Yozo Kawano

Clinical Significance of p21 Expression in Non–Small-Cell Lung Cancer

PURPOSE The clinical significance of p21 expression remains unclear, whereas many experimental studies have demonstrated that p21, the product of the WAF1/CIP1/SDI1 gene, plays an important role in regulation of the cell cycle as an inhibitor of cyclin-dependent kinases. The purpose of this study was to clarify the clinical significance in resected non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS A total of 233 consecutive patients with completely resected pathologic stage I to IIIA NSCLC were retrospectively reviewed. Expression of p21 and the status of p53 were examined immunohistochemically. Proliferative activity was also evaluated immunohistochemically. The incidence of apoptotic cell death was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling staining. RESULTS Expression of p21 was positive in 120 patients (51.5%). The 5-year survival rate of p21-positive patients was 73.8%, significantly higher than that of p21-negative patients (60.7%; P =.006). Aberrant expression of p53 was positive in 98 patients (42.1%). When combined with p53 status, the prognostic value of p21 status was enhanced: the 5-year survival rate of p21-positive and p53-negative patients was 80.7%, markedly higher than that of p21-negative and p53-positive patients (50.0% for both; P =.001). Multivariate analysis confirmed that positive expression of p21 was a significant factor for predicting a favorable prognosis. There was no significant correlation between p21 expression and p53 status, proliferative activity, or incidence of apoptosis. CONCLUSION p21 expression was shown to be an independent prognostic factor in NSCLC.

Matrix metalloproteinase-2 status in stromal fibroblasts, not in tumor cells, is a significant prognostic factor in non-small-cell lung cancer

Purpose: The purpose is to assess clinical significance of matrix metalloproteinase (MMP)-2 and MMP-9 status, especially MMP-2 status, in stromal cells in non–small-cell lung cancer (NSCLC) because experimental studies have revealed that stromal MMP-2 plays important roles in progression of malignant tumors, but most clinical studies focused on tumoral MMP-2 expression, not stromal MMP-2 expression. Experimental Design: We conducted a retrospective study on MMP-2 and MMP-9 expression as evaluated immunohistochemically in a total of 218 consecutive patients with completely resected pathological stage I–IIIA, NSCLC. Results: Strong MMP-2 expression in tumor cells and stromal fibroblasts were documented in 54 (24.8%) and 132 (60.6%) patients, respectively. Strong MMP-2 expression in stromal fibroblasts was more frequently seen in squamous cell carcinoma (72.7%) than in adenocarcinoma (54.9%; P = 0.016). Tumors showing strong MMP-2 expression in stromal fibroblasts showed a significantly higher intratumoral microvessel density (IMVD) than weak stromal MMP-2 tumors (mean intratumoral microvessel density, 50.9 versus 32.4, P = 0.003). In addition, postoperative prognosis of strong stromal MMP-2 patients was significantly poorer than that of weak stromal MMP-2 patients (5-year survival rate, 77.5 versus 60.2%, P = 0.032), and the prognostic significance was enhanced in squamous cell carcinoma patients but disappeared in adenocarcinoma patients. Multivariate analyses confirmed that strong stromal MMP-2 expression was a significant factor to predict a poor prognosis in squamous cell carcinoma patients, not in adenocarcinoma patients. In contrast, MMP-2 or MMP-9 status in tumor cells was not a significant prognostic factor. Conclusions: MMP-2 status in stromal fibroblasts, not in tumor cells, was a significant prognostic factor associated with angiogenesis in NSCLC.

Prognostic Significance of Apoptotic Index in Completely Resected Non–Small-Cell Lung Cancer

PURPOSE To evaluate the significance of apoptotic index (AI) as a prognostic factor after surgery for non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS A total of 236 patients who underwent surgery for previously untreated pathologic stage I to IIIa NSCLC between 1985 and 1990 were reviewed. AI was defined as the number of apoptotic cells, detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling, per 1,000 tumor cells. Proliferative index (PI) and aberrant p53 expression were also evaluated immunohistochemically. RESULTS The 5-year survival rate for the lowest-AI group (AI < 5.0) was 74.7%; those for the lower-AI group (5.0 < or = AI < 11.0) and the higher-AI group (11.0 < or = AI < 25.0) were 51.6% and 57.8%, respectively. These survival rates were significantly lower than that of the lowest-AI group (P =.021 and P =.043, respectively). The highest-AI group (25.0 < or = AI), however, showed the most favorable prognosis, with a 5-year survival rate of 83.2%. Multivariate analysis confirmed that a moderate AI (5.0 < or = AI < 11.0 or 11.0 < or = AI < 25.0) was a significant factor to predict poor prognosis. The PIs for the lowest-, the lower-, the higher-, and the highest-AI groups were 32.3%, 48.0%, 54.3%, and 50.7%, respectively. The lowest-AI group showed a favorable prognosis because of its low PI, whereas the lower- and the higher-AI groups had a poor prognosis caused by increased cancer-cell proliferation. The highest-AI group showed the most favorable prognosis because apoptotic cell death overcame cell proliferation. No significant correlation was observed between AI and aberrant p53 expression. CONCLUSION AI proved to be an independent prognostic factor in NSCLC.

Correlation between apoptotic index and angiogenesis in non-small cell lung cancer: comparison between CD105 and CD34 as a marker of angiogenesis.

Only a few clinical studies have documented a significant correlation between intratumoral microvessel density (IMVD), a measurement of angiogenesis, and apoptotic index (AI), an incidence of apoptosis, although many experimental studies have confirmed that insufficient angiogenesis induces accelerated apoptotic cell death. In the present study, therefore, to assess AI in correlation with IMVD in resected non-small cell lung cancer, a total of 236 patients with pathologic stage I to IIIa were reviewed. IMVDs were determined immunohistochemically with an antibody against a pan-endothelial marker, CD34 (CD34-IMVD), and an antibody against a proliferation-related endothelial marker, CD105 (CD105-IMVD). AI was defined as the number of tumor cells positive for the terminal deoxynucleotidyl tranferase-mediated dUTP-biotin nick end-labeling staining per 1000 tumor cells. When CD34 was used as a marker of angiogenesis, the mean AIs for the lower-IMVD and the higher-IMVD patients were 20.1 and 17.5, respectively, demonstrating no significant difference between the lower- and the higher-IMVD patients. In contrast, when CD105 was used, the mean AI for the lower-IMVD patients was significantly higher than that for the higher-IMVD patients (22.0 and 15.6, respectively; P=0.019). There was no significant correlation between proliferative activity and CD34-IMVD or CD105-IMVD. These results demonstrated that that decreased angiogenesis may induce enhanced apoptotic tumor-cell death without affecting cell proliferation.

Prognostic Factors in Resected Pathologic (p-) Stage IIIA-N2, Non-Small-Cell Lung Cancer

BackgroundPostoperative prognosis for patients with pathologic (p-) stage IIIA-N2 non-small-cell lung cancer (NSCLC) is poor, and significant factors that influence the prognosis remain unclear.MethodsA total of 99 patients who underwent complete resection for p-stage IIIA-N2 NSCLC without any preoperative therapy were retrospectively reviewed. Biological features such as tumor angiogenesis (intratumoral microvessel density [IMVD]), proliferative activity (proliferative index [PI]), and p53 status were also evaluated immunohistochemically.ResultsUnivariate analysis revealed that the number of involved N2 stations was a significant prognostic factor; 5-year survival rates for a tumor with metastases in single N2 stations, tumor with metastases in two N2 stations, and tumor with metastases in 3 or more N2 stations were 41.6%, 35.3%, and 0.0%, respectively (P = .041) In addition, the 5-year survival rate for cN0-1 disease was significantly higher than that for cN2 disease (41.9% and 25.5%, respectively; P = .048) Tumor angiogenesis and proliferative activity were the most significant prognostic factors; 5-year survival rates for lower-IMDV tumor and higher-IMVD tumor were 53.6% and 15.9%, respectively (P = .002), and those for lower-PI tumor and higher-PI tumor were 47.0% and 20.4%, respectively (P = .019) There was no difference in the postoperative survival between tumor showing aberrant p53 expression and tumor showing no aberrant p53 expression. These results were confirmed by a multivariate analysis.ConclusionsP-stage IIIA-N2 NSCLC cases represented a mixture of heterogeneous prognostic subgroups, and the number of involved N2 stations, cN status, PI, and IMVD were significant predictors of the survival.

Apoptosis and p53 status predict the efficacy of postoperative administration of UFT in non-small cell lung cancer

To examine whether efficacy of postoperative oral administration of UFT, a 5-fluorouracil derivative chemotherapeutic agent, may be influenced by incidence of apoptosis (apoptosis index) or apoptosis-related gene status (p53 and bcl-2) of the tumour, a total of 162 patients with pathologic stage I non-small cell lung cancer were retrospectively reviewed. UFT was administrated postoperatively to 44 patients (UFT group), and not to the other 118 patients (Control group). For all patients, 5-year survival rate of the UFT group (79.9%) seemed higher than that of the Control group (69.8%), although without significant difference (P=0.054). For patients with higher apoptotic index, 5-year survival rate of the UFT group (83.3%) was significantly higher than that of the Control group (67.6%, P=0.039); for patients with lower apoptotic index, however, there was no difference in the prognosis between these two groups. Similarly, UFT was effective for patients without p53 aberrant expression (5-year survival rates: 95.2% for the UFT group and 74.3% for the Control group, P=0.022), whereas not effective for patients with p53 aberrant expression. Bcl-2 status did not influence the efficacy of UFT. In conclusion, apoptotic index and p53 status are useful factors to predict the efficacy of postoperative adjuvant therapy using UFT.

Primary lung carcinoma arising from emphysematous bullae

To clarify clinical characteristics and biological features of primary lung carcinoma arising from emphysematous bullae (EB), a total of 50 patients (49 males and one female) among all 1478 patients who underwent operation for primary lung carcinoma cases were reviewed; biological features were examined in 31 patients whose resected specimens were available for immunohistochemical staining (IHS). Thirty-one patients (62.0%) had pathologic stage I disease, and 30 cases (60.0%) had poorly differentiated tumor, demonstrating earlier pathologic stages and poorer cell differentiation of lung carcinoma with EB as compared with that without EB. The mean proliferative index (PI) for carcinoma with EB was 64.0%, which was significantly higher than that for carcinoma without EB (47.2%, P = 0.001); no significant difference in Apoptotic index (AI) was demonstrated. Aberrant p53 expression was less frequent in carcinoma with EB (29.0%) than in carcinoma without EB (47.9%, P = 0.043). Five-year survival rates for carcinoma with and that without EB were 50.3 and 46.9%, respectively, showing no significant difference. Multivariate analysis did not demonstrate that association of EB was a significant prognostic factor. In conclusion, although with the poorer cell differentiation and accelerated proliferative activity of lung carcinoma arising from EB, this does not have a significantly different prognosis than primary lung carcinoma not associated with bullae.

Apoptotic tumor-cell death in response to cell proliferation is influenced by p53 status in resected non-small cell lung cancer

To evaluate the influence of p53 status on postoperative survival and incidence of apoptosis (apoptotic index, AI) in non-small cell lung cancer (NSCLC), a total of 185 pathologic stage I patients were retrospectively analyzed. It was demonstrated by univariate and multivariate analyses that aberrant expression of p53 was a significant factor to predict a poor prognosis, which was caused by a significantly higher proliferative index (PI) in tumor with aberrant expression of p53 (52.7%) than that in tumor without aberrant expression of p53 (37.9%, P < 0.001). In addition, for tumor without aberrant expression of p53, mean AIs of the lowest-, the lower-, the higher-, and the highest-PI groups were 12.6, 12.9, 31.3, and 35.1, respectively, showing that incidence of apoptosis was markedly increased in response to cell proliferation (P = 0.002). In contrast, for tumor with aberrant expression of p53, no increase in incidence of apoptosis in response to cell proliferation was documented. These results clearly demonstrated that active cell proliferation and reduced apoptotic cell death in response to cell proliferation resulted in the poor postoperative prognosis in tumor with aberrant expression of p53.

Structural characterization and chromosomal localization of the MAGE-E1 gene

Genes of the melanoma-associated antigen (MAGE) family are characterized by the expression of tumor antigens on a malignant melanoma recognized by autologous cytolytic T lymphocytes. We have previously identified novel members of the MAGE gene family expressed in human glioma and named them MAGE-E1a-c. In the present study, we have revealed the genomic structure of MAGE-E1 by sequence analysis of a human chromosome bacterial artificial chromosome clone containing the MAGE-E1 gene. The MAGE-E1 gene is composed of 13 exons, and three of these (exon 2, exon 3 and exon 12) are alternatively spliced in each variant (E1a-c). The open reading frame encoding the MAGE-E1 peptides initiates in exon 2 and ends in exon 13. We have also demonstrated that the MAGE-E1 gene is located in Xp11 through the analysis of radiation hybrid panels. The genomic structure of MAGE-E1 is markedly similar to that of MAGE-D and its chromosomal locus is also identical to that of MAGE-D, but these features contrast with those of other MAGEs. These results suggest that MAGE-D and -E1 may be evolutionarily distant from other members of the MAGE family, and the two may be ancestral genes for the others.

P-132 RECK expression in non-small cell lung cancer

Background: The RECK (reversion-inducing cysteine-rich protein with Kazal motifs) gene isolated as a reversion-inducing gene encodes a membraneanchored glycoprotein of about 110 kDa. RECK negatively regulates matrix metallogroteinases (MMP-2, MMP-9, MT1 -MMP), and restored expression of RECK gene inhibits the invasive or metastatic activities of tumor cells (PNAS 95:13221-6,1998; Cell 107: 789-800, 2001). The RECK gene is widely expressed in non-malignant cell lines and various human organs, but its expression is low or undetectable in a number of transformed or tumor-derived cell lines. In our study, we have focused on the expression levels of RECK in lung cancer cell lines and tissues. Methods: Western blot assay and real-time RT-PCR assay were used to assess the amount of the RECK protein and mRNA, respectively, in eight cell lines derived from human lung cancer (H146, H1299, H460, H209, H441, H522, A549, PC14) and normal human lung fibroblasts (NHLF). RECK gene expression in paired tumor and normal lung tissues obtained from 32 patients with NSCLC were also analyzed by RT-PCR. GAPDH mRNA was used as an internal control for RT-PCR. Results: Western blot assay revealed that the level of RECK expressed in NHLF was as high as in MRC-5, a normal human fibroblast cell strain known to express RECK at a high level. By contrast, the RECK protein was barely detectable in all eight lung cancer cell lines. The RT-PCR assay also revealed that RECK gene expression was low in lung cancer cell lines and high in NHLF; the amounts of RECK mRNA detected in the lung cancer cell lines ranged 0.1 to 13.2% of that detected in NHLF. The amounts of RECK mRNA in tumor tissues from lung cancer patients (RECWGAPDH: 0.0015~0.0013) were significantly lower than those in the surrounding non-tumorous tissues (REUVGAPDH: 0.0054~0.0032) (P<O.OOOl). Conclusions: RECK expression was down-regulated in lung cancer cell lines and surgical specimens from lung cancer patients. These results suggest that RECK may serve as a useful marker to distinguish between tumor cells and normal tissues in lung cancer patients.