UHRF1 is a novel diagnostic marker of lung cancer.

BACKGROUND: Lung cancer is the leading cause of cancer deaths worldwide. As the sensitivity and specificity of current diagnostic markers are not perfect, we examined whether ubiquitin-like with PHD and ring finger domains 1 (UHRF1), which is overexpressed in various cancers but not yet examined in lung cancer in large scale, can be a novel diagnostic marker of lung cancer. METHODS: Immunohistochemical analysis using surgical specimens obtained from 56 US and 322 Japanese patients with lung cancer was performed. RESULTS: The UHRF1 was stained specifically in the nuclei of cancer cells, but not in the other cells. The UHRF1 expression was observed in all histological types of lung cancer, especially in non-adenocarcinomas (non-ADCs), both in the US and Japanese cases. In 322 Japanese non-small cell lung cancer (NSCLC) cases, UHRF1 expression was associated with the histological type (higher in non-ADCs; P<0.00001), gender (higher in male; P=0.00082), smoking (higher in smokers; P=0.00004), pT factor (higher in advanced stage; P=0.00010), and pN factor (higher in cancers with metastasis in regional lymph nodes; P=0.00018). The UHRF1 expression was also associated with poor prognosis for NSCLC patients (P=0.0364). Although UHRF1 overexpression was associated with these malignant indicators, UHRF1 was detectable in half of lung cancer patients in an early pathological stage. CONCLUSION: The UHRF1 is overexpressed in various types of lung cancer from early pathological stage. Therefore, detection of UHRF1 expression in tissue specimens by immunohistochemistry can be useful for diagnosis of lung cancer in all pathological stages.

Self-production of tissue factor-coagulation factor VII complex by ovarian cancer cells.

BACKGROUND: Thromboembolic events are a major complication in ovarian cancer patients. Tissue factor (TF) is frequently overexpressed in ovarian cancer tissue and correlates with intravascular thrombosis. TF binds to coagulation factor VII (fVII), changing it to its active form, fVIIa. This leads to activation of the extrinsic coagulation cascade. fVII is produced by the liver and believed to be supplied from blood plasma at the site of coagulation. However, we recently showed that ovarian cancer cells express fVII transcripts under normoxia and that this transcription is inducible under hypoxia. These findings led us to hypothesise that ovarian cancer cells are intrinsically associated with TF-fVIIa coagulation activity, which could result in thrombosis. METHODS: In this study, we examined whether ectopically expressed fVII could cause thrombosis by means of immunohistochemistry, RT-PCR, western blotting and flow cytometry. RESULTS: Ectopic fVII expression occurs frequently in ovarian cancers, particularly in clear cell carcinoma. We further showed that ovarian cancer cells express TF-fVIIa on the cell surface under normoxia and that this procoagulant activity is enhanced by hypoxic stimuli. Moreover, we showed that ovarian cancer cells secrete microparticles (MPs) with TF-fVIIa activity. Production of this procoagulant secretion is enhanced under hypoxia. CONCLUSION: These results raise the possibility that cancer cell-derived TF-fVIIa could cause thrombotic events in ovarian cancer patients.

Novel heteroduplex method using small cytology specimens with a remarkably high success rate for analysing EGFR gene mutations with a significant correlation to gefitinib efficacy in non-small-cell lung cancer.

We conducted a feasibility study to examine whether small numbers of cancer cells could be utilised for analysis of the EGFR gene status using the loop-hybrid mobility shift assay, which is a modified heteroduplex technique. Cytology specimens obtained by transbronchial abrasion were successfully used for analysis of the EGFR gene status in 50 of 52 (96.2%) patients diagnosed with class V non-small-cell carcinoma. Furthermore, the relationship between the EGFR gene status and clinical outcome was analysed in 25 patients treated with gefitinib. Overall, 10 of 11 patients with EGFR mutations in exon 19 or 21 showed tumour regression with gefitinib treatment, compared to only two of 14 patients with wild-type EGFR. The response rate was significantly higher in the EGFR mutation group than in the wild-type EGFR group (90.9 vs 14.3%, P=0.00014). Logistic regression analysis revealed that EGFR mutations in cytology specimens represented an independent predictor of the gefitinib response. The overall and progression-free survivals were significantly longer in the EGFR mutation group than in the wild-type EGFR group (P<0.05). In conclusion, cytology specimens could be useful for analysing the EGFR status in the majority of patients with non-small-cell lung cancer to determine whether they are likely to benefit from gefitinib treatment.

Genetic instability in lung cancer: concurrent analysis of chromosomal, mini- and microsatellite instability and loss of heterozygosity.

To investigate what kind of genetic instability plays important roles in lung carcinogenesis, we analyzed micro- and minisatellite instability, loss of heterozygosity (LOH) and chromosome instability in 55 cases of lung cancer, including, 10 squamous cell, 5 large cell, and 3 small cell carcinomas, and 37 adenocarcinomas. Analysis of minisatellite instability, the mechanism of which is different from microsatellite instability, has not been reported previously. Minisatellite instability was detected in only one case (1/55, 1.8%), and the frequency of microsatellite instability was low, being found only in three cases (3/55, 5.5%). In contrast, LOH, for at least in one locus, was observed in 27 cases (49.1%). In adenocarcinomas, the frequency of LOH was higher in poorly differentiated compared to more differentiated carcinomas. For chromosome instability, a similar correlation between differentiation grade and instability was observed in adenocarcinomas. And instability was more common in large cell and small cell carcinomas than in adenocarcinomas. Our analysis showed that chromosome instability and LOH, rather than mini- and microsatellite instability, play significant roles in the development of lung cancer.

Frequent reassortment among influenza C viruses.

In a 9-year survey from December 1990 to December 1999 in Sendai City, Japan, we succeeded in isolating a total of 45 strains of influenza C virus. These 45 strains were isolated in clusters within 4 months in a year, especially from winter to early summer. Previous studies of the hemagglutinin-esterase genes of various influenza C virus isolates revealed the existence of five distinct virus lineages (Aichi/1/81-, Yamagata/26/81-, Mississippi/80-, Sao Paulo/82-, and Kanagawa/1/76-related lineage) in Japan between 1970 and the early 1990s (Y. Matsuzaki, K. Mizuta, H. Kimura, K. Sugawara, E. Tsuchiya, H. Suzuki, S. Hongo, and K. Nakamura, J. Gen. Virol. 81:1447-1452, 2000). Antigenic and genetic analyses of the 45 strains showed that they could be divided into these five virus lineages and a few antigenic groups were cocirculating in Sendai City. In 1990 and 1991 the dominant antigenic group was the Aichi/1/81 virus group, and in 1992 it was Yamagata/26/81 virus group. The Mississippi/80 virus group was isolated from 1993 to 1996, and the Yamagata/26/81 virus group reemerged in 1996 and continued to circulate until 1999. This finding led us to a speculation that the replacement of the dominant antigenic groups had occurred by immune selection within the human population in the restricted area. Phylogenetic analysis of seven RNA segments showed that 44 viruses among the 45 strains isolated in our surveillance work were reassortant viruses that have various genome compositions distinguishable from those of the reference strains of the each lineage. This observation suggests that the reassortment between two different influenza C virus strains occurs frequently in nature and the genome composition of influenza C viruses may influence their ability to spread in humans.

Antigenic and genetic characterization of influenza C viruses which caused two outbreaks in Yamagata City, Japan, in 1996 and 1998.

During the 3 years from January 1996 to December 1998, a total of 33 strains of influenza C virus were isolated from 10,726 throat swab specimens collected from children with acute respiratory illness who visited two pediatric clinics in Yamagata City, Japan. These 33 strains were isolated in clusters during two different periods, 20 strains in May to August 1996 and the remaining 13 in March to June 1998. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein and phylogenetic analysis of seven RNA segments showed that the 33 influenza C viruses isolated were antigenically and genetically similar and that they were reassortant viruses which had obtained PB2, PB1, HE, M, and NS genes from a C/pig/Beijing/115/81-like virus and P3 and NP genes from a C/Mississippi/80-like virus. These observations suggest strongly that during the survey period of 3 years, two outbreaks of influenza C occurred in Yamagata City, both of which were caused by a reassortant virus having the genome composition described above.

Antigenic and genetic analyses of influenza B viruses isolated in Lusaka, Zambia in 1999.

Previous studies of the hemagglutinin (HA) genes of various influenza B virus isolates demonstrated the existence of two antigenically distinct virus lineages represented by B/Victoria/2/87 and B/Yamagata/16/88, respectively. Here, we investigated the antigenic and genetic characteristics of influenza B viruses isolated from children living in Lusaka, Zambia between January and May 1999. Antigenic analysis with chicken antiviral sera showed that all the Zambian isolates had the HA protein belonging to B/Yamagata/16/88-related lineage. Furthermore, phylogenetic analyses of the eight RNA segments performed by using the total or partial nucleotide sequences of the two representative Zambian strains (B/Lusaka/270/99 and B/Lusaka/432/99) as well as the previously reported sequences suggested that the Zambian viruses are closely related to the recently circulating reassortants represented by B/Shiga/T30/98 and B/Yamanashi/166/98 which acquired the genes coding for three polymerase proteins (PB2, PB1, and PA), HA, nucleoprotein, and matrix protein from a B/Yamagata/16/88-like parent and the gene encoding nonstructural proteins (NS1 and NS2) from a B/Guandong/8/93-like parent.

PKC1, a protein kinase C homologue of Saccharomyces cerevisiae, participates in microtubule function through the yeast EB1 homologue, BIM1.

BACKGROUND: RSC is a chromatin-remodelling complex of Saccharomyces cerevisiae and essential for growth. Its catalytic subunit is encoded by the NPS1/STH1 gene. At the present time, little is known regarding the cellular function of RSC. RESULTS: To identify genes with functions related to NPS1, we screened high-copy suppressor genes for the temperature- and thiabendazole (TBZ)-sensitive mutant allele of NPS1, nps1-105. Amongst the suppressors we cloned PKC1/STT1 and BIM1 that encoded a homologue of mammalian protein kinase C and a conserved microtubule binding protein homologous to human EB1, respectively. Both the temperature sensitive mutation of PKC1, stt1, and the bim1 null mutation caused synthetic growth defects with nps1-105. A genetic analysis of the functional relationships between these genes revealed that PKC1 suppressed the defect of nps1-105 through the BIM1 function but not by the activation of the MPK1/MAPK pathway. The stt1 mutation alone showed TBZ sensitivity and delayed the G2-phase progression at semi-permissive temperatures. Both of these stt1 phenotypes were suppressed by the over-expression of BIM1. In addition, stt1 as well as nps1-105, mis-segregated a mini-chromosome at frequencies higher than the wild-type at a permissive temperature. The mis-segregation was enhanced in the nps1-105 stt1 double mutant. CONCLUSION: These results suggest that Pkc1p plays a role which is relevant to microtubule functions and that this role is mediated by a hitherto unknown PKC signalling pathway and by Bim1p

Expression, function, and clinical implications of the estrogen receptor beta in human lung cancers.

The higher frequency of human lung adenocarcinoma in females than in males, strongly suggests the involvement of gender dependent factors in the etiology of this disease. This is the first investigation of estrogen receptor (ER) beta in human lung. Immunohistochemical staining revealed ERbeta expression in normal lung and in atypical adenomatous hyperplasia (AAH), considered as a precancerous lesion for adenocarcinomas. Adenocarcinomas showed significantly higher expression of ERbeta than squamous cell carcinomas. On the contrary, ERalpha expression was not detected in all cases. The functional integrity of ERbeta such as the binding ability to estrogen responsive element (ERE) and transcriptional activity was confirmed using a human lung cancer cell line, RERF-LC-OK. Colony formation of this cell was significantly reduced in the presence of pure antiestrogen. We conclude that ERbeta, but not ERalpha, is present in lung tissues with an important physiological function in normal lung. Furthermore, ERbeta may play a role in growth and development of adenocarcinomas.

Role of individual oligosaccharide chains in antigenic properties, intracellular transport, and biological activities of influenza C virus hemagglutinin-esterase protein.

The hemagglutinin-esterase (HE) glycoprotein of influenza C virus is composed of three domains: a stem domain active in membrane fusion (F), an acetylesterase domain (E), and a receptor-binding domain (R). The protein contains eight N-linked glycosylation sites, four (positions 26, 395, 552, and 603) in the F domain, three (positions 61, 131, and 144) in the E domain, and one (position 189) in the R domain. Here, we investigated the role of the individual oligosaccharide chains in antigenic properties, intracellular transport, and biological activities of the HE protein by eliminating each of the glycosylation sites by site-specific mutagenesis. Comparison of electrophoretic mobility between the wild-type and the mutant proteins showed that while seven of the glycosylation sites are used, one (position 131) is not. Analysis of reactivity of the mutants with anti-HE monoclonal antibodies demonstrated that glycosylation at position 144 is essential for the formation of conformation-dependent epitopes. It was also evident that glycosylation at the two sites in the F domain (positions 26 and 603), in addition to that in the E domain (position 144), is required for the HE molecule to be transported from the endoplasmic reticulum and that mutant HEs lacking one of these three sites failed to undergo the trimer assembly. Removal of an oligosaccharide chain at position 144 or 189 resulted in a decrease in the esterase activity. By contrast, two mutants lacking an oligosaccharide chain at position 26 or 603, which were defective not only in cell surface expression but in trimerization, possessed full-enzyme activity, suggesting that the HE monomers present within the cell have acetylesterase activity. Fusion activity of cells expressing each of mutant HEs was found to be comparable with the ability of the protein to be transported to the cell surface, suggesting that there is no specific oligosaccharide chain that plays a critical role in promoting membrane fusion.

Reduced HIC-1 gene expression in non-small cell lung cancer and its clinical significance.

BACKGROUND: HIC-1 (hypermethylated in cancer-1) is a candidate tumor suppressor gene, identified in a region of frequent loss of heterozygosity on chromosome 17p13.3, which is telomeric from TP53 and often deleted in surgically resected lung cancers. To determine the significance of HIC-1 in lung cancer, we assessed its expression status and prognostic association in 47 adenocarcinomas and squamous cell carcinomas. MATERIALS AND METHODS: RNA was extracted from tumors and corresponding normal tissues of surgically resected lungs, and the amount of HIC-1 mRNA was determined by means of semi-quantitative reverse transcriptase-polymerase chain reaction. RESULTS: HIC-1 expression in tumors was less than that in normal lung tissues in 40 of 47 patients (85%), indicating frequent partial silencing. Median tumor/normal lung tissue (T/L) ratios for HIC-1 expression were 0.51 and 0.75 for adenocarcinomas and squamous cell carcinomas, respectively. No significant difference of median T/L ratio was observed between the two histological types, or among clinical stages of the patients. However, the reduced expression of HIC-1 gene in the tumor had a direct link with the clinical outcome: lower T/L ratios (< 0.5) were significantly associated with short survival (P = 0.034), an association also observed in cases restricted to stage I (P = 0.047). CONCLUSIONS: The results suggest that low HIC-1 expression is involved in malignant progression of non-small cell lung cancer.

Borrelidin inhibits a cyclin-dependent kinase (CDK), Cdc28/Cln2, of Saccharomyces cerevisiae.

We identified borrrelidin, a member of macrolide antibiotic, as an inhibitor of a cyclin-dependent kinase of the budding yeast, Cdc28/Cln2. A 50% inhibition concentration (IC50) of borrelidin for Cdc28/Cln2 was 24 microM. In addition, borrelidin arrests both haploid and diploid cells in G1 phase at the point indistinguishable from that of alpha-mating pheromone, at concentrations not affecting the gross protein synthesis. Although the inhibition of CDK activity may not be a solo cause of the G1 arrest, our results indicate that borrelidin is a potential lead compound for developing novel CDK inhibitors of higher eukaryotes.

Different subtypes of human lung adenocarcinoma caused by different etiological factors. Evidence from p53 mutational spectra.

Human lung adenocarcinomas are only relatively weakly associated with tobacco smoke, and other etiological factors need to be clarified. These may also vary with the histopathology. Because the p53 mutation status (frequency and spectrum) of a carcinoma can provide clues to causative agents, we subclassified 113 adenocarcinomas into five cell types: hobnail, columnar/cuboidal, mixed, polygonal, and goblet (54, 23, 18, 13, and 5, respectively) and investigated relationships with p53 mutations and smoking history. In the hobnail cell type, a low mutational frequency (37%) and a high proportion of transitions (65%), especially G:C to A:T transitions at CpG dinucleotides (45%) associated with spontaneous mutations, were found with a weak relation to tobacco smoke. In contrast, a high mutation frequency (70%) with a higher proportion of transversions (50%), especially G:C to T:A (44%) on the nontranscribed DNA strand, caused by exogenous carcinogenic agents like tobacco smoke, were observed for the columnar cell type, as with squamous cell carcinomas. These results indicate that two major subtypes of lung adenocarcinoma exist, one probably caused by tobacco smoke, and the other possibly due to spontaneous mutations. For the prevention of lung adenocarcinomas, in addition to stopping tobacco smoking, the elucidation of endogenous mechanisms is important.

Prognostic value of genetically diagnosed lymph node micrometastasis in non-small cell lung carcinoma cases.

The predictive value of lymph node micrometastasis, detected by immunohistochemical or genetic methods, is well appreciated in terms of prognosis. However, a major problem is high false-positive rates, because most methods focus on cytokeratin, which is a component not only of carcinoma but also normal epithelial and nonepithelial cells. Mutant allele-specific amplification (MASA) can detect DNAs derived from cancer cells itself, reportedly with high sensitivity. It was, therefore, used with nested-PCR using p53 or K-ras mutation for analysis of lymph node micrometastasis in non-small cell lung carcinoma (NSCLC) patients in the present study, in comparison with the immunohistochemical method using an anti-cytokeratin reagent for the same samples. Lymph nodes from 31 NSCLC patients with p53 and K-ras mutated tumors (30 and 1, respectively) staged as pathological (p)-T1-4 N0-1 and M0 were examined. Genetic and immunohistochemical methods demonstrated positive reactions in 34 (15%) and 61 (27%) of 229 lymph nodes, respectively (9 cases, 29%, and 24 cases, 77%). The concordance with the two methods was 77%, but 13 (39%) of 34 genetically positive lymph nodes could not be detected by immunohistochemistry (IHC). Of 22 cases with p-N0 disease, 6 (27%) were genetically positive in hilar and/or mediastinal lymph nodes, and 4 (67%) of them died after cancer relapse. In contrast, none of the patients without micrometastasis died of cancer (P < 0.001, log rank analysis). Of the same p-N0 patients, 17 (77%) were positive by IHC, and 4 (24%) of them died of cancer, whereas 5 negative patients did not suffer cancer relapse. Survival did not significantly differ between cases positive and negative (P = 0.246) by IHC. According to the g-N (N factor restaged by a genetic method), patients with g-N1 and g-N2 disease had a shorter survival than those with g-N0 disease (P = 0.042 and P < 0.001, respectively). However, no significant difference was observed with grading by IHC. Thus, detection of micrometastasis in regional lymph nodes with the MASA method, in other words with a carcinoma-specific marker, is of greater prognostic significance for early stage NSCLC patients than immunohistochemical results. This approach should facilitate selection of patients for whom postoperative adjuvant chemotherapy should be performed.

Three new regions on chromosome 17p13.3 distal to p53 with possible tumor suppressor gene involvement in lung cancer.

We investigated loss of heterozygosity (LOH) at the distal portion of the p53 gene on the short arm of chromosome 17 in lung cancers in order to search for new tumor suppressor genes. The roles of the putative genes were also studied in terms of pathological features. One hundred and forty-five resected non-small cell lung cancers were examined for LOH using 11 markers mapped on, and distal to the p53 locus, and deletion maps were constructed. Four commonly deleted regions were found: one from TP53 to ENO3, where the p53 gene resides, and three others from ENO3 to D17S1566, D17S379 to D17S1574 and distal to ABR, with LOH frequencies almost the same as, or higher than, at the TP53 locus. Examination of the relationship between LOH of the latter three regions and histopathological parameters of adenocarcinomas (genetically negative for p53 mutation) revealed allelic losses on D17S379 to be associated with advanced lesions, while D17S513 was more frequently deleted in poorly differentiated tumors. These results indicate that new tumor suppressor gene(s) may reside on these three distinctly deleted regions on chromosome 17p13.3 distal to the p53 gene in lung cancer, with possible roles in progression and differentiation of adenocarcinomas.

Characterization of antigenically unique influenza C virus strains isolated in Yamagata and Sendai cities, Japan, during 1992-1993.

Three influenza C virus strains (C/Yamagata/1/92, C/Yamagata/1/93 and C/Miyagi/5/93) isolated in Yamagata and Sendai Cities, Japan, between June 1992 and May 1993 were found to possess haemagglutinin-esterase glycoproteins that were antigenically indistinguishable from one another but were clearly different from any previous Japanese isolates. To investigate the origin of the 1992/1993 strains, their antigenic and genetic properties were compared with those of eight strains isolated outside Japan between 1967 and 1982. The results showed that the 1992/1993 isolates were closely related to a virus isolated in Brazil in 1982 (C/SaoPaulo/378/82) and that these viruses (including C/SaoPaulo/378/82) are reassortants that had obtained PB1 and NP genes from a C/Yamagata/26/81-like parent and the other genes from another as yet unidentified parent.

Hilar lymph node metastasis in renal cell carcinoma.

A 48-year-old man, who underwent a right nephrectomy for renal cell carcinoma 7 years earlier, was found to have hilar lymph node metastasis alone, without lesions, in the pulmonary parenchyma. Chest X-ray and CT findings showed a left hilar mass about 4 x 2.5 cm in diameter. Left bronchial arteriography showed a hypervascular mass lesion in the left hilum. Macroscopic tumor invasion of the pulmonary artery and left main bronchus indicated left pneumonectomy. The resected specimen was found histologically to involve metastatic renal cell carcinoma in the left hilar lymph node about 3 cm in diameter. Tumor metastasis was limited to the lymph node. The metastatic pathway of renal cell carcinoma to the hilar lymph node was considered lymphogenous via either retrograde lymphatic flow from the thoracic duct or through the lymphatics in the inferior pulmonary ligament.

Practical utility of the revised European-American classification of lymphoid neoplasms for Japanese non-Hodgkin's lymphomas.

A clinicopathological study of 515 non-Hodgkin's lymphoma (NHL) cases was performed using the revised European-American classification of lymphoid neoplasms (REAL classification) in an HTLV1-nonendemic area of Japan. The following characteristics were revealed: 1) frequency of extranodal lymphomas was high (59%) with 79% B-cell lymphomas in this series, while the overall ratio of B:T/NK lineage was 3.7:1; 2) the most common type was the diffuse large B-cell lymphoma (46%), follicle center lymphomas occurred at an incidence lower (15%) than that in European and American populations, and marginal zone B-cell lymphomas accounted for as much as 12%; 3) peripheral T-cell lymphomas were common (19%), with the unspecified type predominant (11%), while adult T-cell lymphomas were present at a level equivalent to that among European and American patients (1%). Clear segregation of survival curves was rated according to cell lineage and B-cell lymphomas had a better prognosis than T / NK-cell lymphomas. Furthermore, new subtypes in the REAL classification, such as marginal zone B-cell and mantle cell lymphomas, exhibited distinct curves. Taken altogether, the REAL classification demonstrated advantages for assessment of Japanese NHL cases.

Abnormalities of the FHIT gene in human oral carcinogenesis.

The abnormalities of the fragile histidine triad (FHIT) gene in tissue samples of oral squamous cell carcinomas (SCCs) along with several leukoplakias and an erythroplakia were examined to determine whether the FHIT gene is actually a frequent target in vivo for alteration during oral carcinogenesis. Abnormal transcripts of the FHIT gene were found in eight of 15 oral SCCs. Although these abnormal transcripts varied widely, deletion patterns incorporating a deletion of exon 5 were the most common. Loss of heterozygosity (LOH) analysis demonstrated that the abnormal FHIT transcripts found in cancer cells were attributable to abnormalities of the FHIT gene. Abnormal FHIT transcripts were also observed in two of seven premalignant lesions. Interestingly, in the case of one patient with a premalignant lesion showing an abnormal FHIT transcript, subsequent oral SCC developed during a 3-year follow-up period. On the other hand, in the two patients from whom both leukoplakia and SCC samples were taken simultaneously, abnormal FHIT transcripts were found only in the SCCs. Although the functional role of FHIT remains to be clarified, these results suggest that the FHIT alteration is actually involved in carcinogenesis of the oral epithelium.

p53 null mutations undetected by immunohistochemical staining predict a poor outcome with early-stage non-small cell lung carcinomas.

The importance of p53 mutations in the pathogenesis of human lung carcinoma is well established, but it is still controversial whether the presence of p53 mutations or overexpression of p53 protein adversely affects an individual patient's chances of survival. The controversy may be partially due to the methodological differences in examination for p53 alterations: gene analysis or immunohistochemical staining. Furthermore, recent studies have suggested that different types of mutations of the p53 tumor suppressor gene confer different biological properties. To clarify the relationship between immunohistochemical staining and prognosis, we investigated mutations using single-strand conformation polymorphism followed by sequencing for exons 4-8 and 10 in 144 surgically treated non-small cell lung carcinoma patients with intensive clinical follow-up. Of 144 cases, 107 adenocarcinomas were examined for immunohistochemical staining with RSP53 antibody. p53 gene mutations were observed in 65 tumors (45%), including 44 missense and 21 null mutations, the latter comprising 7 nonsense mutations, 8 deletions, 2 insertions, and 4 splicing junction mutations. Presence of p53 mutations was an independent prognostic factor with a statistical trend (P = 0.14) in stage I patients but not in all cases. When examined by mutational pattern, null mutation was a significant indicator of poor outcome by multivariate analysis (P = 0.03) in stage I patients, whereas cases with missense mutations and without mutations did not differ (P = 0.76). Forty (37%) tumors demonstrated overexpression of the p53 protein but without any survival difference. Most tumors (76%) with missense mutations were immunopositive, but those with null mutations with one exception (93%) were not, and the concordance between the mutations and immunohistochemical staining was rather low at 65%. These data suggest that the type of p53 mutation is important for prediction of outcome in early-stage non-small cell lung carcinoma patients, whereas immunohistochemical staining for abnormal p53 gene products is nonpredictive. Furthermore, null mutations causing loss of function of the gene product may play more important roles than missense mutations in tumor progression.