B7-H6 expression in non-small cell lung cancers

B7 family has been known to be a negative regulator of immunity response in patients with lung cancer. B7-H6 as a novel identified member of B7 family is found to trigger natural killer (NK) cell cytotoxicity and cytokine secretion by binding natural cytotoxicity receptor NKp30. Up until now, no investigations have been made about B7-H6 expression in lung cancer. We present the result of the B7-H6 prognostic value in 65 non-small cell lung cancer (NSCLC) tissues and 65 matched adjacent non-tumor tissues by Immunohistochemistry (IHC). Meanwhile, fluorescence activated cell sorter (FACS) analysis was used to detect B7-H6 receptor NKp30 expression in 7 non-small cell lung cancer tissues and 7 adjacent non-tumor tissues. Here, the result showed B7-H6 immunoreactivity in 6/65 (9.23%) lung cancer patients and 4/65 (6.15%) in adjacent non-tumor tissues. No relationship was found between B7-H6 expression and clinic pathological features. Similarly, no relevance was found for NKp30 expression in lung cancer tissues and non-tumor tissues. However, B7-H6 positive carcinomas were significantly correlated with degree of differentiation (P = 0.044). Three year survival rate after operation did not show the prognostic value for B7-H6 expression. Our study suggests that B7-H6 has a limited value as a prognostic marker in the patients of lung cancer.     

The candidate oncogene ZNF217 is frequently amplified in colon cancer

In this study we have defined the changes in gene copy number of the candidate oncogene ZNF217 during colon cancer development and progression. This gene is mapped to chromosome 20q and lies within 20q13.2, a region which we have previously shown to be highly amplified in colorectal cancer by comparative genomic hybridization. The gene copy number of ZNF217 was assessed in 100 colon carcinomas (19 Dukes' A, 42 Dukes' B and 39 Dukes' C), 13 colonic adenomas and 10 normal colon samples. DNA extracted from laser microdissected cells was amplified by multiplex real-time PCR at two distinct gene loci--ZNF217 and beta-globin (control gene)--on an ABI7700 sequence detection system. Of the 100 colon cancers studied, 61 showed some level of amplification of ZNF217, 15 had loss of ZNF217, while 24 were diploid. All the adenomas except one were diploid. In this study we have found that ZNF217 amplification is a frequent event in colon cancer and that the extent of its amplification varies markedly between tumours (range 3-13 copies). There was a trend toward poorer survival in patients whose cancers had either gain or loss of ZNF217.     

Mutation of FADD gene is rare in human colon and stomach cancers

Failure of apoptosis is one of the hallmarks of cancer. As an adaptor, FADD plays a crucial role during death receptor-mediated apoptosis. We previously reported that the FADD gene is somatically mutated in non-small cell lung cancers. To explore the possibility that the genetic alterations of the FADD gene might be involved in the development of other human cancers, we analyzed the entire coding region and all splice sites of the human FADD gene to detect somatic mutations in 116 stomach cancers and 98 colon cancers. Overall, we detected a somatic missense mutation of the FADD gene in a colon carcinoma, which was predicted to change an amino acid (R140H) in the death domain (DD) of the FADD protein. This is the first report of FADD gene mutation in gastrointestinal cancers, and our data suggest that the FADD gene is rarely mutated in human colon and stomach cancers.     

Non-small cell lung cancers frequently express phosphorylated Akt; an immunohistochemical study

Mounting evidence suggests that the alterations of Akt/protein kinase B (PKB) play an important role in tumorigenesis. Phosphorylated Akt regulates many of the key effector molecules involved in apoptosis, angiogenesis, and cell cycle progression during tumorigenesis. The expression of phosphorylated Akt has been described in some human malignancies, but not in primary human lung cancer. In this study, to understand the role of Akt in lung tumorigenesis we analyzed the expression of phosphorylated Akt in 43 non-small cell lung cancers (NSCLCs) by immunohistochemistry. Phosphorylated Akt was detected either in the cytoplasm (23 cases) or nucleus (6 cases) in 29 of 43 NSCLCs (67.4%). Squamous cell carcinomas, adenocarcinomas, and bronchioloalveolar carcinomas expressed phosphorylated Akt in 68.2%, 61.5% and 75%, respectively. We also analyzed the phosphorylated Akt expression between primary NSCLCs and their corresponding nodal metastasis; the expression was not, however, different between the primary and metastatic lesions. Taken together, these results indicate that Akt 1 is frequently activated in NSCLCs, irrespective of the histological subtypes, and suggest that phosphorylated Akt may play a role in the development of NSCLC rather than in the progression of NSCLC.     

Increased β1-adrenoceptor density after 6-hydroxydopamine pretreatment in rat colon and lung

Estimation of β-adrenoceptor-binding sites with ¹²⁵I-(-)-pindolol in rat colon show a proportion of 30%β₁,-adrenoceptors and 70%β₂-adrenoceptors. Studies on the isolated colon strip have revealed a neuronal β-adrenoceptor involved in the inhibitory response of colon motility to β-adrenoceptor stimulation. In order to further characterize the β-adrenoceptors in the colon, acute and chronic treatments with 6-hydroxydopamine were made. Both acute pretreatment of rats with 6-hydroxydopamine for 8 and 24 h (one intravenous injection) and chronic treatment for 3 days (implanted osmotic mini-pumps), reduced the noradrenaline tissue content by 90%, and successively increased the β-adrenoceptor-binding sites from 14.3 to 21.7 fmol mg^_1 P^_1 in colon and from 158 to 240 fmol mg^-1 P^_1 in lung membranes. Displacement of the radiolabelled ligand by the selective β-adrenoceptor antagonists, pafenolol and ICI 118.551 showed that the density of β₁,-adrenoceptor binding sites was more than doubled, whereas the density of β₂-adrenoceptor-binding sites was only marginally increased by chronic treatment with 6-hydroxydopamine. Thus sympathetic denervation by 6-hydroxydopamine treatment produced a selective increase in β₁-adrenoceptors in the rat colon. These results may indicate that stimulation of β₁-adrenoceptors in both colon and lung have a neuronal linkage.     

Vascular invasion is an adverse prognostic factor in resected non–small‐cell lung cancer

Lung cancer is a leading cause of death, and there is a need for better prognostic factors in treatment decisions. Vascular invasion is a known negative prognosticator, but it is not clear how to evaluate this feature. Here, we studied the prevalence and prognostic impact of blood and lymphatic vascular invasion (BVI, LVI), tumour grade, necrosis, inflammation and pleural invasion on cancer‐specific survival (LCSS) and time to recurrence (TTR) in non–small‐cell lung cancer (NSCLC). A total of 438 patients surgically treated for NSCLC (1993–2010) were examined, including 213 adenocarcinomas (AC), 135 squamous cell carcinomas (SCC) and 90 other NSCLC. BVI and LVI were found in 25% and 21% of the cases, with reduced LCSS and TTR for both markers in AC and SCC (p < 0.005 for all). BVI and LVI remained independent prognostic factors for LCSS and TTR in separate multivariate models for AC and SCC. Combined BVI/LVI (7%) showed significantly reduced LCSS and TTR (p < 0.001), also by multivariate analysis. Our results support that BVI and LVI are valuable for prognostic staging. Vascular invasion identifies a group of patients at higher risk of recurrence and lung cancer–related death, and this could influence stratification of patients for treatment and follow‐up.     

Prognostic significance of circulating IL-10 and IL-6 serum levels in colon cancer patients undergoing surgery

The prognostic significance of IL-10 and IL-6 serum levels in colon cancer patients undergoing surgery was investigated. To this end, 50 candidate patients with colon cancer for surgery were admitted to the study. Of these, 30 could be subjected to a potentially curative surgery. Cytokine serum levels at several time points before and after surgery were measured by ELISA. Circulating levels of IL-10 and IL-6 were found to be elevated in cancer patients with respect to controls. Both IL-10 and IL-6 serum levels were demonstrated to predict the likelihood of curative surgery (predictive accuracy, 83.3%). IL-10 serum levels returned to normal in all but 6 patients who underwent curative surgery. These latter had tumor recurrence (predictive accuracy, 100%). In contrast, IL-6 serum levels significantly decreased in all patients, regardless of whether cure was surgically achieved, but did not normalize. On multivariate analysis, basal IL-10 serum levels were found to be among the variables significantly predicting the disease-free survival rate. Stepwise regression selected tumor stage, basal IL-10 serum level, and basal CEA serum level as the best combination of variables for prediction of the likelihood of tumor recurrence. In conclusion, preoperative serum levels of IL-10 were shown to be useful markers for predicting both likelihood to perform curative surgery and, in combination with the 16th postoperative day IL-10 serum levels, tumor recurrence (predictive accuracy, 73.6 and 96%, respectively). IL-6 serum levels were found to have a more limited prognostic role.     

Fibronectin is chemotactic for CT 26 colon carcinoma cells: sub-lines selected for increased chemotaxis to fibronectin display decreased tumorigenicity and lung colonization

CT 26 murine colon carcinoma cells demonstrated directional migration (chemotaxis) in response to fibronectin (FN). Sub-lines were derived by positive and negative selection to FN across Transwell filters of 8 mm pore size. The FL6 sub-line (positively selected) demonstrated a significantly increased chemotactic response (P < 0.01) to FN compared with parental CT 26 cells, while the FU7 sub-line (negatively selected) showed a reduced chemotactic response to FN (P < 0.01). Comparable levels of a4, a5, av and b1 integrins, which mediate FN attachment, were expressed on positively and negatively selected sub-lines and parental CT 26 cells. Activation of integrins with Mn 2+ suggested that the integrins expressed on FL6 cells were in the fully activated state; in contrast FU7 cells displayed only partially activated integrins. Cell attachment and integrin activation status of the sub-lines correlated with their chemotactic response to FN. In vivo FL6 cells showed a significantly reduced tumour growth rate s.c. and a reduction in the number of lung colonies formed following i.v. injection compared with parental CT 26 and FU7 cells. In contrast FU7 cells displayed a sig-nificant increase in s.c. tumour growth and the number of lung colonies when compared with the parental line and FL6 sub-line. The results indicate that interaction between integrin receptors expressed on cancer cells and FN plays a central role in the chemotactic response of CT 26 colon carcinoma cells, and that in this model cells selected for chemotaxis to FN displayed a reduced malignant potential.© Kluwer Academic Publishers 1998     

Glucose-6-phosphatase gene (727G→T) splicing mutation is prevalent in Hong Kong Chinese patients with glycogen storage disease type la

Glycogen storage disease type la (GSD1a) is an autosomal recessive metabolic disorder caused by a deficiency in glucose-6-phosphatase (GóPase). We analyzed the GóPase genes of two unrelated Chinese families with GSD1a. DNA sequencing of all five exons and the exonintron boundaries revealed a G → T transversion at nucleotide 727 (727G→T) in exon 5, which has previously been reported to cause abnormal splicing. In one family, the subject and her affected sister were confirmed to be homozygous for this mutation and their parents to be heterozygotes. In the other family, the proband was identified to be heterozygous for this mutation, and a novel mutation, the 341delG in exon 2, was identified. This mutation alters the reading frame and creates a stop codon TAA 15 codons downstream from the mutation, resulting in a truncated protein. Family studies revealed that the father was heterozygous for the 727G → T mutation and that the mother was heterozygous for the 341delG mutation. This is the first time that the 727G→T mutation has been found in Chinese patients or outside Japan. Since we only tested two GSDla families and found 727G→T in both, we believe that this mutation may also be prevalent in our local Chinese population. To investigate allele frequencies, we screened 385 Chinese healthy volunteers and found two asymptomatic carriers. Our findings suggest that the 727G → T mutation is indeed prevalent in Hong Kong.     

Clinical significance of combined detection of interleukin-6 and tumour markers in lung cancer

Tumour markers play an important role in the early diagnosis and guidance of prognosis of lung cancer. This study focused on the significance of combined monitoring of interleukin (IL)-6 and tumour markers in improving the specificity and sensitivity of lung cancer diagnosis and disease. The expression of IL-6, carcinoembryonic antigen (CEA), neuron-specific enolase (NSE) and cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) in serum of patients with non-small cell lung cancer (NSCLC) (n?=?138) was significantly higher compared to patients with benign pulmonary lesions (BPL) (n?=?60) and was associated with the clinicopathological features of NSCLC patients. The simultaneous increase in the expression of IL-6 and tumour markers worsened the prognosis of patients. Lung cancer cells were grouped into the blank control group, IL-6 group, niclosamide group (IL-6 pathway inhibitor, NIC) and IL-6?+?NIC group. The expression of CEA, NSE, CYFRA21-1, p-Erk1/2 and p-AKT in the IL-6 group was significantly higher compared to the other groups. Therefore, the combined detection of IL-6 and tumour markers is critical to improve the specificity and sensitivity of lung cancer diagnosis. This in-depth study not only helped to elucidate the mechanism of how IL-6 promotes lung cancer but also provided new ideas and entry points to resolve the low specificity and sensitivity of lung cancer-related tumour markers.     

RNA-based mutation analysis identifies an unusual MSH6 splicing defect and circumvents PMS2 pseudogene interference

Heterozygous germline mutations in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 cause hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome, a dominantly inherited cancer susceptibility syndrome. Recent reports provide evidence for a novel recessively inherited cancer syndrome with constitutive MMR deficiency due to biallelic germline mutations in one of the MMR genes. MMR-deficiency (MMR-D) syndrome is characterized by childhood brain tumors, hematological and/or gastrointestinal malignancies, and signs of neurofibromatosis type 1 (NF1). We established an RNA-based mutation detection assay for the four MMR genes, since 1) a number of splicing defects may escape detection by the analysis of genomic DNA, and 2) DNA-based mutation detection in the PMS2 gene is severely hampered by the presence of multiple highly similar pseudogenes, including PMS2CL. Using this assay, which is based on direct cDNA sequencing of RT-PCR products, we investigated two families with children suspected to suffer from MMR-D syndrome. We identified a homozygous complex MSH6 splicing alteration in the index patients of the first family and a novel homozygous PMS2 mutation (c.182delA) in the index patient of the second family. Furthermore, we demonstrate, by the analysis of a PMS2/PMS2CL "hybrid" allele carrier, that RNA-based PMS2 testing effectively avoids the caveats of genomic DNA amplification approaches; i.e., pseudogene coamplification as well as allelic dropout, and will, thus, allow more sensitive mutation analysis in MMR deficiency and in HNPCC patients with PMS2 defects.     

非小细胞肺癌中KLF6-SV1的表达及临床病理学意义

目的观察抑癌基因KLF6的剪切变异体KLF6-SV1在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的表达特点,探讨其与临床病理学参数的关系。方法应用免疫组化检测42例NSCLC和40例良性肺疾病(肺的炎性肌纤维母细胞瘤、肺脓肿、肺结核、肺的硬化性血管瘤等)中KLF6-SV1蛋白的表达水平,原位杂交检测42例NSCLC和40例良性肺疾病中KLF6-SV1基因mRNA的表达。结果 KLF6-SV1在NSCLC中的蛋白阳性率为71.4%(30/42),表达水平均高于良性肺疾病(P<0.05)。KLF6-SV1基因mRNA的阳性率为69.0%(29/42),表达水平高于良性肺疾病(P<0.05)。KLF6-SV1的蛋白表达水平与其mRNA表达水平之间具有较好的一致性。KLF6-SV1的表达与有无淋巴结转移及肿瘤的分化程度有关,组间差异具有统计学意义(P<0.05),与病理分型、组织大小和临床分期无关,组间差异不具有统计学意义(P>0.05)。结论 KLF6-SV1的过表达可能与NSCLC的发生、发展及早期淋巴结转移密切相关,有望成为今后诊断的参考指标之一,并有可能成为肿瘤基因治疗的新靶点之一。