p16 protein expression is associated with a poor prognosis in squamous cell carcinoma of the lung

An immunohistochemical analysis for p16 protein was performed in 171 patients with non-small-cell lung cancer (NSCLC). Sixty-two carcinomas (36.3%) were classified as p16-negative. p16-negative tumours in squamous cell carcinomas (SCCs) were significantly more than those in adenocarcinomas (P = 0.039). There was no significant difference in survival according to tumour p16 status in patients with NSCLCs or in patients with adenocarcinomas. In contrast, of patients with SCCs, the 5-year survival rate of patients with p16-negative tumours was significantly lower than those with p16-positive tumours (P = 0.001). Especially, the survival of patients with p16-negative tumours was significantly worse than that of patients with p16-positive tumours in the early stage of the SCC, e.g. stage I (P = 0.005). Multivariate analysis showed that p16 status and nodal status were significant prognostic factors for the survival of patients with SCCs of the lung (P = 0.024 and P = 0.008 respectively). In conclusion, our study showed that alteration of p16 was one of the significant factors of a poor prognosis in SCCs of the lung, and that p16 might play an important role in some SCCs of the lung due to its high prevalence and prognostic value.     

Aberrant promoter methylation in Chinese patients with non-small cell lung cancer: patterns in primary tumors and potential diagnostic application in bronchoalevolar lavage.

This study was aimed at defining patterns of aberrant gene methylation in non-small cell lung cancer (NSCLC) in Chinese patients and its use in detecting cancer cells in bronchoalveolar lavage (BAL). The methylation-specific PCR (MSP) was used to study methylation of the p16, retinoic acid receptor-beta (RARbeta), death-associated protein (DAP) kinase, and O(6)-methylguanine-DNA-methyltransferase (MGMT) genes in 75 NSCLCs [44 adenocarcinomas and 31 squamous cell carcinomas (SCCs)] and 68 BALs from suspected lung cancers. More females had adenocarcinoma than SCC (11 of 44 versus 2 of 31, P = 0.04). Aberrant methylation in at least one gene was found in 63 of 75 (84%) NSCLCs. p16, RARbeta, DAP kinase, and MGMT methylation was similar in adenocarcinoma and SCC. However, females with NSCLC showed more frequent p16 methylation than males (12 of 13 versus 36 of 62, P = 0.02), because of more frequent p16 methylation in female adenocarcinomas (10 of 11 versus 17 of 33, P = 0.02). This sexual difference was not observed in RARbeta, DAP kinase, and MGMT. At 92%, the frequency of p16 methylation in Chinese female NSCLC is one of the highest known. For BAL, MSP and cytological analysis showed concordant and discordant results in 25 of 68 and 43 of 68 samples. Of 41 MSP+/cytology- cases, 35 were eventually shown to have malignant lung lesions, 4 were at high risk but had no evidence of lung cancer, and 2 were lost to follow-up. There were two MSP-/cytology+ cases. Frequent gene methylations were seen in Chinese NSCLC patients. More frequent p16 methylation was seen in female patients. MSP is a useful molecular adjunct for cancer cell detection in BAL samples.     

甲状腺转录因子1在肺癌中的表达及应用

目的观察甲状腺转录因子1(TTF1)在肺肿瘤中的表达差异,探讨其在鉴别诊断中的应用。方法134例肺叶切除标本中,肺原发癌105例,肺转移性腺癌29例。原发癌中,非小细胞癌(NSCLC)76例,其中腺癌42例,鳞状细胞癌30例,大细胞癌4例;小细胞癌(SCLC)28例;复合性癌(小细胞癌与鳞状细胞癌复合存在)1例。通过免疫组织化学染色,观察TTF1在肿瘤中的表达情况。结果判定 阳性瘤细胞占6%～25%; 阳性瘤细胞占26%～50%; 阳性瘤细胞占51%～75%; 阳性瘤细胞>76%。结果TTF1在小细胞肺癌的表达率最高,为82.1%(23/28),而在鳞状细胞肺癌的表达率为0(0/30),两者之间差异有显著性(P<0.001)。TTF1在肺腺癌的表达率为73.8%(31/42),其表达与分化程度及亚型分类无关(P均>0.05)。在29例肺转移腺癌中,除1例甲状腺滤泡癌TTF1表达为阳性外,其余均为阴性。组织自身对照中的间质成分及淋巴细胞等TTF1染色全部为阴性。结论根据TTF1在肺不同肿瘤内的差异表达结果,可将其用于小细胞肺癌与鳞状细胞肺癌、小细胞肺癌与肺炎症或淋巴瘤的鉴别;TTF1对肺组织的特异性表达,有助于对肺原发腺癌及转移腺癌的鉴别。     

Expression of MET and SOX2 genes in non-small cell lung carcinoma with EGFR mutation

Non-small cell lung carcinoma (NSCLC) is a leading cause of cancer-related deaths. Aberrance of the two oncogenes MET and SOX2 are frequently encountered in NSCLC. Exons?18 through 21 of the EGFR gene were screened and MET and SOX2 immunostaining was conducted to analyze the immunohistological staining of MET and SOX2 and the EGFR mutation status. One hundred and fifty tissue samples were examined including 57 squamous cell carcinomas (SCCs), 80 adenocarcinomas (ADCs), 9 adenosquamous carcinomas (ADSCs) and 4 large cell carcinomas (LCCs). The 32 NSCLCs harboring an EGFR mutation included 28 ADCs, 3 SCCs and 1 ADSC. A higher level of SOX2 expression appeared in NSCLCs without the EGFR mutation compared to those with EGFR mutation (χ2=9.02, P=0.0027). Of the 28 ADCs, 24 (85.7%) with an EGFR mutation showed low level of SOX2 expression. ADCs with deletion in exon?19 overexpressed MET and showed low levels of SOX2. SOX2 expression was inversely correlated to the expression of MET in NSCLC and mainly present in non-mutated NSCLC (r=-0.42, P<0.0001). There was a tendency for SOX2 to be expressed in SCCs and particularly in the part of SCC among ADSCs, whereas MET was mainly expressed in the part of ADC among ADSCs and ADCs. High level of MET and SOX2 expression were respectively demonstrated in ADCs and SCCs; MET activation was accompanied with exon?19 deletion in ADCs. EGFR and MET coordinate to drive tumorigenesis. Detection of the activation of MET and EGFR may be used for targeted drug therapy.     

A chemically induced model for squamous cell carcinoma of the lung in mice: histopathology and strain susceptibility

Lung cancer, primarily associated with tobacco use, is the leading cause of cancer morbidity and mortality in the United States. Squamous cell carcinoma (SCC) is one of the four major histological types of lung cancer. Although there are several established models for lung adenoma and adenocarcinomas, there is no well-established mouse model for lung SCC. We treated eight different inbred strains of mice with N-nitroso-tris-chloroethylurea by skin painting and found that this regimen induced lung SCCs in five strains of mouse (SWR/J, NIH Swiss, A/J, BALB/cJ, and FVB/J) but not in the others (AKR/J, 129/svJ, and C57BL/6J). Mouse lung SCCs have similar histopathological features and keratin staining to human SCC. Moreover, a wide spectrum of abnormal lung squamous phenotypes including hyperplasia, metaplasia, carcinoma in situ, and invasive carcinoma, were observed. There are strain-specific differences in susceptibility to Lscc induction by N-nitroso-tris-chloroethylurea with NIH Swiss, A/J, and SWR/J mice developing scores of SCCs whereas the resistant strains AKR/J, 129/svJ, and C57BL/6J failed to develop any SCCs. FVB/J and BALB/cJ mice had an intermediate response. We conducted whole-genome linkage disequilibrium analysis in seven strains of mice, divided into three phenotype categories of susceptibility, using Fisher's exact test applied to 6,128 markers in publically available databases. Three markers were found significantly associated with susceptibility to SCC with the P < 0.05. They were D1Mit169, D3Mit178, and D18Mit91. Interestingly, none of these sites overlap with the major susceptibility loci associated with lung adenoma/adenocarcinoma development in mice. The mouse SCC described here is highly significant for preclinical studies of lung cancer chemopreventive agents because most human trials have been conducted against precancerous lesions for SCC. Furthermore, this model can be used in determining genetic modifiers that contribute to susceptibility or resistance to lung SCC development.     

osteopontin在非小细胞肺癌中的异常表达及其临床意义

目的探讨细胞外基质蛋白分子osteopontin(OPN)在非小细胞肺癌(NSCLC)中的表达状况及特点,以及其在肺癌发生演进中的作用及其临床意义。方法35例新鲜NSCLC肿瘤及其对照肺组织,采用逆转录PCR(RT-PCR)技术,分析OPN基因在RNA水平的表达改变。大样本石蜡包埋组织(662个有效样品的组织微阵列)以免疫组织化学染色,检测OPN蛋白在NSCLC中的表达状况,并分析其与肿瘤的组织学及临床特征的关系。结果RT-PCR分析显示,OPN在80.0%的NSCLC肿瘤组织中转录上调。免疫组化染色分析显示,OPN蛋白在肿瘤组织中的表达(59.6%)显著高于正常肺组织(25.2%,P<0.001),且OPN在鳞癌中的表达高于腺癌。通过对鳞癌的深入分析则表明,OPN蛋白表达水平在有淋巴结转移和无淋巴结转移的原发癌间有统计学意义(68.6%和49.7%,P= 0.001),而在同一组患者的原发癌和淋巴结转移癌之间的表达水平则趋于一致(68.6%和75.5%,P=0.171)。结论OPN在NSCLC(尤其是鳞癌)中表达水平明显升高,其过表达与肺鳞癌淋巴结转移密切相关,增加了肿瘤转移的风险(OR=2.212);OPN有促进肺癌恶性演进的作用。     

Difference of caveolin-1 expression pattern in human lung neoplastic tissue. Atypical adenomatous hyperplasia, adenocarcinoma and squamous cell carcinoma

Caveolin-1 has been implicated in cellular transformation and tumorigenesis. We assessed lung cancer specimens for caveolin-1 expression immunohistochemistry. A majority of the cell types in the lung and the bronchial epithelium normally exhibited positive staining for caveolin-1. In adenocarcinomas (ADs) of positive staining for caveolin-1, pT1 tumors exhibited significantly higher staining than pT2-pT4 tumors (P=0.0240). In squamous cell carcinomas (SCCs), pT1-pT2 tumors expressed significantly lower expression levels than pT3-pT4 tumors (P=0.0175). In AD, loss of caveolin-1 may be essential for tumor extension and dedifferentiation. In contrast, caveolin-1 overexpression may be correlated with tumor extension in SCC.     

Loss of heterozygosity and the smoking index increase with decrease in differentiation of lung adenocarcinomas: etiologic implications

To better understand causative relations of smoking to lung adenocarcinomas, the frequency of loss of heterozygosity (LOH) of all autosomal chromosomes was compared among the three grades of histological differentiation with 119 pulmonary adenocarcinomas (AC) and 41 squamous cell carcinomas (SCC), using Southern blotting. The fractional allelic loss (FAL) values, defined as (number of chromosome arms with LOH)/(number of informative arms), and smoking index (a product of number of cigarettes per day and duration in years) for all ACs were 0.19 and 520 whereas those for SCCs were 0.34 and 1,160, respectively. Those for well- (n=33), moderately (n=63) and poorly (n=23) differentiated ACs were 0.100, 0.197, 0.295 and 310, 480, 1,010, respectively. These results showed that less differentiated ACs are more similar to SCC in terms of LOH frequency and smoking.     

KIF5B/RET fusion gene in surgically-treated adenocarcinoma of the lung

Recently, a novel fusion gene resulting from a linkage between the kinesin family member 5B gene (KIF5B; 10p11.22) and the rearranged during transfection gene (RET; 10q11.21) was identified in non-small cell lung cancer (NSCLC). However, the correlation between the KIF5B/RET fusion gene status and the clinicopathological features of surgically-treated lung cancer has not been well characterized. In this study, we have independently investigated the KIF5B/RET fusion gene status in 371?surgically-treated NSCLCs (270 were adenocarcinomas and 101 were squamous cell carcinomas), 60 breast cancers, 11?metastatic lung cancers from colon cancers and thyroid papillary adenocarcinoma cases at the Nagoya City University Hospital. The fusion gene status was analyzed by an RT-PCR-based assay and by using direct sequencing. We detected 3 of 270 cases of KIF5B/RET fusion genes in adenocarcinomas (1.1%) consisting of female and never smokers with mixed subtype adenocarcinomas. The fusion genes were detected exclusively with other mutations, such as EGFR, Kras, Braf, erbB2 mutations, and EML4/ALK fusion. KIF5B/RET fusion was not detected in the cases with squamous cell carcinoma or other types of cancers. From the 3?cases, 2?were KIF5B (exon 15); RET (exon 12) fusions with papillary dominant and 1?case was KIF5B (exon 22); RET (exon?12) fusion with solid dominant adenocarcinoma. The matched normal lung tissues did not display translocation. We reported KIF5B/RET fusion genes as a driver somatic mutation of lung adenocarcinomas. The cinicopathological backgrounds of the KIF5B/RET fusion-positive patients were similar with those of the EML4/ALK fusion-positive patients. The chimeric oncogene may be a promising molecular target for the personalized diagnosis and treatment of NSCLC.     

Frequent allelic deletion at the FHIT locus associated with p53 overexpression in squamous cell carcinoma subtype of Taiwanese non-small-cell lung cancers

The fragile histidine triad (FHIT) gene, encompassing the FRA3B fragile site at chromosome 3p14.2, is a tumour suppressor gene involved in different tumour types including non-small-cell lung cancers (NSCLCs). In the current study, we examined for allelic deletion at the FHIT locus in 58 primary and microdissected NSCLCs, for which a clinicopathologic profile was available. We found a loss of 87.7% in heterozygosity (LOH) frequency at one or more microsatellite markers (D3S1289, D3S2408, D3S1766, D3S1312, D3S1600). Allelic deletion of D3S1766 was related to tumour histology in 10 of 11 squamous cell carcinomas (90.9%) displaying LOH compared with nine of 17 adenocarcinomas (52.9%; P=0.049). Besides, in the subset of adenocarcinomas, a higher rate of LOH at D3S1289 was observed in male (six out of eight, 75%) than in female patients (four out of 17, 23.5%; P=0.028). However, FHIT LOH was not correlated overall with a variety of clinical parameters including sex, smoking status, staging, lymph node metastasis and survival. These results indicated that the high frequency of FHIT gene disruption was important in the development of both squamous cell carcinomas and adenocarcinomas. Furthermore, there was no association between LOH at FHIT and protein expression, suggesting the presence of complex mechanisms of Fhit inactivation. On the other hand, the association between FHIT LOH and p53 protein overexpression assessment reached statistical significance (P=0.026), implying that common alterations affect the two genes in tumour progression.     

Stat3 downstream genes serve as biomarkers in human lung carcinomas and chronic obstructive pulmonary disease

Smoking causes lung cancer and chronic obstructive pulmonary disease (COPD) that impose severe health problem to humans. Both diseases are related to each other and can be induced by chronic inflammation in the lung. To identify the molecular mechanism for lung cancer formation, a CCSP-rtTA/(teto)(7)Stat3C bitransgenic model was generated recently. In this model, persistent activation of the Stat3 signaling pathway induced pulmonary inflammation and adenocarcinoma formation in the lung. A group of Stat3 downstream genes were identified by Affymetrix GeneChip microarray analysis that can be used as biomarkers for lung cancer diagnosis and prognosis. To determine which human lung cancers are related to the Stat3 pathway, multiple Stat3 downstream genes were screened in human lung cancers (adenocarcinomas and squamous cell carcinomas) and lung tissue with COPD. In both cancer and COPD, the Stat3 gene was up-regulated. A panel of Stat3-up-regulated downstream genes in mice was up-regulated in human adenocarcinomas, but not in human squamous cell carcinomas. This panel of genes was also modestly up-regulated in lung tissue with COPD from patients with a history of smoking and not up-regulated in those without histories of smoking. Several Stat3-down-regulated downstream genes also showed differential expression patterns in carcinoma and COPD. These studies support a concept that Stat3 is a potent oncogenic molecule that plays a role in formation of lung adenocarcinomas in both mice and humans. The carcinogenesis of adenocarcinoma and squamous cell carcinoma is mediated by different molecular mechanisms and pathways in vivo. Stat3 and its downstream genes can serve as biomarkers for lung adenocarcinoma and COPD diagnosis and prognosis in mice and humans.     

Expression pattern of the scaffold protein IQGAP1 in lung cancer

IQGAP1 is a scaffold protein whose function relates to signal transduction, cell adhesion, local invasion, and distant metastasis of cancer cells. We examined the expression patterns of this protein and clinicopathologic features of lung cancer, and the antibody against IQGAP1 was used for immunohistochemical analysis. Of the 70 surgical specimens examined, there were 40 adenocarcinomas, 19 squamous cell carcinomas, 5 large cell carcinomas, 3 small cell carcinomas, 2 carcinoid tumors, and 1 mucoepidermoid carcinoma. The localization of IQGAP1 was classified into three types: 1) cytoplasmic, 2) membranous, and 3) reduced expression. In adenocarcinoma, the 3 types were observed equally, and differentiation grade was related to the expression pattern. The cytoplasmic type was common in well-differentiated adenocarcinomas, and membranous or reduced expression was frequently seen in moderately- or poorly-differentiated adenocarcinomas. In squamous cell carcinoma, the membranous type was most common. Although the staining pattern of IQGAP1 did not correlate with the positivity of regional lymph nodes, survival in those patients with a cytoplasmic type was significantly better than others with adenocarcinoma (p=0.0144). Expression typing of IQGAP1 in lung cancer was associated with histologic type and can be used to predict survival in patients with adenocarcinoma of the lung.     

Heterogeneity of lung cancer cells with respect to the amplification and rearrangement of myc family oncogenes

Seventy lung tumors from 53 patients were analysed for alterations of myc family oncogenes, c-myc, N-myc and L-myc, to evaluate when activation of these genes occurs during tumor development. The 53 cases were 17 small cell carcinomas (SCCs), 18 adenocarcinomas, 12 squamous cell carcinomas (SqCs), 4 large cell carcinomas and 2 adenosquamous carcinomas. Either N-myc or L-myc was amplified in 4 of the 17 (one N-myc and 3 L-myc) SCCs (24%), while c-myc was amplified in 3 of the 12 SqCs (25%). In one SCC, amplification of N-myc was found in the primary tumor, a pulmonary hilar lymph node metastasis and a pleural metastasis, but not in a liver metastasis or a para-aortic lymph node metastasis. In one SqC, c-myc was amplified in a pleural metastasis and a lymph node metastasis, but not in the primary tumor. In 2 cases of SCCs, amplification or rearrangement of c-myc was detected only in the cell lines, but not in the original tumors taken from the same individuals. These results indicate that tumor cells were heterogeneous for amplification and rearrangement of myc family oncogenes, and suggest that activation of these oncogenes in SCCs and SqCs occurs not at the time of malignant transformation but during tumor progression.     

Prognostic role of protease-activated receptors 1 and 4 in resected stage IB non-small-cell lung cancer

BACKGROUND: Protease-activated receptor (PAR)-1 and PAR-4 are involved in extracellular matrix invasion and angiogenesis. PATIENTS AND METHODS: A series of 60 resected stage IB non-small-cell lung cancers (NSCLCs), including 30 adenocarcinomas (ADCs) and 30 squamous cell carcinomas (SCCs), were processed by immunohistochemistry with antibodies to PAR-1, PAR-4, vascular endothelial growth factor (VEGF), and CD34. RESULTS: Protease-activated receptor-1 was expressed in 37 cases (62%) and PAR-4 in 39 (65%). Adenocarcinomas were significantly more positive than SCC for PAR-1 (17 vs. 8 cases) and PAR-4 (10 vs. 5 cases). Vascular endothelial growth factor was expressed in 42 cases (70%): 22 ADC and 20 SCC. A significant correlation emerged between PAR-1 and/or PAR-4 expression and VEGF but not with microvessel density. Median follow-up was 38 months; actuarial 5-year survival was 43%. At univariate analysis, 3-year survival was shorter in patients expressing PAR-4 versus negative cases (29% vs. 60%; P = 0.002). In 46 patients expressing PAR-1 and/or PAR-4, 3-year survival was 30% versus 68% in 14 patients with no PAR expression (P = 0.002). A trend toward shorter 3-year survival was seen in PAR-1-positive versus PAR-1-negative cases (34% vs. 46%; P = 0.06). Multivariate analysis identified expression of PAR-1 and/or PAR-4 as an independent prognostic factor for reduced survival in resected stage IB NSCLC. CONCLUSION: Expression of PAR-1 and PAR-4 in early-stage NSCLC could be included in a molecular algorithm for the selection of patients eligible for adjuvant studies.     

Detection of MET and SOX2 amplification by quantitative real-time PCR in non-small cell lung carcinoma

Non-small cell lung carcinoma is a leading cause of cancer-related death. Amplification of the two oncogenes MET and SOX2 is frequently encountered in non-small-cell lung carcinoma. This study aimed to use real-time quantitative PCR to assess the correlation of MET and SOX2 amplification with clinicopathological factors. This study was conducted using 115 tissue samples including 57 squamous cell carcinomas (SCCs), 50 adenocarcinomas (ADCs) and 8 adenosquamous carcinomas (ADSCs). A total of 67 patients (58.3%) had a history of smoking. Our results showed that the frequency of MET amplification in SCCs was significantly higher compared to ADCs (χ(2)=8.0, P=0.005). SOX2 showed a markedly preferential amplification in SCCs compared to ADCs in the smoking group cases (P=0.014). Lymph node invasion correlated with MET amplification in SCCs marginally more significantly compared to ADCs (P=0.02). The amplified MET occurred more frequently in SCCs compared to ADCs correlated to tumor dimension at a small scale (<5 cm) (P=0.01). No significant difference in SOX2 amplification was found with regards to lymph node metastasis or tumor dimension. SOX2 and MET amplifications were not associated with gender or age. However, MET amplification in SCCs among patients younger than 64 years of age was higher compared to ADCs and ADSCs (P=0.03). Among ADSCs, MET was not amplified among patients who had never been smokers or were younger than 64 years of age. Neither MET nor SOX2 were amplified in tumors with dimensions <5 cm and without lymph node invasion. Findings of this study showed that MET and SOX2 amplifications are more common in the SCCs of smokers. Moreover, MET amplification is intrinsic in SCCs particularly among smokers, with regards to tumor growth, lymph node invasion and negative correlation to SOX2 amplification. The incidence of discrepancy in the amplifications of MET and SOX2 in SCCs and ADCs suggests that the MET and SOX2 genes play different roles in SCC and ADC tumorigenesis, respectively, particularly among smokers.     

Promoter hypermethylation of tumor suppressor and tumor-related genes in non-small cell lung cancers

Aberrant methylation of promoter CpG islands is known to be a major inactivation mechanism of tumor suppressor and tumor-related genes. To determine the clinicopathological significance of gene promoter methylation in non-small cell lung cancer (NSCLC), we examined the promoter methylation status of the APC, DAP-kinase, E-cadherin, GSTP1, hMLH1, p16, RASSF1A and RUNX3 genes in 75 NSCLCs and corresponding non-neoplastic lung tissues by methylation-specific PCR (MSP). The frequencies of methylation in NSCLCs and corresponding non-neoplastic lung tissues were: 37% (28 of 75) and 48% (36 of 75) for APC , 28% (21 of 75) and 13% (10 of 75) for DAP-kinase , 29% (22 of 75) and 15% (11 of 75) for E-cadherin , 1% (1 of 75) and 0% (0 of 75) for GSTP1 , 7% (5 of 75) and 0% (0 of 75) for hMLH1 , 31% (23 of 75) and 0% (0 of 75) for p16 , 43% (32 of 75) and 4% (3 of 75) for RASSF1A , and 20% (15 of 75) and 3% (2 of 75) for RUNX3 , respectively. Methylation of p16 was more frequent in squamous cell carcinomas than in adenocarcinomas ( P <0.05), and was associated with tobacco smoking ( P <0.05). On the contrary, methylation of APC and RUNX3 was more frequent in adenocarcinomas than in squamous cell carcinomas ( P <0.05). Thus, a different set of genes is thought to undergo promoter methylation, which leads to the development of different histologies. In addition, methylation of p16, RASSF1A and RUNX3 was mostly cancer-specific ( P <0.05), and may be utilized as a molecular diagnostic marker of NSCLCs.