Frequent silencing of DBC1 is by genetic or epigenetic mechanisms in non-small cell lung cancers

Genome-wide screening of DNA copy number aberrations in 27 cell lines derived from non-small cell lung cancers (NSCLCs), using a custom-made comparative genomic hybridization (CGH)-array, identified a homozygous deletion of the deleted in bladder cancer 1 gene (DBC1) in one cell line. Homozygous deletion of DBC1, located at 9q33.1, was also observed in two of 53 primary NSCLC tumors examined. Moreover, 21 of the other 26 cell lines showed complete loss of DBC1 expression, although normal lung tissues express this gene, and treatment with 5-aza-2'-deoxycytidine restored expression of DBC1. Hypermethylation in part of a CpG island around the exon 1 of DBC1 has been reported in urothelial cancers, but the potential association between methylation and expression status was never clarified in that disease. In our experiments, a different part of the same CpG island showed promoter activity in vitro and was frequently methylated in our cell lines and primary tumors of NSCLC, where methylation status correlated inversely with gene expression. Among our primary NSCLC cases, methylation of the DBC1 promoter occurred more frequently in men, elderly patients and smokers than in women, younger patients and nonsmokers respectively, but it was not correlated with tumor stage or histology. Exogenous overexpression of DBC1 in NSCLC cell lines lacking its expression inhibited cell growth. Our results provide the first evidence that DBC1 is a likely tumor suppressor for NSCLC; silencing of the gene through homozygous deletion or methylation of its promoter region may be associated with progression of this disease.     

CD133 positive endothelial progenitor cells contribute to the tumour vasculature in non-small cell lung cancer

AIMS: Recent results generated in a mouse model suggest that tumour angiogenesis/vasculogenesis can be initiated and maintained by bone marrow derived endothelial progenitor cells. This present study investigated the distribution and frequency of CD133 positive endothelial progenitor cells in patients with non-small cell lung cancer (NSCLC) (tumour tissue and tumour free lung regions) and healthy controls using fresh frozen specimens. The novel marker CD133 identifies human haemopoetic precursor cells, in addition to human endothelial progenitor cells. METHODS: Seventy nine lung cancer specimens and 66 adjacent histologically tumour free tissues of the same patient cohort were analysed; 11 postmortem specimens from control patients who did not suffer from malignant disease served as controls. Cryostat sections were stained for CD133, CD31, vascular endothelial growth factor receptor 2 (VEGFR-2; KDR), p53, and the proliferation marker Ki-67, and the correlations were analysed. RESULTS: Forty three of 63 evaluable tumour specimens had increased numbers of CD133 positive cells and in some cases capillary forming CD133 positive structures were detectable. In addition, 30 of 63 specimens had raised expression of KDR and 29 of 63 had increased MVD. Increased CD133 expression marginally correlated with raised KDR expression but not with p53 and Ki-67. CONCLUSION: A significant increase in CD133 positive cells was documented in patients with NSCLC, suggesting an involvement of endothelial progenitor cells in tumour vasculogenesis and tumour growth in these patients.     

Cytogenetics of non-small cell lung cancer: analysis of consistent non-random abnormalities

Cytogenetic analysis of ten primary non-small cell lung carcinomas (NSCLC), including five adenocarcinomas (ADC), three squamous cell (SQC), and two large cell (LCC) carcinomas has been carried out in an attempt to determine karyotype changes involved in the early stage of disease. The tumors were all aneuploid and exhibited complex karyotypes with multiple structural and numerical abnormalities. Clonal structural rearrangements were identified and in particular loss of material from the short arm of chromosome 9 had a 90% incidence. This loss was due to non-reciprocal translocation, deletion, or chromosome loss. Breakpoints were in the region 9q13 to p22. Other chromosome regions that were non-randomly involved are as follows: I cen to p13, 3p, 5q11 to q13, 6p, 6q15 to q27, 7p, 8p, 11q12 to q23, 13p, 14p, 15p, 17p, and 19p. While a primary cytogenetic change in NSCLC has not been identified conclusively, our findings implicate loss of material from 9p as a potentially important event.     

Investigation of genetic susceptibility to non-small cell lung cancer by fragile site expression

Fragile sites are non-staining gaps and breaks in specific points of chromosomes that are inducible by various culture conditions. Previous studies have shown that various clastogenic agents increase expression of fragile sites. In this study, the expression of common fragile sites induced by aphidicolin was evaluated on prometaphase chromosomes obtained from peripheral blood lymphocytes. Chromosomal aberrations and fragile site expression of 60 individuals, including 20 patients with non-small cell lung cancer (NSCLC), 20 of their clinically healthy family members, and 20 age-matched normal healthy controls without history of any cancer type were studied. Both the proportion of damaged cells (P < 0.001) and the mean number of gaps and breaks per cell (P < 0.001) were significantly higher in both the patients and relatives' groups when compared with the control group. However, they were insignificant when the patients were compared to their relatives (P > 0.05). We determined four aphidicolin type common fragile sites in our study. These sites in patients with NSCLC and relatives were the following: 1p21, 2q33, 3p14, and 16q23. In these fragile sites, 2q33, 3p14, and 16q23 sites were statistically significant when compared with control group (P < 0.001, P < 0.0005, and P < 0.05, respectively). Consequently, we believe that fragile site studies may be helpful to detection of cancer risk.     

Chromosome abnormalities in non-small cell lung cancer pleural effusions: Cytogenetic indicators of disease subgroups

A cytogenetic study of pleural effusions (PE) containing metastatic or invasive tumor cells from 11 patients with non-small cell lung cancer (NSCLC) (3 squamous cell carcinomas [SQC] and 8 adenocarcinomas [ADC] including 1 giant cell variant) was performed to identify non-random chromosome abnormalities. Numerical abnormalities seen in ≥ 30% of cases included gain of chromosomes 7 and 20, and loss of chromosomes 4, 9, 10, 13, 15, 16, 18, 19, 21, and 22. The most frequent structural abnormality involved rearrangement in 1p with breakpoints clustering at 1p10-p13. Other recurrent breakpoint regions, seen in ≥ 30% of cases, occurred in chromosome regions 3p10-p21, 3q11-q25, 6p11-p25, 6q13-q23, 7q11-q36, 9q32-q34, 11p11-p13, 11q13-q24, 13p/14p and/or 15p, 17p and 19p, with, in particular, apparent loss of 6q21-q27, 3p21-p26, 7q21-q22, 9p22-p24 (shortest regions of common overlap) and 17p. There was also recurrent gain of 1q23-q44, 8q13-q24, and 11q13-q23. These abnormalities were not restricted to a particular histological subtype, with the exception of + 8 and a breakpoint in 9q32-q34, which were seen only in ADC. The 9q32-q34 breakpoint observed in 4 ADC PE (including 1 giant cell variant) represents a new observation in NSCLC. These findings, when compared to those reported for primary NSCLC indicate cytogenetic differences between the two which may be associated with pleural invasion of NSCLC. © 1993 Wiley-Liss, Inc.     

Stathmin在非小细胞肺癌中表达增强* 基金项目：辽宁省科学技术计划立项课题资助（编号：2007225005-13）

 摘要:目的 探讨Stathmin在非小细胞肺癌（non-small cell lung cancer, NSCLC）组织中和正常组织中的表达,了解其与NSCLC临床病理特征之间的关系。方法 用免疫组化与RT-PCR方法,检测43例NSCLC 术后癌组织及正常组织标本中Stathmin蛋白与mRNA的表达。结果 Stathmin蛋白和基因在NSCLC中阳性表达率分别为62.79%和67.44%,显著高于正常组织的16.28%和20.93%（P＜0.01）,其表达与肿瘤细胞分化程度和有无淋巴结转移有关（P ＜0.05）。结论 Stathmin在NSCLC的发生发展过程中起了重要作用,可能成为预测NSCLC恶性程度的新的生物学及个体化治疗的敏感指标。     

Immunotherapeutic targets in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is one of the most common types of cancer in the world and has a 5-year survival rate of ~20%. Immunotherapies have shown promising results leading to durable responses, however, they are only effective for a subset of patients. To determine the best therapeutic approach, a thorough and in-depth profiling of the tumour microenvironment (TME) is required. The TME is a complex network of cell types that form an interconnected network, promoting tumour cell initiation, growth and dissemination. The stroma, immune cells and endothelial cells that comprise the TME generate a plethora of cytotoxic or cytoprotective signalling pathways. In this review, we discuss immunotherapeutic targets in NSCLC tumours and how the TME may influence patients' response to immunotherapy.     

Centrosome abnormalities in non-small cell lung cancer: correlations with DNA aneuploidy and expression of cell cycle regulatory proteins

The aims of this study were to investigate centrosome abnormalities in non-small cell lung cancer (NSCLC), and to assess their relationship with DNA aneuploidy, the expression of the cell cycle-associated proteins, and clinicopathological profiles. Tissue microarrays were constructed from 175 NSCLCs. We analyzed centrosome abnormalities and the expression of p16^INK4a, p53, and pRb using immunohistochemistry. Centrosome abnormalities were noted in 29% of the tumors and were even observed in the normal cells adjacent to the tumor. The frequency of DNA aneuploidy was significantly higher in the tumors containing centrosome abnormalities than in the tumors with a normal centrosome. p16^INK4a expression and loss of pRb expression, but not p53 expression, were significantly associated with centrosome abnormalities. Clinically, centrosome abnormalities were not found to have any prognostic value for NSCLCs. These results suggest that centrosome abnormalities may be associated with inactive pRb-pathway and contribute to pulmonary carcinogenesis by the level of increasing chromosome instability.     

Frequent loss of heterozygosity on chromosome 5 in non-small cell lung carcinoma.

AIMS: Loss of heterozygosity (LOH) at specific chromosomal regions strongly suggests the existence of tumour suppressor genes at the relevant segment. Frequent LOH on chromosome 5q has been reported in a wide variety of human tumours, including those of the lung. The aim of this study was to screen for LOH and to clarify the location of putative tumour suppressor genes on chromosome 5 implicated in the genesis and/or development of non-small cell lung carcinoma. METHODS: Thirty three patients with advanced non-small cell lung carcinoma were screened for LOH with a panel of 21 microsatellite DNA markers spanning the entire chromosome 5, using semi-automated fluorochrome based methodology. RESULTS: Twenty of the non-small cell lung carcinoma samples displayed LOH for one or more informative locus. LOH involving only 5q was found in 10 of 14 of the informative samples. Deletions involving 5p only were not present in the samples under study. There was no evidence of microsatellite instability in any of the analysed loci. These results indicate the presence of five distinct segments displaying high frequencies of deletion on chromosome 5, namely: 5q11.2-q12.2, 5q15 (D5S644 locus), 5q22.3-q23.1, 5q31.1, and 5q35.3. Eight of 14 samples had simultaneous interstitial deletions in at least two different regions. Moreover, concomitant deletion of three and four distinct regions was displayed in three of 14 and two of 14, respectively, of the informative samples. CONCLUSION: Allelic deletion on chromosome 5 is a frequent event in patients with non-small cell lung carcinoma. These results suggest the involvement of these five regions, either independently or simultaneously, in both lung squamous cell carcinoma and lung adenocarcinoma.     

Molecular pathology of non-small cell lung cancer

Our increasing understanding of the molecular pathogenesis of non-small cell lung cancer (NSCLC), particularly adenocarcinomas, has opened the door to ‘personalised medicine’ and the advent of new therapeutic strategies. Over the last few years new drugs, or classes of drugs, have been licensed and entered clinical practice for use in advanced NSCLC. The activity of these drugs is dependent on the presence of specific molecular or protein changes in cancer cells which are usually identified using ‘companion diagnostic tests’ specifically designed for this purpose. Pathologists and Pathology Departments have had to forge new links with Clinical Scientists in order to facilitate these additional investigations on the, often limited, tissue obtained for diagnosis. This collaboration plays a critical role in providing the link that allows integration of the traditional morphological diagnosis with the results of these new ‘companion diagnostic’ tests to guide patient management.     

Updates in the molecular pathology of non-small cell lung cancer

An understanding of the molecular pathology of non-small cell lung cancer (NSCLC) is important for pathologists as molecular characterization is now required for treatment decisions in advanced stage disease. While assessment for EGFR mutations, ALK and ROS1 fusions, and in some countries BRAF mutations, is now standard practice, other oncogenic mutations are also emerging that may impact routine clinical practice including alterations involving KRAS, NTRK, RET, MET and HER2. In addition, molecular pathology alterations of NSCLC are associated with responses to immune checkpoint therapy and are being increasingly investigated. Finally, specific molecular pathological alterations define some rarer subtypes of NSCLC such as salivary gland tumours, NUT carcinoma and SMARCA4-deficient undifferentiated tumour, and an understanding of the molecular pathology is important for their accurate diagnosis. In this review, the molecular pathology of NSCLC is discussed with a focus on clinically relevant molecular alterations.     

Diversity of the angiogenic phenotype in non-small cell lung cancer

Angiogenesis is crucial for tumor biology. There are many mechanisms by which tumors induce angiogenesis. We hypothesize that each individual tumor develops a unique mechanism to induce angiogenesis, and that activation of a particular angiogenic pathway suppresses the evolution of alternative pathways. We characterized 168 human non-small cell lung cancer (NSCLC) specimens for levels of angiogenic factors (angiogenic CXC chemokines, basic fibroblast growth factor, and vascular endothelial growth factor). We also induced lung tumor formation in A/J mice by injecting the tobacco carcinogen NNK. We dissected individual lung tumors and measured expression of angiogenic factors from three distinct families using real-time PCR. Finally, we controlled the angiogenic milieu using in vivo models to determine the resultant phenotype of the angiogenic factors expressed by NSCLC cells. Human tumors displayed marked variation in the expression of angiogenic factors. Individual mouse tumors, even from within the same mouse, displayed variability in their pattern of expression of angiogenic factors. In a sponge model of angiogenesis using murine lung cancer cells, implanting LLC cells with an angiogenic factor suppressed the expression of other angiogenic factors in implanted sponges. This suppressive effect was not seen in vitro. We conclude that lung cancer tumors evolve a unique and dominant angiogenic phenotype. Once an angiogenic pathway is activated, it may allow for tumor growth to proceed in the absence of a selection pressure to activate a second pathway.     

非小细胞肺癌中MTDH及VEGF表达的临床病理意义

目的探讨MTDH基因表达及血管内皮生长因子(VEGF)表达与非小细胞肺癌(non-small cell lung cancer,NSCLC)临床病理的关系及意义。方法用免疫组化EnVision两步法检测328例NSCLC手术治疗患者癌组织和癌旁组织中MTDH及VEGF蛋白表达,分析其在NSCLC临床病理中的关系、应用价值及两者在癌组织血管形成中的协同作用。结果 328例NSCLC中MTDH表达明显高于癌旁正常肺组织,MTDH过表达和异位表达与NSCLC的VEGF蛋白表达、组织学分级、TNM分期及淋巴结转移呈密切正相关性(P值均<0.05)。结论 NSCLC中MTDH过表达或异位表达提示患者预后差,同时作为血管发生的重要调控因子与VEGF协同参与NSCLC的生长、转移和扩散,联合检测将作为NSCLC转移潜能和预后的指标。     

非小细胞肺癌中Axin与β-连环素异常表达的关系

目的 探讨Axin（axis inhibition protein）和β-连环素（β-catenin）在非小细胞肺癌（NSCLC）中的表达、β-catenin突变及它们与各临床病理因素的关系。方法 采用免疫组织化学（SP法）和直接测序方法对100例NSCLC标本中Axin和β-catenin的表达和β-catenin基因突变进行了检测。结果 β-catenin的细胞膜表达降低率为80．0％,细胞核表达率为26．0％。高、中分化和低分化NSCLC中,β-catenin的表达降低率分别为70．0％（35／50）和90．0％（45／50）,差异有统计学意义（P=0．012）。有淋巴结转移和无淋巴结转移NSCLC中β-catenin表达降低率分别为87．3％（48／55）和71．1％（32／45）,差异有统计学意义（P=0．044）。Axin的阳性表达率为48．0％,高、中分化和低分化NSCLC中,Axin的阳性率分别为60．0％（30／50）和36．0％（18／50）,差异有统计学意义（P=0．016）。在β-catenin核表达阳性的病例中,Axin阳性表达率为15．4％（4／26）,低于β-catenin核表达阴性的病例（59．5％,44／74）（P〈0．001）。100例肺癌新鲜标本中未发现β-catenin基因第三外显子突变。结论 β-catenin的膜表达降低与NSCLC的低分化和淋巴结转移相关,Axin的表达与NSCLC的低分化和β-catenin的细胞核蓄积负相关。β-catenin基因第三外显子的突变可能不是NSCLC中β-catenin蛋白异常表达的主要原因。     

Stathmin在非小细胞肺癌中表达增强

目的探讨stathmin在非小细胞肺癌(NSCLC)组织中和正常肺组织中的表达,了解其与NSCLC临床病理特征之间的关系。方法用免疫组化与RT-PCR方法,检测43例NSCLC术后癌组织及正常肺组织标本中stathmin蛋白与基因的表达。结果 Stathmin蛋白和基因在NSCLC中阳性表达率分别为62.79%和67.44%,显著高于正常肺组织的16.28%和20.93%(P<0.01),其表达与肿瘤细胞分化程度和有无淋巴结转移有关(P<0.05)。结论 Stathmin在NSCLC的发生发展过程中起了重要作用,可能成为预测NSCLC恶性程度的新的生物学及个体化治疗的敏感指标。