肺癌肿瘤干细胞表面标志物的研究

背景：对肿瘤干细胞表面标志物进行检测及跟踪监测研究,可为肿瘤的诊断及生物治疗如免疫治疗等提供重要的帮助。 目的：对肺癌的肿瘤干细胞表面标志物的研究进行文献检索分析,为提高肿瘤的诊断率、改善治疗方法、提高患者的生活质量提供可借鉴的理论依据。 方法：检索SCI数据库2002/2011肺癌肿瘤干细胞表面标志物研究相关文献,采用检索词为“肿瘤干细胞(cancer stem cell);肺癌(lung cancer)”,共检索110篇文献,选取符合标准的18篇文献进行数据分析。 结果与结论：肺癌是目前世界上发病率和死亡率最高的恶性肿瘤之一,检测肺癌肿瘤干细胞表面标志物CD133可深入研究肿瘤发生、发展及增殖、维持过程,同时CD133可以为肺癌的靶向生物治疗提供相关信息。随着研究的不断深入,肿瘤干细胞表面标志物不仅可以应用于肿瘤的早期诊断,还将应用于肿瘤的生物治疗。     

应用荧光原位杂交技术检测肺癌患者染色体数目的改变

目的应用荧光原位杂交(FISH)技术研究肺癌患者染色体数目改变及其意义。方法用人的全套染色体特异探针与肺癌患者中期染色体进行杂交检测。结果肺癌患者细胞为异倍体,染色体数目以亚二倍体居多,常见2、5、7、8、10、11、14和17号染色体增多及1、4号染色体的丢失。结论 FISH技术可检测肺癌患者染色体数目的改变,肺癌遗传学变异主要是2、5、7、8、10、11、14、17号染色体的增加,并对肺癌的发生、发展和预后有一定的提示作用。     

DNA Methylation and Nonsmall Cell Lung Cancer

Lung cancer is the leading cause of cancer‐related death in men and women worldwide. Owing to the scarcity of effective tools for early detection and therapy strategies, the 5‐year survival rate of lung cancer is very poor. Because the accumulation of multiple genetic and/or epigenetic changes, including DNA methylation, has been suggested to contribute to development and progression of human cancers, improved understanding of the relationship between DNA methylation and lung cancer will provide new insights for identifying promising biomarkers for diagnosis, prognosis, and treatment of lung cancer. Here, we present a relatively comprehensive review of DNA methylation and lung cancer, discuss DNA methylation changes in carcinogenesis and metastasis of lung cancer, and explore the association of microRNA with DNA methylation. Additionally, we outline the applications of DNA methylation in clinical practice, such as diagnosis, prognosis, and therapy of lung cancer. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

肺癌干细胞富集及相关标志物的表达

背景：肺癌干细胞与肺癌的治疗和预后等紧密相关,通过对其表面标志物和致瘤性的研究,可以为临床诊治提供更多的参考依据。目的：探讨肺癌球体干细胞的富集方法及其致瘤性。方法：使用含有表皮细胞生长因子、胰岛素样生长因子1以及碱性成纤维细胞生长因子的无血清培养基对肺癌细胞进行诱导,采用免疫荧光检测诱导后细胞相关标志物的表达。小鼠皮下移植诱导后肺球体细胞,以了解其致瘤性。结果与结论：经无血清悬浮培养和诱导,肺癌细胞形成细胞球体,免疫荧光检测显示80%以上的球体细胞呈SP-C、CCSP以及OCT4染色阳性。小鼠皮下移植球体细胞后可形成肿瘤。结果表明,无血清悬浮培养肺癌细胞可增殖形成细胞球体,该细胞球体中肺癌干细胞高度富集并具有致瘤性。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

荧光原位杂交技术检测肺癌患者痰液中部分染色体数量的改变

目的应用染色体特异性探针对痰液脱落细胞进行荧光原位杂交(fluorescence in situ hybridization,FISH)用以分析肺癌的染色体畸变,探讨FISH技术辅助诊断肺癌的可行性和有效性。方法选用3、7、17号染色体着丝粒探针,p16、p53探针对40例疑似肺癌患者痰液的脱落细胞进行FISH研究。结果肺癌患者痰液脱落细胞中3、7、17号染色体数目和p16、p53畸变阳性率分别为41.9%、45.2%、41.9%、54.9%、51.6%,其中p16、p53畸变率较高。FISH联合应用五种探针诊断肺癌的敏感性为80.6%,特异性为77.8%,诊断效率为80.0%。结论肺癌的发生、发展与染色体的畸变有关,应用FISH技术检测肺癌患者痰液细胞染色体数目畸变,可作为肺癌诊断的一项辅助方法。     

Exhaled breath condensate as a source of biomarkers for lung carcinomas. A focus on genetic and epigenetic markers—A mini‐review

Lung carcinoma is one of the most common causes of cancer‐related mortality worldwide. It is an aggressive tumor, often diagnosed at an advanced stage when treatment options are limited. Currently, the importance of detection and assessment of various genetic alterations in cancer is recognized as they can serve as very helpful markers in early diagnosis and follow‐up of treatment regimens. Recently, several therapeutically important genetic markers have been identified. One major problem is that tumor tissue specimens used to assay these genetic biomarkers are not always available, especially in the early stages of the disease. Therefore, exhaled breath condensates (EBC) could represent a good non‐invasive source to allow the evaluation of these important genetic markers; these could help in the diagnosis, follow‐up of the disease and/or assessment of treatment efficacy. The key aims of this review are first to describe the origin and constituents of EBC, as well as the different methodological procedures used in studying EBC biomarkers, and second, to document genetic and epigenetic markers that have been analyzed in EBC from lung cancer patients and to estimate their diagnostic and prognostic value. © 2016 Wiley Periodicals, Inc.     

EGFR expression as an ancillary tool for diagnosing lung cancer in cytology specimens.

Lung cancer evolves in a multistep process, and its early detection portends a better prognosis. Bronchial washings/brushings and fine-needle aspirations are often used as early screening and cytological diagnosis of lung cancer. In some cases, it is difficult to differentiate morphologically malignant from reactive cells. Epidermal growth factor receptor (EGFR) is a transmembrane receptor overexpressed in high percentage lung cancers, and contributes to tumor growth. Assessing EGFR expression levels by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) may provide critical information of tumor marker abnormalities, assist in the cytological diagnosis, and stratify patients for EGFR inhibitor therapy. Fifty patients with bronchial washings/brushings or fine-needle aspiration specimens, and corresponding histologically confirmed lung biopsies, were studied for EGFR expression with FISH and IHC. Copy numbers of the EGFR gene locus were analyzed with those of chromosome 7 by FISH. EGFR and FISH results were compared to our FISH data with combined EGFR, c-myc, 5p15.2, and chromosome 6 probes in selected cases. Cell blocks, if available, and tissue biopsy sections were used for EGFR IHC. The intensity of IHC was scored, and quantified. Only balanced aneuploidy of EGFR was identified by FISH. Gene amplification was not detected. The chromosomal abnormalities of EGFR were often accompanied by other chromosomal aneuploidies demonstrated in c-myc (8q24), 5p15.2 or 6p, indicating a general genomic instability. About half of the specimens with confirmed malignancy showed EGFR balanced aneuploidy by FISH, and gene copy number was not coupled with protein expression in many cases. The benign or reactive cytology specimens confirmed by biopsies had high specificity by FISH (96%) and IHC (88%). FISH and IHC analysis of EGFR, possibly along with other tumor markers, may be a useful ancillary tool to classify difficult cytology cases and inform clinicians arranging targeted chemotherapy.     

Genetic variations as cancer prognostic markers: review and update

Cancer molecular epidemiology traditionally studies the relationship between genetic variations and cancer risk. However, recent studies have also focused on disease outcomes. The application and design of disease outcome studies have been an extension of disease risk assessment. Yet there are a number of unique considerations important in outcome assessments. We review how genetic approaches used for disease susceptibility, such as candidate gene and genome‐wide association study (GWAS) approaches, can be adapted carefully to systematically identify cancer prognostic and predictive alleles. We discuss the interrelatedness among the disease susceptibility, treatment response, and prognosis at the genetic level and focus on how the emerging technologies and approaches can uniquely benefit the genetic prognosis studies. Hum Mutat 30:1–9, 2009. © 2009 Wiley‐Liss, Inc.     

Use of markers for the detection and treatment of lung cancer

The unacceptably high morbidity and mortality associated with the diagnosis of lung cancer mandates new approaches toward the early detection and treatment of this disease. Enhanced understanding of the molecular biology of the carcinogenic process is identifying many potential markers of risk of lung cancer occurrence as well as of poor prognosis. Identification of high risk populations who are at greatest risk of being diagnosed with and dying from lung cancer would allow delivery of more intensive screening and interventions to the individuals who are most likely to benefit from such strategies. This review examines the current status of markers of lung cancer risk, early detection, and prognosis, and their applicability to current standards of clinical care.     

Diagnosis and subclassification of lymphomas and non‐neoplastic lesions involving mediastinal lymph nodes using endobronchial ultrasound‐guided transbronchial needle aspiration

Introduction: The value of endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) has been established for staging mediastinal lymph nodes in lung carcinoma patients with radiologically enlarged lymph nodes, but its utility for evaluation of primary lymph node disorders is not well defined. The objective of this study was to evaluate the usefulness of EBUS‐TBNA with on‐site assessment and triage of sample for multiple ancillary techniques, for the diagnosis and subclassification of lymphomas and non‐neoplastic lesions involving mediastinal lymph nodes. Methods: One hundred and twenty consecutive patients who underwent EBUS‐TBNA between January 2008 and August 2009 were reviewed. The final cytological diagnosis was based on air‐dried Romanowsky and alcohol‐fixed Papanicolaou stained direct smears, immunohistochemistry, immunophenotyping, and fluorescence in situ hybridization (FISH). Results: A total of 38 cases were included in this study consisting of eight reactive lymphoid hyperplasia, 20 granulomatous lymphadenitis (17 non‐necrotizing and 3 necrotizing granulomatous inflammations), 3 Hodgkin lymphomas and 7 non‐Hodgkin lymphomas (1 small lymphocytic lymphoma (SLL), 1 SLL with scattered Reed‐Sternberg cells, 1 marginal zone lymphoma, and 4 large B cell lymphomas). Cultures performed in 13 cases were negative for AFB and fungi. Immunophenotyping and immunohistochemistry for MIB1 in six cases, and FISH in five cases provided necessary information for subclassification. Conclusions: EBUS‐TBNA is a minimally invasive procedure which provides sufficient sample for definitive primary diagnosis and classification of malignant lymphoma and granulomatous inflammation in patients with mediastinal lymphadenopathy. Rapid on‐site specimen assessment is invaluable for appropriate assignment of sample to ancillary studies. Diagn. Cytopathol. 2013;41:1023–1030. © 2011 Wiley Periodicals, Inc.     

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.     

Relationships between adult asthma and oxidative stress markers and pH in exhaled breath condensate: a systematic review

Oxidative stress has a recognized role in the pathophysiology of asthma. Recently, interest has increased in the assessment of pH and airway oxidative stress markers. Collection of exhaled breath condensate (EBC) and quantification of biomarkers in breath samples can potentially indicate lung disease activity and help in the study of airway inflammation, and asthma severity. Levels of oxidative stress markers in the EBC have been systematically evaluated in children with asthma; however, there is no such systematic review conducted for adult asthma. A systematic review of oxidative stress markers measured in EBC of adult asthma was conducted, and studies were identified by searching MEDLINE and SCOPUS databases. Sixteen papers met the inclusion criteria. Concentrations of exhaled hydrogen ions, nitric oxide products, hydrogen peroxide and 8‐isoprostanes were generally elevated and related to lower lung function tests in adults with asthma compared to healthy subjects. Assessment of EBC markers may be a noninvasive approach to evaluate airway inflammation, exacerbations, and disease severity of asthma, and to monitor the effectiveness of anti‐inflammatory treatment regimens. Longitudinal studies, using standardized analytical techniques for EBC collection, are required to establish reference values for the interpretation of EBC markers in the context of asthma.     

Loss of chromosome arms 3p and 9p and inactivation of P16 (INK4a) in normal epithelium of patients with primary lung cancer

The accumulation of genetic alterations in the respiratory epithelium may give rise to cancer and often is accompanied by a series of histologic alterations over a period of several years. Recent studies have identified some molecular alterations in histologically normal-appearing epithelium among patients with lung cancer. To extend these observations, we investigated clonal genetic alterations by using fluorescence in situ hybridization (FISH) analysis and immunohistochemistry in 69 biopsy samples of histologically normal-appearing bronchial epithelium from 22 patients with or without lung cancer. Thirty-seven biopsy specimens from 13 patients were examined for loss of 3p14, and 48 biopsy specimens from 18 patients were examined for loss at 9p21 by FISH. P16(INK4a) expression was analyzed in 54 biopsy samples from 19 patients. In at least one biopsy specimen from five of the 13 patients with primary lung cancer, FISH or immunohistochemistry detected loss of the 3p14 or 9p21 region. In contrast, no alterations were detected for the same regions in the nine patients without primary lung cancer. Our results support the concept that the normal epithelial surface of large bronchi of patients with lung cancer has molecular changes suggestive of the outgrowth of numerous clonal foci.     

AQUA and FISH analysis of HER-2/neu expression and amplification in a small cell lung carcinoma tissue microarray

AIMS: Most small cell lung carcinoma (SCLC) patients have metastatic disease at the time of diagnosis and are faced with poor prognosis and limited treatment options. Reports of HER-2/neu gene amplification and overexpression in this malignancy have raised the possibility of applying targeted immunotherapy with trastuzumab, the monoclonal antibody used to treat metastatic breast cancer. However, a review of the studies measuring HER-2/neu gene amplification and protein expression in SCLC reveals discordant results. The aim of the present study was to re-examine HER-2/neu expression in SCLC in relation to gene copy number using the new, highly sensitive, immunofluorescence automated quantitative analysis (AQUA) technology. METHODS AND RESULTS: Fluorescence in situ hybridization (FISH) was used to measure HER-2/neu gene copy number and amplification status and AQUA was used to measure protein expression in a series of 23 SCLC tumours on a tissue microarray. None of the 17 SCLC specimens assessable by FISH exhibited HER-2/neu gene amplification as defined by a HER-2/neu/chromosome 17 ratio = or > 2. Twelve of 17 (70.1%) SCLC samples were polysomic for chromosome 17 with corresponding increases in HER-2/neu gene copy numbers. Intermediate levels of protein expression corresponding to AQUA scores in the range of 4-24 were detected in all 23 specimens. High protein expression levels corresponding to AQUA scores up to 83, observed previously in association with gene amplification and poor prognosis in breast cancer cases, were not detected in the present study. No statistically significant association was observed between absolute chromosome 17 or HER-2/neu gene copy numbers and protein expression levels in tumour cells (P > 0.45). CONCLUSIONS: The lack of gene amplification and robust HER-2/neu protein expression in SCLC tumour cells in this series does not suggest a prominent role for the HER-2/neu gene in SCLC tumour progression and does not support the general applicability of targeted immunotherapy with trastuzumab to this malignancy.     

Correlation between morphology and human telomerase gene amplification in bronchial brushing cells for the diagnosis of lung cancer

The aim of this study was to investigate the frequency of amplification of the human telomerase gene (TERC), as measured by fluorescence in situ hybridization (FISH), in routine liquid‐based cytological preparations from bronchial brushing specimens, and to assess the associations between TERC amplification, cytological diagnosis, and cytological morphology, in order to obtain further insight into these associations. Bronchial brushings from 102 patients with lung carcinoma (52 squamous‐cell carcinomas, 22 adenocarcinomas, 28 small cell lung carcinomas) and 40 patients with nonmalignant disease were used. Amplification of TERC was performed using a commercially available two‐color FISH probe, and slides were prepared for the SurePath liquid‐based Pap test (LPT) using the same samples. Amplification of TERC was significantly associated with histological diagnoses (P < 0.05). Patients with lung cancer, and especially those with nonsmall cell lung cancer, had significantly higher percentages of cells with amplification of TERC than did patients with nonmalignant disease (P < 0.05). Comparing the FISH and LPT results, there was no significant difference in diagnostic sensitivity between the two methods (P > 0.05). However the difference in diagnostic sensitivity of the two methods for squamous‐cell carcinoma was significant (P < 0.01). FISH can be performed on bronchial brushing specimens to detect amplification of TERC. This test may be an adjunct to cytology screening, especially in squamous‐cell carcinoma, and may provide an indication of the potential of individual lesions to progress. Diagn. Cytopathol. 2010. © 2009 Wiley‐Liss, Inc.     

Glycolytic pathway candidate markers in the prognosis of oral squamous cell carcinoma: a systematic review with meta-analysis

Molecular changes that affect mitochondrial glycolysis have been associated with the maintenance of tumor cells. Some metabolic factors have already been described as predictors of disease severity and outcomes. This systematic review was conducted to answer the question: Is the glycolytic pathway correlated with the prognosis of oral squamous cell carcinoma (OSCC)? A search strategy was developed to retrieve studies in English from PubMed, Scopus, and ISI Web of Science using keywords related to squamous cell carcinoma, survival, and glycolytic pathway, with no restriction of publication date. The search retrieved 1273 publications. After the titles and abstracts were analyzed, 27 studies met inclusion criteria. Studies were divided into groups according to two subtopics, glycolytic pathways and diagnosis, which describe the glycolytic profile of OSCC tumors. Several components of tumor energy metabolism found in this review are important predictors of survival of patients with OSCC.     

Variants in folate pathway genes as modulators of genetic instability and lung cancer risk

Genetic instability plays a crucial role in cancer development. The genetic stability of the cell as well as DNA methylation status could be modulated by folate levels. Several studies suggested associations between polymorphisms in folate genes and alterations in protein expression and variations in serum levels of the folate. The objective of this study was to investigate the effect of folate pathway polymorphisms on modulating genetic instability and lung cancer risk. Genotyping of 5 SNPs in folate pathway genes and cytokinesis‐blocked micronucleus cytome assay analysis (to determine the genetic instability at baseline and following NNK treatment) was conducted on 180 lung cancer cases and 180 age‐, gender‐, and smoking‐matched controls. Our results showed that individually, folate pathway SNPs were not associated with cytogenetic damage or lung cancer risk. However, in a polygenic disease such as lung cancer, gene–gene interactions are expected to play an important role in determining the phenotypic variability of the diseases. We observed that interactions between MTHFR677, MTHFR1298, and SHMT polymorphisms may have a significant impact on genetic instability in lung cancer patients. With regard to cytogenetic alterations, our results showed that lymphocytes from lung cancer patients exposed to the tobacco‐specific carcinogen 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone [NNK] had considerably increased frequency of cytogenetic damage in presence of MTHFR 677, MTHFR 1298, and SHMT allelic variants. These findings support the notion that significant interactions may potentially modulate the lung cancer susceptibility and alter the overall the repair abilities of lung cancer patients when exposed to tobacco carcinogens such as NNK. © 2010 Wiley‐Liss, Inc.     

Multitarget FISH analysis in the diagnosis of lung cancer

The aim of the present study was to explore the diagnostic usefulness of the multitarget fluorescence in situ hybridization (FISH) test, LAVysion (Vysis, Downers Grove, IL), for the detection of lung cancer cells in cytologic specimens. Specimens from bronchial washings, bronchial brushings, and transbronchial fine-needle aspirates (TBNAs) from 100 patients with suspected lung cancer and from a control group of 71 patients with nonneoplastic lung disorders were analyzed. FISH positivity was defined as more than 5 cells with gains of at least 2 chromosomes or gene loci. FISH significantly improved the sensitivity of bronchial brushings from 49% to 73%. The specificities of FISH and cytologic examination were 87% and 100%, respectively. In contrast, FISH provided no additional diagnostic information in TBNAs and bronchial washings. There was no increased prevalence of genetic changes in contralateral bronchial washings from patients with lung cancer compared with the control group. The quantity of previous smoking had no effect on the prevalence of chromosomal changes.     

Fine needle aspiration cytology of histiocytic sarcoma with dendritic cell differentiation: A case of transdifferentiation from low‐grade follicular lymphoma

Histiocytic sarcoma (HS) is a rare malignant tumor, and in extraordinary circumstances it can be transdifferentiated from a low‐grade B‐cell lymphoma. In our report, a 62‐year‐old female was initially diagnosed with a low‐grade follicular lymphoma, and 2 years later she presented with bilateral lung masses and lymphadenopathy. Fine needle aspiration (FNA) of the lung masses revealed solid nests of large pleomorphic epithelioid cells. By immunohistochemical studies, the tumor cells were positive for histiocytic markers (CD68 and CD163) and S100, supporting a diagnosis of HS with dendritic cell differentiation. Further fluorescence in situ hybridization (FISH) analyses detected IGH@/BCL2 rearrangement in both the previous follicular lymphoma and the current HS, which was highly suggestive of the clonal relationship between these two tumors. To the best of our knowledge, this is the first case of transformation of follicular lymphoma into HS diagnosed by FNA of lung masses. Diagn. Cytopathol. 2015;43:659–663. © 2015 Wiley Periodicals, Inc.