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Secreted frizzled related protein 1 (SFRP1) is an antagonist of the transmembrane frizzled receptor, a component of the Wnt signaling pathway, and has been suggested to be a candidate tumor suppressor in several human malignancies. Since SFRP1 is located at chromosome 8p11, where lung cancers also exhibit frequent allelic loss, we hypothesized that the inactivation of SFRP1 is also involved in lung carcinogenesis. To substantiate this, we performed mutational analysis of SFRP1 for 29 nonsmall-cell lung cancer (NSCLC) and 25 small-cell lung cancer (SCLC) cell lines, and expression analysis for the same cell lines. Although somatic mutations were not detected in the coding sequence, downregulation of SFRP1 was observed in 14 (48%) NSCLC and nine (36%) SCLC cell lines. We analysed epigenetic alteration of the SFRP1 promoter region and detected hypermethylation in 15 (52%) of 29 NSCLC cell lines, two (8%) of 25 SCLC cell lines, and 44 (55%) of 80 primary lung tumors. By comparing the methylation status with SFRP1 expression, we found a significant correlation between them. We also performed loss of heterozygosity (LOH) analysis and found that 15 (38%) of 40 informative surgical specimens had LOH in the SFRP1 gene locus. Furthermore, we performed colony formation assay of two NSCLC cell lines (NCI-H460 and NCI-H2009) and found the reduction of colony formation with SFRP1 transfection. In addition, we also detected that SFRP1 inhibits the transcriptional activity of b-catenin, which is thought to be a downstream molecule of SFRP1, with luciferase reporter assay. Our current studies demonstrated that the SFRP1 gene is frequently downregulated by promoter hypermethylation and suppresses tumor growth activity of lung cancer cells, which suggests that SFRP1 is a candidate tumor suppressor gene for lung cancer.Oncogene (

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Activating mutations of EGFR are found frequently in a subgroup of patients with non-small cell lung cancer (NSCLC) and are highly correlated with the response to gefitinib and erlotinib. In the present study, we searched for mutations of EGFR, HER2 and KRAS in 264 resected primary NSCLC from Japanese patients and determined whether there is a correlation between genetic alterations of these genes and clinicopathological factors, together with 85 tumors that we reported previously. EGFR mutations were found in 102 of the total 349 tumors, and seven tumors had two missense mutations. Reverse transcriptionpolymerase chain reaction of EGFR and subsequent subcloning analyses identified that the double mutations occurred in the same allele. Although the prognosis for patients with advanced NSCLC has not improved in the last 20 years, (3) the molecular and biological characteristics of lung cancer have been studied intensively (4,5) and many molecules associated with the pathogenesis of lung cancer have been identified as promising new targets for lung cancer therapy.(6) Among these molecules, EGFR, one of the ERBB family of receptors, is thought to be a promising target because of its frequent overexpression (range 40 -80%) in NSCLC (7) and its relationship with poor prognosis.(8) Small molecules of EGFR-TKI, such as gefitinib and erlotinib, have been developed, examined for the treatment of NSCLC, and shown to have antitumor activity.(9) Several clinical studies have revealed that Japanese female never-smoking patients with adenocarcinoma have a higher response rate to gefitinib. (10,11) Recently, activating mutations of the TK domain of EGFR were found in a unique subgroup of NSCLC and were highly correlated with the response to EGFR-TKI.(12-14) Subsequent analyses using a large series of NSCLC confirmed somatic mutations in the similar subgroup of patients as known clinical predictors of EGFR-TKI sensitivity.(15-18) Moreover, recent studies have shown that increased copy numbers of the EGFR gene are associated with a better response to EGFR-TKI. (17,19) HER2, another member of the ERBB family, can heterodimerize with EGFR and has also been shown to have somatic mutations in the TK domain in several cancers including NSCLC. (20,21) However, the involvement of HER2 alterations in the pathogenesis of NSCLC is not clearly understood.After earlier reporting 17 mutations of EGFR in 85 NSCLC, (22) we further extended our analysis with another 264 NSCLC. In a total of 349 tumors, we found 102 with EGFR mutation, six with HER2 mutation, and 21 with KRAS mutation. We also identified seven tumors with double mutations of EGFR and eight tumors with both mutation and amplification of EGFR. We therefore investigated the double genetic event of EGFR in detail. Our results provide new insights into the involvement of alterations of the receptor TK family in NSCLC development.

BACKGROUND Lung carcinomas show frequent inactivation of the p53 tumor suppressor, which regulates an apoptotic pathway. The objective of the current study was to assess how the p53 apoptotic pathway is altered in nonsmall cell lung carcinoma (NSCLC), especially in tumors without p53 alterations. METHODS p53, its upstream regulators (p14ARF and HDM2), and downstream effectors of the apoptotic pathway (BAX and BCL2) were studied in 118 NSCLC specimens. p53 was analyzed by single‐stranded conformation polymorphism analysis covering exons 2–11 and by immunohistochemistry (IHC). p14ARF was analyzed by methylation‐specific polymerase chain reaction and IHC. HDM2 was analyzed using Southern blot analysis and IHC. BAX and BCL2 were analyzed by IHC. Two other upstream regulators that regulate the stability of HDM2, PTEN and HAUSP, also were studied. RESULTS Of 118 NSCLC specimens that were analyzed, p53 alterations were detected in 74 tumors (63%), p14ARF inactivation was detected in 53 tumors (45%), and overexpression of HDM2 was found in 31 tumors (26%), including 6 tumors with gene amplification. Although p53 inactivation and HDM2 overexpression were detected simultaneously, HDM2 gene amplification was observed only in tumor specimens without p53 mutations. IHC revealed PTEN down‐regulation in 22 of 88 tumors (25%). HAUSP Northern blot analysis demonstrated several‐fold differences in gene expression that did not correlate with p53 alterations. Of 118 NSCLC specimens, expression of BAX and BCL2 expression were detected in 46 tumors (39%) and 17 tumors (14%), respectively. Finally, ASPP1 and ASPP2, molecules involved in mediating the transcription function of p53, were not found to be aberrantly expressed when tested by Northern blot analysis. CONCLUSIONS Overall, two or more p53 pathway components were found to be frequently altered in patients with NSCLC. Greater than 90% of the alterations were due to abnormalities of p53, p14ARF, or HDM2. Therefore, the inactivation of one or more components of the p53 pathway appears to be a prerequisite for the development of most NSCLCs. Cancer 2004. © 2004 American Cancer Society.

CT-guided hookwire marking appears to be useful for VATS, but the procedure may, although rarely, cause severe complications such as air embolism.

Activating mutations of RAS gene families have been found in a variety of human malignancies, including lung cancer, suggesting their dominant role in tumorigenesis. However, several studies have shown a frequent loss of the wild-type KRAS allele in the tumors of murine models and an inhibition of oncogenic phenotype in tumor cell lines by transfection of wild-type RAS, indicating that wild-type RAS may have oncosuppressive properties. To determine whether loss of wildtype KRAS is involved in the development of human lung cancer, we investigated the mutations of KRAS, NRAS and BRAF in 154 primary non-small cell lung cancers (NSCLCs) as well as 10 NSCLC cell lines that have been shown to have KRAS mutations. We also determined the loss of heterozygosity status of KRAS alleles in these tumors. We detected point mutations of KRAS in 11 (7%) of 154 NSCLCs, with 10 cases at codon 12 and 1 at codon 61, but no mutations of NRAS or BRAF were found. Using the laser capture microdissection technique, we confirmed that 9 of the 11 tumors and 7 of the 10 NSCLC cell lines retained the wild-type KRAS allele. Among the cell lines with heterozygosity of mutant and wild-type KRAS, all of the cell lines tested for expression were shown to express more mutated KRAS than wild-type mRNA, with higher amounts of KRAS protein also being expressed compared to the cell lines with a loss of wild-type KRAS allele. In addition, among 148 specimens available for immunohistochemical analysis, 113 (76%) showed positive staining of KRAS, indicating that the vast majority of NSCLCs continue to express wild-type KRAS. Our findings indicate that the wild-type KRAS allele is occasionally lost in human lung cancer, and that the oncogenic activation of mutant KRAS is more frequently associated with an overexpression of the mutant allele than with a loss of the wild-type allele in human NSCLC development. © 2003 Wiley-Liss, Inc. Key words: protooncogene; NRAS; BRAF; immunohistochemistry RAS, a well-known dominant oncogene, encodes a small GTPbinding protein involved in many cellular processes, including proliferation, differentiation and apoptosis. 1 The RAS gene family members (KRAS, HRAS and NRAS) are activated by point mutations at codons 12, 13 or 61 in approximately 20 -30% of lung adenocarcinomas and 15-20% of all non-small cell lung cancers (NSCLCs), but very rarely in small cell lung cancers (SCLCs). 2 Mutations in KRAS account for approximately 90% of RAS mutations of lung adenocarcinomas with 85% of the KRAS mutations affecting codon 12. Characteristically, approximately 70% of KRAS mutations are G-to-T transversions, involving the substitution of the normal glycine (GGT) with either cysteine (TGT) or valine (GTT). 3 Although the frequency of RAS mutations has been extensively studied in a variety of human malignancies, 4,5 several lines of evidence have suggested that the remaining allele of wild-type RAS may play a key role in controlling tumorigenesis. Homologous recombination has been used to replace one wild-type Hras1 allele with a ...