Absence of Fhit protein in primary lung tumors and cell lines with FHIT gene abnormalities

Genomic alterations and abnormal expression of the FHIT gene at 3p14.2 have been observed in cell lines and primary tumors of the lung. To correlate FHIT locus DNA and RNA lesions with effects on Fhit protein expression, we have analyzed 11 lung cancer cell lines, 15 small cell lung carcinomas, and 38 pairs of non-small cell primary tumors and bronchial mucosa specimens by molecular genetic and immunocytochemical methods. Using specific antibodies against the Fhit protein, we observed concordance between RNA abnormalities and lack of Fhit protein expression in lung tumors and cell lines. In addition, absence of Fhit protein in some precancerous dysplastic lesions suggested that FHIT inactivation may occur at an early phase of lung carcinogenesis.     

Absence or reduction of Fhit expression in most clear cell renal carcinomas

The FHIT gene at human chromosome region 3p14.2 straddles the common fragile site, FRA3B, and numerous homozygous deletions in cancer cell lines and primary tumors. Also, the 3p14.2 chromosome breakpoint of the familial clear cell kidney carcinoma-associated translocation, t(3;8)(p14.2;q24), disrupts one FHIT allele between exons 3 and 4, fulfilling one criterion for a familial tumor suppressor gene: that one allele is constitutionally inactivated. Because the FHIT gene sustains biallelic intragenic deletions rather than mutations, there has not been evidence that the FHIT gene frequently plays a role in kidney cancer, although replacement of Fhit expression in a Fhit-negative renal carcinoma cell line suppressed tumor growth in nude mice. We have now assessed 41 clear cell renal carcinomas for expression of Fhit by immunohistochemistry. Normal renal tubule epithelial cells express Fhit uniformly and strongly, whereas 51% of the tumors are completely negative, 34% of tumors show a mixture of positive and negative cells, and 14% are uniformly positive, although usually less strongly positive than the normal epithelial cells. Most interestingly, there was a correlation between complete absence of Fhit and the G1 morphological grade and early clinical stage. Morphological grades G2 and G3 exhibited a mixture of positive and negative cells with a tendency for a higher fraction of negative cells in G3. Fhit inactivation is likely to be an early event in G1 tumors and may be associated with progression in G2 and G3 tumors.     

The murine Fhit locus: isolation, characterization, and expression in normal and tumor cells

The murine Fhit locus maps near the centromere nu proximal Ptprg locus on mouse chromosome 14. The cDNA sequence and structure are similar to those of the human gene, with exons 5-9 encoding the protein. The predominant mRNA in the tissues and cell lines tested was an alternatively spliced form missing exon 3. Most murine cell lines tested, including lines established from normal mouse embryos and tumors, expressed very low or undetectable levels of Fhit mRNA. Most normal mouse tissues expressed wild-type Fhit mRNA, whereas approximately 40% of murine lung carcinomas expressed wild-type and aberrant Fhit RT-PCR products that lacked various exons. Several tumorigenic mouse cell lines exhibited homozygous deletions of Fhit exons. We conclude that the murine Fhit gene, like its human counterpart, is a target of alterations involved in murine carcinogenesis.     

Nephrotoxicity of cadmium-metallothionein: protection by zinc and role of glutathione

BACKGROUND: In vitro studies have shown an antiproliferative effect of tumor necrosis factor (TNF) against various nonsmall cell lung carcinoma (NSCLC) cell lines. However, clinical trials of combined interleukin-2 and TNF-alpha in patients with advanced NSCLC have demonstrated both conflicting and disappointing results. METHODS: Immunohistochemical (IHC) staining was performed on formalin fixed, paraffin embedded tissues from 39 bronchogenic adenocarcinomas and 32 squamous cell carcinomas using polyclonal antibodies against TNF-alpha, TNF-beta, TNF-R1, and TNF-R2 proteins. IHC positivity was correlated with tumor stage, grade, and patient survival. RESULTS: Significant coexpression of TNF-alpha, TNF-beta, TNF-R1, and TNF-R2 was observed in NSCLC (significance range, P < 0.001-0.02). Although immunoreactivity for TNFs remained high in all tumor stages, a loss of TNF-R expression was found in advanced NSCLC (P < 0.006 for TNF-R1 and P < 0.003 for TNF-R2), suggesting down-regulation of TNF-Rs in the process of tumor progression. When all stages were considered together, immunoreactivity for TNF-beta(P < 0.001), TNF-R1, and TNF-R2 (both P < 0.001) significantly correlated with favorable outcome in univariate analysis. However, when stages were studied separately, an association between immunopositivity for TNF-Rs and favorable prognosis was found only in NSCLC without distant metastasis (P < 0.04 and P < 0.005 for TNF-R1 and TNF-R2 in Stage I [according to the American Joint Committee on Cancer staging system] disease, and P < 0.03 and P < 0.02 for TNF-R1 and TNF-R2 in Stage III disease). On multivariate analysis, increased expression of TNF-R1 (P < 0.003) and TNF-R2 (P < 0.001) as well as tumor stage (P < 0.001) independently predicted favorable outcome in patients with NSCLC. CONCLUSIONS: Although NSCLC exhibits strong coexpression of TNF-alpha, TNF-beta, TNF-R1, and TNF-R2, there is a loss/down-regulation of TNF receptors in high stage tumors. TNF-R1 and TNF-R2 positivity independently predicts favorable outcome in NSCLC, particularly in tumors with no clinically distant metastasis. The current study supports a role for TNFs and their receptors in the evolution and progression of NSCLC.     

Mutations in exon 7 and 8 of p53 as poor prognostic factors in patients with non-small cell lung cancer

This study was performed to clarify the different effects of each mutant exon of p53 as indicators of a poor prognosis in patients with non-small cell lung cancer (NSCLC). Tumor tissues of 204 patients with NSCLC were analysed; 96 tumors were stage I, 22 stage II, and 86 stage III. DNA was extracted from frozen specimens and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to investigate mutations of p53 from exon 5 to exon 8. Seventy-five patients with NSCLC (36.8%) had mutations in p53 which included 72 cases of missense mutations and three cases of non-missense mutations. The overall survival rate of patients with mutant p53 adenocarcinomas was strikingly worse than that of patients whose tumors had wild-type p53 (35.7% vs 53.8%; P=0.041), but no significant difference in survival was found in the patients with NSCLC and squamous cell carcinoma. Mutations in exon 5 of p53 occurred in 33 cases (16.2%), mutation in exon 6 was detected in only one case (0.5%), mutations in exon 7 in 20 cases (9.8%), and mutations in exon 8 in 18 cases (8.8%). The overall survival rate of patients with mutations in exon 7 was worse than that of patients with wild-type p53 in NSCLCs and adenocarcinomas (42.9% vs 56.0%; P=0.025 and 33.3% vs 53.8%; P=0.048, respectively), whereas the overall survival of patients with mutations in exon 5 was almost the same as that of patients with wild-type p53. In addition, the overall survival rate of patients with mutations in exon 8 was strikingly worse than that of patients with wild-type p53 in NSCLCs, adenocarcinomas and squamous cell carcinomas (22.9% vs 56.0%; P<0.001, 19.0% vs 53.8%; P=0.004 and 33.3% vs 62.5%; P=0.042, respectively). Multivariate analysis with the Cox regression model of patients with NSCLC, adenocarcinoma and squamous cell carcinoma indicated that mutations in exon 8 were best correlated with the overall survival rate, followed by lymph node status (P<0.001, P=0.015 and P=0.006, respectively), and mutations in exon 7 of NSCLC were also revealed to have good correlation, followed by lymph node status and mutations in exon 8 (P=0.031). Mutation of p53 was a poor prognostic factor for adenocarcinoma as described previously. Moreover, mutations in exon 8 were more useful indicators of prognosis not only for adenocarcinoma but also for NSCLC. Worse overall survival of the patients with mutations in exon 8 of p53 was suggested to be associated with codon 273 mutations as well as mutations between codon 280 and 285 included into the H2 alpha helix corresponding to residues 278-286. These results suggested that abnormal conformation of H2 alpha helix might play an important role not only in the loss of normal function but also in the acquisition of tumorigenesis. Investigation of mutations in exon 8, especially codon 273 mutation and mutant H2 alpha helix was considered to be a clinically useful approach for determining the prognosis of patients with NSCLC.     

Replacement of Fhit in cancer cells suppresses tumorigenicity

The candidate tumor suppressor gene, FHIT, encompasses the common human chromosomal fragile site at 3p14.2, the hereditary renal cancer translocation breakpoint, and cancer cell homozygous deletions. Fhit hydrolyzes dinucleotide 5',5"'-P1,P3-triphosphate in vitro and mutation of a central histidine abolishes hydrolase activity. To study Fhit function, wild-type and mutant FHIT genes were transfected into cancer cell lines that lacked endogenous Fhit. No consistent effect of exogenous Fhit on growth in culture was observed, but Fhit and hydrolase "dead" Fhit mutant proteins suppressed tumorigenicity in nude mice, indicating that 5',5"'-P1, P3-triphosphate hydrolysis is not required for tumor suppression.     

Acute hemorrhagic pulmonary infarction and necrotizing pneumonia in horses: 21 cases (1967-1993)

Specimens of formalin-fixed, paraffin-embedded non-small-cell lung carcinomas (NSCLCs; n = 187) were analysed immunohistochemically for expression of cyclin A. The analysis was intended to determine whether cyclin A has additional prognostic value for predicting patients' survival and drug response. Of the 187 NSCLCs, 141 cases (75%) showed expression of cyclin A. Patients with cyclin A-positive carcinomas had significantly shorter median survival times than patients with cyclin A-negative carcinomas (79 vs 129 weeks, P = 0.045). Similar results were obtained with more homogeneous groups of patients: patients with only T3 tumours, patients with epidermoid carcinomas and patients with lymph node involvement. The clinical parameters (age, stage, histology, extent of tumour size, lymph node involvement) had no influence on expression of cyclin A. A direct correlation between cyclin A and the proportion of S-phase cells (P = 0.08) and an inverse relationship between cyclin A and the proportion of G0/G1-phase cells (P = 0.04) were found. Furthermore, a significant correlation between the expression of cyclin A and the response of NSCLC to doxorubicin in vitro was detected (P = 0.026).     

Association between cigarette smoking and FHIT gene alterations in lung cancer

Epidemiologic data have strongly indicated that cigarette smoking is linked to the development of lung cancer. However, little is known of the molecular targets of carcinogens contained in tobacco smoke. To identify genetic lesions characteristic of tobacco damage, we undertook a molecular analysis of microsatellite alterations within the FHIT gene and FRA3B, as well as at an independent locus on chromosome 10, D10S197, in lung tumors from heavy smokers and in tumors from never smokers. Loss of heterozygosity affecting at least one locus of the FHIT gene was observed in 41 of 51 tumors in the smokers group (80%) but in only 9 of 40 tumors in nonsmokers (22%). The comparison between the frequency of losses in FHIT in smokers and nonsmokers was statistically significant (P = 0.0001), whereas no difference in loss of heterozygosity rate was observed at D10S197 locus. These findings suggest that FHIT is a candidate molecular target of carcinogens contained in tobacco smoke.     

The utility of p53 immunostaining of transbronchial biopsy specimens of lung cancer: p53 overexpression predicts poor prognosis and chemoresistance in advanced non-small cell lung cancer

There are few reports on the p53 status of small cell lung cancer (SCLC) and advanced non-SCLC (NSCLC) because surgically resected specimens are generally not available. Therefore, we evaluated p53 immunostaining in 175 transbronchial biopsy (TBB) specimens obtained from patients with all stages of lung cancer and retrospectively evaluated the relationship between p53 status and clinical parameters. All of the specimens were obtained prior to therapy. Formalin-fixed, paraffin-embedded TBB specimens were immunostained using an anti-p53 antibody (DO-1). p53 protein was detected in 55% (61 of 111) of NSCLCs and 58% (37 of 64) of SCLCs. The rate of positivity increased significantly with increasing stage (stages I and II, 45%; stage III, 54%; stage IV, 66%), but not with other clinical parameters. Ninety-five patients were evaluated for their response to chemotherapy. Positive staining for p53 correlated significantly with unresponsiveness to chemotherapy in NSCLC (response rate of 13 versus 60%; P = 0.006), but not in SCLC (80 versus 57%; P = 0.22). p53 positivity was a statistically significant negative prognostic factor for stage III and stage IV NSCLC (P = 0.02), but not for stage I and stage II NSCLC (P = 0.79). There was no survival difference relative to p53 status in SCLC (P = 0.35). These results indicate that p53 overexpression in TBB specimens predicts poor prognosis and chemoresistance in advanced stage NSCLC.     

Apoptotic index in ovarian carcinoma: correlation with clinicopathologic factors and prognosis

The apoptotic index (apoptotic cells/1000 tumor cells, AI) was evaluated in 71 ovarian carcinomas, all surgically resected. Apoptosis was examined by modified terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) method in histologic sections. High AI (>/=2.8) significantly correlated with high mitotic index (P = 0.05), high histologic grade of the tumor (P = 0. 018), and short overall survival (P = 0.017). An inverse relationship between AI and bcl-2 protein expression was also observed (P = 0.007). In addition, AI was assessed in 5 ovarian epithelial tumors of borderline malignancy, and all were categorized as low AI (<2.8). No significant correlation was found between AI and other clinicopathologic factors, such as age, clinical stage, lymph node metastasis, tumor size, histology of the tumor, and expression of p53 protein. Multivariate survival analysis showed that only clinical stage (P = 0.0395) and mitotic index (P = 0.0387) had independent prognostic value, whereas AI did not. Our results suggest that counting apoptosis can be useful for predicting the patient survival in ovarian carcinoma, although AI is not an independent prognostic factor. It is also suggested that bcl-2 protein is an important regulator of apoptosis in ovarian carcinoma.     

Altered retinoblastoma and p53 protein status in non-small cell carcinoma of the lung: potential synergistic effects on prognosis

Routinely processed pathological specimens from 119 patients with stage I and II adenocarcinomas or squamous cell carcinomas were examined by immunohistochemical analysis for altered retinoblastoma (RB) and/or p53 protein expression. Absent RB nuclear staining (RB-) indicating loss of RB function occurred in 19 (16%) of the cases studied, whereas expression of a putative mutant p53 nuclear protein (p53(+)) was found in 54 (45%) of the tumors. The median survival was 39 versus 12 months for patients with RB+ and RB- tumors, respectively (P = 0.05 by log rank analysis). Similarly, the median survival was 41 months for patients whose tumors had no expression of mutant p53 (p53(-)) compared with 24 months for individuals with p53 (+) tumors (P = 0.01). These differences in survival, however, were not statistically significant by multivariate analysis. Nevertheless, individuals with RB-/p53(+) tumors had a significantly shorter median survival (12 months) than those with RB+/p53(-) tumors (41 months), as determined by both log rank and multivariate analyses (P = 0.005 and 0.03, respectively). In addition, 66 large cell carcinomas from all stages were examined. Again, a more significant difference in survival (48 versus 8 months) was found between patients with RB+/p53(-) versus RB-/p53(+) tumors (P = 0. 006). These results suggest that RB and p53 status might be used synergistically as prognostic factors in a subset of non-small cell lung carcinomas.     

Rb and p16INK4a expression in resected non-small cell lung tumors

Inactivation of the cyclin-dependent kinase inhibitor p16INK4a (CDKN2/MTS1) is documented in a wide variety of cancer cell lines and tumors. We have shown that loss of p16INK4a protein expression is a common event in early stage non-small cell lung cancer (NSCLC), correlates with a significantly worse survival, and is more common in higher stage disease. One hundred NSCLC tumors from patients undergoing definitive thoracotomies at a single institution were examined for p16INK4a and retinoblastoma protein (pRB) expression. Abnormal pRB staining was identified in 15% of the tumors, whereas 51% possessed aberrant p16INK4a protein expression. Tumors with aberrant expression of p16INK4a by immunohistochemistry were associated with a significantly worse survival (P=0.04). Additionally, the inverse correlation of pRB and p16INK4a expression previously noted in lung cancer cell lines and tumors was confirmed in this large cohort of patients, with 65% of the tumors demonstrating inverse expression of pRB and p16INK4a (p=0.00019). A statistically significant increase in aberrant p16INK4a expression, as well as inverse expression of p16INK4a and pRB, was seen with increasing pathological stage of disease. These findings establish the prognostic significance (of the absence of p16INK4, in resected NSCLC and confirm the critical importance of disrupting the pathway of cyclin-dependent kinase-mediated phosphorylation of pRB in the molecular oncogenesis and progression of NSCLC.     

p53 and thymidylate synthase expression in untreated stage II colon cancer: associations with recurrence, survival, and site

We initiated a retrospective study to determine whether p53 status and thymidylate synthase (TS) protein expression in primary colon tumors influence recurrence and survival for patients with stage II colon cancer. Tumor specimens from 45 consecutive untreated patients with stage II colon cancer were examined for p53 and TS protein expression using immunohistochemistry. The median follow-up was 5.1 years. Eighteen patients had left-sided tumors, and 27 had right-sided tumors. Fourteen of 45 patients (31%) developed recurrence. p53 overexpression was detected in the tumors of 18 patients (40%); 10 patients (55%) with p53 overexpression recurred; and 4 of 27 (15%) without evidence of p53 overexpression recurred (P = 0.002). High TS expression was detected in the tumors of 16 patients (36%): 8 patients (50%) with high TS expression recurred, and 6 patients (21%) with low TS expression recurred (P = 0.027). Patients with p53 overexpression had a significantly poorer survival than did those patients without p53 overexpression (P < 0.001). High TS expression was associated with poor survival (P = 0.004). p53 overexpression and high TS expression were significantly associated with left-sided tumors (P = 0.003 and P = 0.022). Thirteen of 16 patients (81%) with high TS expression also overexpressed p53, and 24 of 29 patients (81%) with low TS expression did not manifest p53 overexpression (P < 0.001). p53 and TS expression in primary stage II colon cancer are associated and appear to influence recurrence and survival. In this pilot study, left-sided tumors demonstrate significantly more p53 overexpression and significantly higher TS expression than do right-sided tumors, which may explain the significantly poorer survival for patients with left-sided tumors.     

Neural networks as a prognostic tool for patients with non-small cell carcinoma of the lung

Patients with non-small cell carcinoma of the lung (NSCLC) have a poor prognosis (64 and 41% survival rates in Stages I and II). It is currently not possible to predict which patients with Stage I or II NSCLC will survive the disease. Sixty-seven patients with NSCLC, including 49 patients with Stage I NSCLC and 18 with Stage II disease (11 with squamous cell carcinomas, 35 with adenocarcinomas, and 21 with large cell carcinomas) were treated with lobectomy and followed for a minimum of 5 years. The tumors were studied with DNA flow cytometry and quantitative immunocytochemical studies for proliferation cell nuclear antigen, p53 protein, and MIB-1. The data were analyzed with backpropagation neural networks, univariate analysis of variance, the Kaplan-Meier survival method, and Cox proportional hazards model. The dependent variables were "free of disease" and "recurrence or dead from disease." Twenty neural network models were trained, using all cases but one, after 1883 to 2000 training cycles. At 5 years, 30 patients were free of disease and 37 were dead or had recurrence. Proliferating cell nuclear antigen was the only statistically significant prognostic factor by univariate analysis of variance and Cox proportional hazards analysis. The S phase was statistically significant by univariate analysis of variance (P <.05). All of the 20 models classified the test cases correctly. Study with backpropagation neural networks using multiple prognostic features from patients with NSCLC suggests that this technology might be useful for prediction of survival. This preliminary study must be validated with data from a larger group of patients with NSCLC before its clinical adequacy is established.     

Smoking-related increase of O6-methylguanine-DNA methyltransferase expression in human lung carcinomas

Eighty-three non-small cell lung carcinomas (NSCLC) of previously untreated patients were analysed for expression of O6-methylguanine-DNA methyltransferase (MGMT) by means of immunohistochemistry. Expression of MGMT was detected in 62 of 83 tumours (75%). There was a significant difference in MGMT staining between smokers and non-smokers (P = 0.001). Tumours of smokers expressed more frequently MGMT than tumours of non-smokers. There was a trend of MGMT expression to be higher in tumours of patients smoking >20 cigarettes/day than patients smoking <20 cigarettes/day. Abstinence from smoking resulted in a significant decrease in MGMT expression (lin reg r = -0.59, P < 0.05). These results demonstrate that MGMT expression in human lung carcinomas is influenced by smoking habits of the patients.