[Advanced thymic carcinoma effectively treated by surgical resection and postoperative radiation therapy: report of a case].

Thymic carcinomas are rare neoplasms, and standard treatment has not yet been established. We reported a case of advanced thymic carcinoma effectively treated by surgical resection and postoperative radiation therapy. A 71-year-old man was pointed out an abnormal shadow on chest X-ray. Chest computed tomography (CT) scan demonstrated an anterior mediastinal tumor. The tumor was diagnosed as carcinoma by CT-guided tumor biopsy and was extirpated completely with combined partial resection of the left lung. Microscopically, the tumor was diagnosed as thymic carcinoma with direct invasion to the left lung. Following postoperative radiation therapy, the patient is doing well without apparent recurrence 5 years after surgery.

[Peripheral small squamous cell carcinoma (9 mm) of the lung with metastasis to mediastinal lymph nodes].

A 61-year-old man was pointed out a small peripheral lung nodule and mediastinal lymph node swelling on the chest computed tomography (CT). At the operation, it was diagnosed squamous cell carcinoma and right upper lobectomy and nodal dissection were done. The tumor was 9 mm in size and diagnosed as well differentiated squamous cell carcinoma with metastasis to mediastinal lymph nodes. Postoperative radiotherapy was done (50 Gy). The patient is doing well without apparent recurrence 33 months after surgery. We reported a case of peripheral small squamous cell carcinoma (9 mm) of the lung with metastasis to mediastinal lymph nodes.

Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor gene expression.

BACKGROUND: Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene is frequently associated with malignant gliomas. One type of EGFR mutation in primary gliomas results in overexpression of an aberrant EGFR messenger RNA (mRNA) that lacks sequences of exons II through VI of the human EGFR gene. We observed that the aberrantly spliced EGFR mRNA contains a ribozyme cleavable sequence (5'-AAG GUA AUU-3') created by the joining of EGFR exon I to exon VII. We hypothesized that an appropriately designed ribozyme RNA could mediate site-specific cleavage of the aberrant EGFR mRNA and reduce the growth of aberrant EGFR-producing tumor cells. METHODS: We synthesized aberrant EGFR mRNA substrates and a sequence-specific hammerhead ribozyme (abEGFR-rib) to examine the ribozyme's activity in vitro. We also constructed an abEGFR-rib plasmid and introduced it into ERM5-1 cells, which are murine NIH3T3 cells transfected to express an aberrant EGFR complementary DNA. We measured the growth potential of the cotransfected cells in culture and in nude mice. RESULTS: The synthesized abEGFR-rib efficiently and specifically cleaved aberrant EGFR mRNA substrates in vitro. Expression of the transfected abEGFR-rib suppressed expression of aberrant EGFR mRNA in ERM5-1 cells and reduced the growth of tumors formed by the cotransfected cells in nude mice. Finally, the incorporation of bromodeoxyuridine, a measure of mitotic activity, was also decreased in abEGFR-rib-producing ERM5-1 cells in vivo. CONCLUSION: Ribozymes targeted to aberrant EGFR mRNA can inhibit the growth of tumors formed by cells that express this mRNA.

Thrombospondin 2 gene expression is correlated with decreased vascularity in non-small cell lung cancer.

Stromal vascularity is thought to be a major factor involved in the progression of carcinoma. However, the crucial mechanisms of vascularization in the stroma are not well understood. Vascularity could be regulated by various cytokines produced by neoplastic or stromal cells in carcinoma. Thrombospondin (TSP) has an inhibitory role against vascularization in vitro, although the biological significance of TSP has not been characterized in vivo. We examined expression of TSP1 and TSP2 genes in 78 non-small cell lung cancers (NSCLCs) and 33 extraneoplastic lung tissue samples by reverse transcription-PCR. TSP1 expression was detected in 66.7% (52 of 78) of NSCLCs and in 69.7% (23 of 33) of extraneoplastic lung tissue specimens. TSP2 expression was seen in 48.7% (38 of 78) of NSCLCs, whereas 72.7% (24 of 33) of extraneoplastic lung tissue samples showed TSP2 gene expression. TSP2 expression was significantly decreased in NSCLC as compared with extraneoplastic lung tissue (chi2 test, P=0.019). Vascularity in the NSCLC was inversely correlated with TSP2 gene expression (Mann-Whitney U test, P=0.009). Patients with adenocarcinoma positive for TSP2 gene expression (22 of 49) showed significantly better prognosis than those without TSP2 (27 of 49; Cox-Mantel test, P=0.034). TSP1 expression showed no apparent correlation with these factors. These results suggested that TSP2 had an inhibitory role against vascularization and progression of NSCLC.

Significant correlation between interleukin 10 expression and vascularization through angiopoietin/TIE2 networks in non-small cell lung cancer.

The expression of interleukin 10 (IL-10) is correlated with clinical prognosis in non-small cell lung cancer [NSCLC (H. Hatanaka et al., ANN: ONCOL:, 11: 815--819, 2000)]. However, the effects of IL-10 expression on vascularization in NSCLC are not apparent. We examined the gene expression of IL-10/IL-10 receptor and various angiogenic/angioinhibitory factors in 95 NSCLC samples to determine the correlation between IL-10 production and vascularization. Vascular endothelial growth factor, angiopoietin [Ang (Ang-1 and Ang-2)], thrombospondin, brain-specific angiogenesis inhibitor 1, vascular endothelial growth factor receptors (KDR and flt-1), and Ang receptor (TIE2) gene expression were evaluated by reverse transcription-PCR. The cellular localization of these factors and vascularity in the cancer stroma were examined immunohistochemically. Seventy-eight (82.1%) and 93 (97.9%) of these 95 NSCLCs were positive for IL-10 and IL-10 receptor, respectively. Ang-1, Ang-2, and TIE2 gene expression was seen in 76 (97.4%), 73 (93.6%), and 78 (100%) of 78 IL-10-positive NSCLCs, respectively, and was significantly correlated with IL-10 gene expression (P < 0.0088, <0.0008, and 0.0305, respectively; Fisher's exact method). The localizations of Ang-1, Ang-2, and TIE2 were confirmed within tumor cells immunohistochemically. Vascular number and measurement area were significantly higher in the IL-10-positive NSCLCs (33.500 +/- 9.299/microm(2) and 4.742 +/- 1.287%) as compared with IL-10-negative NSCLCs (10.611 +/- 2.839/microm(2) and 0.718 +/- 0.331%; Mann-Whitney U test, P = 0.0039). The IL-10 expression did not show any significant correlation with the expression of other factors. These results suggested that tumor-produced IL-10 promotes stromal vascularization through expression of Ang-1, Ang-2, and TIE2.

Adenovirus-mediated anti-K-ras ribozyme induces apoptosis and growth suppression of human pancreatic carcinoma.

Human pancreatic cancer is a lethal malignancy, and the lesions show a very high incidence of point mutations of the K-ras oncogene. These alterations can be used as potential targets for specific ribozyme (Rz)-mediated growth suppression of the cancer cells. We designed an anti-K-ras Rz against mutant K-ras gene transcripts (codon 12, GGT to GTT) and generated a recombinant adenovirus (rAd) to express the Rz (rAd/anti-K-ras Rz). More than 95% of Capan-1 human pancreatic cells were infected with rAd/anti-K-ras Rz when treated with the virus at 200 plaque-forming units/cell. The virus, rAd/anti-K-ras Rz, significantly suppressed mutant K-ras gene expression and inhibited the growth of Capan-1 cells. At 3 days postinfection, we observed maximum growth suppression of the cells, characteristic morphological changes of apoptosis such as nuclear condensation and oligonucleosomal DNA fragmentation, and suppression of bcl-2 oncoprotein. These changes were not found in control virus-infected cells. Our results indicated that the virus rAd/anti-K-ras Rz specifically down-regulated the K-ras/bcl-2 pathway and induced apoptotic changes in Capan-1 pancreatic carcinoma cells. High-efficiency adenovirus-mediated delivery of anti-K-ras Rz could become a significant gene therapy strategy against human pancreatic cancer.

Thrombospondin-2 (TSP2) expression is inversely correlated with vascularity in glioma.

Thrombospondins (TSPs) are angiostatic factors in various cancers. However, the significance of TSPs has not been well characterised in glioma. We examined TSP1, TSP2 and vascular endothelial growth factor (VEGF) gene expression by reverse transcription-polymerase chain reaction (RT-PCR) in 37 gliomas. Thirty of the 37 glioma specimens showed VEGF gene expression. Eighteen of the 37 gliomas expressed the TSP1 gene. Seven gliomas lacked TSP2 gene expression, while the other 30 expressed TSP2. The lack of TSP2 gene expression was significantly associated with higher histological grade (Fisher's test, P = 0.0019) and increased vessel counts and density (Student's t-test, P < 0.0001), while there were no correlations between TSP1 and VEGF gene expression and clinicopathological features. These results indicate that the lack of TSP2 gene expression is a potent factor for enhancement of angiogenesis in glioma.

Expression pattern of vascular endothelial growth factor isoform is closely correlated with tumour stage and vascularisation in renal cell carcinoma.

Vascular endothelial growth factor (VEGF) has five isoforms (VEGF206, 189, 165, 145 and 121). Increased VEGF expression in renal cell carcinoma (RCC) is associated with angiogenesis, but, it is not apparent which isoform is involved in this effect. We examined the isoform patterns of VEGF by reverse transcription-polymerase chain reaction (RT-PCR) in 47 RCCs. All showed increased VEGF expression as compared with extraneoplastic renal tissue. Four of the 47 RCCs showed VEGF121 alone, 10 showed VEGF121 + 165, and 33 showed the VEGF121 + 165 + 189 pattern. Patients with pathological stage pT3-4 RCC showed the VEGF121 + 165 + 189 isoform pattern at a significantly higher incidence (10/10, 100%) than those with pT0-2 (23/37, 62%) (P < 0.022). The VEGF121 + 165 + 189 isoform pattern was also significantly associated with high vessel counts and density (P = 0.0002, Mann-Whitney U test). These observations suggested that the VEGF189 mRNA isoform is closely associated with angiogenesis and results in the growth of RCC.

Ribozyme approach to downregulate vascular endothelial growth factor (VEGF) 189 expression in non-small cell lung cancer (NSCLC).

The aim of this study was to further clarify the role of the cell-associated isoform of vascular endothelial growth factor (VEGF189) on tumour growth and vascularity. Five isoforms of VEGF have been identified with different biological activities. VEGF121, VEGF145, VEGF165, VEGF189, VEGF206 are generated by alternative splicing. We used a hammerhead-type ribozyme (V189Rz) to suppress VEGF189 mRNA. The V189Rz specifically cleaved exon 6 of VEGF189 mRNA, but showed no activity against the VEGF121 or VEGF165 isoforms. The V189Rz was introduced into the human non-small cell lung cancer (NSCLC) cell line (OZ-6/VR). The expression level of VEGF189 mRNA was decreased in the OZ-6/VR cells, while VEGF121 and 165 expression was unaltered. The OZ-6/VR cells xenotransplanted into nude mice showed markedly reduced vascularisation and growth, whereas the cell line did not show any decreased growth under tissue culture conditions. The OZ-6/VR cells (1 x 10(5) cells/mouse) formed no tumours, whereas the parental OZ-6 cells formed large tumours within 8 weeks. The specific suppression of VEGF189 by the ribozyme decreased vascularity and xenotransplantability of the lung cancer cell line. Thus, the cell-associated isoform of VEGF, VEGF189, might have a key role in stromal vascularisation and the growth of NSCLC xenografts in vivo.

Growth stimulation of non-small cell lung cancer xenografts by granulocyte-macrophage colony-stimulating factor (GM-CSF).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been suggested to be involved in the carcinogenesis of some types of tumours by autocrine or paracrine mechanisms. We examined GM-CSF/GM-CSF receptor (GM-CSFR) gene expression in 20 human non-small cell lung cancer (NSCLC) xenografts. The stimulatory effects of GM-CSF were examined using GM-CSF transgenic severe combined immunodeficient (SCID) mice (GM-Tg-SCID), which produce abundant human GM-CSF. A NSCLC xenograft (LC11-JCK), expressed GM-CSFR but not GM-CSF, and showed more rapid growth in GM-Tg-SCID than non-GM-CSF transgenic SCID mice (non-Tg-SCID). GM-CSF gene expression was detected in 48 of 90 (53%) primary NSCLC human specimens and GM-CSFR gene expression was detected in 42 specimens (47%). GM-CSF expression was detected in 13 of 30 squamous cell carcinoma specimens (43%) and GM-CSFR expression was detected in 10 specimens (33%). Patients with squamous cell carcinoma coexpressing GM-CSF and GM-CSFR showed significantly poorer prognosis than those expressing neither GM-CSF nor GM-CSFR (P < 0.05, Cox-Mantel test). These results suggest that GM-CSF can have a stimulatory effect on some NSCLC.

Cell-retained isoforms of vascular endothelial growth factor (VEGF) are correlated with poor prognosis in osteosarcoma.

Vascular endothelial growth factor (VEGF) is a major angiogenic factor. Osteosarcoma is characterised by hypervascularity and metastatic potential. We examined VEGF mRNA expression, VEGF isoform pattern and VEGF receptor (flt-1 and KDR) by RT-PCR analysis in 30 osteosarcomas. All 30 osteosarcomas expressed VEGF mRNA. 17 osteosarcomas (57%) expressed flt-1 mRNA, whilst 20 (67%) expressed KDR mRNA. 6/30 (20%) osteosarcomas were positive for VEGF121 only, 8 (27%) for VEGF121 + VEGF165, and 16 (53%) for VEGF121 + VEGF165 + VEGF189. Patients with osteosarcomas with VEGF165 (n = 24) had significantly poorer prognosis in comparison with those without VEGF165 (P = 0.022, Wilcoxon's test). The osteosarcomas with VEGF165 had significantly increased vascularity assessed on sections immunostained for CD34 (P < 0.001, Mann-Whitney U test). Although VEGF165 is a soluble isoform, it is also retained on the cellular surface. These results suggest that cell-retained VEGF isoforms (VEGF165, VEGF189) might be essential for neovascularisation in osteosarcoma, whilst the soluble VEGF121 isoform is not sufficient to stimulate neovascularisation in this type of neoplasm.

Multidrug resistance-associated protein (MRP) expression is correlated with expression of aberrant p53 protein in colorectal cancer.

Multidrug resistance-associated protein (MRP) is one of the major factors responsible for non-P-glycoprotein (Pgp)-mediated multidrug resistance of human tumour cells. In this study, we examined MRP and aberrant p53 expression in 54 colorectal cancers (CRC), 35 carcinoma in adenomas (CIA) and 40 adenomatous polyps by immunohistochemical procedures. 38 of 54 (70%) CRCs, 16 of 35 (46%) CIAs and 3 of 40 (8%) adenomatous polyps were MRP positive (chi 2 test, P < 0.0001). 36/54 (67%) CRCs, 10/35 (29%) CIAs and 0/40 adenomatous polyps were p53 positive. 30 of the 36 p53-positive CRCs were also MRP positive and 8/10 CIAs were both p53 and MRP positive. MRP overexpression correlated with aberrant p53 accumulation in CRCs and CIAs (chi 2 test, P < = or 0.01). Coexpression of MRP and p53 in the same cells was confirmed in the CRCs and CIAs by double staining procedures. These results suggested that MRP overexpression is related to aberrant p53 expression in CRC.

Expression of cell-associated isoform of vascular endothelial growth factor 189 and its prognostic relevance in non-small cell lung cancer.

Vascular endothelial growth factor (VEGF) has four isoforms (VEGF121, VEGF165, VEGF189, VEGF206). We examined the expression patterns of VEGF isoforms in non-small cell lung cancer (NSCLC) by RT-PCR. The cell-associated isoform VEGF189 was more frequently expressed in NSCLC (90.5%) than in extraneoplastic lung tissue (57.6%, p=0.00004). Immunohistochemical examination confirmed VEGF protein expression in 33 of 76 NSCLC expressing VEGF189 (V189p+ group). The V189p+ group showed significantly poorer prognosis than those without VEGF189 mRNA and protein (V189p- group, p=0.01722). These results suggest that expression of the cell-associated isoform VEGF189 is closely associated with progression of NSCLC.

Expression of angiostatic factors in colorectal cancer.

Angiogenesis plays an important role in growth and proliferation of cancer. Various angiogenic and angiostatic factors regulate angiogenesis. We examined expression of genes encoding various angiostatic factors: thrombospondin 1 (TSP1), thrombospondin 2 (TSP2), brain-specific angiogenesis inhibitor 1 (BAI1) and angiopoietin 2 (AGP2) in 62 colorectal cancers and 40 samples of extraneoplastic colon mucosa. The expression of the angiostatic factors TSP2 and AGP2 were significantly increased in the cancerous mucosa as compared to these in extraneoplastic mucosa (o2 test; p<0. 0001, and Fisher's exact test; p<0.0001), while the increase in TSP1 expression was not significant. BAI1 expression was slightly decreased in the cancer tissue. These results suggested that specific types of angiostatic factors might have protective roles against cancer cell proliferation via dormancy due to hyponutrition caused by decreased vascularity.

Brain-specific angiogenesis inhibitor 1 expression is inversely correlated with vascularity and distant metastasis of colorectal cancer.

Brain-specific angiogenesis inhibitor (BAI) 1 was recently isolated as a novel p53 inducible gene. BAI1 has been suggested to play a significant role in angiostasis. We studied the expression of BAI1 in 49 colorectal cancer specimens by RT-PCR. BAI1 expression was significantly reduced in colorectal cancers as compared to the extraneoplastic tissues (X(2) test, p=0.041). BAI1 expression was inversely correlated with vascular invasion and metastasis (Fisher's exact test, p 0.045). Moreover, vascularity in the colorectal cancer was inversely correlated with BAI1 gene expression (Mann-Whitney U-test, p=0.0003). These observations suggested that BAI1 expression might inhibit angiogenesis and metastasis of colorectal cancer.

Expression of the thrombospondin 1 receptor CD36 is correlated with decreased stromal vascularisation in colon cancer.

Vascularisation is determined by the balance between various angiogenic and angio-inhibitory factors in cancer stroma. CD36 is a cell adhesion receptor shown to interact with the ligand thrombospondin 1 (TSP1). It has not been determined whether the interaction of TSP1 and its receptor CD36 is correlated with vascularisation or clinical outcome of malignant tumours in vivo. We studied the correlations between expression of TSP1, CD36 and vascularisation and prognosis in 65 colon cancers. Various levels of TSP1 gene expression were observed in 27 of 65 colon cancers. CD36 expression was detected in 33 of 65 cancers, and was significantly correlated with decreased stromal vascularisation (P<0.001). The colon cancers expressing CD36 showed better prognosis (P<0.01). TSP1 expression levels did not affect CD36 expression. These results suggested that CD36 expression which decreased stromal vascularisation is correlated with better prognosis of colon cancer.

Multidrug resistance mediated by overexpression of P-glycoprotein in human osteosarcoma in vivo.

We examined the expression levels of P-glycoprotein (P-Gp)/the human multidrug resistance gene (MDR1) and in vivo chemosensitivity in the 7 osteosarcoma xenografts. Three of seven (43%) osteosarcoma xenografts expressed MDR1 by reverse transcriptase-polymerase chain reaction (RT-PCR). The OSS-516R xenograft selected with vincristine (VCR) from the MDR1-negative xenograft OSS-516, which was sensitive to VCR and doxorubicin (DOX), acquired cross-resistance to DOX. In the OSS-516R, RT-PCR assay showed definite MDR1 expression and immunohistochemical analysis demonstrated P-Gp-positive tumor cells. These results suggest that P-Gp/MDR1 overexpression is related to multidrug resistance in human osteosarcoma in vivo.

A xenograft line of human teratocarcinoma established by serial transplantation in severe combined immunodeficient (SCID) mice.

We established a xenograft line of human teratocarcinoma (TC-1) and characterized the pluripotency of differentiation of the neoplastic cells. A teratocarcinoma specimen obtained from a primary mediastinal lesion (22-year-old male patient) was inoculated subcutaneously into severe combined immunodeficient (SCID) mice. The carcinoma formed tumors in the mice. We established a xenograft line by serial passage of the tumor in vivo. The primary tumor was composed of papillary and pseudoglandular nests of highly atypical epithelial cells with foci of glomeruloid structures. The metastatic cells showed apparent production of mucin and differentiation to striated muscle. The xenograft line TC-1 retained the basic histopathological features seen in the primary and metastatic cells. The xenograft line showed focal differentiation to cartilage through serial passages. Immunohistochemical studies with anti-alpha-fetoprotein (AFP) demonstrated positive immunoreactivity on the TC-1 cells. Serum AFP levels were also elevated in the TC-1-bearing SCID mice. The human teratocarcinoma xenograft line TC-1 will be useful for studying the differentiation mechanism in human totipotent stem cells.

Establishment of an adrenocortical carcinoma xenograft with normotensive hyperaldosteronism in vivo.

We established a xenograft line of human adrenocortical carcinoma (ADR-1), and analyzed the hyperaldosteronism induced by the xenograft in vivo. Adrenocortical carcinoma specimens from a 25-year-old woman were subcutaneously inoculated into nude mice (BALB/c-nu/nu) followed by serial passages in vivo. ADR-1 retained the histopathological features (trabecular and sinusoid nests) seen in the primary carcinoma. The patient showed hyperaldosteronism (serum aldosterone >4000 pg/ml) and hypokalemia (serum K 2.1 mEq/l), but did not show hypertension. The nude rat (F344-rnu/rnu) bearing ADR-1 showed hyperaldosteronism (serum aldosterone 3320+/-1420 pg/ml; control 191+/-130 pg/ml) and hypokalemia (serum K 3.4+/-0.4 mEq/l; control 5.2+/-1.0 mEq/l) in vivo, and hypertension was not obvious. ADR-1 was shown immunohistochemically to retain production of human-specific corticosteroid synthetase. The xenograft ADR-1 will be useful to elucidate the regulatory mechanism of normotensive hyperaldosteronism.

Alterations in tumour suppressor gene p53 correlate with inhibition of thrombospondin-1 gene expression in colon cancer cells.

If activation of the p53 gene is involved in the progression or metastasis of colon cancer, it may affect the angiogenic phenotype in vivo. To verify this hypothesis, we studied the correlation between p53 accumulation and expression of thrombospondin-1 (TSP1) in colon cancer specimens. Levels of TSP1 gene expression were estimated by Northern blotting in 65 colon cancers. Accumulation of p53 and the distribution of TSP1 protein were evaluated immunohistochemically. Various levels of TSP1 gene expression were seen in colon cancers, while p53 accumulation was confirmed in 42 of the 65 colon cancers. The level of TSP1 gene expression demonstrated a significant inverse correlation with p53 accumulation in colon cancer. Colon cancer cells expressed TSP1 protein and p53 accumulation reciprocally in the same nests. These results suggest that alterations in the tumour suppressor gene p53 may inhibit TSP1 expression in colon cancer.