describe protein growth factors that have been shown to induce endothelial cell proliferation and angiogenesis. We also discuss the signal transduction cascades that result from growth factor receptor binding in light of drugs that are know to inhibit these cascades. Finally, we discuss the potential use of antagonists of lipid second messengers. In particular BN-50730, a PAF antagonist shows promise in preliminary anti-tumor therapy in vitro and in vivo in athymic nude mice by specifically inhibiting angiogenesis.

Suppression of tumor cell growth both in nude mice and in culture by n-3 polyunsaturated fatty acids: mediation through cyclooxygenase-independent pathways.

Dietary n-3 polyunsaturated fatty acids (PUFAs), as compared with n-6 PUFAs, suppress cellular production of prostaglandins and tumor cell growth both in vitro and in vivo. However, the mechanism by which n-3 PUFAs suppress tumor growth is not understood. We investigated whether the suppression of tumor cell growth by dietary n-3 PUFAs is mediated through inhibition of cyclooxygenase (COX). A colon tumor cell line, HCT-116, that does not express COX was stably transfected with the constitutively expressed COX-1 or the inducible COX-2 cDNA using a retroviral transfection and infection system. Athymic nude mice transplanted with the cells expressing enzymatically active COX were fed isocaloric diets containing either safflower oil or fish oil for 2 weeks before the start of the experiment and for an additional 21 days after transplantation. Both tumor volume and tumor burden (tumor volume/body weight) were significantly reduced in mice fed the fish oil diet as compared with safflower oil-fed mice. This reduction occurred even in control mice that received injections with cells infected with the retroviral vector without COX-1 or COX-2 cDNA. The growth of tumor cells expressing COX was not different from the growth of those transfected with the vector alone in the nude mice and in soft agar. N-3 PUFAs, as compared with linoleic acid, also inhibited the growth of these cells in culture. This growth inhibition by n-3 PUFAs was not affected by COX-1 or COX-2 overexpression. Contrary to general belief, these results indicate that the suppression of tumor growth by dietary n-3 PUFAs is mediated through COX-independent pathways.

KRAS mutations are not predictive for progression of preneoplastic gastric lesions.

Individuals with atrophic gastritis (n = 863) were recruited to participate in a chemoprevention trial in Nariño, Columbia. The volunteers were randomly assigned to intervention therapies, which included antibiotic treatment for Helicobacter pylori infection, and then daily dietary supplementation with antioxidant micronutrients in a 2(3) factorial design. Biopsies were obtained according to a specified protocol from designated areas in the stomach for each individual at baseline (before intervention therapy), at year 3, and at year 6. A systematic sample of 160 participants was selected from each of the eight treatment combinations, and the first exon of KRAS was examined for mutations. At year 3, the data indicated that individuals with KRAS mutations in their baseline premalignant stomach biopsies were 3.74 times as likely to progress to a higher premalignant stage than those who lacked baseline mutations (P = 0.04; C. Gong et al., Cancer Epidemiol. Biomark. Prevy. 8:167-171, 1999). However, after 6 years, baseline KRAS mutations failed to predict histological progression. Also, KRAS mutation in 72-month biopsies did not predict histological progression.

Orthotopic human lung carcinoma xenografts in BALB/c mice immunosuppressed with anti-CD4 monoclonal antibodies and chronic alcohol consumption.

BACKGROUND: The role of the immune system in the surveillance of the body for cancer cells is well established. Human tumor cells do not survive in mice with intact immune systems, but they propagate in athymic nude mice. Presumably, the lack of a thymus gland and consequent loss of T lymphocytes results in a seriously compromised immune system without adequate cell-mediated immunity and tumor surveillance. In patients infected with the human immunodeficiency virus (HIV), a progressive loss of cell-mediated immunity is associated with the development of malignancies and opportunistic infections. This effect may be exacerbated in patients who chronically consume alcohol. METHODS: Normal and alcoholic BALB/c mice were treated with a monoclonal antibody to deplete CD4(+) lymphocytes before orthotopic implantation of human lung adenocarcinoma xenografts. Tumor volume and weight were measured and compared between groups. RESULTS: The authors' data show that a single treatment of anti-CD4 antibody causes almost complete depletion of CD4(+) lymphocytes and permits the formation of large intrapulmonary human nonsmall lung carcinoma xenografts in 100% of treated mice. All control animals injected with heat-denatured antibody failed to produce tumors. Chronic alcohol consumption by CD4-depleted mice resulted in larger tumors, compared with mice that did not receive ethanol in their diet (P = 0.05). CONCLUSIONS: Depletion of CD4(+) lymphocytes allows for the orthotopic growth of human lung adenocarcinoma xenografts in BALB/c mice. Furthermore, the consumption of alcohol reduces the ability of the impaired immune system to reject tumors.

Evidence for autocrine actions of neuromedin B and gastrin-releasing peptide in non-small cell lung cancer.

Gastrin-releasing peptide (GRP), a member of the bombesin family of peptides, has been shown to have mitogenic activity in small cell lung carcinoma (SCLC), and to be produced by SCLC in an autocrine fashion. In this report, we demonstrate that both GRP and another member of the bombesin family of peptides, neuromedin B (NMB), are also autocrine growth factors for non-small cell lung carcinoma (NSCLC). Using the reverse transcription-polymerase chain reaction (RT-PCR), we have detected mRNA for the neuromedin B receptor (NMBR) in all 14 of the NSCLC cell lines examined. GRP receptor (GRPR) mRNA was also expressed in the majority of NSCLC cell lines (nine of 14). By immunoblotting using SDS-PAGE gradient gels fixed in trichloroacetic acid, GRP and NMB were found in fractions of culture medium that had been purified by high pressure liquid chromatography (HPLC) from NSCLC cell lines. NMB was detected in the conditioned medium of seven of nine cell lines and GRP in seven of nine cell lines; both peptides were produced in six cell lines. In four of the cell lines where both peptides were produced, the relative amount of NMB secreted into the medium was 7-15 times that of GRP; in the other two cases, the relative amounts of GRP and NMB were equivalent. Cultured human bronchial epithelial (HBE) cells expressed the GRPR and NMBR but did not produce either peptide. A subline of A549 cells that was adapted to grow in serum-free and growth factor-free conditions, termed A549-R(0), secreted both bombesin-like peptides (BLPs) into the culture medium. Using either a colony-forming assay or a BrDU incorporation assay, both NMB and GRP were found to be mitogens for three NSCLC cell lines that express mRNA for BLP receptors and secrete BLPs, regardless of which peptide and/or receptor subtype was detected. The monoclonal antibody 2A11, which preferentially recognizes GRP, was able to block the in vitro proliferative response to GRP in the BrDU incorporation assay, and partially blocked the response to NMB. The 2A11 antibody could only partially block the in vivo growth of cell lines that showed proliferative responses to BLPs. 2A11 antibody was more effective against the 239T cell line, which secreted a low amount of GRP into the medium (0.6 nM), compared to the 201T cell line, which secreted a higher amount of both GRP and NMB (4.2 nM and 36.6 nM, respectively). These results suggest that both NMB and GRP are autocrine growth factors for NSCLC, but that the production of NMB and expression of the NMBR may be more prominent than the production of GRP and expression of the GRP receptor. If BLP ligand-receptor systems are to be targeted therapeutically in NSCLC, it will be necessary to inhibit both NMB and GRP.

Propylthiouracil-induced hypothyroidism reduces xenograft tumor growth in athymic nude mice.

BACKGROUND: Thyroid hormones are endocrine modulators of several vital processes that are crucial to tumor growth and differentiation. Several anecdotal reports in the literature suggest that some histologic types of carcinoma may remain in a dormant state for prolonged periods of time in patients with hypothyroidism, with eventual progression of the disease once the decreased thyroid function is identified and corrected. METHODS: Oral propylthiouracil (PTU) was used to induce hypothyroidism in athymic nude mice that were subsequently inoculated with lung adenocarcinoma and prostate adenocarcinoma cells. Mice were also treated with a combination of PTU and thyroxine, which resulted in hyperthyroid levels of T(4). RESULTS: Subcutaneous lung and prostate xenografts grew significantly more slowly in hypothyroid mice treated with PTU than in euthyroid or hyperthyroid mice, regardless of treatment with PTU. Tumors grew well in groups of mice that were changed from a hypothyroid state to a euthyroid state by withdrawal of oral PTU. Administration of PTU 3 weeks after tumor inoculation also caused the tumor growth to slow significantly compared with tumors in mice that did not receive PTU. Mice that received PTU and thyroxine had tumors that grew as well as the tumors in euthyroid control animals. CONCLUSIONS: Our study indicates that human lung and prostate tumors do not grow well in hypothyroid nude mice, and that rendering these animals euthyroid has a significant impact on the growth rate of these tumors. Furthermore, in vitro and in vivo data indicated that this was not a result of an interaction of the tumor cells with PTU, but rather a result of the hypothyroid state.

Cloning and characterization of the prostate-specific membrane antigen promoter.

Prostate-specific membrane antigen (PSMA) is a protein that is expressed predominantly in normal prostate epithelial cells and in most adenocarcinomas of the prostate (Cap) and in virtually all Cap metastases. In this article we describe the cloning of a 2-kb human genomic DNA fragment containing the 5' upstream untranslated region of the PSMA gene and present evidence that it provides promoter activity. When the DNA fragment was cloned into transient expression vectors to examine promoter activity, the vectors were functional in promoting expression in several prostate and nonprostate cell lines in transient transfection assays. A 614-bp fragment derived from the 3' end of the 2-kb fragment may represent the minimal PSMA promoter as determined by deletion mutagenesis. The 2-kb fragment compared with the 614-bp fragment provided higher expression levels when using prostate-derived cell lines (DU 145 and LNCaP). The increased transcription using the 2-kb fragment was not as great in non-prostate cell lines. Little or no transcription over basal levels was seen with a 232-bp promoter fragment. When the concentration of dihydrotestosterone was depleted or supplemented in the growth medium, no significant effect was seen on PSMA-promoted transient expression in LNCaP cells, a prostate cell line. J. Cell. Biochem. 74:395-405, 1999. Published 1999 Wiley-Liss, Inc.

KRAS mutations predict progression of preneoplastic gastric lesions.

Eight hundred sixty-three subjects with atrophic gastritis were recruited to participate in an ongoing chemoprevention trial in Nariño, Colombia. The participants were randomly assigned to intervention therapies, which included treatment to eradicate Helicobacter pylori infection followed by daily dietary supplementation with antioxidant micronutrients in a 2 x 2 x 2 factorial design. A series of biopsies of gastric mucosa were obtained according to a specified protocol from designated locations in the stomach for each participant at baseline (before intervention therapy) and at year three. A systematic sample of 160 participants was selected from each of the eight treatment combinations. DNA was isolated from each of these biopsies (n = 320), and the first exon of KRAS was amplified using PCR. Mutations in the KRAS gene were detected using denaturing gradient gel electrophoresis and confirmed by sequence analysis. Of all baseline biopsies, 14.4% (23 of 160) contained KRAS mutations. Among those participants with atrophic gastritis without metaplasia, 19.4% (6 of 25) contained KRAS mutations, indicating that mutation of this important gene is likely an early event in the etiology of gastric carcinoma. An important association was found between the presence of KRAS mutations in baseline biopsies and the progression of preneoplastic lesions. Only 14.6% (20 of 137) of participants without baseline KRAS mutations progressed from atrophic gastritis to intestinal metaplasia or from small intestinal metaplasia to colonic metaplasia; however, 39.1% (9 of 23) with baseline KRAS mutations progressed to a more advanced lesion after 3 years [univariate odds ratio (OR), 3.76 (P = 0.05); multivariate OR adjusted for treatment, 3.74 (P = 0.04)]. In addition, the specificity of the KRAS mutation predicted progression. For those participants with G-->T transversions at position 1 of codon 12 (GGT-->TGT), 19.4% (5 of 17) progressed (univariate OR, 2.4); however, 60.0% (3 of 5) of participants with G-->A transitions at position 1 of codon 12 (GGT-->AGT) progressed (univariate OR, 8.7; P = 0.004 using chi2 test).

Recombinant oncotoxin AR209 (anti-P185erbB-2) diminishes human prostate carcinoma xenografts.

PURPOSE: Prostate cancer is the most common malignancy of males in the United States. Although the overall survival rate for early stage prostate cancer is good, if cancer recurs following curative therapies there is no adequate salvage therapy. Systemic chemotherapy has never been associated with any meaningful improvement in overall survival or overall objective benefit. There is a need to develop novel therapies for prostate cancer. MATERIALS AND METHODS: Two prostatic cancer cell lines, DU-145 and PC-3, were grown as subcutaneous xenografts in athymic nude mice. The recombinant oncotoxin AR209, formerly OLX-209 [e23(Fv)PE38KDEL]), has the specificity of an anti-p185erbB-2 antibody contained within a single-chain antibody domain (e23Fv) coupled to a portion of the Pseudomonas exotoxin A (PE38KDEL). Using Western blot analysis, the cell lines were shown to express p185erbB-2. The mice received either 3 i.v. injections, one every 2 days, of the recombinant oncotoxin AR209 or PBS, or were implanted with osmotic pumps that delivered a constant s.c. amount of AR209 or PBS. RESULTS: The oncotoxin was effective in reducing the size of s.c. prostatic xenografts in athymic nude mice. The data demonstrated that small tumors (<200 mm.3) were effectively reduced in size. However, larger tumors (>500 mm.3) were not effectively diminished. CONCLUSIONS: This study provides preliminary evidence for the utility of a recombinant oncotoxin in the treatment of prostate carcinoma. Recombinant oncotoxins may be an effective clinical addition for the management of metastatic prostate lesions in patients treated with conventional therapy.

Aggressive administration of recombinant oncotoxin AR209 (anti-ErbB-2) in athymic nude mice implanted with orthotopic human non-small cell lung tumours.

Lung cancer remains a significant public health problem in the U.S.A. and will result in an estimated 160,400 deaths in 1997. This appalling number is due in large part to the lack of adequate treatment for tumours that are refractory to surgery with curable intent, or of an adequate salvage therapy for those patients who recur after surgical resection. Because non-small cell lung cancer is refractory to traditional chemotherapy, non-traditional therapies have been developed to treat patients with this disease. Recombinant oncotoxins have been designed to target cells that express certain proteins as part of their cellular membrane. One such oncotoxin, AR209 (formerly OLX-209 [e23(Fv)PE38KDEL]), has the specificity of an anti-ErbB-2 antibody contained within a single-chain antibody domain (e23v) coupled to a portion of the Pseudomonas exotoxin A (PE38KDEL). Previous studies demonstrate that this drug is capable of significantly reducing the size of orthotopic lung tumour xenografts. However, most of the treated mice developed tumours once therapy was removed. In this study, mice were treated aggressively using one of four drug treatment schedules. Mice were treated with either intravenous or subcutaneous injections of AR209 over a 2 week period. The data indicate that AR209 significantly reduced the size of tumours and upon microscopic analysis at necropsy, some mice were cured. However, despite the treatment schedule used, many mice contained residual tumour. Residual tumours expressed the ErbB-2 protein, indicating that more aggressive treatment with AR209 may have resulted in higher rates of cure.

Trinucleotide repeat length variation in the human ribosomal protein L14 gene (RPL14): localization to 3p21.3 and loss of heterozygosity in lung and oral cancers.

Chromosome 3p is consistently deleted in lung cancer, oral squamous cell carcinoma, and renal cell carcinoma, and is believed to contain several tumor suppressor genes. We have shown a role for chromosome 3 in tumor suppression by microcell-mediated chromosome transfer experiments. We have isolated a gene that is located at 3p21.3 within the smallest region of deletion overlap in lung tumors and is the human homolog of the ribosomal protein L14 gene (RPL14). The RPL14 sequence contains a highly polymorphic trinucleotide repeat array which encodes a variable-length polyalanine tract. Genotype analysis of RPL14 shows that this locus is 68% heterozygous in the normal population, compared with 25% in non-small cell lung cancer (NSCLC) cell lines (p = 0.008). Cell cultures derived from normal bronchial epithelium show a 65% level of heterozygosity, reflecting that of the normal population. Squamous cell carcinoma of the head and neck (SCCHN), which has the same risk factors as lung cancer and is hypothesized to have a similar etiology, demonstrates 54% loss of heterozygosity at the RNA level, suggesting that transcriptional loss may be a primary mechanism of RPL14 alteration in SCCHN. In addition, RPL14 shows significant differences in allele frequency distribution in ethnically-defined populations, making this sequence a useful marker for the study of ethnicity-adjusted lung cancer risk.

Receptor-targeted recombinant adenovirus conglomerates: a novel molecular conjugate vector with improved expression characteristics.

To develop improved strategies for gene transfer to hematopoietic cells, we have explored targeted gene transfer using molecular conjugate vectors (MCVs). MCVs are constructed by condensing plasmid DNA containing the gene of interest with polylysine (PL), PL linked to a replication-incompetent adenovirus (endosomolytic agent), and PL linked to streptavidin for targeting with biotinylated ligands. In this report, we compare gene transfer to K562 cells by using the previously described transferrin-targeted MCV (Trans-MCV) to a novel transferrin-targeted MCV. In the novel MCV, the transferred gene (luciferase) is in the genome of recombinant replication-incompetent adenovirus (recMCV), which also acts as the endosomolytic agent. The level of luciferase gene expression was fivefold higher in K562 cells transfected with Trans-recMCV than in cells transfected with Trans-MCV. Furthermore, targeted transfection with recMCV resulted in prolonged luciferase expression that declined 14 to 20 days after transfection, in comparison with Trans-MCV, where luciferase expression declined by 4 to 8 days. Moreover, targeted transfection of K562 cells with the Trans-recMCV resulted in persistent luciferase gene expression for 6 months. Analysis of luciferase gene expression in K562 single-cell clones that were subcloned 5 weeks after transfection with Trans-recMCV showed that 35 to 50% of the single-cell clones had intermediate to high levels of luciferase gene expression that was stable for 6 months, with the remaining clones showing low or no luciferase gene expression. Stable gene expression was associated with integration of adenovirus sequences into genomic DNA.

Prognostic value of specific KRAS mutations in lung adenocarcinomas.

Adenocarcinomas of the lung remain a significant public health problem. Locally defined (stage I) tumors are considered amenable to resection with curative intent. However, only about 45% of these patients survive for 5 years. The median survival for more advanced tumors is drastically lower. Much research has been focused on identifying a valid genetic biomarker of prognosis. Mutations of the proto-oncogene KRAS have been identified by some groups as being a valid prognostic indicator for adenocarcinoma of the lung. To evaluate the effect of KRAS gene mutation on the survival of patients with lung adenocarcinoma, 181 archival tumors were examined by PCR and denaturing gradient gel electrophoresis. Mutations in either codon 12 or 13 were found in 31.5% of the samples. The most common mutation was a G-->T transversion in codon 12, representing 66.7% of the mutations. No difference was observed in the survival of patients with a KRAS mutation versus those whose tumors contained wild-type KRAS. This lack of difference was also observed when the analysis was restricted to those with stage I tumors or when patients with stage I or II disease were grouped together. However, certain amino acid substitutions, including cysteine, arginine, and aspartate, indicated a significantly poorer prognosis, whereas hydrophobic amino acid substitutions showed a significantly better prognosis than wild-type (P = 0.04). Sample sizes were small for this analysis due to the number of possible mutations. As expected, the stage of tumor at resection was the most significant predictor of outcome. Based on this study of 181 patients from two major medical centers located in different cities, we conclude that KRAS mutation status is not a satisfactory predictor of prognosis in lung adenocarcinoma, but the substitution of a polar or charged amino acid for the wild-type glycine residue may be a negative prognostic indicator.

Percutaneous implantation of non-small-cell lung carcinoma: technique and observations.

RATIONALE AND OBJECTIVES: The authors performed this study to determine whether intrathoracic inoculation of non-small-cell lung carcinoma with fluoroscopic guidance would provide for more accurate implantation. MATERIALS AND METHODS: A tumor cell inoculum (2 x 10(6) cells per 0.15 mL) was injected percutaneously under fluoroscopic guidance at the posterior midaxillary line in 22 athymic nude mice. The mice underwent imaging with a mammographic unit at 3, 5, and 8 weeks after implantation. The mice were sacrificed at 8 weeks, and autopsy was performed to determine tumor yield. RESULTS: The use of a percutaneous technique under fluoroscopic guidance greatly facilitated the accurate implantation of xenografts. Tumor growth was seen at radiography in 18 of the 22 (82%) mice at 8 weeks. Necropsy revealed a 100% tumor yield. Histologic examination confirmed adenocarcinoma of the lung. The average number of tumors found in the lung parenchyma was 1.05 +/- 0.35; the average number of tumors found in the mediastinum was 0.59 +/- 0.67. The average tumor weight was 389 mg +/- 64.3. The average tumor size was 300 mm3 +/- 66.23. CONCLUSION: With fluoroscopic guidance, percutaneous implantation of tumor cells in athymic nude mice is simple and effective.

Expression of mRNA for gastrin-releasing peptide receptor by human bronchial epithelial cells. Association with prolonged tobacco exposure and responsiveness to bombesin-like peptides.

Bombesin-like peptides (BLPs) are important regulators of lung development and may also act as autocrine growth factors in lung tumors. We have previously demonstrated expression of mRNA for the three BLP receptor subtypes (neuromedin B [NMB]) receptor, gastrin-releasing peptide [GRP] receptor, and bombesin receptor subtype 3 [BRS-3]) in human non-small cell lung carcinoma (NSCLC) cell lines and bronchial biopsies using the reverse transcription-polymerase chain reaction (RT-PCR; DeMichele, et al. Am. J. Respir. Cell Mol. Biol. 1994; 11:66-74). We have also previously found that growth responses to BLPs could be elicited in some, but not all, cultures of human bronchial epithelial (HBE) cells (Siegfried, et al. Anat. Rec. 1993; 236:241-247). In this report, we utilized RT-PCR to demonstrate mRNA expression of BLP receptor subtypes in cultured HBE cells and also assessed the response of these cultures to BLPs in proliferation assays. The pattern of mRNA expression was correlated with proliferative response, and the results were also analyzed in relation to smoking history and pulmonary function of the subjects studied. Our results suggest that expression of mRNA for the GRP receptor is associated with a long smoking history (> 25 pack-years [PY], p = 0.02). This association was related to past tobacco exposure, regardless of whether the subjects were still active smokers at the time of tissue procurement. Responsiveness to GRP and NMB in proliferation assays was also found only in those HBE cultures with expression of mRNA for at least one of the known receptors for BLPs, and there was a significant association between expression of mRNA for the GRP receptor and proliferative response to both GRP and NMB (p = 0.048). HBE cultures from subjects with a greater than 25 PY smoking history were also more likely to respond to BLPs in the proliferation assays than cells from subjects with less than a 25 PY history (10 of 16 versus 1 of 7, p = 0.06). Cultures of HBE cells from four of the five subjects with severe obstructive lung disease gave a positive response to GRP and NMB in proliferation assays, compared to five of fifteen without severe obstructive lung disease, but this difference was not significant (p = 0.13). These results suggest there is an increased likelihood of expression of the GRP receptor mRNA in the respiratory epithelium of some individuals with a history of prolonged tobacco exposure, and that expression of the GRP receptor mRNA is accompanied by responsiveness to the mitogenic effects of BLPs. These effects appear to persist after smoking cessation.

Tumor specific Epstein-Barr virus infection is not associated with leiomyosarcoma in human immunodeficiency virus negative individuals.

BACKGROUND: Recent studies have suggested that the Epstein-Barr virus (EBV) is associated with leiomyosarcoma in children with human immunodeficiency virus (HIV) and in organ transplant recipients. To determine whether EBV is associated with leiomyosarcoma in HIV negative patients, the authors examined resected leiomyosarcomas for EBV and HIV. METHODS: Twenty-four leiomyosarcomas were studied and their diagnosis confirmed on pathologic review. From these specimens DNA was isolated. Tumor samples were analyzed for EBV and HIV using a polymerase chain reaction (PCR) technique followed by gel electrophoresis and Southern blot analysis. DNA from an EBV-infected human Burkitt's lymphoma cell line and peripheral blood from an HIV positive patient were used as positive controls for the presence of EBV and HIV, respectively. Immunohistochemistry was performed using an antibody to Epstein-Barr nuclear antigen. RESULTS: HIV was not present in any of the patients analyzed. EBV DNA was detected in tumor tissue; however, 80 cycles of PCR were used before EBV sequences were detected. Therefore, the data indicate that tumor tissue was not infected with EBV. The positive results observed after 80 cycles of PCR were likely due to infiltrating lymphocytes. Immunohistochemistry confirmed the lack of active or latent EBV infection in tumor cells. CONCLUSIONS: The results indicate that EBV is not associated with sporadic leiomyosarcoma in HIV negative patients. Therefore, the biology of leiomyosarcoma associated with HIV may be substantially different from the more common sporadic form.

Detection of K-ras mutations in resected primary leiomyosarcoma.

Mutation of the K-ras oncogene occurs frequently in human malignancy. However, there are few reports concerning K-ras mutations in soft-tissue sarcoma, including leiomyosarcoma. We therefore designed a study to determine the prevalence of mutations in the first exon of K-ras in leiomyosarcoma and to evaluate its prognostic potential. Fifty-one leiomyosarcomas were reviewed, and their diagnoses were confirmed on pathological review. Tissue blocks were retrieved, and new sections were prepared for confirmation of diagnosis. Additional tissue sections were used for DNA isolation. PCR and denaturing gradient gel electrophoresis (DGGE) were used to detect K-ras mutations in the first exon of genomic DNA isolated from the specimens. Seven (14%) K-ras mutations were detected using DGGE. Subsequent sequencing of the K-ras gene from each of the mutated tumors confirmed the DGGE results in each case. The median survival for patients whose tumors did not contain mutations of K-ras was 42 months (n = 42) versus 25 months (n = 7) for those with mutations (P = 0.06). However, patients with stages I and II tumors had a median survival of 82 months (n = 28) compared to 28 months for those with stages III and IV disease (n = 20, P = 0.02). The results suggest that K-ras codon 12 mutations are uncommon in leiomyosarcoma; however, when such mutations are found, there is a trend toward worse survival. Furthermore, the data confirm that stage is a significant prognostic indicator.

Effects of anti-erbB-2 (HER-2/neu) recombinant oncotoxin AR209 on human non-small cell lung carcinoma grown orthotopically in athymic nude mice.

The recombinant oncotoxin AR209 [e23(Fv)PE38KDEL; formerly OLX-209] was developed to treat neoplasia that expresses the c-erbB-2 (HER-2/neu) protein product p185(erbB-2). The AR209 compound contains a single-chain antibody domain specific for p185(erbB-2), coupled with a portion of the Pseudomonas exotoxin. The drug has been shown to be effective in inhibiting cells that overexpress erbB-2 due to gene amplification and in cells that do not contain amplified erbB-2 but express slightly higher levels of the protein than normal cells do. To test the efficacy of AR209 on human lung tumors, athymic nude mice were inoculated intrathoracically with a cell line derived from a poorly differentiated lung adenocarcinoma. This cell line, termed 201T, expresses moderately elevated levels of p185(erbB-2) 7.6-fold over normal bronchial epithelium. Mice treated with i.v. injections of AR209 for 5 weeks after orthotopic tumor implantation had smaller tumors and in 20% of cases showed no evidence of disease. The data from this study indicate that AR209 may be an effective treatment for patients with non-small cell lung cancers that express p185(erbB-2).

Activity of anti-erbB-2 recombinant toxin OLX-209 on lung cancer cell lines in the absence of erbB-2 gene amplification.

The recombinant oncotoxin OLX-209 [e23(Fv)PE38KDEL] has been developed to target cancers with erbB-2 expression and is nearing a clinical trial. Important in clinical planning is the selection of patients on the basis of tumor expression of erbB-2. ErbB-2 gene amplification occurs in cancers of the breast, stomach, and ovary. Patients with these diseases and evident overexpression are candidates for OLX-209 therapy. In lung cancer, overexpression of erbB-2 is also frequent, but in most cases, it is not caused by gene amplification. This study demonstrates that OLX-209 has activity on lung cancer cells with varying levels of erbB-2 expression in the presence and absence of gene amplification. In vitro sensitivity of cell lines to OLX-209 is related to erbB-2 expression level. Normal bronchial epithelial cells were not sensitive. Effective treatment of lung cancer cell lines growing as xenografts in nude mice was shown with Calu-3 (a lung adenocarcinoma line with high levels of p185(erbB-2) caused by gene amplification) and three other lung adenocarcinomas (A549, NCI-H1466, and 201T) with lower levels of p185(erbB-2) and no gene amplification. The 201T cell line was isolated recently from a lung tumor with erbB-2 expression in the original tumor. The results of this study indicate that patients with erbB-2-positive, non-small cell lung cancer should be included in clinical trials of OLX-209.

Cytogenetic analysis of 63 non-small cell lung carcinomas: recurrent chromosome alterations amid frequent and widespread genomic upheaval.

A detailed cytogenetic analysis of 63 non-small cell lung carcinomas (NSCLCs) was carried out for identification of recurrent chromosomal alterations. Most specimens displayed very complex karyotypes with multiple numerical and structural changes (median number, 31). Losses of chromosomes 9 (65% of cases) and 13 (71%) were the most frequent numerical changes. Loss of the Y was often observed in tumors from males. Gain of chromosome 7 was also frequent (41%). Chromosome arms 1p, 1q, 3p, 3q, 6q, 7q, 8q, 9p, 11q, 17p, and 19q were particularly prone to rearrangement. The chromosome arm most often contributing to losses was 9p (79%). Other arms that were frequently lost included 3p, 6q, 8p, 9q, 13q, 17p, 18q, 19p, 21q, 22q, and the short arm of each acrocentric chromosome. The percentage of cases with loss of 3p was significantly higher in squamous cell carcinomas (94%) than in adenocarcinomas (60%). There was also a statistically significant increase in the proportion of cases with gains of 1q, 7p, and 11q in adenocarcinomas compared to squamous cell carcinomas. Several recurrent isochromosomes and unbalanced exchanges were found. Among these was i(5p), which was observed in nine tumors, eight of which displayed adenomatous features. An i(8q) was identified in six cases, including five adenocarcinomas. Double minutes and/or homogeneously staining regions were seen in seven specimens. These data indicate that numerous chromosome alterations contribute to the pathogenesis of NSCLC and that, amid this widespread genomic disarray, recurrent abnormalities exist that could have biological and clinical implications.