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1.  Integrative Genomic Analyses Identify BRF2 as a Novel Lineage-Specific Oncogene in Lung Squamous Cell Carcinoma 
PLoS Medicine  2010;7(7):e1000315.
William Lockwood and colleagues show that the focal amplification of a gene, BRF2, on Chromosome 8p12 plays a key role in squamous cell carcinoma of the lung.
Background
Traditionally, non-small cell lung cancer is treated as a single disease entity in terms of systemic therapy. Emerging evidence suggests the major subtypes—adenocarcinoma (AC) and squamous cell carcinoma (SqCC)—respond differently to therapy. Identification of the molecular differences between these tumor types will have a significant impact in designing novel therapies that can improve the treatment outcome.
Methods and Findings
We used an integrative genomics approach, combing high-resolution comparative genomic hybridization and gene expression microarray profiles, to compare AC and SqCC tumors in order to uncover alterations at the DNA level, with corresponding gene transcription changes, which are selected for during development of lung cancer subtypes. Through the analysis of multiple independent cohorts of clinical tumor samples (>330), normal lung tissues and bronchial epithelial cells obtained by bronchial brushing in smokers without lung cancer, we identified the overexpression of BRF2, a gene on Chromosome 8p12, which is specific for development of SqCC of lung. Genetic activation of BRF2, which encodes a RNA polymerase III (Pol III) transcription initiation factor, was found to be associated with increased expression of small nuclear RNAs (snRNAs) that are involved in processes essential for cell growth, such as RNA splicing. Ectopic expression of BRF2 in human bronchial epithelial cells induced a transformed phenotype and demonstrates downstream oncogenic effects, whereas RNA interference (RNAi)-mediated knockdown suppressed growth and colony formation of SqCC cells overexpressing BRF2, but not AC cells. Frequent activation of BRF2 in >35% preinvasive bronchial carcinoma in situ, as well as in dysplastic lesions, provides evidence that BRF2 expression is an early event in cancer development of this cell lineage.
Conclusions
This is the first study, to our knowledge, to show that the focal amplification of a gene in Chromosome 8p12, plays a key role in squamous cell lineage specificity of the disease. Our data suggest that genetic activation of BRF2 represents a unique mechanism of SqCC lung tumorigenesis through the increase of Pol III-mediated transcription. It can serve as a marker for lung SqCC and may provide a novel target for therapy.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Lung cancer is the commonest cause of cancer-related death. Every year, 1.3 million people die from this disease, which is mainly caused by smoking. Most cases of lung cancer are “non-small cell lung cancers” (NSCLCs). Like all cancers, NSCLC starts when cells begin to divide uncontrollably and to move round the body (metastasize) because of changes (mutations) in their genes. These mutations are often in “oncogenes,” genes that, when activated, encourage cell division. Oncogenes can be activated by mutations that alter the properties of the proteins they encode or by mutations that increase the amount of protein made from them, such as gene amplification (an increase in the number of copies of a gene). If NSCLC is diagnosed before it has spread from the lungs (stage I disease), it can be surgically removed and many patients with stage I NSCLC survive for more than 5 years after their diagnosis. Unfortunately, in more than half of patients, NSCLC has metastasized before it is diagnosed. This stage IV NSCLC can be treated with chemotherapy (toxic chemicals that kill fast-growing cancer cells) but only 2% of patients with stage IV lung cancer are alive 5 years after diagnosis.
Why Was This Study Done?
Traditionally, NSCLC has been regarded as a single disease in terms of treatment. However, emerging evidence suggests that the two major subtypes of NSCLC—adenocarcinoma and squamous cell carcinoma (SqCC)—respond differently to chemotherapy. Adenocarcinoma and SqCC start in different types of lung cell and experts think that for each cell type in the body, specific combinations of mutations interact with the cell type's own unique characteristics to provide the growth and survival advantage needed for cancer development. If this is true, then identifying the molecular differences between adenocarcinoma and SqCC could provide targets for more effective therapies for these major subtypes of NSCLC. Amplification of a chromosome region called 8p12 is very common in NSCLC, which suggests that an oncogene that drives lung cancer development is present in this chromosome region. In this study, the researchers investigate this possibility by looking for an amplified gene in the 8p12 chromosome region that makes increased amounts of protein in lung SqCC but not in lung adenocarcinoma.
What Did the Researchers Do and Find?
The researchers used a technique called comparative genomic hybridization to show that focal regions of Chromosome 8p are amplified in about 40% of lung SqCCs, but that DNA loss in this region is the most common alteration in lung adenocarcinomas. Ten genes in the 8p12 chromosome region were expressed at higher levels in the SqCC samples that they examined than in adenocarcinoma samples, they report, and overexpression of five of these genes correlated with amplification of the 8p12 region in the SqCC samples. Only one of the genes—BRF2—was more highly expressed in squamous carcinoma cells than in normal bronchial epithelial cells (the cell type that lines the tubes that take air into the lungs and from which SqCC develops). Artificially induced expression of BRF2 in bronchial epithelial cells made these normal cells behave like tumor cells, whereas reduction of BRF2 expression in squamous carcinoma cells made them behave more like normal bronchial epithelial cells. Finally, BRF2 was frequently activated in two early stages of squamous cell carcinoma—bronchial carcinoma in situ and dysplastic lesions.
What Do These Findings Mean?
Together, these findings show that the focal amplification of chromosome region 8p12 plays a role in the development of lung SqCC but not in the development of lung adenocarcinoma, the other major subtype of NSCLC. These findings identify BRF2 (which encodes a RNA polymerase III transcription initiation factor, a protein that is required for the synthesis of RNA molecules that help to control cell growth) as a lung SqCC-specific oncogene and uncover a unique mechanism for lung SqCC development. Most importantly, these findings suggest that genetic activation of BRF2 could be used as a marker for lung SqCC, which might facilitate the early detection of this type of NSCLC and that BRF2 might provide a new target for therapy.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000315.
The US National Cancer Institute provides detailed information for patients and professionals about all aspects of lung cancer, including information on non-small cell carcinoma (in English and Spanish)
Cancer Research UK also provides information about lung cancer and information on how cancer starts
MedlinePlus has links to other resources about lung cancer (in English and Spanish)
doi:10.1371/journal.pmed.1000315
PMCID: PMC2910599  PMID: 20668658
2.  Comprehensive genomic characterization of squamous cell lung cancers 
Nature  2012;489(7417):519-525.
Summary
Lung squamous cell carcinoma (lung SqCC) is a common type of lung cancer, causing approximately 400,000 deaths per year worldwide. Genomic alterations in lung SqCC have not been comprehensively characterized and no molecularly targeted agents have been developed specifically for its treatment. As part of The Cancer Genome Atlas (TCGA), we profiled 178 lung SqCCs to provide a comprehensive landscape of genomic and epigenomic alterations. Lung SqCC is characterized by complex genomic alterations, with a mean of 360 exonic mutations, 165 genomic rearrangements, and 323 segments of copy number alteration per tumor. We found statistically recurrent mutations in 18 genes in including mutation of TP53 in nearly all specimens. Previously unreported loss-of-function mutations were seen in the HLA-A class I major histocompatibility gene. Significantly altered pathways included NFE2L2/KEAP1 in 34%, squamous differentiation genes in 44%, PI3K/AKT in 47%, and CDKN2A/RB1 in 72% of tumors. We identified a potential therapeutic target in the majority of tumors, offering new avenues of investigation for lung SqCC treatment.
doi:10.1038/nature11404
PMCID: PMC3466113  PMID: 22960745
3.  Genome-Wide Association Study Identifies a Novel Susceptibility Locus at 12q23.1 for Lung Squamous Cell Carcinoma in Han Chinese 
PLoS Genetics  2013;9(1):e1003190.
Adenocarcinoma (AC) and squamous cell carcinoma (SqCC) are two major histological subtypes of lung cancer. Genome-wide association studies (GWAS) have made considerable advances in the understanding of lung cancer susceptibility. Obvious heterogeneity has been observed between different histological subtypes of lung cancer, but genetic determinants in specific to lung SqCC have not been systematically investigated. Here, we performed the GWAS analysis specifically for lung SqCC in 833 SqCC cases and 3,094 controls followed by a two-stage replication in additional 2,223 lung SqCC cases and 6,409 controls from Chinese populations. We found that rs12296850 in SLC17A8-NR1H4 gene region at12q23.1 was significantly associated with risk of lung SqCC at genome-wide significance level [additive model: odds ratio (OR) = 0.78, 95% confidence interval (CI) = 0.72–0.84, P = 1.19×10−10]. Subjects carrying AG or GG genotype had a 26% (OR = 0.74, 95% CI = 0.67–0.81) or 32% (OR = 0.68, 95% CI = 0.56–0.83) decreased risk of lung SqCC, respectively, as compared with AA genotype. However, we did not observe significant association between rs12296850 and risk of lung AC in a total of 4,368 cases with lung AC and 9,486 controls (OR = 0.96, 95% CI = 0.90–1.02, P = 0.173). These results indicate that genetic variations on chromosome 12q23.1 may specifically contribute to lung SqCC susceptibility in Chinese population.
Author Summary
Previous genome-wide association studies (GWAS) strongly suggested the importance of genetic susceptibility for lung cancer. However, the studies specific to different histological subtypes of lung cancer were limited. We performed the GWAS analysis specifically for lung squamous cell carcinoma (SqCC) with 570,009 autosomal SNPs in 833 SqCC cases and 3,094 controls and replicated in additional 2,223 lung SqCC cases and 6,409 controls from Chinese populations (822 SqCC cases and 2,243 controls for the first replication stage and 1,401 SqCC cases and 4,166 controls for the second replication stage). We found a novel association at rs12296850 (SLC17A8-NR1H4) on12q23.1. However, rs12296850 didn't show significant association with risk of lung adenocacinoma (AC) in 4,368 lung AC cases and 9,486 controls. These results indicate that genetic variations on chromosome 12q23.1 may specifically contribute to lung SqCC susceptibility in Chinese population.
doi:10.1371/journal.pgen.1003190
PMCID: PMC3547794  PMID: 23341777
4.  Genomics of Squamous Cell Lung Cancer 
The Oncologist  2013;18(6):707-716.
Approximately 30% of patients with non-small cell lung cancer have the squamous cell carcinoma (SQCC) histological subtype. This review discusses key molecular aberrations reported by The Cancer Genome Atlas and other investigators and their potential therapeutic implications for patients with SQCC of lung.
Learning Objectives
Describe important molecular aberrations associated with squamous cell carcinoma.Identify molecular aberrations that may have therapeutic implications.
Approximately 30% of patients with non-small cell lung cancer have the squamous cell carcinoma (SQCC) histological subtype. Although targeted therapies have improved outcomes in patients with adenocarcinoma, no agents are currently approved specifically for use in SQCC. The Cancer Genome Atlas (TCGA) recently published the results of comprehensive genomic analyses of tumor samples from 178 patients with SQCC of the lung. In this review, we briefly discuss key molecular aberrations reported by TCGA and other investigators and their potential therapeutic implications. Carefully designed preclinical and clinical studies based on these large-scale genomic analyses are critical to improve the outcomes of patients with SQCC of lung in the near future.
doi:10.1634/theoncologist.2013-0063
PMCID: PMC4063398  PMID: 23728941
5.  Arsenic-related DNA copy-number alterations in lung squamous cell carcinomas 
British Journal of Cancer  2010;103(8):1277-1283.
Background:
Lung squamous cell carcinomas (SqCCs) occur at higher rates following arsenic exposure. Somatic DNA copy-number alterations (CNAs) are understood to be critical drivers in several tumour types. We have assembled a rare panel of lung tumours from a population with chronic arsenic exposure, including SqCC tumours from patients with no smoking history.
Methods:
Fifty-two lung SqCCs were analysed by whole-genome tiling-set array comparative genomic hybridisation. Twenty-two were derived from arsenic-exposed patients from Northern Chile (10 never smokers and 12 smokers). Thirty additional cases were obtained for comparison from North American smokers without arsenic exposure. Twenty-two blood samples from healthy individuals from Northern Chile were examined to identify germline DNA copy-number variations (CNVs) that could be excluded from analysis.
Results:
We identified multiple CNAs associated with arsenic exposure. These alterations were not attributable to either smoking status or CNVs. DNA losses at chromosomes 1q21.1, 7p22.3, 9q12, and 19q13.31 represented the most recurrent events. An arsenic-associated gain at 19q13.33 contains genes previously identified as oncogene candidates.
Conclusions:
Our results provide a comprehensive approach to molecular characteristics of the arsenic-exposed lung cancer genome and the non-smoking lung SqCC genome. The distinct and recurrent arsenic-related alterations suggest that this group of tumours may be considered as a separate disease subclass.
doi:10.1038/sj.bjc.6605879
PMCID: PMC2967055  PMID: 20842114
arsenic; lung squamous cell carcinoma; never smokers; copy-number alterations; copy-number variation; CGH
6.  Role of the extracellular matrix in variations of invasive pathways in lung cancers 
Among the most common features of highly invasive tumors, such as lung adenocarcinomas (AD) and squamous cell carcinomas (SqCC), is the massive degradation of the extracellular matrix. The remarkable qualitative and quantitative modifications of hyaluronidases (HAases), hyaluronan synthases (HAS), E-cadherin adhesion molecules, and the transforming growth factor β (TGF-β) may favor invasion, cellular motility, and proliferation. We examined HAase proteins (Hyal), HAS, E-cadherin, and TGF-β profiles in lung AD subtypes and SqCC obtained from smokers and non-smokers. Fifty-six patients, median age 64 years, who underwent lobectomy for AD (N = 31) and SqCC (N = 25) were included in the study. HAS-1, -2 and -3, and Hyal-1 and -3 were significantly more expressed by tumor cells than normal and stroma cells (P < 0.01). When stratified according to histologic types, HAS-3 and Hyal-1 immunoreactivity was significantly increased in tumor cells of AD (P = 0.01) and stroma of SqCC (P = 0.002), respectively. Tobacco history in patients with AD was significantly associated with increased HAS-3 immunoreactivity in tumor cells (P < 0.01). Stroma cells of SqCC from non-smokers presented a significant association with HAS-3 (P < 0.01). Hyal, HAS, E-cadherin, and TGF-β modulate a different tumor-induced invasive pathway in lung AD subgroups and SqCC. HAases in resected AD and SqCC were strongly related to the prognosis. Therefore, our findings suggest that strategies aimed at preventing high HAS-3 and Hyal-1 synthesis, or local responses to low TGF-β and E-cadherin, may have a greater impact in lung cancer prognosis.
doi:10.1590/1414-431X20122263
PMCID: PMC3854345  PMID: 23314337
Lung cancer; E-cadherin; TGF-β; HAS-1, HAS-2 and HAS-3; Hyal-1 and Hyal-3; Immunohistochemistry; Prognosis and morphometry
7.  Aberrant DNA Methylation of OLIG1, a Novel Prognostic Factor in Non-Small Cell Lung Cancer 
PLoS Medicine  2007;4(3):e108.
Background
Lung cancer is the leading cause of cancer-related death worldwide. Currently, tumor, node, metastasis (TNM) staging provides the most accurate prognostic parameter for patients with non-small cell lung cancer (NSCLC). However, the overall survival of patients with resectable tumors varies significantly, indicating the need for additional prognostic factors to better predict the outcome of the disease, particularly within a given TNM subset.
Methods and Findings
In this study, we investigated whether adenocarcinomas and squamous cell carcinomas could be differentiated based on their global aberrant DNA methylation patterns. We performed restriction landmark genomic scanning on 40 patient samples and identified 47 DNA methylation targets that together could distinguish the two lung cancer subgroups. The protein expression of one of those targets, oligodendrocyte transcription factor 1 (OLIG1), significantly correlated with survival in NSCLC patients, as shown by univariate and multivariate analyses. Furthermore, the hazard ratio for patients negative for OLIG1 protein was significantly higher than the one for those patients expressing the protein, even at low levels.
Conclusions
Multivariate analyses of our data confirmed that OLIG1 protein expression significantly correlates with overall survival in NSCLC patients, with a relative risk of 0.84 (95% confidence interval 0.77–0.91, p < 0.001) along with T and N stages, as indicated by a Cox proportional hazard model. Taken together, our results suggests that OLIG1 protein expression could be utilized as a novel prognostic factor, which could aid in deciding which NSCLC patients might benefit from more aggressive therapy. This is potentially of great significance, as the addition of postoperative adjuvant chemotherapy in T2N0 NSCLC patients is still controversial.
Christopher Plass and colleagues find thatOLIG1 expression correlates with survival in lung cancer patients and suggest that it could be used in deciding which patients are likely to benefit from more aggressive therapy.
Editors' Summary
Background.
Lung cancer is the commonest cause of cancer-related death worldwide. Most cases are of a type called non-small cell lung cancer (NSCLC). Like other cancers, treatment of NCSLC depends on the “TNM stage” at which the cancer is detected. Staging takes into account the size and local spread of the tumor (its T classification), whether nearby lymph nodes contain tumor cells (its N classification), and whether tumor cells have spread (metastasized) throughout the body (its M classification). Stage I tumors are confined to the lung and are removed surgically. Stage II tumors have spread to nearby lymph nodes and are treated with a combination of surgery and chemotherapy. Stage III tumors have spread throughout the chest, and stage IV tumors have metastasized around the body; patients with both of these stages are treated with chemotherapy alone. About 70% of patients with stage I or II lung cancer, but only 2% of patients with stage IV lung cancer, survive for five years after diagnosis.
Why Was This Study Done?
TNM staging is the best way to predict the likely outcome (prognosis) for patients with NSCLC, but survival times for patients with stage I and II tumors vary widely. Another prognostic marker—maybe a “molecular signature”—that could distinguish patients who are likely to respond to treatment from those whose cancer will inevitably progress would be very useful. Unlike normal cells, cancer cells divide uncontrollably and can move around the body. These behavioral changes are caused by alterations in the pattern of proteins expressed by the cells. But what causes these alterations? The answer in some cases is “epigenetic changes” or chemical modifications of genes. In cancer cells, methyl groups are aberrantly added to GC-rich gene regions. These so-called “CpG islands” lie near gene promoters (sequences that control the transcription of DNA into mRNA, the template for protein production), and their methylation stops the promoters working and silences the gene. In this study, the researchers have investigated whether aberrant methylation patterns vary between NSCLC subtypes and whether specific aberrant methylations are associated with survival and can, therefore, be used prognostically.
What Did the Researchers Do and Find?
The researchers used “restriction landmark genomic scanning” (RLGS) to catalog global aberrant DNA methylation patterns in human lung tumor samples. In RLGS, DNA is cut into fragments with a restriction enzyme (a protein that cuts at specific DNA sequences), end-labeled, and separated using two-dimensional gel electrophoresis to give a pattern of spots. Because methylation stops some restriction enzymes cutting their target sequence, normal lung tissue and lung tumor samples yield different patterns of spots. The researchers used these patterns to identify 47 DNA methylation targets (many in CpG islands) that together distinguished between adenocarcinomas and squamous cell carcinomas, two major types of NSCLCs. Next, they measured mRNA production from the genes with the greatest difference in methylation between adenocarcinomas and squamous cell carcinomas. OLIG1 (the gene that encodes a protein involved in nerve cell development) had one of the highest differences in mRNA production between these tumor types. Furthermore, three-quarters of NSCLCs had reduced or no expression of OLIG1 protein and, when the researchers analyzed the association between OLIG1 protein expression and overall survival in patients with NSCLC, reduced OLIG1 protein expression was associated with reduced survival.
What Do These Findings Mean?
These findings indicate that different types of NSCLC can be distinguished by examining their aberrant methylation patterns. This suggests that the establishment of different DNA methylation patterns might be related to the cell type from which the tumors developed. Alternatively, the different aberrant methylation patterns might reflect the different routes that these cells take to becoming tumor cells. This research identifies a potential new prognostic marker for NSCLC by showing that OLIG1 protein expression correlates with overall survival in patients with NSCLC. This correlation needs to be tested in a clinical setting to see if adding OLIG1 expression to the current prognostic parameters can lead to better treatment choices for early-stage lung cancer patients and ultimately improve these patients' overall survival.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040108.
Patient and professional information on lung cancer, including staging (in English and Spanish), is available from the US National Cancer Institute
The MedlinePlus encyclopedia has pages on non-small cell lung cancer (in English and Spanish)
Cancerbackup provides patient information on lung cancer
CancerQuest, provided by Emory University, has information about how cancer develops (in English, Spanish, Chinese and Russian)
Wikipedia pages on epigenetics (note that Wikipedia is a free online encyclopedia that anyone can edit)
The Epigenome Network of Excellence gives background information and the latest news about epigenetics (in several European languages)
doi:10.1371/journal.pmed.0040108
PMCID: PMC1831740  PMID: 17388669
8.  EGFR-Targeted Therapy for Non-Small Cell Lung Cancer: Focus on EGFR Oncogenic Mutation 
The two essential requirements for pathologic specimens in the era of personalized therapies for non-small cell lung carcinoma (NSCLC) are accurate subtyping as adenocarcinoma (ADC) versus squamous cell carcinoma (SqCC) and suitability for EGFR molecular testing, as well as for testing of other oncogenes such as EML4-ALK and KRAS. Actually, the value of EGFR expressed in patients with NSCLC in predicting a benefit in terms of survival from treatment with an epidermal growth factor receptor targeted therapy is still in debate, while there is a convincing evidence on the predictive role of the EGFR mutational status with regard to the response to tyrosine kinase inhibitors (TKIs).
This is a literature overview on the state-of-the-art of EGFR oncogenic mutation in NSCLC. It is designed to highlight the preclinical rationale driving the molecular footprint assessment, the progressive development of a specific pharmacological treatment and the best method to identify those NSCLC who would most likely benefit from treatment with EGFR-targeted therapy. This is supported by the belief that a rationale for the prioritization of specific regimens based on patient-tailored therapy could be closer than commonly expected.
doi:10.7150/ijms.4609
PMCID: PMC3575628  PMID: 23423768
EGFR targeted therapy; NSCLC; advanced; mutation; TKIs; resistance.
9.  Targeted therapy for squamous cell lung cancer 
Lung cancer management  2012;1(4):293-300.
SUMMARY
Lung squamous cell carcinoma (SqCC) is the second most common subtype of non-small-cell lung cancer and leads to 40,000–50,000 deaths per year in the USA. Management of non-small-cell lung cancer has dramatically changed over the past decade with the introduction of targeted therapeutic agents for genotypically selected individuals with lung adenocarcinoma. These agents lead to improved outcomes, and it has now become the standard of care to perform routine molecular genotyping of lung adenocarcinomas. By contrast, progress in lung SqCC has been modest, and there has yet to be a successful demonstration of targeted therapy in this disease. Here, we review exciting work from ongoing genomic characterization and biomarker validation efforts that have nominated several likely therapeutic targets in lung SqCCs. These studies suggest that targeted therapies are likely to be successful in the treatment of lung SqCCs and should be further explored in both preclinical models and in clinical trials.
doi:10.2217/lmt.12.40
PMCID: PMC3744181  PMID: 23956794
10.  Prognostic value of FGFR1 gene copy number in patients with non-small cell lung cancer: a meta-analysis 
Journal of Thoracic Disease  2014;6(6):803-809.
Background
A number of studies have investigated the relationship between fibroblast growth factor receptor1 (FGFR1) gene copy number and survival in non-small cell lung cancer (NSCLC) patients. However, conclusions reported by different parties seem to be inconsistent, especially regarding the differences among different histopathologic subtypes. To derive a more precise estimate of the prognostic significance of FGFR1 gene copy number, we have reviewed published studies and carried out a meta-analysis.
Methods
The meta-analysis was conducted in accordance with PRISMA guidelines. The required data for estimation of individual hazard ratios (HRs) for survival were extracted from the publications and an overall HR was calculated.
Results
We identified 6 eligible studies, all dealing with NSCLC. The global quality score ranged 32.5-80%, with a median of 53.33%. For FGFR1 amplification in three studies including differed according to histological type, the overall RR was 0.86 which 95% confidence interval (CI) was 0.75 to 0.99 and P value was 0.048. Combined HR for the six evaluable studies was 1.17 (95% CI: 0.95 to 1.43). In the subgroup of squamous cell lung cancer (SQCC), the combined HR was 1.24 (95% CI: 0.89 to 1.73). For the Asian populations’ studies, the combined HR was 1.67 (95% CI: 1.1 to 2.52).
Conclusions
FGFR1 amplification significantly was more frequent in SQCC. FGFR1 was not associated with poorer survival in patients with NSCLC. Furthermore studies will be needed in terms of survival implications.
doi:10.3978/j.issn.2072-1439.2014.05.02
PMCID: PMC4073380  PMID: 24977006
Lung cancer; meta-analysis; fibroblast growth factor receptor1 (FGFR1); prognosis; survival; systemic review
11.  Tumor budding is a significant indicator of a poor prognosis in lung squamous cell carcinoma patients 
Molecular Medicine Reports  2012;6(5):937-943.
Lung cancer is a leading cause of cancer mortality worldwide and patients occasionally develop local recurrence or distant metastasis soon after curative resection. Reports of new therapeutic strategies for lung squamous cell carcinoma (SqCC) are extremely rare, while selective anticancer therapy has been reported for lung adenocarcinoma. The aim of this study was to identify clinicopathological prognostic factors for SqCC. We analyzed tumor budding and infiltrative patterns (INF) in 103 cases of surgically-resected SqCC. Tumor infiltrative patterns were classified into three groups (INFa, b and c) and INFc was infiltrative growth at the tumor invasive front. The cases with an INFc component [INFc(+)]were significantly associated with venous invasion (P=0.014) and the scirrhous stromal type (P<0.001). The overall survival rate of patients with INFc(+) was significantly lower than that of patients without the INFc component [INFc(−); P=0.003]. Tumor budding was defined as a single cancer cell or a small nest of up to four cancer cells within stromal tissue. The cases with tumor budding [Bud(+)] were significantly associated with lymph node metastasis (P=0.001), lymphatic invasion (P=0.002), INFc(+) (P<0.001) and the scirrhous stromal type (P=0.014). Patients with the Bud(+) type had a lower overall survival rate than patients with the Bud(−) type (P<0.001). Multivariate analysis demonstrated that tumor budding [hazard ratio (HR), 2.766; 95% confidence interval (CI), 1.497–5.109] and lymph node metastasis (HR, 1.937; 95% CI, 1.097–3.419) were independent predictors of mortality. In conclusion, tumor budding is a significant indicator of a high malignant potential and poor prognosis in SqCC of the lung.
doi:10.3892/mmr.2012.1048
PMCID: PMC3493086  PMID: 22940760
lung cancer; squamous cell carcinoma; patient prognosis; tumor budding
12.  Clarifying the spectrum of driver oncogene mutations in biomarker-verified squamous carcinoma of lung: lack of EGFR/KRAS and presence of PIK3CA/AKT1 mutations 
Purpose
There is persistent controversy as to whether EGFR/KRAS mutations occur in pulmonary squamous cell carcinoma (SQCC). We hypothesized that the reported variability may reflect difficulties in the pathologic distinction of true SQCC from adenosquamous carcinoma (AD-SQC) and poorly-differentiated adenocarcinoma (ADC) due to incomplete sampling or morphologic overlap. The recent development of a robust immunohistochemical approach for distinguishing squamous vs glandular differentiation provides an opportunity to reassess EGFR/KRAS and other targetable kinase mutation frequencies in a pathologically homogeneous series of SQCC.
Experimental Design
Ninety-five resected SQCC, verified by immunohistochemistry as ΔNp63+/TTF-1−, were tested for activating mutations in EGFR, KRAS, BRAF, PIK3CA, NRAS, AKT1, ERBB2/HER2, and MAP2K1/MEK1. Additionally, all tissue samples from rare patients with the diagnosis of EGFR/KRAS-mutant “SQCC” encountered during5 years of routine clinical genotyping were reassessed pathologically.
Results
The screen of 95biomarker-verified SQCC revealed no EGFR/KRAS (0%; 95%CI 0–3.8%), 4 PIK3CA (4%; 95% CI 1–10%) and 1 AKT1 (1%; 95% CI 0–5.7%) mutations. Detailed morphologic and immunohistochemical reevaluation of EGFR/KRAS-mutant SQCC” identified during clinical genotyping (n=16) resulted in reclassification of 10 (63%)cases as AD-SQC and 5 (31%) cases as poorly-differentiated ADC morphologically mimicking SQCC (i.e. ADC with “squamoid” morphology). One (6%) case had no follow-up.
Conclusions
Our findings suggest that EGFR/KRAS mutations do not occur in pure pulmonary SQCC, and occasional detection of these mutations in samples diagnosed as “SQCC” is due to challenges with the diagnosis of AD-SQC and ADC, which can be largely resolved by comprehensive pathologic assessment incorporating immunohistochemical biomarkers.
doi:10.1158/1078-0432.CCR-11-2109
PMCID: PMC3487403  PMID: 22228640
EGFR; KRAS; TTF-1; p63; squamous cell carcinoma
13.  HPV Analysis in Distinguishing Second Primary Tumors from Lung Metastases in Patients with Head and Neck Squamous Cell Carcinoma 
For patients with head and neck squamous cell carcinoma (HNSqCC), the development of squamous cell carcinoma (SqCC) in the lung may signal a new primary or the onset of metastatic dissemination. Although the distinction influences prognosis and therapy, it may not be straightforward on histological or clinical grounds. Human papillomavirus (HPV) is an etiologic agent for SqCCs arising from the oropharynx, but not for SqCCs arising from other head and neck sites. For patients with HNSqCC who develop a lung SqCC, HPV analysis could be useful in establishing tumor relationships. High risk HPV in-situ hybridization was performed on 54 lung SqCCs from patients with a prior HNSqCC, and on 166 primary lung carcinomas from patients without prior HNSqCC. HPV was detected in 11 of 220 (5%) cases. All HPV-positive cases were from patients with a prior oropharyngeal SqCC. For the paired oropharyngeal and lung SqCCs, HPV status was concordant in 95% of cases. Time from treatment of the HPV-positive oropharyngeal carcinomas to detection of the lung carcinoma ranged from 1 to 97 months (mean 36 months). Two HPV-positive cancers were detected in the lung 8 years after treatment of the oropharyngeal primary. Despite the long interval, E6 sequencing analysis of one of these paired samples confirmed that the tumors harbored the same HPV-16 variant. HPV does not appear to play a role in the development of primary lung cancer. For patients with oropharyngeal SqCC who develop lung SqCCs, HPV analysis may be helpful in clarifying tumor relationships. These relationships may not be obvious on clinical grounds as HPV-related HNSqCC may metastasize long after treatment of the primary tumor.
doi:10.1097/PAS.0b013e3182395c7b
PMCID: PMC3273906  PMID: 22173119
Human papillomavirus; lung carcinoma; oropharyngeal squamous cell carcinoma
14.  Deciphering Squamous Cell Carcinoma Using Multidimensional Genomic Approaches 
Journal of Skin Cancer  2010;2011:541405.
Squamous cell carcinomas (SqCCs) arise in a wide range of tissues including skin, lung, and oral mucosa. Although all SqCCs are epithelial in origin and share common nomenclature, these cancers differ greatly with respect to incidence, prognosis, and treatment. Current knowledge of genetic similarities and differences between SqCCs is insufficient to describe the biology of these cancers, which arise from diverse tissue origins. In this paper we provide a general overview of whole genome approaches for gene and pathway discovery and highlight the advancement of integrative genomics as a state-of-the-art technology in the study of SqCC genetics.
doi:10.1155/2011/541405
PMCID: PMC3017908  PMID: 21234096
15.  A Gene Expression Signature Predicts Survival of Patients with Stage I Non-Small Cell Lung Cancer 
PLoS Medicine  2006;3(12):e467.
Background
Lung cancer is the leading cause of cancer-related death in the United States. Nearly 50% of patients with stages I and II non-small cell lung cancer (NSCLC) will die from recurrent disease despite surgical resection. No reliable clinical or molecular predictors are currently available for identifying those at high risk for developing recurrent disease. As a consequence, it is not possible to select those high-risk patients for more aggressive therapies and assign less aggressive treatments to patients at low risk for recurrence.
Methods and Findings
In this study, we applied a meta-analysis of datasets from seven different microarray studies on NSCLC for differentially expressed genes related to survival time (under 2 y and over 5 y). A consensus set of 4,905 genes from these studies was selected, and systematic bias adjustment in the datasets was performed by distance-weighted discrimination (DWD). We identified a gene expression signature consisting of 64 genes that is highly predictive of which stage I lung cancer patients may benefit from more aggressive therapy. Kaplan-Meier analysis of the overall survival of stage I NSCLC patients with the 64-gene expression signature demonstrated that the high- and low-risk groups are significantly different in their overall survival. Of the 64 genes, 11 are related to cancer metastasis (APC, CDH8, IL8RB, LY6D, PCDHGA12, DSP, NID, ENPP2, CCR2, CASP8, and CASP10) and eight are involved in apoptosis (CASP8, CASP10, PIK3R1, BCL2, SON, INHA, PSEN1, and BIK).
Conclusions
Our results indicate that gene expression signatures from several datasets can be reconciled. The resulting signature is useful in predicting survival of stage I NSCLC and might be useful in informing treatment decisions.
Meta-analysis of several lung cancer gene expression studies yields a set of 64 genes whose expression profile is useful in predicting survival of patients with early-stage lung cancer and possibly informing treatment decisions.
Editors' Summary
Background.
Lung cancer is the commonest cause of cancer-related death worldwide. Most cases are of a type called non-small cell lung cancer (NSCLC) and are mainly caused by smoking. Like other cancers, how NSCLC is treated depends on the “stage” at which it is detected. Stage IA NSCLCs are small and confined to the lung and can be removed surgically; patients with slightly larger stage IB tumors often receive chemotherapy after surgery. In stage II NSCLC, cancer cells may be present in lymph nodes near the tumor. Surgery plus chemotherapy is the usual treatment for this stage and for some stage III NSCLCs. However, in this stage, the tumor can be present throughout the chest and surgery is not always possible. For such cases and in stage IV NSCLC, where the tumor has spread throughout the body, patients are treated with chemotherapy alone. The stage at which NSCLC is detected also determines how well patients respond to treatment. Those who can be treated surgically do much better than those who can't. So, whereas only 2% of patients with stage IV lung cancer survive for 5 years after diagnosis, about 70% of patients with stage I or II lung cancer live at least this long.
Why Was This Study Done?
Even stage I and II lung cancers often recur and there is no accurate way to identify the patients in which this will happen. If there was, these patients could be given aggressive chemotherapy, so the search is on for a “molecular signature” to help identify which NSCLCs are likely to recur. Unlike normal cells, cancer cells divide uncontrollably and can move around the body. These behavioral differences are caused by changes in their genetic material that alter their patterns of RNA transcription and protein expression. In this study, the researchers have investigated whether data from several microarray studies (a technique used to catalog the genes expressed in cells) can be pooled to construct a gene expression signature that predicts the survival of patients with stage I NSCLC.
What Did the Researchers Do and Find?
The researchers took the data from seven independent microarray studies (including a new study of their own) that recorded gene expression profiles related to survival time (less than 2 years and greater than 5 years) for stage I NSCLC. Because these studies had been done in different places with slightly different techniques, the researchers applied a statistical tool called distance-weighted discrimination to smooth out any systematic differences among the studies before identifying 64 genes whose expression was associated with survival. Most of these genes are involved in cell adhesion, cell motility, cell proliferation, and cell death, all processes that are altered in cancer cells. The researchers then developed a statistical model that allowed them to use the gene expression and survival data to calculate risk scores for nearly 200 patients in five of the datasets. When they separated the patients into high and low risk groups on the basis of these scores, the two groups were significantly different in terms of survival time. Indeed, the gene expression signature was better at predicting outcome than routine staging. Finally, the researchers validated the gene expression signature by showing that it predicted survival with more than 85% accuracy in two independent datasets.
What Do These Findings Mean?
The 64 gene expression signature identified here could help clinicians prepare treatment plans for patients with stage I NSCLC. Because it accurately predicts survival in patients with adenocarcinoma or squamous cell cancer (the two major subtypes of NSCLC), it potentially indicates which of these patients should receive aggressive chemotherapy and which can be spared this unpleasant treatment. Previous attempts to establish gene expression signatures to predict outcome have used data from small groups of patients and have failed when tested in additional patients. In contrast, this new signature seems to be generalizable. Nevertheless, its ability to predict outcomes must be confirmed in further studies before it is routinely adopted by oncologists for treatment planning.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030467.
US National Cancer Institute information on lung cancer for patients and health professionals.
MedlinePlus encyclopedia entries on small-cell and non-small-cell lung cancer.
Cancer Research UK, information on patients about all aspects of lung cancer.
Wikipedia pages on DNA microarrays and expression profiling (note that Wikipedia is a free online encyclopedia that anyone can edit).
doi:10.1371/journal.pmed.0030467
PMCID: PMC1716187  PMID: 17194181
16.  Low levels of p27 in association with deregulated p53-pRb protein status enhance tumor proliferation and chromosomal instability in non-small cell lung carcinomas. 
Molecular Medicine  2001;7(6):418-429.
BACKGROUND: Down-regulation or overexpression of the cyclin-dependent kinase inhibitor p27 have been observed in a range of malignancies, including lung cancer. To further elucidate the role of the molecule in tumor growth regulation, we evaluated p27 expression in a series of non-small cell lung carcinomas (NSCLCs), and examined its relation with histology, kinetic parameters, ploidy, and overall survival. We extended our investigation into the association of p27 levels with the presence of Ki-ras mutations, as well as with the expression status of p53 and pRb in tumor cells. MATERIAL AND METHODS: p27, p53, and pRb status were immunohistochemically evaluated in a total of 69 NSCLCs. In situ assays were employed to assess the kinetic parameters (Ki-67 immunohistochemistry for proliferation index, Tdt-mediated dUTP nick end labeling assay for apoptotic index). The ploidy status of the tumors was assessed after staining nuclei with the Feulgen procedure, and the presence of Ki-ras mutations was examined by restriction fragment length polymorphisms. All possible associations were assessed with a series of statistical methods. RESULTS: Immunoreactivity for p27 was observed in the entire series of specimens, with the mean percentage of positive cells being 33%. Adenocarcinomas (AdCs) exhibited higher p27 levels compared to squamous cell carcinomas (SqCCs) (p < 0.01). An inverse correlation was established between p27 expression and proliferation index (PI) (r = -0.834, p < 0.01) but not with apoptotic index (AI), whereas aneuploid tumors were characterized by lower p27 levels than diploid ones (p < 0.01). No difference in p27 immunostaining was observed with regard to the presence of Ki-ras mutations, whereas aberrant p53 and/or pRb expression patterns were associated with p27 underexpression (p < 0.01 for p53 status, p < 0.05 regarding pRb levels, and p < 0.01 for a combined deregulation of both proteins). Two or more alterations in the p27/p53/pRb protein network (i.e., p27 levels lower than the estimated mean value, overexpressed p53, and/or aberrant pRb) were associated with increased PI and aneuploidy (p < 0.001 and p < 0.01, respectively). A powerful trend was found between p27 expression and overall survival (p = 0.066). CONCLUSIONS: Our findings confirm the heterogeneity between AdCs and SqCCs, and are suggestive of an increased proliferative activity in NSCLCs underexpressing p27. Furthermore, our analysis supports the concept of p27 forming a functionally compact network with p53 and pRb, which is actively involved in the regulation of cellular proliferation and chromosomal stability.
PMCID: PMC1950049  PMID: 11474135
17.  Can quantifying free-circulating DNA in plasma be used to identify subjects with high-grade pre-invasive endobronchial lesions? 
Oncology Letters  2013;5(5):1591-1594.
Increased concentrations of free-circulating plasma DNA (cpDNA) are observed in patients with invasive cancer, including lung cancer. Whether cpDNA levels are elevated in subjects with high-grade pre-invasive lesions of lung squamous cell carcinoma (SqCC) and whether its detection may be of value for identifying subjects at the highest risk of developing lung SqCC is currently unknown. The present study assessed cpDNA levels in subjects with high- and low-grade pre-invasive squamous endobronchial lesions relative to patients with clinically overt lung SqCC and healthy controls using real-time quantitative PCR methodology. The median cpDNA levels of the patients with invasive lung SqCC (n=16) were significantly higher compared with those of the healthy controls (n=16; P<0.01), whereas the cpDNA levels in the subjects with pre-invasive lesions (n=20) did not differ from those of the controls (P=0.29). The cpDNA levels in subjects with high-grade pre-invasive lesions were highly similar to those diagnosed with low-grade pre-invasive lesions (P=0.85). Our data suggest that cpDNA levels are not increased during the pre-invasive stages of lung squamous carcinogenesis.
doi:10.3892/ol.2013.1262
PMCID: PMC3678857  PMID: 23761824
circulating plasma DNA; autofluorescence bronchoscopy; premalignant endobronchial lesion
18.  Deep Sequence Analysis of Non-Small Cell Lung Cancer: Integrated Analysis of Gene Expression, Alternative Splicing, and Single Nucleotide Variations in Lung Adenocarcinomas with and without Oncogenic KRAS Mutations 
KRAS mutations are highly prevalent in non-small cell lung cancer (NSCLC), and tumors harboring these mutations tend to be aggressive and resistant to chemotherapy. We used next-generation sequencing technology to identify pathways that are specifically altered in lung tumors harboring a KRAS mutation. Paired-end RNA-sequencing of 15 primary lung adenocarcinoma tumors (8 harboring mutant KRAS and 7 with wild-type KRAS) were performed. Sequences were mapped to the human genome, and genomic features, including differentially expressed genes, alternate splicing isoforms and single nucleotide variants, were determined for tumors with and without KRAS mutation using a variety of computational methods. Network analysis was carried out on genes showing differential expression (374 genes), alternate splicing (259 genes), and SNV-related changes (65 genes) in NSCLC tumors harboring a KRAS mutation. Genes exhibiting two or more connections from the lung adenocarcinoma network were used to carry out integrated pathway analysis. The most significant signaling pathways identified through this analysis were the NFκB, ERK1/2, and AKT pathways. A 27 gene mutant KRAS-specific sub network was extracted based on gene–gene connections from the integrated network, and interrogated for druggable targets. Our results confirm previous evidence that mutant KRAS tumors exhibit activated NFκB, ERK1/2, and AKT pathways and may be preferentially sensitive to target therapeutics toward these pathways. In addition, our analysis indicates novel, previously unappreciated links between mutant KRAS and the TNFR and PPARγ signaling pathways, suggesting that targeted PPARγ antagonists and TNFR inhibitors may be useful therapeutic strategies for treatment of mutant KRAS lung tumors. Our study is the first to integrate genomic features from RNA-Seq data from NSCLC and to define a first draft genomic landscape model that is unique to tumors with oncogenic KRAS mutations.
doi:10.3389/fonc.2012.00012
PMCID: PMC3356053  PMID: 22655260
transcriptome sequencing; RNA-Seq; KRAS mutation; NSCLC; bioinformatics; network analysis; data integration and computational methods
19.  The Natural Tumor Suppressor Protein Maspin and Potential Application in Non Small Cell Lung Cancer 
Current pharmaceutical design  2010;16(16):1877-1881.
The grim prognosis of lung cancer, that has an overall 10–15% survival at 5 years, remains in the US the leading cause of cancer mortality, providing a compelling rationale for studying the molecular basis of this malignancy. Surmising the common, general association with smoking, lung cancers differ at the microscopic, anatomical, epidemiological and clinical level and harbor complex genetic and epigenetic alterations. Currently, lung cancer is divided into small cell lung carcinoma (SCLC) and non small cell lung carcinoma (NSCLC) for the purpose of clinical management. NSCLC constitutes 80–85% of lung cancers and is further divided into histological subtypes such as adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, etc.
The ultimate goal for lung cancer research is to develop a strategy to block the tumor progression and improve the prognosis of lung cancer. This goal can realistically be achieved only when the biological complexity of this disease is taken into account. To this end, identification and understanding of molecular markers that are mechanistically involved in tumor progression are needed. Our recent studies suggest histological subtype-dependent distinct correlations between the expression and/or subcellular localization of tumor suppressive maspin with the progression and prognosis of NSCLC. Maspin is an epithelial specific member of the serine protease inhibitor (serpin) superfamily but recently identified as an endogenous inhibitor of histone deacetylase 1 (HDAC1). This novel biochemical activity coincides with a consensus emerged recently from the evidence that nuclear maspin confers better differentiated epithelial phenotypes, decreased tumor angiogenesis, increased tumor sensitivity to drug-induced apoptosis, and a more favorable prognosis. In the current review, we discuss the evidence that maspin may be a marker that stratifies the progression and prognosis of different subtypes of NSCLC.
PMCID: PMC2908495  PMID: 20337574
lung cancer; molecular marker; prognosis; histological subtypes; histone deacetylase 1; natural HDAC inhibitor; subcellular localization; differential expression; drug discovery
20.  Differential disruption of cell cycle pathways in small cell and non-small cell lung cancer 
British Journal of Cancer  2006;94(12):1927-1935.
Lung cancer is the leading cause of cancer-related mortality in the world, with small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) comprising the two major cell types. Although these cell types can be distinguished readily at the histological level, knowledge of their underlying molecular differences is very limited. In this study, we compared 14 SCLC cell lines against 27 NSCLC cell lines using an integrated array comparative genomic hybridisation and gene expression profiling approach to identify subtype-specific disruptions. Using stringent criteria, we have identified 159 of the genes that are responsible for the different biology of these cell types. Sorting of these genes by their biological functions revealed the differential disruption of key components involved in cell cycle pathways. Our novel comparative combined genome and transcriptome analysis not only identified differentially altered genes, but also revealed that certain shared pathways are preferentially disrupted at different steps in these cell types. Small cell lung cancer exhibited increased expression of MRP5, activation of Wnt pathway inhibitors, and upregulation of p38 MAPK activating genes, while NSCLC showed downregulation of CDKN2A, and upregulation of MAPK9 and EGFR. This information suggests that cell cycle upregulation in SCLC and NSCLC occurs through drastically different mechanisms, highlighting the need for differential molecular target selection in the treatment of these cancers.
doi:10.1038/sj.bjc.6603167
PMCID: PMC2361340  PMID: 16705311
array CGH; gene expression analysis; cancer genome; non-small cell lung cancer; small cell lung cancer
21.  Efficacy of epidermal growth factor receptor-tyrosine kinase inhibitors for Chinese patients with squamous cell carcinoma of lung harboring EGFR mutation 
Journal of Thoracic Disease  2013;5(5):585-592.
Objective
Epidermal growth factor receptor (EGFR) mutation mostly occurred in lung adenocarcinoma, rarely in squamous cell carcinoma (SQCC). EGFR mutation rate in SQCC varied in previous reports, and the efficacy of EGFR tyrosine kinase inhibitors (TKIs) in SQCC harboring EGFR mutation has not yet been fully evaluated. The aim of this study was to investigate the efficacy EGFR-TKIs for Chinese patients with SQCC of lung harboring EGFR mutation.
Patients and methods
Two cohorts of patients were analyzed. The first cohort included 146 consecutive post-operation SQCC patients from January 2008 to October 2012. The second cohort included 63 patients with advanced SQCC receiving EGFR-TKIs treatment. EGFR mutation analysis was performed with Real-time PCR method. The pathologic diagnosis was validated with immunohistochemistry (IHC) for patients harboring activated EGFR mutation. And the efficacy of EGFR-TKIs in squamous cell carcinoma of lung (SQCC) was evaluated in patients with activated EGFR mutations.
Results
In the first cohort, 146 resected patients, EGFR mutations were detected in 3 patients, with the mutation rate of 2.0%. In cohort two, 63 patients treated with EGFR-TKIs, 15 patients possessed activated EGFR mutations. The response rate and disease control rate in these patients was 26.7% and 66.7% respectively. 5 patients had disease control over 6 months. The progression free survival (PFS) in EGFR-mutated patients was 3.9 months.
Conclusions
In Chinese SQCC patients, EGFR mutation rate was extremely low. EGFR-TKIs seemed to be less effective in EGFR-mutated SQCC patients, but some patients could still obtain benefit from EGFR-TKIs. To identify this part of patients, further study was warranted in the future.
doi:10.3978/j.issn.2072-1439.2013.09.15
PMCID: PMC3815724  PMID: 24255770
Squamous cell carcinoma of lung (SQCC); immunohistochemistry (IHC); epidermal growth factor receptor mutation (EGFR mutation); epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs)
22.  Polymorphisms, Mutations, and Amplification of the EGFR Gene in Non-Small Cell Lung Cancers 
PLoS Medicine  2007;4(4):e125.
Background
The epidermal growth factor receptor (EGFR) gene is the prototype member of the type I receptor tyrosine kinase (TK) family and plays a pivotal role in cell proliferation and differentiation. There are three well described polymorphisms that are associated with increased protein production in experimental systems: a polymorphic dinucleotide repeat (CA simple sequence repeat 1 [CA-SSR1]) in intron one (lower number of repeats) and two single nucleotide polymorphisms (SNPs) in the promoter region, −216 (G/T or T/T) and −191 (C/A or A/A). The objective of this study was to examine distributions of these three polymorphisms and their relationships to each other and to EGFR gene mutations and allelic imbalance (AI) in non-small cell lung cancers.
Methods and Findings
We examined the frequencies of the three polymorphisms of EGFR in 556 resected lung cancers and corresponding non-malignant lung tissues from 336 East Asians, 213 individuals of Northern European descent, and seven of other ethnicities. We also studied the EGFR gene in 93 corresponding non-malignant lung tissue samples from European-descent patients from Italy and in peripheral blood mononuclear cells from 250 normal healthy US individuals enrolled in epidemiological studies including individuals of European descent, African–Americans, and Mexican–Americans. We sequenced the four exons (18–21) of the TK domain known to harbor activating mutations in tumors and examined the status of the CA-SSR1 alleles (presence of heterozygosity, repeat number of the alleles, and relative amplification of one allele) and allele-specific amplification of mutant tumors as determined by a standardized semiautomated method of microsatellite analysis. Variant forms of SNP −216 (G/T or T/T) and SNP −191 (C/A or A/A) (associated with higher protein production in experimental systems) were less frequent in East Asians than in individuals of other ethnicities (p < 0.001). Both alleles of CA-SSR1 were significantly longer in East Asians than in individuals of other ethnicities (p < 0.001). Expression studies using bronchial epithelial cultures demonstrated a trend towards increased mRNA expression in cultures having the variant SNP −216 G/T or T/T genotypes. Monoallelic amplification of the CA-SSR1 locus was present in 30.6% of the informative cases and occurred more often in individuals of East Asian ethnicity. AI was present in 44.4% (95% confidence interval: 34.1%–54.7%) of mutant tumors compared with 25.9% (20.6%–31.2%) of wild-type tumors (p = 0.002). The shorter allele in tumors with AI in East Asian individuals was selectively amplified (shorter allele dominant) more often in mutant tumors (75.0%, 61.6%–88.4%) than in wild-type tumors (43.5%, 31.8%–55.2%, p = 0.003). In addition, there was a strong positive association between AI ratios of CA-SSR1 alleles and AI of mutant alleles.
Conclusions
The three polymorphisms associated with increased EGFR protein production (shorter CA-SSR1 length and variant forms of SNPs −216 and −191) were found to be rare in East Asians as compared to other ethnicities, suggesting that the cells of East Asians may make relatively less intrinsic EGFR protein. Interestingly, especially in tumors from patients of East Asian ethnicity, EGFR mutations were found to favor the shorter allele of CA-SSR1, and selective amplification of the shorter allele of CA-SSR1 occurred frequently in tumors harboring a mutation. These distinct molecular events targeting the same allele would both be predicted to result in greater EGFR protein production and/or activity. Our findings may help explain to some of the ethnic differences observed in mutational frequencies and responses to TK inhibitors.
Masaharu Nomura and colleagues examine the distribution ofEGFR polymorphisms in different populations and find differences that might explain different responses to tyrosine kinase inhibitors in lung cancer patients.
Editors' Summary
Background.
Most cases of lung cancer—the leading cause of cancer deaths worldwide—are “non-small cell lung cancer” (NSCLC), which has a very low cure rate. Recently, however, “targeted” therapies have brought new hope to patients with NSCLC. Like all cancers, NSCLC occurs when cells begin to divide uncontrollably because of changes (mutations) in their genetic material. Chemotherapy drugs treat cancer by killing these rapidly dividing cells, but, because some normal tissues are sensitive to these agents, it is hard to kill the cancer completely without causing serious side effects. Targeted therapies specifically attack the changes in cancer cells that allow them to divide uncontrollably, so it might be possible to kill the cancer cells selectively without damaging normal tissues. Epidermal growth factor receptor (EGRF) was one of the first molecules for which a targeted therapy was developed. In normal cells, messenger proteins bind to EGFR and activate its “tyrosine kinase,” an enzyme that sticks phosphate groups on tyrosine (an amino acid) in other proteins. These proteins then tell the cell to divide. Alterations to this signaling system drive the uncontrolled growth of some cancers, including NSCLC.
Why Was This Study Done?
Molecules that inhibit the tyrosine kinase activity of EGFR (for example, gefitinib) dramatically shrink some NSCLCs, particularly those in East Asian patients. Tumors shrunk by tyrosine kinase inhibitors (TKIs) often (but not always) have mutations in EGFR's tyrosine kinase. However, not all tumors with these mutations respond to TKIs, and other genetic changes—for example, amplification (multiple copies) of the EGFR gene—also affect tumor responses to TKIs. It would be useful to know which genetic changes predict these responses when planning treatments for NSCLC and to understand why the frequency of these changes varies between ethnic groups. In this study, the researchers have examined three polymorphisms—differences in DNA sequences that occur between individuals—in the EGFR gene in people with and without NSCLC. In addition, they have looked for associations between these polymorphisms, which are present in every cell of the body, and the EGFR gene mutations and allelic imbalances (genes occur in pairs but amplification or loss of one copy, or allele, often causes allelic imbalance in tumors) that occur in NSCLCs.
What Did the Researchers Do and Find?
The researchers measured how often three EGFR polymorphisms (the length of a repeat sequence called CA-SSR1, and two single nucleotide variations [SNPs])—all of which probably affect how much protein is made from the EGFR gene—occurred in normal tissue and NSCLC tissue from East Asians and individuals of European descent. They also looked for mutations in the EGFR tyrosine kinase and allelic imbalance in the tumors, and then determined which genetic variations and alterations tended to occur together in people with the same ethnicity. Among many associations, the researchers found that shorter alleles of CA-SSR1 and the minor forms of the two SNPs occurred less often in East Asians than in individuals of European descent. They also confirmed that EGFR kinase mutations were more common in NSCLCs in East Asians than in European-descent individuals. Furthermore, mutations occurred more often in tumors with allelic imbalance, and in tumors where there was allelic imbalance and an EGFR mutation, the mutant allele was amplified more often than the wild-type allele.
What Do These Findings Mean?
The researchers use these associations between gene variants and tumor-associated alterations to propose a model to explain the ethnic differences in mutational frequencies and responses to TKIs seen in NSCLC. They suggest that because of the polymorphisms in the EGFR gene commonly seen in East Asians, people from this ethnic group make less EGFR protein than people from other ethnic groups. This would explain why, if a threshold level of EGFR is needed to drive cells towards malignancy, East Asians have a high frequency of amplified EGFR tyrosine kinase mutations in their tumors—mutation followed by amplification would be needed to activate EGFR signaling. This model, though speculative, helps to explain some clinical findings, such as the frequency of EGFR mutations and of TKI sensitivity in NSCLCs in East Asians. Further studies of this type in different ethnic groups and in different tumors, as well as with other genes for which targeted therapies are available, should help oncologists provide personalized cancer therapies for their patients.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040125.
US National Cancer Institute information on lung cancer and on cancer treatment for patients and professionals
MedlinePlus encyclopedia entries on NSCLC
Cancer Research UK information for patients about all aspects of lung cancer, including treatment with TKIs
Wikipedia pages on lung cancer, EGFR, and gefitinib (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0040125
PMCID: PMC1876407  PMID: 17455987
23.  Preoperative CYFRA 21-1 level as a prognostic indicator in resected primary squamous cell lung cancer. 
British Journal of Cancer  1996;74(6):956-960.
The CYFRA 21-1 assay is a test that has been developed recently for detection of a cytokeratin 19 fragment in serum. A diagnostic role for CYFRA 21-1 has already been proposed. The question of whether this marker is prognostically significant is important in clarifying the role of CYFRA 21-1 in clinical practice. The aim of this study was to evaluate the prognostic significance of elevated preoperative CYFRA 21-1 levels in patients with resected primary squamous-cell lung cancer (SqCC). Serum levels of CYFRA 21-1 were measured using an immunoradiometric assay (CIS bio) in 91 patients with operable SqCC. Survival and disease-free survival curves related to initial levels of this marker were estimated using the Kaplan-Meier method. In the univariate analysis the log-rank test and the log-rank test for trend were used. In the multivariate analysis the stratified log-rank test and the proportional hazard model were used. Elevated preoperative CYFRA 21-1 levels were identified in 55% of patients with SqCC. The number of patients with elevated levels of this marker increased with TNM stage (P = 0.0001). In univariate analysis elevated levels of CYFRA 21-1 were significantly associated with poor overall survival (P < 0.00005) and with disease-free survival (P < 0.00005). In multivariate analysis elevated levels of this marker were also found to be associated with poor overall and disease-free survival (P = 0.01 and P = 0.003 respectively). In conclusion, CYFRA 21-1 may be an independent prognostic parameter of survival and tumour relapse in SqCC and may be useful in identifying resected SqCC patients at high risk of treatment failure.
PMCID: PMC2074750  PMID: 8826865
24.  Impact of Histopathological Diagnosis with Ancillary Immunohistochemical Studies on Lung Cancer Subtypes Incidence and Survival: A Population-Based Study 
Journal of Cancer Epidemiology  2011;2011:275758.
Purpose. The aim of this study was to assess the impact of immunohistochemical- (IHC-) studies on incidence and survival of lung cancer histotypes. Patients and Methods. Lung cancers occurred in southern Switzerland between 1996 and 2010 were selected by the Ticino Cancer Registry and categorised into adenocarcinoma (AC), squamous-cell-carcinoma (SqCC), small-cell-carcinoma (SmCC), and large-cell carcinoma/non-small-cell lung cancer (LCC/NSCLC). Incidence rates, annual-percentage-change (APC), and two-year overall survival (OS) (follow-up: 31.12.2010) were performed. Results. 2467 cases were selected: 997 (40.4%) AC; 522 (21.2%) LCC/NSCLC, 378 (15.3%) SmCC, and 570 (23.1%) SqCC. Trend-analysis showed significant increase in AC (APC: 4.6; 95% CI: 3.1; 6.0) and decrease of LCC/NSCLC, with significant joinpoint in 2003 (APC: −14.7; 95% CI: −21.6; −7.1). Improved OS and decreased OS were detected in SqCC and LCC/NSCLC, respectively. Conclusions. This study highlights that diagnosis with ancillary immunohistochemical studies will change incidence and survival of precisely defined lung cancer subtypes. It calls attention to the need for cautious interpretation of studies and clinical trials, where the diagnosis was based on histology unaccompanied by IHC studies, and to the need of standardised diagnostic procedures.
doi:10.1155/2011/275758
PMCID: PMC3255178  PMID: 22253626
25.  Nuclear Receptor Expression Defines a Set of Prognostic Biomarkers for Lung Cancer 
PLoS Medicine  2010;7(12):e1000378.
David Mangelsdorf and colleagues show that nuclear receptor expression is strongly associated with clinical outcomes of lung cancer patients, and this expression profile is a potential prognostic signature for lung cancer patient survival time, particularly for individuals with early stage disease.
Background
The identification of prognostic tumor biomarkers that also would have potential as therapeutic targets, particularly in patients with early stage disease, has been a long sought-after goal in the management and treatment of lung cancer. The nuclear receptor (NR) superfamily, which is composed of 48 transcription factors that govern complex physiologic and pathophysiologic processes, could represent a unique subset of these biomarkers. In fact, many members of this family are the targets of already identified selective receptor modulators, providing a direct link between individual tumor NR quantitation and selection of therapy. The goal of this study, which begins this overall strategy, was to investigate the association between mRNA expression of the NR superfamily and the clinical outcome for patients with lung cancer, and to test whether a tumor NR gene signature provided useful information (over available clinical data) for patients with lung cancer.
Methods and Findings
Using quantitative real-time PCR to study NR expression in 30 microdissected non-small-cell lung cancers (NSCLCs) and their pair-matched normal lung epithelium, we found great variability in NR expression among patients' tumor and non-involved lung epithelium, found a strong association between NR expression and clinical outcome, and identified an NR gene signature from both normal and tumor tissues that predicted patient survival time and disease recurrence. The NR signature derived from the initial 30 NSCLC samples was validated in two independent microarray datasets derived from 442 and 117 resected lung adenocarcinomas. The NR gene signature was also validated in 130 squamous cell carcinomas. The prognostic signature in tumors could be distilled to expression of two NRs, short heterodimer partner and progesterone receptor, as single gene predictors of NSCLC patient survival time, including for patients with stage I disease. Of equal interest, the studies of microdissected histologically normal epithelium and matched tumors identified expression in normal (but not tumor) epithelium of NGFIB3 and mineralocorticoid receptor as single gene predictors of good prognosis.
Conclusions
NR expression is strongly associated with clinical outcomes for patients with lung cancer, and this expression profile provides a unique prognostic signature for lung cancer patient survival time, particularly for those with early stage disease. This study highlights the potential use of NRs as a rational set of therapeutically tractable genes as theragnostic biomarkers, and specifically identifies short heterodimer partner and progesterone receptor in tumors, and NGFIB3 and MR in non-neoplastic lung epithelium, for future detailed translational study in lung cancer.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Lung cancer, the most common cause of cancer-related death, kills 1.3 million people annually. Most lung cancers are “non-small-cell lung cancers” (NSCLCs), and most are caused by smoking. Exposure to chemicals in smoke causes changes in the genes of the cells lining the lungs that allow the cells to grow uncontrollably and to move around the body. How NSCLC is treated and responds to treatment depends on its “stage.” Stage I tumors, which are small and confined to the lung, are removed surgically, although chemotherapy is also sometimes given. Stage II tumors have spread to nearby lymph nodes and are treated with surgery and chemotherapy, as are some stage III tumors. However, because cancer cells in stage III tumors can be present throughout the chest, surgery is not always possible. For such cases, and for stage IV NSCLC, where the tumor has spread around the body, patients are treated with chemotherapy alone. About 70% of patients with stage I and II NSCLC but only 2% of patients with stage IV NSCLC survive for five years after diagnosis; more than 50% of patients have stage IV NSCLC at diagnosis.
Why Was This Study Done?
Patient responses to treatment vary considerably. Oncologists (doctors who treat cancer) would like to know which patients have a good prognosis (are likely to do well) to help them individualize their treatment. Consequently, the search is on for “prognostic tumor biomarkers,” molecules made by cancer cells that can be used to predict likely clinical outcomes. Such biomarkers, which may also be potential therapeutic targets, can be identified by analyzing the overall pattern of gene expression in a panel of tumors using a technique called microarray analysis and looking for associations between the expression of sets of genes and clinical outcomes. In this study, the researchers take a more directed approach to identifying prognostic biomarkers by investigating the association between the expression of the genes encoding nuclear receptors (NRs) and clinical outcome in patients with lung cancer. The NR superfamily contains 48 transcription factors (proteins that control the expression of other genes) that respond to several hormones and to diet-derived fats. NRs control many biological processes and are targets for several successful drugs, including some used to treat cancer.
What Did the Researchers Do and Find?
The researchers analyzed the expression of NR mRNAs using “quantitative real-time PCR” in 30 microdissected NSCLCs and in matched normal lung tissue samples (mRNA is the blueprint for protein production). They then used an approach called standard classification and regression tree analysis to build a prognostic model for NSCLC based on the expression data. This model predicted both survival time and disease recurrence among the patients from whom the tumors had been taken. The researchers validated their prognostic model in two large independent lung adenocarcinoma microarray datasets and in a squamous cell carcinoma dataset (adenocarcinomas and squamous cell carcinomas are two major NSCLC subtypes). Finally, they explored the roles of specific NRs in the prediction model. This analysis revealed that the ability of the NR signature in tumors to predict outcomes was mainly due to the expression of two NRs—the short heterodimer partner (SHP) and the progesterone receptor (PR). Expression of either gene could be used as a single gene predictor of the survival time of patients, including those with stage I disease. Similarly, the expression of either nerve growth factor induced gene B3 (NGFIB3) or mineralocorticoid receptor (MR) in normal tissue was a single gene predictor of a good prognosis.
What Do These Findings Mean?
These findings indicate that the expression of NR mRNA is strongly associated with clinical outcomes in patients with NSCLC. Furthermore, they identify a prognostic NR expression signature that provides information on the survival time of patients, including those with early stage disease. The signature needs to be confirmed in more patients before it can be used clinically, and researchers would like to establish whether changes in mRNA expression are reflected in changes in protein expression if NRs are to be targeted therapeutically. Nevertheless, these findings highlight the potential use of NRs as prognostic tumor biomarkers. Furthermore, they identify SHP and PR in tumors and two NRs in normal lung tissue as molecules that might provide new targets for the treatment of lung cancer and new insights into the early diagnosis, pathogenesis, and chemoprevention of lung cancer.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000378.
The Nuclear Receptor Signaling Atlas (NURSA) is consortium of scientists sponsored by the US National Institutes of Health that provides scientific reagents, datasets, and educational material on nuclear receptors and their co-regulators to the scientific community through a Web-based portal
The Cancer Prevention and Research Institute of Texas (CPRIT) provides information and resources to anyone interested in the prevention and treatment of lung and other cancers
The US National Cancer Institute provides detailed information for patients and professionals about all aspects of lung cancer, including information on non-small-cell carcinoma and on tumor markers (in English and Spanish)
Cancer Research UK also provides information about lung cancer and information on how cancer starts
MedlinePlus has links to other resources about lung cancer (in English and Spanish)
Wikipedia has a page on nuclear receptors (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.1000378
PMCID: PMC3001894  PMID: 21179495

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