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BACKGROUND The ras oncogene and the p53 tumor suppressor gene play important roles in the carcinogenic process of lung carcinoma. The authors evaluated whether alterations of the ras and p53 proteins may contribute to the development of lung carcinoma in patients with idiopathic pulmonary fibrosis (IPF) and whether such alterations may explain the high incidence of lung carcinoma among patients with IPF. METHODS Lung tissues were obtained from 35 patients who had IPF without complications of lung carcinoma and from 36 patients who had IPF with complications of lung carcinoma. Altered expression of ras and p53 proteins was evaluated by immunohistochemistry, and mutations of both genes were evaluated by polymerase chain reaction-single strand conformation polymorphism and sequencing analyses. RESULTS The frequency of expression of ras protein in type II alveolar pneumocytes was significantly greater in lung tissues from patients with IPF who had lung carcinoma compared with lung tissues from patients with IPF who did not have lung carcinoma (75% vs. 40%, respectively; P < 0.01). K-ras point mutation in codon 12 (GGT to GTT transversion) was detected in lung tissue with interstitial pneumonia, in which ras protein was overexpressed in type II alveolar pneumocytes obtained from 2 of 41 patients with IPF complicated by lung carcinoma, causing amino acid substitution (Gly to Val) in both patients. A p53 mutation was detected in three of six lung tissue samples from patients who had IPF lung with positive p53 immunoreactivity, and multiple mutations were detected in two samples. CONCLUSIONS Expression of ras protein in type II alveolar pneumocytes and mutation in the codon 12 of K-ras gene in lung tissue may contribute to the induction of lung carcinoma in patients with IPF. Furthermore, the presence of multiple mutations in the p53 gene may explain the high incidence lung carcinoma in patients with IPF. Cancer 2002;95:624–33. © 2002 American Cancer Society. DOI 10.1002/cncr.10708

BACKGROUND With the recent development of minimal treatment for early stage gastric carcinoma, identifying specific indicators of the metastatic potential of primary tumors has become more important. Cathepsin B and cathepsin L, both lysosomal cysteine proteases, degrade the extracellular matrix during tumor progression. Although many studies have shown their relation to human cancer progression, little is known about their roles in the early stage. The clinicopathologic significance of cathepsins was therefore studied in early stage gastric carcinoma. METHODS Expression of both cathepsins was studied immunohistochemically in 51 tissue specimens from gastric carcinomas that invaded the submucosal layer or muscularis propria. The relation between their expression and clinicopathologic factors was analyzed. RESULTS Both cathepsins were expressed at higher levels in tumors that invaded the muscularis propria than in those within the submucosa (P < 0.05). In addition, tumors with lymphatic invasion showed higher cathepsin B expression than those without it (P < 0.05), whereas tumors with venous invasion showed higher cathepsin L expression than those without it (P < 0.05). No other clinicopathologic factors correlated with expression of either cathepsin. CONCLUSIONS Tumors with overexpression of cathepsins have powerful potential for invasiveness in the early stage of gastric carcinoma. Moreover, the authors hypothesize that cathepsins may be one of the determinants of the metastatic route. To the authors' knowledge, this is the first report on specific proteases concerning the mode of metastasis, and the results of this study suggest that therapeutic strategies for early stage gastric carcinoma might need to be changed according to the status of cathepsins. Cancer 2000;89:482–7. © 2000 American Cancer Society.

BACKGROUND Macrophage migration inhibitory factor (MIF) is known to be a proinflammatory cytokine and glucocorticoid-induced immunomodulator as well as a regulator of tumor growth. Although positive and negative effects of MIF on tumor cell growth have been reported, to the authors' knowledge the precise role of MIF in tumorigenesis remains unclear. In the current study the authors assessed expression of MIF protein and mRNA in lung adenocarcinomas with regard to patient prognosis. METHODS Immunohistochemical analysis was performed on tissue specimens surgically obtained from 74 patients with primary lung adenocarcinoma (American Joint Committee on Cancer pathologic Stages I, II, and IIIa). In addition, expression of MIF mRNA in the cancerous tissue was investigated using in situ hybridization. Patient prognosis was evaluated with regard to MIF expression levels and its distribution was analyzed with the Kaplan–Meier method. RESULTS MIF mRNA and MIF protein were observed in the bronchial epithelium, alveolar epithelium, vascular smooth muscle, and alveolar macrophages in the normal lung tissue. In tumor tissue from lung adenocarcinoma specimens, both MIF mRNA and protein were observed at much higher levels than in the normal alveolar epithelium. MIF protein was observed diffusely in the cytoplasm of tumor cells in all tumor specimens examined. MIF protein also was observed in the nuclei of tumor cells from 59 patients (79.7%), whereas it was not observed in the nuclei of tumor cells from 15 patients (20.3%). The patients without nuclear MIF expression had a worse prognosis compared with those patients with MIF expression in the nuclei (P = 0.04). CONCLUSIONS The results of the current study suggest that intracellular MIF distribution predicts patient prognosis in individuals with adenocarcinoma of the lung. Cancer 2000;89:334–41. © 2000 American Cancer Society.

BACKGROUND The uptake of thallium-201 (Tl-201) in malignant tumors is associated with malignant potential (metastatic potential and proliferative activity). The grade of accumulation of Tl-201 in malignant tumors may provide information regarding prognosis. METHODS A Tl-201 single photon emission computed tomography was conducted 120 minutes after intravenous injection of 111 megabecquerel of Tl-201 chloride. The authors calculated the uptake ratio to evaluate the degree of Tl-201 uptake in the primary tumor. This ratio was compared with survival time and other prognostic factors. RESULTS The authors studied 152 patients (125 men and 27 women). The group of patients with the low Tl-201 uptake ratio survived longer than the group of patients with the high Tl-201 uptake ratio (median survival, 58 weeks vs. 33 weeks; P = 0.0138 by the log rank test). The multivariate analysis confirmed that the Tl-201 uptake ratio was an independent prognostic factor for survival. CONCLUSIONS These results suggest that the Tl-201 uptake ratio provides independent and objective prognostic information for patients with lung carcinoma. Cancer 1997; 80:198-203. © 1997 American Cancer Society.

BACKGROUND Inactivation of the retinoblastoma (Rb) gene has been documented in various types of cancer, including lung cancer. Alterations of the p53 and ras genes are also common features in the molecular biology of lung carcinoma, and the authors of this article have reported previously on the prognostic significance of both of them. In the present study, the authors evaluated the prognostic significance of the loss of Rb protein expression alone, then performed a combined analysis of Rb protein and ras p21 status (Rb/ras) as well as an analysis of Rb and p53 protein status (Rb/p53) in patients with nonsmall cell lung cancer (NSCLC). METHODS Ninety-one patients with NSCLC underwent potentially curative resection between 1977 and 1988, 65 of whom received postoperative combination chemotherapy. Tumor specimens were analyzed for Rb protein expression by immunohistochemistry. Univariate and multivariate analyses were performed to assess the association between Rb protein expression and survival. RESULTS Nineteen (21%) of the 91 NSCLCs showed negative Rb protein expression. Positive or negative Rb protein expression (Rb+ or Rb-) as an individual factor was not statistically correlated with survival or prognosis in this cohort of NSCLC patients, although a tendency among Rb- patients to do worse was observed. The authors then combined the Rb protein status with previously studied results of ras p21 and p53 protein expression in the same tumor specimens, and compared the prognosis between the individuals with theoretically the best pattern of gene expression in their tumors and those with theoretically the worst pattern of expression, i.e., Rb+/ras- versus Rb-/ras+ and Rb+/p53- versus Rb-/p53+. In patients with adenocarcinoma, those with Rb-/ras+ tumors survived for a significantly shorter period after surgery (13% 5-year survival) than those with Rb+/ras- tumors (82% 5-year survival) (P = 0.01). Similarly, patients with Rb-/p53+ tumors survived for a significantly shorter period (20% 5-year survival) compared with those who had Rb+/p53- tumors (73% 5-year survival) (P = 0.008). Rb/ras status was a significant prognostic factor (P = 0.02 by univariate analysis, P = 0.048 by multivariate analysis), and Rb/p53 status tended to be significant as a prognostic factor (P = 0.04 by univariate analysis, P = 0.08 by multivariate analysis). In patients with squamous cell carcinoma, neither Rb/ras nor Rb/p53 status was a significant prognostic factor in this cohort. CONCLUSIONS These results suggest that combined immunohistochemical analyses of Rb and ras p21 proteins and of Rb and p53 proteins may indicate their potentially synergistic effects on survival and prognosis. These analyses may also be useful for stratifying patients with adenocarcinoma of the lung into different prognostic groups and identifying populations with different risks of recurrence. Larger prospective studies with Stage I NSCLC patients are necessary to confirm the current findings. Cancer 1997; 79:1329-37. © 1997 American Cancer Society.

Background. Alterations of the p53 gene are one of the most common genetic changes in various types of cancer, including lung cancer. Abnormalities in the ras genes, including point mutations and overexpression, are another common feature in the molecular biology of lung cancer and are associated with a poorer prognosis. The authors' purpose was to determine expression of the mutated p53 gene in nonsmall cell lung cancer (NSCLC) specimens that were studied for expression of ras p21 and to document whether altered p53 expression was also an important factor for survival. Methods. Ninety-six patients with NSCLC underwent surgical resection between 1977 and 1985, 63 of whom received postoperative combination chemotherapy. None received radiation therapy. Tumor specimens were analyzed for altered p53 expression by immunohistochemistry. Univariate and multivariate analyses were performed to assess the association between p53 expression and survival. Results. Fifty-six (58%) of 96 tumor specimens showed altered p53 expression, and 91 patients were analyzed for survival. Altered p53 expression did not correlate with clinicopathologic characteristics except for postsurgical pathologic tumor (pT) classification. The patients with altered p53 expression survived for a significantly shorter period after surgery than those without p53 expression, including all patients who underwent resection and potentially curative resection (P = 0.02 and P = 0.048, respectively, generalized [Wilcoxon test]. Multivariate analysis showed independent prognostic significance for altered p53 expression (hazard ratio [HR] = 1.72, P = 0.04) and surgical cure (HR = 4.69, P < 0.001). The combined analysis of mutated p53 and ras p21 expression in the same tumor specimens revealed that patients with p53- and ras p21-negative tumors survived the longest among those with different p53 and ras p21 features (P = 0.005, generalized Wilcoxon test). Conclusion. Altered p53 expression is a significant and independent negative prognostic factor for patients with surgically resected NSCLC. Combined immunohistochemical analysis of mutated p53 and ras p21 expression can divide patients with NSCLC into more accurate prognostic groups. If the current findings can be confirmed in larger prospective studies, combined immunohistochemical analysis of mutated p53 and ras p21 expression can be a useful clinical tool for stratifying patients with NSCLC into accurate prognostic groups and for identifying the population with a different risk of recurrence.

BACKGROUND Hematogenous metastasis requires angiogenesis within the tumor. Previous studies have shown that microvessel counts in histologic sections of the primary tumor, which reflect angiogenesis, are correlated with metastasis in breast, prostate and Stage I nonsmall cell lung carcinoma. In this study, the authors investigated the association between angiogenesis, hematogenous metastasis and lymph node metastasis in all stages of lung adenocarcinoma. METHODS Microvessels were highlighted by immunostaining endothelial cells for factor VIII. We counted microvessels within the tumors of 42 patients who had surgical resection (25 with relapse and 11 without relapse more than 5 years after surgical resection). Without knowledge of patient outcome, microvessels were counted on a 200x field (0.723 mm2) in the most active areas of neovascularization. RESULTS The microvessel counts from patients with relapse after surgical resection (mean +/- standard deviation, 75.4 +/- 64.3) were significantly higher than those without relapse more than 5 years after surgical resection (42.6 +/- 26.0) (P = 0.027). Analysis of regional lymph node metastases (factor N) revealed that the microvessel counts were 62.6 +/- 35.1 for N0 (no regional lymph node metastasis), 51.7 +/- 22.2 for N1 (metastasis in ipsilateral, peribronchial and/or ipsilateral hilar lymph nodes, including direct extension), 75.4 +/- 75.3 for N2 (metastasis in ipsilateral mediastinal and/or subcarinal lymph nodes), and 74.0 for N3 (metastasis in contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene or supraclavicular lymph node[s]), and these values were not significantly different from each other. CONCLUSIONS Angiogenesis assessed by microvessel counts, correlated positively with relapse after surgical resection and hematogenous metastasis in all stages of lung adenocarcinoma; there was no correlation with lymph node metastasis in lung adenocarcinoma.

BACKGROUND The value of nucleolar organizer regions (NOR) visualized by silver staining (AgNOR) for the histologic differentiation, pathologic staging, and estimation of growth rate was assessed by the investigation of paraffin sections from 58 lung adenocarcinomas. AgNOR consist of NOR-associated proteins, and the number of AgNOR might be related to proliferative activity. METHODS In lung adenocarcinoma, the growth rate can be measured by means of chest radiographs. Using this technique, the authors studied the correlation between the mean number of AgNOR and growth rate. RESULTS The mean number of AgNOR ranged from 1.8 to 6.3 (mean +/- standard deviation, 4.0 +/- 0.8). Neither the degree of histologic differentiation nor the pathologic staging was related to the AgNOR count. The tumor growth rate was estimated on the basis of the doubling time in the chest radiographs of 13 patients. The doubling time ranged from 80 to 760 days. There was a high inverse correlation between the AgNOR count and the doubling time (r = -0.910; P less than 0.001). CONCLUSIONS Thus, it appears to be possible to use the mean number of AgNOR as an index of proliferative activity.

The clinical significance of ras oncogene expression in non-small cell lung cancer was evaluated in 116 surgically treated patients. Archival paraffin sections of the tumors were analyzed immunohistochemically using anti-ras p21 monoclonal antibody (MoAb) rp-35, and p21 staining was correlated with clinicopathologic parameters and survival. Positive reactions (+ and ++) were observed in 72.5% of the adenocarcinomas and 55.6% of the squamous cell carcinomas studied. The T1 tumors showed a ++ reaction less frequently than T2 and T3 tumors (P less than 0.05). Stage I tumors also were less reactive with MoAb rp-35 than tumors in more advanced stages (P less than 0.05). Survival analysis showed that patients with p21-negative tumors had significantly longer survival times (a 5-year survival rate of 64.1%) than those with p21 + tumors (38.0%, P less than 0.05) or those with p21 ++ tumors (11.5%, P less than 0.005). The significant correlation between p21 staining and patient survival was independent of histologic type, stage of disease, tumor or node status, and the resectability of tumors. On Cox's multivariate analysis, p21 staining was a major and independent prognostic determinant of survival. These results suggest that enhanced ras p21 expression may be one of the important biologic and clinical markers indicating the malignant potential of non-small cell lung cancer.

BACKGROUND Neuroendocrine neoplasms of the lung represent a wide spectrum of phenotypically and biologically distinct entities. Their histopathologic diagnosis, which carries therapeutic and prognostic significance, may sometimes be difficult because of their overlapping features. We previously demonstrated that large cell neuroendocrine carcinomas (LCNECs) and small cell lung carcinomas (SCLCs) failed to show positive nuclear staining of RB protein (RB−), whereas typical and atypical carcinoids (TCs and ACs) showed nuclear RB immunostaining (RB+). METHODS In the current study, a series of 58 surgically resected lung tumors, of which 33 tumors were initially diagnosed as SCLCs and 25 as TCs or ACs, were studied for RB and p16 protein expression by immunohistochemistry. They were also reviewed for their pathologic diagnosis; the reviewers were blinded to the RB and p16 protein status. RESULTS Nineteen tumors were diagnosed as TCs, 5 as ACs, 7 as LCNECs, and 27 as SCLCs. Three of seven LCNECs were RB+, whereas the other four were RB−. In contrast, all 19 TCs were RB+ and all 27 SCLCs were RB−. In addition, two of five ACs were RB+, whereas the other three were RB−. Interestingly, all 3 RB+ LCNECs and the 1 RB+ AC tested failed to show nuclear staining of p16 protein in any tumor cells (p16−), although some normal stromal cells showed nuclear staining of p16 protein (p16+) as positive internal controls, indicating loss of p16 function in these tumors. It is also noteworthy that the three RB+ LCNECs were initially diagnosed as SCLCs and one of the RB− ACs was initially considered a TC. With the exception of TCs, tumors were significantly more prevalent among heavy smokers with >20 pack-years compared with nonsmokers and light smokers with ≤20 pack-years (P < 0.01). CONCLUSIONS These findings suggest that all SCLCs and LCNECs have abnormalities in the p16:RB pathway, as do at least certain ACs, whereas the p16:RB pathway is normal in TCs. Cancer 2000;88:550–56. © 2000 American Cancer Society.

BACKGROUND Thallium-201 (201Tl) scintigraphy has been used to detect malignant pulmonary disease. The mechanism of Tl influx in tumor cells is believed to be similar to that of cisplatin (CDDP) mediated by sodium- and potassium-activated adenosine triphosphatase (Na-K ATPase), and the Na-K ATPase activity may determine the cellular CDDP accumulation and sensitivity to CDDP. The objective of this study was to determine the accumulation of CDDP and Tl in vitro by using inductively coupled plasma mass spectrometry (ICP-MS), a new analytic technique for detecting ultra trace elements, and to evaluate the correlations between cellular CDDP and Tl accumulation, between CDDP 50% inhibitory concentration (IC50) values and cellular CDDP accumulation, and between CDDP IC50 values and cellular Tl accumulation. METHODS Eight nonsmall cell lung carcinoma (NSCLC) cell lines were used (five adenocarcinomas and three squamous cell carcinomas). The cell lines were exposed to CDDP or Tl for 1 hour, and the resulting cellular accumulation of platinum and Tl was determined by ICP-MS. CDDP IC50 values were determined by a soluble tetrazolium/formazan assay. RESULTS The authors were able to measure cellular CDDP and Tl accumulation precisely, and heterogeneity in the cellular accumulation of CDDP and Tl existed among the NSCLC cell lines. A significant inverse correlation was observed between CDDP IC50 values and the cellular accumulation of both CDDP and Tl. CONCLUSIONS ICP-MS is suitable for the determination of cellular CDDP and Tl accumulation in NSCLC cell lines. Cellular Tl accumulation determined by ICP-MS may reflect CDDP cytotoxicity rather than cellular CDDP accumulation. Cancer 1998;83:930-935. © 1998 American Cancer Society.

To evaluate prognostic and therapeutic significance, tumor DNA content was determined by flow cytometry in 310 paraffin-embedded tissue samples obtained surgically from 130 patients with non-small cell lung cancer. Ninety-six (76.8%) patients had DNA aneuploid patterns that were statistically higher in adenocarcinoma than in squamous cell carcinoma. A better 5-year survival rate was observed in Group A (DNA diploidy, 69.6%) than in Group B (DNA aneuploidy and DNA peridiploidy, 33.2%; P less than 0.001). The survival curves of the patients in Group B continued to decrease during the next 2.5 years. Cox's model analysis showed that both the pathologic stage and the DNA content were the significant prognostic factors for survival. However, the DNA content was an independent prognostic factor in squamous cell carcinoma, but not in adenocarcinoma. These results indicate that DNA content analysis is useful for the evaluation of clinical behavior and prognosis, and that the clinical value of the DNA content must be differentiated between squamous cell carcinoma and adenocarcinoma.

Whether or not serum selenium and vitamin E (alpha-tocopherol) concentrations were changed was examined among healthy families of lung cancer patients. Family members as a whole (115 sons and daughters of 55 patients with primary lung cancer) were found to have a trend to lower serum selenium levels (0.116 +/- SD 0.024 microgram/ml, 0.05 less than P less than 0.1). Particularly among families of adenocarcinoma patients, the mean level was significantly lower (0.111 +/- 0.019 microgram/ml, P less than 0.05) than that (0.122 +/- 0.014 microgram/ml) in age-ratio matched controls who did not have cancer patients among their second-degree relatives. Serum vitamin E levels (11.85 +/- 2.85 micrograms/ml) were significantly lower among family members of adenocarcinoma patients than the controls (14.1 +/- 3.1 micrograms/ml, P less than 0.01). Serum selenium and vitamin E levels were significantly lower in lung cancer patients (n = 37, mean age, 63.9 +/- 11.2 yr) than in the controls (P less than 0.001). These data suggest that there are familial factors in serum selenium and vitamin E levels among families of lung cancer patients.