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1.  Loss of Fhit expression in non-small-cell lung cancer: correlation with molecular genetic abnormalities and clinicopathological features 
British Journal of Cancer  2000;82(6):1191-1197.
The FHIT gene is located at a chromosomal site (3p14.2) which is commonly affected by translocations and deletions in human neoplasia. Although FHIT alterations at the DNA and RNA level are frequent in many types of tumours, the biological and clinical significance of these changes is not clear. In this study we aimed at correlating loss of Fhit protein expression with a large number of molecular genetic and clinical parameters in a well-characterized cohort of non-small-cell lung cancers (NSCLCs). Paraffin sections of 99 non-small-cell carcinomas were reacted with an anti-Fhit polyclonal antibody in a standard immunohistochemical reaction. Abnormal cases were characterized by complete loss of cytoplasmic Fhit staining. The Fhit staining results were then correlated with previously obtained clinical and molecular data. Fifty-two of 99 tumours lacked cytoplasmic Fhit staining, with preserved reactivity in adjacent normal cells. Lack of Fhit staining correlated with: loss of heterozygosity (LOH) at the FHIT 3p14.2 locus, but not at other loci on 3p; squamous histology; LOH at 17p13 and 5q but not with LOH at multiple other suspected tumour suppressor gene loci; and was inversely correlated with codon 12 mutations in K- ras. Fhit expression was not correlated overall with a variety of clinical parameters including survival and was not associated with abnormalities of immunohistochemical expression of p53, RB, and p16. All of these findings are consistent with loss of Fhit protein expression being as frequent an abnormality in lung cancer pathogenesis as are p53 and p16 protein abnormalities and that such loss occurs independently of the commitment to the metastatic state and of most other molecular abnormalities. © 2000 Cancer Research Campaign
doi:10.1054/bjoc.1999.1062
PMCID: PMC2363352  PMID: 10735505
FHIT; immunohistochemistry; lung cancer; prognosis; loss of heterozygosity
2.  Fragile histidine triad gene alterations are not essential for hepatocellular carcinoma development in South Korea 
AIM: To establish the role of FHIT in the pathogenesis hepatocellular carcinoma (HCC).
METHODS: We examined genomic alterations, as well as, mRNA and protein expression patterns from the FHIT gene, in 48 surgically resected hepatocellular carcinoma (HCC) tissues. Additionally, p53 mutations were analyzed.
RESULTS: Aberrant FHIT transcripts were detected in 11 of 48 surrounding non-tumor liver tissues and 27 of 48 HCC samples (22.9% vs 56.3%, P = 0.002). No point mutations were identified within the open reading frame region of FHIT. Loss of heterozygosity (LOH) of the FHIT locus was detected in 4 of 42 informative cases for D3S1300, and 3 of 29 informative cases for D3S1313. Reduced expression of FHIT protein (Fhit) was observed in 8 (16.7%) of 48 HCC samples, with complete loss of Fhit in only 1 case. There were no associations with abnormal transcripts, LOH, and Fhit expression. p53 mutations were identified in 9 of the 48 HCC cases. However, none of the cases displayed a G to T transversion at p53 codon 249.
CONCLUSION: Aberrant FHIT transcripts were more common in HCC tissues as compared to non-cancerous liver tissues. However, Fhit expression was lost or reduced in a minor fraction of HCC tissues, while it was strongly expressed in non-cancerous liver tissues. Therefore, our study suggests that FHIT plays a role in relatively few HCC cases in South Korea.
doi:10.3748/wjg.14.3526
PMCID: PMC2716616  PMID: 18567082
Fragile histidine triad; Aberrant transcripts; Microsatellite instability; Protein expression; Hepatocellular carcinoma
3.  p53 gene aberrations in non-small-cell lung carcinomas from a smoking population. 
British Journal of Cancer  1997;75(8):1119-1124.
We examined 46 non-small-cell lung carcinomas (NSCLCs) for the presence of p53 mutations in exons 4-9, positive p53 immunostaining and loss of heterozygosity (LOH) in the TP53 locus. p53 mutations were detected in 13 tumour samples (28.3%), whereas overexpression of the p53 protein was found in 30 of 45 (67%) samples. Allelic loss was found in 9 of 38 (23.6%) informative cases. The statistical analysis revealed no significant correlation between p53 mutations and clinicopathological data, although mutations appear to occur more frequently in squamous cell carcinomas (7 of 18) than in adenocarcinomas (2 of 15). All but three individuals in this study group smoked. In contrast to previous reports, we found a higher prevalence of GC-->AT transitions than of GC-->TA transversions, as expected in a smoking population. A trend was found between p53-positive immunostaining and a history of heavy smoking (76-126 pack-years) and was inversely correlated with allelic deletion (LOH) at the TP53 locus. Eight of the 12 NSCLCs containing p53 mutations also had concomitant p53 overexpression, and it is of specific note that three of the four tumours containing p53 'mutations' with no overexpression of the p53 protein had either insertions or deletions in the p53 gene. No correlation was found between p53 mutations and fractional allele loss or ras mutations. p53 mutations in this Merseyside population in the UK do not appear to be as common as in other reports for NSCLC and exhibit predominance of GC-->AT transitions preferentially at non-CpG sites, suggesting that other carcinogens in addition to those in tobacco smoke may be involved in NSCLC in the Merseyside area of the UK.
Images
PMCID: PMC2222790  PMID: 9099958
4.  Loss of heterozygosity is related to p53 mutations and smoking in lung cancer 
British Journal of Cancer  2001;84(2):226-231.
Carcinogenesis results from an accumulation of several genetic alterations. Mutations in the p53 gene are frequent and occur at an early stage of lung carcinogenesis. Loss of multiple chromosomal regions is another genetic alteration frequently found in lung tumours. We have examined the association between p53 mutations, loss of heterozygosity (LOH) at frequently deleted loci in lung cancer, and tobacco exposure in 165 tumours from non-small cell lung cancer (NSCLC) patients. A highly significant association between p53 mutations and deletions on 3p, 5q, 9p, 11p and 17p was found. There was also a significant correlation between deletions at these loci. 86% of the tumours with concordant deletion in the 4 most involved loci (3p21, 5q11–13, 9p21 and 17p13) had p53 mutations as compared to only 8% of the tumours without deletions at the corresponding loci (P< 0.0001). Data were also examined in relation to smoking status of the patients and histology of the tumours. The frequency of deletions was significantly higher among smokers as compared to non-smokers. This difference was significant for the 3p21.3 (hMLH1 locus), 3p14.2 (FHIT locus), 5q11–13 (hMSH3 locus) and 9p21 (D9S157 locus). Tumours with deletions at the hMLH1 locus had higher levels of hydrophobic DNA adducts. Deletions were more common in squamous cell carcinomas than in adenocarcinomas. Covariate analysis revealed that histological type and p53 mutations were significant and independent parameters for predicting LOH status at several loci. In the pathogenesis of NSCLC exposure to tobacco carcinogens in addition to clonal selection may be the driving force in these alterations. © 2001 Cancer Research Campaign http://www.bjcancer.com
doi:10.1054/bjoc.2000.1528
PMCID: PMC2363705  PMID: 11161381
non-small cell lung cancer; LOH; p53 mutations; smoking; DNA-adducts
5.  Aberration of FHIT gene is associated with increased tumor proliferation and decreased apoptosis-clinical evidence in lung and head and neck carcinomas. 
Molecular Medicine  2001;7(7):442-453.
BACKGROUND: Human FHIT (fragile histidine triad) gene is highly conserved gene homologous to a group of genes identified in prokaryotes and eukaryotes. Loss of FHIT function may be important in the development and/or progression of various types of cancer. MATERIALS AND METHODS: We undertook a clinical study to analyze the relation between aberrant function of FHIT gene, tumor cell proliferation, and intensity of apoptosis as well as prognostic output in lung and squamous cell head and neck carcinoma (HNSCC). Status of FHIT gene, expression of p21waf1, intensity of apoptosis, and cell proliferation were analyzed in HNSCC and lung carcinoma tissues by molecular genetic methods, immunohistochemistry, [3H]-thymidine labeling method, and FACScan analysis in frozen and paraffin-embedded tissue sections. RESULTS: The majority of the malignant lung and HNSCC lesions displayed aberrant expression of FHIT gene, followed by low or negative expression of p21waf1, and increased intensity of cell proliferation. Similar results were obtained on synchronous combinations of normal, precancerous, and cancerous head and neck tissues. The observed changes increased with progression of these lesions. We examined tumor and corresponding normal tissue samples for microsatellite markers D3S1300 and D3S4103 to evaluate the loss of heterozygosity (LOH) at the FHIT gene loci. We found high percentage of LOH in both lung tumors and HNSCC (75% for D3S1300 and 79% for D3S4103 in lung cancer, and 87% for D3S1300 and 78% for D3S4103 in HNSCC). The median survival time of the patients suffering from lung cancer without FHIT protein expression was 22.46 months and that of the patients with FHIT expression 36.04 months. FHIT-negative cases tended to correlate with a worse prognosis, but this was not statistically significant. Median survival time of HNSCC patients without FHIT protein expression was 30.86 months and that of the patients with FHIT expression was 64.04 months (p < 0.05). CONCLUSIONS: Our results show a correlation between aberrant FHIT expression, a low rate of apoptosis, and high tumor cell proliferation. Aberrant FHIT gene could be a prognostic marker in lung cancer.
PMCID: PMC1950052  PMID: 11683369
6.  Genetic pathways and mutation profiles of human cancers: site- and exposure-specific patterns 
Carcinogenesis  2007;28(9):1851-1858.
Cancer is a complex disease that involves the accumulation of both genetic and epigenetic alterations of numerous genes. Data in the Genetic Alterations in Cancer database for gene mutations and allelic loss [loss of heterozygosity (LOH)] in human tumors (e.g. lung, oral, esophagus, stomach and colon/rectum) were reviewed. Results for the genes and pathways implicated in tumor development at these sites are presented. Mutation incidence, spectra and codon specificity are described for lung, larynx and oral tumors. LOH occurred more frequently than gene mutations in tumors from all sites examined. The cell cycle gene, TP53 (all sites), and cell signaling gene, APC (colorectal and gastric cancers), were the only genes with similar incidences of LOH and mutation. Alterations of one or more cell cycle and cell signaling genes were reported for tumors from each site. Site-specific activation was apparent in the cell signaling mitogen-activated protein kinase oncogenes (KRAS in lung, HRAS in oral cancers and BRAF in esophageal and colorectal cancers). Analysis of genetic changes in lung tumors showed that the incidence of mutations in the TP53 and KRAS genes and the incidence of LOH in the FHIT gene were significantly greater in smokers versus non-smokers (P < 0.01). In lung and oral cancers, the TP53 GC → TA transversion frequency increased with tobacco smoke exposure (P < 0.05). Furthermore, the TP53 mutational hot spots for lung and laryngeal cancers from smokers included codons 157, 245 and 273, whereas for oral tumors included codons 280 and 281.
doi:10.1093/carcin/bgm176
PMCID: PMC2131731  PMID: 17693665
7.  Initiation of Genome Instability and Preneoplastic Processes through Loss of Fhit Expression 
PLoS Genetics  2012;8(11):e1003077.
Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.
Author Summary
Normal cells have robust mechanisms to maintain the proper sequence of their DNA; in cancer cells these mechanisms are compromised, resulting in complex changes in the DNA of tumors. How this genome instability begins has not been defined, except in cases of familial cancers, which often have mutations in genes called “caretaker” genes, necessary to preserve DNA stability. We have defined a mechanism for genome instability in non-familial tumors that occur sporadically in the population. Certain fragile regions of our DNA are more difficult to duplicate during cell division and are prone to breakage. A fragile region, FRA3B, lies within the FHIT gene, and deletions within FRA3B are common in precancer cells, causing loss of Fhit protein expression. We find that loss of Fhit protein causes defective DNA replication, leading to further DNA breaks. Cells that continue DNA replication in the absence of Fhit develop numerous chromosomal aberrations. Importantly, cells established from tissues of mice that are missing Fhit undergo selection for increasing DNA alterations that can promote immortality, a cancer cell hallmark. Thus, loss of Fhit expression in precancer cells is the first step in the initiation of genomic instability and facilitates cancer development.
doi:10.1371/journal.pgen.1003077
PMCID: PMC3510054  PMID: 23209436
8.  Current and Former Smoking and Risk for Venous Thromboembolism: A Systematic Review and Meta-Analysis 
PLoS Medicine  2013;10(9):e1001515.
In a meta-analysis of 32 observational studies involving 3,966,184 participants and 35,151 events, Suhua Wu and colleagues found that current, ever, and former smoking was associated with risk of venous thromboembolism.
Please see later in the article for the Editors' Summary
Background
Smoking is a well-established risk factor for atherosclerotic disease, but its role as an independent risk factor for venous thromboembolism (VTE) remains controversial. We conducted a meta-analysis to summarize all published prospective studies and case-control studies to update the risk for VTE in smokers and determine whether a dose–response relationship exists.
Methods and Findings
We performed a literature search using MEDLINE (source PubMed, January 1, 1966 to June 15, 2013) and EMBASE (January 1, 1980 to June 15, 2013) with no restrictions. Pooled effect estimates were obtained by using random-effects meta-analysis. Thirty-two observational studies involving 3,966,184 participants and 35,151 VTE events were identified. Compared with never smokers, the overall combined relative risks (RRs) for developing VTE were 1.17 (95% CI 1.09–1.25) for ever smokers, 1.23 (95% CI 1.14–1.33) for current smokers, and 1.10 (95% CI 1.03–1.17) for former smokers, respectively. The risk increased by 10.2% (95% CI 8.6%–11.8%) for every additional ten cigarettes per day smoked or by 6.1% (95% CI 3.8%–8.5%) for every additional ten pack-years. Analysis of 13 studies adjusted for body mass index (BMI) yielded a relatively higher RR (1.30; 95% CI 1.24–1.37) for current smokers. The population attributable fractions of VTE were 8.7% (95% CI 4.8%–12.3%) for ever smoking, 5.8% (95% CI 3.6%–8.2%) for current smoking, and 2.7% (95% CI 0.8%–4.5%) for former smoking. Smoking was associated with an absolute risk increase of 24.3 (95% CI 15.4–26.7) cases per 100,000 person-years.
Conclusions
Cigarette smoking is associated with a slightly increased risk for VTE. BMI appears to be a confounding factor in the risk estimates. The relationship between VTE and smoking has clinical relevance with respect to individual screening, risk factor modification, and the primary and secondary prevention of VTE.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Blood normally flows throughout the human body, supplying its organs and tissues with oxygen and nutrients. But, when an injury occurs, proteins called clotting factors make the blood gel (coagulate) at the injury site. The resultant clot (thrombus) plugs the wound and prevents blood loss. Occasionally, a thrombus forms inside an uninjured blood vessel and partly or completely blocks the blood flow. Clot formation inside one of the veins deep within the body, usually in a leg, is called deep vein thrombosis (DVT) and can cause pain, swelling, and redness in the affected limb. DVT can be treated with drugs that stop the blood clot from getting larger (anticoagulants) but, if left untreated, part of the clot can break off and travel to the lungs, where it can cause a life-threatening pulmonary embolism. DVT and pulmonary embolism are collectively known as venous thromboembolism (VTE). Risk factors for VTE include having an inherited blood clotting disorder, oral contraceptive use, prolonged inactivity (for example, during a long-haul plane flight), and having surgery. VTEs are present in about a third of all people who die in hospital and, in non-bedridden populations, about 10% of people die within 28 days of a first VTE event.
Why Was This Study Done?
Some but not all studies have reported that smoking is also a risk factor for VTE. A clear demonstration of a significant association (a relationship unlikely to have occurred by chance) between smoking and VTE might help to reduce the burden of VTE because smoking can potentially be reduced by encouraging individuals to quit smoking and through taxation policies and other measures designed to reduce tobacco consumption. In this systematic review and meta-analysis, the researchers examine the link between smoking and the risk of VTE in the general population and investigate whether heavy smokers have a higher risk of VTE than light smokers. A systematic review uses predefined criteria to identify all the research on a given topic; meta-analysis is a statistical method for combining the results of several studies.
What Did the Researchers Do and Find?
The researchers identified 32 observational studies (investigations that record a population's baseline characteristics and subsequent disease development) that provided data on smoking and VTE. Together, the studies involved nearly 4 million participants and recorded 35,151 VTE events. Compared with never smokers, ever smokers (current and former smokers combined) had a relative risk (RR) of developing VTE of 1.17. That is, ever smokers were 17% more likely to develop VTE than never smokers. For current smokers and former smokers, RRs were 1.23 and 1.10, respectively. Analysis of only studies that adjusted for body mass index (a measure of body fat and a known risk factor for conditions that affect the heart and circulation) yielded a slightly higher RR (1.30) for current smokers compared with never smokers. For ever smokers, the population attributable fraction (the proportional reduction in VTE that would accrue in the population if no one smoked) was 8.7%. Notably, the risk of VTE increased by 10.2% for every additional ten cigarettes smoked per day and by 6.1% for every additional ten pack-years. Thus, an individual who smoked one pack of cigarettes per day for 40 years had a 26.7% higher risk of developing VTE than someone who had never smoked. Finally, smoking was associated with an absolute risk increase of 24.3 cases of VTE per 100,000 person-years.
What Do These Findings Mean?
These findings indicate that cigarette smoking is associated with a statistically significant, slightly increased risk for VTE among the general population and reveal a dose-relationship between smoking and VTE risk. They cannot prove that smoking causes VTE—people who smoke may share other unknown characteristics (confounding factors) that are actually responsible for their increased risk of VTE. Indeed, these findings identify body mass index as a potential confounding factor that might affect the accuracy of estimates of the association between smoking and VTE risk. Although the risk of VTE associated with smoking is smaller than the risk associated with some well-established VTE risk factors, smoking is more common (globally, there are 1.1 billion smokers) and may act synergistically with some of these risk factors. Thus, smoking behavior should be considered when screening individuals for VTE and in the prevention of first and subsequent VTE events.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001515.
The US National Heart Lung and Blood Institute provides information on deep vein thrombosis (including an animation about how DVT causes pulmonary embolism), and information on pulmonary embolism
The UK National Health Service Choices website has information on deep vein thrombosis, including personal stories, and on pulmonary embolism; SmokeFree is a website provided by the UK National Health Service that offers advice on quitting smoking
The non-profit organization US National Blood Clot Alliance provides detailed information about deep vein thrombosis and pulmonary embolism for patients and professionals and includes a selection of personal stories about these conditions
The World Health Organization provides information about the dangers of tobacco (in several languages)
Smokefree.gov, from the US National Cancer Institute, offers online tools and resources to help people quit smoking
MedlinePlus has links to further information about deep vein thrombosis, pulmonary embolism, and the dangers of smoking (in English and Spanish)
doi:10.1371/journal.pmed.1001515
PMCID: PMC3775725  PMID: 24068896
9.  Lung Cancer Occurrence in Never-Smokers: An Analysis of 13 Cohorts and 22 Cancer Registry Studies  
PLoS Medicine  2008;5(9):e185.
Background
Better information on lung cancer occurrence in lifelong nonsmokers is needed to understand gender and racial disparities and to examine how factors other than active smoking influence risk in different time periods and geographic regions.
Methods and Findings
We pooled information on lung cancer incidence and/or death rates among self-reported never-smokers from 13 large cohort studies, representing over 630,000 and 1.8 million persons for incidence and mortality, respectively. We also abstracted population-based data for women from 22 cancer registries and ten countries in time periods and geographic regions where few women smoked. Our main findings were: (1) Men had higher death rates from lung cancer than women in all age and racial groups studied; (2) male and female incidence rates were similar when standardized across all ages 40+ y, albeit with some variation by age; (3) African Americans and Asians living in Korea and Japan (but not in the US) had higher death rates from lung cancer than individuals of European descent; (4) no temporal trends were seen when comparing incidence and death rates among US women age 40–69 y during the 1930s to contemporary populations where few women smoke, or in temporal comparisons of never-smokers in two large American Cancer Society cohorts from 1959 to 2004; and (5) lung cancer incidence rates were higher and more variable among women in East Asia than in other geographic areas with low female smoking.
Conclusions
These comprehensive analyses support claims that the death rate from lung cancer among never-smokers is higher in men than in women, and in African Americans and Asians residing in Asia than in individuals of European descent, but contradict assertions that risk is increasing or that women have a higher incidence rate than men. Further research is needed on the high and variable lung cancer rates among women in Pacific Rim countries.
Michael Thun and colleagues pooled and analyzed comprehensive data on lung cancer incidence and death rates among never-smokers to examine what factors other than active smoking affect lung cancer risk.
Editors' Summary
Background.
Every year, more than 1.4 million people die from lung cancer, a leading cause of cancer deaths worldwide. In the US alone, more than 161,000 people will die from lung cancer this year. Like all cancers, lung cancer occurs when cells begin to divide uncontrollably because of changes in their genes. The main trigger for these changes in lung cancer is exposure to the chemicals in cigarette smoke—either directly through smoking cigarettes or indirectly through exposure to secondhand smoke. Eighty-five to 90% of lung cancer deaths are caused by exposure to cigarette smoke and, on average, current smokers are 15 times more likely to die from lung cancer than lifelong nonsmokers (never smokers). Furthermore, a person's cumulative lifetime risk of developing lung cancer is related to how much they smoke, to how many years they are a smoker, and—if they give up smoking—to the age at which they stop smoking.
Why Was This Study Done?
Because lung cancer is so common, even the small fraction of lung cancer that occurs in lifelong nonsmokers represents a large number of people. For example, about 20,000 of this year's US lung cancer deaths will be in never-smokers. However, very little is known about how age, sex, or race affects the incidence (the annual number of new cases of diseases in a population) or death rates from lung cancer among never-smokers. A better understanding of the patterns of lung cancer incidence and death rates among never-smokers could provide useful information about the factors other than cigarette smoke that increase the likelihood of not only never-smokers, but also former smokers and current smokers developing lung cancer. In this study, therefore, the researchers pooled and analyzed a large amount of information about lung cancer incidence and death rates among never smokers to examine what factors other than active smoking affect lung cancer risk.
What Did the Researchers Do and Find?
The researchers analyzed information on lung cancer incidence and/or death rates among nearly 2.5 million self-reported never smokers (men and women) from 13 large studies investigating the health of people in North America, Europe, and Asia. They also analyzed similar information for women taken from cancer registries in ten countries at times when very few women were smokers (for example, the US in the late 1930s). The researchers' detailed statistical analyses reveal, for example, that lung cancer death rates in African Americans and in Asians living in Korea and Japan (but not among Asians living in the US) are higher than those in people of the European continental ancestry group. They also show that men have higher death rates from lung cancer than women irrespective of racial group, but that women aged 40–59 years have a slightly higher incidence of lung cancer than men of a similar age. This difference disappears at older ages. Finally, an analysis of lung cancer incidence and death rates at different times during the past 70 years shows no evidence of an increase in the lung cancer burden among never smokers over time.
What Do These Findings Mean?
Although some of the findings described above have been hinted at in previous, smaller studies, these and other findings provide a much more accurate picture of lung cancer incidence and death rates among never smokers. Most importantly the underlying data used in these analyses are now freely available and should provide an excellent resource for future studies of lung cancer in never smokers.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050185.
The US National Cancer Institute provides detailed information for patients and health professionals about all aspects of lung cancer and information on smoking and cancer (in English and Spanish)
Links to other US-based resources dealing with lung cancer are provided by MedlinePlus (in English and Spanish)
Cancer Research UK provides key facts about the link between lung cancer and smoking and information about all other aspects of lung cancer
doi:10.1371/journal.pmed.0050185
PMCID: PMC2531137  PMID: 18788891
10.  Aberrant transcripts of the FHIT gene are expressed in normal and leukaemic haemopoietic cells. 
British Journal of Cancer  1998;78(5):601-605.
Deletions and apparent transcriptional abnormalities of the FHIT gene at 3p14.2 have recently been reported in a wide variety of solid tumours. To determine whether lesions of this gene also occur in leukaemia, we have analysed a total of 97 patients (chronic myeloid leukaemia, CML, in chronic phase or blast crisis, n = 71; de novo acute leukaemia, n = 26) and 16 normal individuals. Intact FHIT transcripts from all cases were amplified using RT-PCR. In addition, smaller size bands that were less intense than the full-length products were amplified from several samples from patients with leukaemia and also from normal leucocytes. Sequencing of the small products revealed that they were derived from FHIT transcripts lacking whole exons. Using single-strand conformation polymorphism analysis, no mutations in the coding sequence were detected in any patient. Furthermore, loss of heterozygosity was not seen in any of 36 informative patients at D3S1300 or D3S1481, markers located within the FHIT locus. We conclude that the FHIT gene and other uncharacterized tumour-suppressor genes at 3p14.2 are unlikely to be involved in the pathogenesis of acute leukaemia or progression of CML from chronic phase to blast crisis. Moreover, low-abundance FHIT transcripts that lack whole exons are not specific to malignant cells and should not be taken as evidence of an abnormality in the absence of demonstrable genomic DNA lesions.
Images
PMCID: PMC2063063  PMID: 9744498
11.  Combined effects of asbestos and cigarette smoke on the development of lung adenocarcinoma: Different carcinogens may cause different genomic changes 
Oncology Reports  2014;32(2):475-482.
The carcinogens in cigarette smoke are distinct from asbestos. However, an understanding of their differential effects on lung adenocarcinoma development remains elusive. We investigated loss of heterozygosity (LOH) and the p53 mutation in 132 lung adenocarcinomas, for which asbestos body burden (AB; in numbers per gram of dry lung) was measured using adjacent normal lung. All cases were classified into 9 groups based on a matrix of cumulative smoking (CS in pack-years; CS=0, 00 groups, LOH frequency increased as AB and/or CS was elevated and was significantly higher in the ≥1,000 AB, ≥25 CS group (p=0.032). p53 mutation frequency was the lowest in the AB=0, CS=0 group, increased as AB and/or CS rose, and was significantly higher in the ≥1,000 AB, ≥25 CS group (p=0.039). p53 mutations characteristic of smoking were frequently observed in the CS>0 groups contrary to non-specific mutations in the CS=0, AB>0 groups. Combined effects of asbestos and smoking were suggested by LOH and p53 analyses. Sole exposure to asbestos did not increase LOH frequency but increased non-specific p53 mutations. These findings indicate that the major carcinogenic mechanism of asbestos may be tumor promotion, acting in an additive or synergistic manner, contributing to the genotoxic effect of smoking. Since this study was based on a general cancer center’s experience, the limited sample size did not permit the consideration that the result was conclusive. Further investigation with a large sample size is needed to establish the mechanism of asbestos-induced lung carcinogenesis.
doi:10.3892/or.2014.3263
PMCID: PMC4091886  PMID: 24926563
lung cancer; asbestos; smoking; loss of heterozygosity; p53 mutation
12.  Loss of heterozygosity of the BRCA1 and FHIT genes in patients with sporadic breast cancer from Southern Brazil 
Journal of Clinical Pathology  2004;57(4):374-377.
Aim: The evaluation of allelic losses at the FHIT and the BRCA1 genes and at three other loci at the 17q region in a series of 34 sporadic breast cancer cases from Southern Brazil.
Methods: The samples were evaluated for loss of heterozygosity (LOH) at the FHIT and the BRCA1 genes and at three other microsatellite markers at 17q, and the findings were correlated with clinicopathological parameters.
Results: The BRCA1 intragenic marker, D17S855, had the highest frequency of LOH, detected in 10 of 24 informative cases, followed by the D17S579 (six of 23 informative cases), D17S806 (five of 21 informative cases), and D17S785 markers (five of 21 informative cases). LOH at the FHIT intragenic marker, D3S1300, was found in six of 25 informative cases. In four of the six cases with LOH of the FHIT gene, there was concomitant loss of the BRCA1 intragenic marker.
Conclusions: The frequency of allelic losses in the FHIT and BRCA1 loci in the Southern Brazilian population is similar to that described in the general population. No correlations were found when the total LOH frequency was compared with tumour size, grade, or presence of axillary lymph node metastasis. Further studies using larger sporadic breast cancer samples and additional markers would be useful to confirm these findings, in addition to establishing more specific associations with clinicopathological parameters in this specific population.
doi:10.1136/jcp.2003.013490
PMCID: PMC1770278  PMID: 15047740
loss of heterozygosity; sporadic breast cancer; BRCA1 gene; FHIT gene
13.  Investigation of recurrent deletion loci specific to conventional renal cell carcinoma by comparative allelotyping in major epithelial carcinomas 
Objective:
Loss of heterozygosity (LOH) studies were undertaken to investigate the consistently deleted loci/? tumor suppressor gene loci (TSG) on 3p in conventional renal cell carcinoma (cRCC).
Materials and Methods:
LOH studies were performed by polymerase chain reaction (PCR) using 15 micro satellite markers mapped in region 3p12-p26 on 40 paired cRCC tumors and normal kidney at Stages I-IV. Simultaneously, fluorescent in-situ hybridization (FISH) studies were performed to investigate the allelic deletion of fragile histidine triad (FHIT).
Results:
Our studies revealed three affected regions; 3p12.2-p14.1, 3p14.2-p21.1, and 3p24.2-p26.1 with differential frequencies in Group I (Stage I and II) and Group II (Stage III and IV). Incidence for D3S1234 (FHIT locus) and D3S2454 (3p13) was 75% and 83% in Group I and II, respectively. Comparative allelotyping in epithelial malignancies like lung, bladder, and breast tumors revealed LOH (frequency 14–20%) only in breast tumors for D3S2406, D3S1766 (distal to FHIT), and D3S1560 (distal to VHL, Von-Hippal Lindau). FISH using FHIT gene probe revealed deletions in cRCC (88%), breast (30%), and lung tumors (10%) with no deletions in bladder tumors and leukemias, signifying the importance of FHIT in the pathogenesis of tumors of epithelial origin.
Conclusion:
Our findings suggested FHIT deletion as an early and VHL deletion as an early and/or late event in cRCC. Additionally, studies also disclosed the recurrent deletions of flanking loci to FHIT and VHL in cRCC. The dilemma of interstitial or continuous deletion on 3p needs to be resolved by implementation of latest sensitive molecular techniques that would further help to narrow down search for TSG loci specific to cRCC, other than VHL and FHIT.
doi:10.4103/0970-1591.94956
PMCID: PMC3339786  PMID: 22557717
3p; Conventional renal cell carcinoma; comparative allelotyping; fragile histidine triad; interstitial deletion; von hippal lindau
14.  Smoking Cessation for Patients With Chronic Obstructive Pulmonary Disease (COPD) 
Executive Summary
In July 2010, the Medical Advisory Secretariat (MAS) began work on a Chronic Obstructive Pulmonary Disease (COPD) evidentiary framework, an evidence-based review of the literature surrounding treatment strategies for patients with COPD. This project emerged from a request by the Health System Strategy Division of the Ministry of Health and Long-Term Care that MAS provide them with an evidentiary platform on the effectiveness and cost-effectiveness of COPD interventions.
After an initial review of health technology assessments and systematic reviews of COPD literature, and consultation with experts, MAS identified the following topics for analysis: vaccinations (influenza and pneumococcal), smoking cessation, multidisciplinary care, pulmonary rehabilitation, long-term oxygen therapy, noninvasive positive pressure ventilation for acute and chronic respiratory failure, hospital-at-home for acute exacerbations of COPD, and telehealth (including telemonitoring and telephone support). Evidence-based analyses were prepared for each of these topics. For each technology, an economic analysis was also completed where appropriate. In addition, a review of the qualitative literature on patient, caregiver, and provider perspectives on living and dying with COPD was conducted, as were reviews of the qualitative literature on each of the technologies included in these analyses.
The Chronic Obstructive Pulmonary Disease Mega-Analysis series is made up of the following reports, which can be publicly accessed at the MAS website at: http://www.hqontario.ca/en/mas/mas_ohtas_mn.html.
Chronic Obstructive Pulmonary Disease (COPD) Evidentiary Framework
Influenza and Pneumococcal Vaccinations for Patients With Chronic Obstructive Pulmonary Disease (COPD): An Evidence-Based Analysis
Smoking Cessation for Patients With Chronic Obstructive Pulmonary Disease (COPD): An Evidence-Based Analysis
Community-Based Multidisciplinary Care for Patients With Stable Chronic Obstructive Pulmonary Disease (COPD): An Evidence-Based Analysis
Pulmonary Rehabilitation for Patients With Chronic Obstructive Pulmonary Disease (COPD): An Evidence-Based Analysis
Long-term Oxygen Therapy for Patients With Chronic Obstructive Pulmonary Disease (COPD): An Evidence-Based Analysis
Noninvasive Positive Pressure Ventilation for Acute Respiratory Failure Patients With Chronic Obstructive Pulmonary Disease (COPD): An Evidence-Based Analysis
Noninvasive Positive Pressure Ventilation for Chronic Respiratory Failure Patients With Stable Chronic Obstructive Pulmonary Disease (COPD): An Evidence-Based Analysis
Hospital-at-Home Programs for Patients With Acute Exacerbations of Chronic Obstructive Pulmonary Disease (COPD): An Evidence-Based Analysis
Home Telehealth for Patients With Chronic Obstructive Pulmonary Disease (COPD): An Evidence-Based Analysis
Cost-Effectiveness of Interventions for Chronic Obstructive Pulmonary Disease Using an Ontario Policy Model
Experiences of Living and Dying With COPD: A Systematic Review and Synthesis of the Qualitative Empirical Literature
For more information on the qualitative review, please contact Mita Giacomini at: http://fhs.mcmaster.ca/ceb/faculty member_giacomini.htm.
For more information on the economic analysis, please visit the PATH website: http://www.path-hta.ca/About-Us/Contact-Us.aspx.
The Toronto Health Economics and Technology Assessment (THETA) collaborative has produced an associated report on patient preference for mechanical ventilation. For more information, please visit the THETA website: http://theta.utoronto.ca/static/contact.
Objective
The objective of this evidence-based analysis was to determine the effectiveness and cost-effectiveness of smoking cessation interventions in the management of chronic obstructive pulmonary disease (COPD).
Clinical Need: Condition and Target Population
Tobacco smoking is the main risk factor for COPD. It is estimated that 50% of older smokers develop COPD and more than 80% of COPD-associated morbidity is attributed to tobacco smoking. According to the Canadian Community Health Survey, 38.5% of Ontarians who smoke have COPD. In patients with a significant history of smoking, COPD is usually present with symptoms of progressive dyspnea (shortness of breath), cough, and sputum production. Patients with COPD who smoke have a particularly high level of nicotine dependence, and about 30.4% to 43% of patients with moderate to severe COPD continue to smoke. Despite the severe symptoms that COPD patients suffer, the majority of patients with COPD are unable to quit smoking on their own; each year only about 1% of smokers succeed in quitting on their own initiative.
Technology
Smoking cessation is the process of discontinuing the practice of inhaling a smoked substance. Smoking cessation can help to slow or halt the progression of COPD. Smoking cessation programs mainly target tobacco smoking, but may also encompass other substances that can be difficult to stop smoking due to the development of strong physical addictions or psychological dependencies resulting from their habitual use.
Smoking cessation strategies include both pharmacological and nonpharmacological (behavioural or psychosocial) approaches. The basic components of smoking cessation interventions include simple advice, written self-help materials, individual and group behavioural support, telephone quit lines, nicotine replacement therapy (NRT), and antidepressants. As nicotine addiction is a chronic, relapsing condition that usually requires several attempts to overcome, cessation support is often tailored to individual needs, while recognizing that in general, the more intensive the support, the greater the chance of success. Success at quitting smoking decreases in relation to:
a lack of motivation to quit,
a history of smoking more than a pack of cigarettes a day for more than 10 years,
a lack of social support, such as from family and friends, and
the presence of mental health disorders (such as depression).
Research Question
What are the effectiveness and cost-effectiveness of smoking cessation interventions compared with usual care for patients with COPD?
Research Methods
Literature Search
Search Strategy
A literature search was performed on June 24, 2010 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations (1950 to June Week 3 2010), EMBASE (1980 to 2010 Week 24), the Cumulative Index to Nursing and Allied Health Literature (CINAHL), the Cochrane Library, and the Centre for Reviews and Dissemination for studies published between 1950 and June 2010. A single reviewer reviewed the abstracts and obtained full-text articles for those studies meeting the eligibility criteria. Reference lists were also examined for any additional relevant studies not identified through the search. Data were extracted using a standardized data abstraction form.
Inclusion Criteria
English-language, full reports from 1950 to week 3 of June, 2010;
either randomized controlled trials (RCTs), systematic reviews and meta-analyses, or non-RCTs with controls;
a proven diagnosis of COPD;
adult patients (≥ 18 years);
a smoking cessation intervention that comprised at least one of the treatment arms;
≥ 6 months’ abstinence as an outcome; and
patients followed for ≥ 6 months.
Exclusion Criteria
case reports
case series
Outcomes of Interest
≥ 6 months’ abstinence
Quality of Evidence
The quality of each included study was assessed taking into consideration allocation concealment, randomization, blinding, power/sample size, withdrawals/dropouts, and intention-to-treat analyses.
The quality of the body of evidence was assessed as high, moderate, low, or very low according to the GRADE Working Group criteria. The following definitions of quality were used in grading the quality of the evidence:
Summary of Findings
Nine RCTs were identified from the literature search. The sample sizes ranged from 74 to 5,887 participants. A total of 8,291 participants were included in the nine studies. The mean age of the patients in the studies ranged from 54 to 64 years. The majority of studies used the Global Initiative for Chronic Obstructive Lung Disease (GOLD) COPD staging criteria to stage the disease in study subjects. Studies included patients with mild COPD (2 studies), mild-moderate COPD (3 studies), moderate–severe COPD (1 study) and severe–very severe COPD (1 study). One study included persons at risk of COPD in addition to those with mild, moderate, or severe COPD, and 1 study did not define the stages of COPD. The individual quality of the studies was high. Smoking cessation interventions varied across studies and included counselling or pharmacotherapy or a combination of both. Two studies were delivered in a hospital setting, whereas the remaining 7 studies were delivered in an outpatient setting. All studies reported a usual care group or a placebo-controlled group (for the drug-only trials). The follow-up periods ranged from 6 months to 5 years. Due to excessive clinical heterogeneity in the interventions, studies were first grouped into categories of similar interventions; statistical pooling was subsequently performed, where appropriate. When possible, pooled estimates using relative risks for abstinence rates with 95% confidence intervals were calculated. The remaining studies were reported separately.
Abstinence Rates
Table ES1 provides a summary of the pooled estimates for abstinence, at longest follow-up, from the trials included in this review. It also shows the respective GRADE qualities of evidence.
Summary of Results*
Abbreviations: CI, confidence interval; NRT, nicotine replacement therapy.
Statistically significant (P < 0.05).
One trial used in this comparison had 2 treatment arms each examining a different antidepressant.
Conclusions
Based on a moderate quality of evidence, compared with usual care, abstinence rates are significantly higher in COPD patients receiving intensive counselling or a combination of intensive counselling and NRT.
Based on limited and moderate quality of evidence, abstinence rates are significantly higher in COPD patients receiving NRT compared with placebo.
Based on a moderate quality of evidence, abstinence rates are significantly higher in COPD patients receiving the antidepressant bupropion compared to placebo.
PMCID: PMC3384371  PMID: 23074432
15.  Impaired FHIT expression characterizes serous ovarian carcinoma 
British Journal of Cancer  2001;85(2):247-254.
The FHIT (fragile histidine triad) gene on chromosome 3p14.2 is a candidate tumour suppressor gene. To define the role of the FHIT gene in the development of ovarian cancer, we have examined 33 ovarian carcinomas, 2 borderline tumours and 10 benign adenomas for the presence of FHIT gene alterations. FHIT transcripts were analysed by RT-PCR and sequencing. Aberrant FHIT transcripts were observed in 5/33 carcinomas (15%) and in 1 of 2 borderline tumours. Loss of normal FHIT transcript was observed in 5/33 carcinomas (15%) but not in 2 borderline tumours or 10 benign adenomas. Allelic losses at D3S1300 and D3S4103, both located within intron 5 of FHIT were detected in 5/24 (21%) and 5/25 (20%) informative ovarian carcinomas, respectively. Expression of Fhit protein was analysed by immunohistochemistry in 44 carcinomas, 19 borderline tumours and 16 benign adenomas. Loss or significantly reduced expression of Fhit protein was observed in 6/44 (14%) ovarian carcinomas but not in any of 19 borderline tumours or 16 benign adenomas. The impaired Fhit protein expression was significantly correlated with the loss of normal FHIT transcription. Most notably, loss of normal FHIT transcript and impaired expression of Fhit protein occurred only in serous adenocarcinomas of grade 2 and 3 (5/15; 33% and 6/19; 32%, respectively). The present data suggest that inactivation of the FHIT gene by loss of expression is one of the important molecular events associated with the genesis of ovarian carcinoma, especially of high-grade serous carcinoma. © 2001 Cancer Research Campaign http://www.bjcancer.com
doi:10.1054/bjoc.2001.1886
PMCID: PMC2364051  PMID: 11461085
FHIT (fragile histidine triad) gene; ovarian tumour; serous carcinoma
16.  Abnormalities of the FHIT gene in human oral carcinogenesis 
British Journal of Cancer  2000;82(4):838-843.
The abnormalities of the fragile histidine triad (FHIT) gene in tissue samples of oral squamous cell carcinomas (SCCs) along with several leukoplakias and an erythroplakia were examined to determine whether the FHIT gene is actually a frequent target in vivo for alteration during oral carcinogenesis. Abnormal transcripts of the FHIT gene were found in eight of 15 oral SCCs. Although these abnormal transcripts varied widely, deletion patterns incorporating a deletion of exon 5 were the most common. Loss of heterozygosity (LOH) analysis demonstrated that the abnormal FHIT transcripts found in cancer cells were attributable to abnormalities of the FHIT gene. Abnormal FHIT transcripts were also observed in two of seven premalignant lesions. Interestingly, in the case of one patient with a premalignant lesion showing an abnormal FHIT transcript, subsequent oral SCC developed during a 3-year follow-up period. On the other hand, in the two patients from whom both leukoplakia and SCC samples were taken simultaneously, abnormal FHIT transcripts were found only in the SCCs. Although the functional role of FHIT remains to be clarified, these results suggest that the FHIT alteration is actually involved in carcinogenesis of the oral epithelium. © 2000 Cancer Research Campaign
doi:10.1054/bjoc.1999.1009
PMCID: PMC2374395  PMID: 10732756
FHIT; gene alteration; microdissection; oral SCC; oral leukoplakia
17.  Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer 
Executive Summary
In February 2010, the Medical Advisory Secretariat (MAS) began work on evidence-based reviews of the literature surrounding three pharmacogenomic tests. This project came about when Cancer Care Ontario (CCO) asked MAS to provide evidence-based analyses on the effectiveness and cost-effectiveness of three oncology pharmacogenomic tests currently in use in Ontario.
Evidence-based analyses have been prepared for each of these technologies. These have been completed in conjunction with internal and external stakeholders, including a Provincial Expert Panel on Pharmacogenetics (PEPP). Within the PEPP, subgroup committees were developed for each disease area. For each technology, an economic analysis was also completed by the Toronto Health Economics and Technology Assessment Collaborative (THETA) and is summarized within the reports.
The following reports can be publicly accessed at the MAS website at: http://www.health.gov.on.ca/mas or at www.health.gov.on.ca/english/providers/program/mas/mas_about.html
Gene Expression Profiling for Guiding Adjuvant Chemotherapy Decisions in Women with Early Breast Cancer: An Evidence-Based Analysis
Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: an Evidence-Based Analysis
K-RAS testing in Treatment Decisions for Advanced Colorectal Cancer: an Evidence-Based Analysis
Objective
The Medical Advisory Secretariat undertook a systematic review of the evidence on the clinical effectiveness and cost-effectiveness of epidermal growth factor receptor (EGFR) mutation testing compared with no EGFR mutation testing to predict response to tyrosine kinase inhibitors (TKIs), gefitinib (Iressa®) or erlotinib (Tarceva®) in patients with advanced non-small cell lung cancer (NSCLC).
Clinical Need: Target Population and Condition
With an estimated 7,800 new cases and 7,000 deaths last year, lung cancer is the leading cause of cancer deaths in Ontario. Those with unresectable or advanced disease are commonly treated with concurrent chemoradiation or platinum-based combination chemotherapy. Although response rates to cytotoxic chemotherapy for advanced NSCLC are approximately 30 to 40%, all patients eventually develop resistance and have a median survival of only 8 to 10 months. Treatment for refractory or relapsed disease includes single-agent treatment with docetaxel, pemetrexed or EGFR-targeting TKIs (gefitinib, erlotinib). TKIs disrupt EGFR signaling by competing with adenosine triphosphate (ATP) for the binding sites at the tyrosine kinase (TK) domain, thus inhibiting the phosphorylation and activation of EGFRs and the downstream signaling network. Gefitinib and erlotinib have been shown to be either non-inferior or superior to chemotherapy in the first- or second-line setting (gefitinib), or superior to placebo in the second- or third-line setting (erlotinib).
Certain patient characteristics (adenocarcinoma, non-smoking history, Asian ethnicity, female gender) predict for better survival benefit and response to therapy with TKIs. In addition, the current body of evidence shows that somatic mutations in the EGFR gene are the most robust biomarkers for EGFR-targeting therapy selection. Drugs used in this therapy, however, can be costly, up to C$ 2000 to C$ 3000 per month, and they have only approximately a 10% chance of benefiting unselected patients. For these reasons, the predictive value of EGFR mutation testing for TKIs in patients with advanced NSCLC needs to be determined.
The Technology: EGFR mutation testing
The EGFR gene sequencing by polymerase chain reaction (PCR) assays is the most widely used method for EGFR mutation testing. PCR assays can be performed at pathology laboratories across Ontario. According to experts in the province, sequencing is not currently done in Ontario due to lack of adequate measurement sensitivity. A variety of new methods have been introduced to increase the measurement sensitivity of the mutation assay. Some technologies such as single-stranded conformational polymorphism, denaturing high-performance liquid chromatography, and high-resolution melting analysis have the advantage of facilitating rapid mutation screening of large numbers of samples with high measurement sensitivity but require direct sequencing to confirm the identity of the detected mutations. Other techniques have been developed for the simple, but highly sensitive detection of specific EGFR mutations, such as the amplification refractory mutations system (ARMS) and the peptide nucleic acid-locked PCR clamping. Others selectively digest wild-type DNA templates with restriction endonucleases to enrich mutant alleles by PCR. Experts in the province of Ontario have commented that currently PCR fragment analysis for deletion and point mutation conducts in Ontario, with measurement sensitivity of 1% to 5%.
Research Questions
In patients with locally-advanced or metastatic NSCLC, what is the clinical effectiveness of EGFR mutation testing for prediction of response to treatment with TKIs (gefitinib, erlotinib) in terms of progression-free survival (PFS), objective response rates (ORR), overall survival (OS), and quality of life (QoL)?
What is the impact of EGFR mutation testing on overall clinical decision-making for patients with advanced or metastatic NSCLC?
What is the cost-effectiveness of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the first-line setting?
What is the budget impact of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the second- or third-line setting?
Methods
A literature search was performed on March 9, 2010 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, OVID EMBASE, Wiley Cochrane, CINAHL, Centre for Reviews and Dissemination/International Agency for Health Technology Assessment for studies published from January 1, 2004 until February 28, 2010 using the following terms:
Non-Small-Cell Lung Carcinoma
Epidermal Growth Factor Receptor
An automatic literature update program also extracted all papers published from February 2010 until August 2010. Abstracts were reviewed by a single reviewer and for those studies meeting the eligibility criteria full-text articles were obtained. Reference lists were also examined for any additional relevant studies not identified through the search. Articles with unknown eligibility were reviewed with a second clinical epidemiologist, and then a group of epidemiologists, until consensus was established. The quality of evidence was assessed as high, moderate, low or very low according to GRADE methodology.
The inclusion criteria were as follows:
Population: patients with locally advanced or metastatic NSCLC (stage IIIB or IV)
Procedure: EGFR mutation testing before treatment with gefitinib or erlotinib
Language: publication in English
Published health technology assessments, guidelines, and peer-reviewed literature (abstracts, full text, conference abstract)
Outcomes: progression-free survival (PFS), Objective response rate (ORR), overall survival (OS), quality of life (QoL).
The exclusion criteria were as follows:
Studies lacking outcomes specific to those of interest
Studies focused on erlotinib maintenance therapy
Studies focused on gefitinib or erlotinib use in combination with cytotoxic agents or any other drug
Grey literature, where relevant, was also reviewed.
Outcomes of Interest
PFS
ORR determined by means of the Response Evaluation Criteria in Solid Tumours (RECIST)
OS
QoL
Quality of Evidence
The quality of the Phase II trials and observational studies was based on the method of subject recruitment and sampling, possibility of selection bias, and generalizability to the source population. The overall quality of evidence was assessed as high, moderate, low or very low according to the GRADE Working Group criteria.
Summary of Findings
Since the last published health technology assessment by Blue Cross Blue Shield Association in 2007 there have been a number of phase III trials which provide evidence of predictive value of EGFR mutation testing in patients who were treated with gefitinib compared to chemotherapy in the first- or second-line setting. The Iressa Pan Asian Study (IPASS) trial showed the superiority of gefitinib in terms of PFS in patients with EGFR mutations versus patients with wild-type EGFR (Hazard ratio [HR], 0.48, 95%CI; 0.36-0.64 versus HR, 2.85; 95%CI, 2.05-3.98). Moreover, there was a statistically significant increased ORR in patients who received gefitinib and had EGFR mutations compared to patients with wild-type EGFR (71% versus 1%). The First-SIGNAL trial in patients with similar clinical characteristics as IPASS as well as the NEJ002 and WJTOG3405 trials that included only patients with EGFR mutations, provide confirmation that gefitinib is superior to chemotherapy in terms of improved PFS or higher ORR in patients with EGFR mutations. The INTEREST trial further indicated that patients with EGFR mutations had prolonged PFS and higher ORR when treated with gefitinib compared with docetaxel.
In contrast, there is still a paucity of strong evidence regarding the predictive value of EGFR mutation testing for response to erlotinib in the second- or third-line setting. The BR.21 trial randomized 731 patients with NSCLC who were refractory or intolerant to prior first- or second-line chemotherapy to receive erlotinib or placebo. While the HR of 0.61 (95%CI, 0.51-0.74) favored erlotinib in the overall population, this was not a significant in the subsequent retrospective subgroup analysis. A retrospective evaluation of 116 of the BR.21 tumor samples demonstrated that patients with EGFR mutations had significantly higher ORRs when treated with erlotinib compared with placebo (27% versus 7%; P=0.03). However, erlotinib did not confer a significant survival benefit compared with placebo in patients with EGFR mutations (HR, 0.55; 95%CI, 0.25-1.19) versus wild-type (HR, 0.74; 95%CI, 0.52-1.05). The interaction between EGFR mutation status and erlotinib use was not significant (P=0.47). The lack of significance could be attributable to a type II error since there was a low sample size that was available for subgroup analysis.
A series of phase II studies have examined the clinical effectiveness of erlotinib in patients known to have EGFR mutations. Evidence from these studies has consistently shown that erlotinib yields a very high ORR (typically 70% vs. 4%) and a prolonged PFS (9 months vs. 2 months) in patients with EGFR mutations compared with patients with wild-type EGFR. Although having a prolonged PFS and higher respond in EGFR mutated patients might be due to a better prognostic profile regardless of the treatment received. In the absence of a comparative treatment or placebo control group, it is difficult to determine if the observed differences in survival benefit in patients with EGFR mutation is attributed to prognostic or predictive value of EGFR mutation status.
Conclusions
Based on moderate quality of evidence, patients with locally advanced or metastatic NSCLC with adenocarcinoma histology being treated with gefitinib in the first-line setting are highly likely to benefit from gefitinib if they have EGFR mutations compared to those with wild-type EGFR. This advantage is reflected in improved PFS, ORR and QoL in patients with EGFR mutation who are being treated with gefitinib relative to patients treated with chemotherapy.
Based on low quality of evidence, in patients with locally advanced or metastatic NSCLC who are being treated with erlotinib, the identification of EGFR mutation status selects those who are most likely to benefit from erlotinib relative to patients treated with placebo in the second or third-line setting.
PMCID: PMC3377519  PMID: 23074402
18.  Burden of Total and Cause-Specific Mortality Related to Tobacco Smoking among Adults Aged ≥45 Years in Asia: A Pooled Analysis of 21 Cohorts 
PLoS Medicine  2014;11(4):e1001631.
Wei Zheng and colleagues quantify the burden of tobacco-smoking-related deaths for adults in Asia.
Please see later in the article for the Editors' Summary
Background
Tobacco smoking is a major risk factor for many diseases. We sought to quantify the burden of tobacco-smoking-related deaths in Asia, in parts of which men's smoking prevalence is among the world's highest.
Methods and Findings
We performed pooled analyses of data from 1,049,929 participants in 21 cohorts in Asia to quantify the risks of total and cause-specific mortality associated with tobacco smoking using adjusted hazard ratios and their 95% confidence intervals. We then estimated smoking-related deaths among adults aged ≥45 y in 2004 in Bangladesh, India, mainland China, Japan, Republic of Korea, Singapore, and Taiwan—accounting for ∼71% of Asia's total population. An approximately 1.44-fold (95% CI = 1.37–1.51) and 1.48-fold (1.38–1.58) elevated risk of death from any cause was found in male and female ever-smokers, respectively. In 2004, active tobacco smoking accounted for approximately 15.8% (95% CI = 14.3%–17.2%) and 3.3% (2.6%–4.0%) of deaths, respectively, in men and women aged ≥45 y in the seven countries/regions combined, with a total number of estimated deaths of ∼1,575,500 (95% CI = 1,398,000–1,744,700). Among men, approximately 11.4%, 30.5%, and 19.8% of deaths due to cardiovascular diseases, cancer, and respiratory diseases, respectively, were attributable to tobacco smoking. Corresponding proportions for East Asian women were 3.7%, 4.6%, and 1.7%, respectively. The strongest association with tobacco smoking was found for lung cancer: a 3- to 4-fold elevated risk, accounting for 60.5% and 16.7% of lung cancer deaths, respectively, in Asian men and East Asian women aged ≥45 y.
Conclusions
Tobacco smoking is associated with a substantially elevated risk of mortality, accounting for approximately 2 million deaths in adults aged ≥45 y throughout Asia in 2004. It is likely that smoking-related deaths in Asia will continue to rise over the next few decades if no effective smoking control programs are implemented.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Every year, more than 5 million smokers die from tobacco-related diseases. Tobacco smoking is a major risk factor for cardiovascular disease (conditions that affect the heart and the circulation), respiratory disease (conditions that affect breathing), lung cancer, and several other types of cancer. All told, tobacco smoking kills up to half its users. The ongoing global “epidemic” of tobacco smoking and tobacco-related diseases initially affected people living in the US and other Western countries, where the prevalence of smoking (the proportion of the population that smokes) in men began to rise in the early 1900s, peaking in the 1960s. A similar epidemic occurred in women about 40 years later. Smoking-related deaths began to increase in the second half of the 20th century, and by the 1990s, tobacco smoking accounted for a third of all deaths and about half of cancer deaths among men in the US and other Western countries. More recently, increased awareness of the risks of smoking and the introduction of various tobacco control measures has led to a steady decline in tobacco use and in smoking-related diseases in many developed countries.
Why Was This Study Done?
Unfortunately, less well-developed tobacco control programs, inadequate public awareness of smoking risks, and tobacco company marketing have recently led to sharp increases in the prevalence of smoking in many low- and middle-income countries, particularly in Asia. More than 50% of men in many Asian countries are now smokers, about twice the prevalence in many Western countries, and more women in some Asian countries are smoking than previously. More than half of the world's billion smokers now live in Asia. However, little is known about the burden of tobacco-related mortality (deaths) in this region. In this study, the researchers quantify the risk of total and cause-specific mortality associated with tobacco use among adults aged 45 years or older by undertaking a pooled statistical analysis of data collected from 21 Asian cohorts (groups) about their smoking history and health.
What Did the Researchers Do and Find?
For their study, the researchers used data from more than 1 million participants enrolled in studies undertaken in Bangladesh, India, mainland China, Japan, the Republic of Korea, Singapore, and Taiwan (which together account for 71% of Asia's total population). Smoking prevalences among male and female participants were 65.1% and 7.1%, respectively. Compared with never-smokers, ever-smokers had a higher risk of death from any cause in pooled analyses of all the cohorts (adjusted hazard ratios [HRs] of 1.44 and 1.48 for men and women, respectively; an adjusted HR indicates how often an event occurs in one group compared to another group after adjustment for other characteristics that affect an individual's risk of the event). Compared with never smoking, ever smoking was associated with a higher risk of death due to cardiovascular disease, cancer (particularly lung cancer), and respiratory disease among Asian men and among East Asian women. Moreover, the researchers estimate that, in the countries included in this study, tobacco smoking accounted for 15.8% of all deaths among men and 3.3% of deaths among women in 2004—a total of about 1.5 million deaths, which scales up to 2 million deaths for the population of the whole of Asia. Notably, in 2004, tobacco smoking accounted for 60.5% of lung-cancer deaths among Asian men and 16.7% of lung-cancer deaths among East Asian women.
What Do These Findings Mean?
These findings provide strong evidence that tobacco smoking is associated with a substantially raised risk of death among adults aged 45 years or older throughout Asia. The association between smoking and mortality risk in Asia reported here is weaker than that previously reported for Western countries, possibly because widespread tobacco smoking started several decades later in most Asian countries than in Europe and North America and the deleterious effects of smoking take some years to become evident. The researchers note that certain limitations of their analysis are likely to affect the accuracy of its findings. For example, because no data were available to estimate the impact of secondhand smoke, the estimate of deaths attributable to smoking is likely to be an underestimate. However, the finding that nearly 45% of the global deaths from active tobacco smoking occur in Asia highlights the urgent need to implement comprehensive tobacco control programs in Asia to reduce the burden of tobacco-related disease.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001631.
The World Health Organization provides information about the dangers of tobacco (in several languages) and about the WHO Framework Convention on Tobacco Control, an international instrument for tobacco control that came into force in February 2005 and requires parties to implement a set of core tobacco control provisions including legislation to ban tobacco advertising and to increase tobacco taxes; its 2013 report on the global tobacco epidemic is available
The US Centers for Disease Control and Prevention provides detailed information about all aspects of smoking and tobacco use
The UK National Health Services Choices website provides information about the health risks associated with smoking
MedlinePlus has links to further information about the dangers of smoking (in English and Spanish)
SmokeFree, a website provided by the UK National Health Service, offers advice on quitting smoking and includes personal stories from people who have stopped smoking
Smokefree.gov, from the US National Cancer Institute, offers online tools and resources to help people quit smoking
doi:10.1371/journal.pmed.1001631
PMCID: PMC3995657  PMID: 24756146
19.  Significant frequency of allelic imbalance in 3p region covering RARβ and MLH1 loci seems to be essential in molecular non-small cell lung cancer diagnosis 
The aim of the study was to investigate the influence of allelic imbalance (AI) in several loci of tumor suppressor genes in 3p region on the non-small cell lung cancer (NSCLC) development. We evaluated the frequency of loss of heterozygosity and/or microsatellite imbalance (LOH/MSI) and assessed their association with patients’ characteristics (age, gender, tobacco addiction) and NSCLC classification according to TNM/AJCC staging. To analyze the potential role of AI involved in NSCLC pathogenesis, we allelotyped a group of 74 NSCLC patients using 7 microsatellite markers. The highest frequency of LOH/MSI, however, not statistically significant, was observed in RARβ and MLH1 (p = 0.104 and p = 0.216, respectively) loci. The association between high LOH/MSI frequency in 3p region with male gender (p = 0.041) as well as with age (especially >60 years) for RARβ and MLH1 genes (p = 0.0001 and p = 0.020, respectively) was documented. Statistically significant increased frequency of MLH1 allelic loss in squamous cell carcinoma (SCC) versus non-squamous cell carcinoma (non-SCC) was observed (p = 0.01). Significant increase in LOH/MSI frequency in 3p region (mainly in FHIT and MLH1loci) in correlation with cigarette addiction in a lifetime (≥40 years and ≥40 Pack Years) was also documented (p < 0.05). The highest LOH/MSI was revealed in RARβ locus in IA tumors (p = 0.0001), while the similarly high allelic loss of MLH1 correlated with III A/B tumors (p = 0.0002), according to AJCC staging. The obtained results demonstrate that AI is influenced by tobacco smoking and seems to be vital in the molecular diagnosis of NSCLC, especially of SCC subtype.
doi:10.1007/s12032-013-0532-9
PMCID: PMC3667378  PMID: 23504373
Non-small cell lung carcinoma (NSCLC); Loss of heterozygosity (LOH); Microsatellite instability (MSI); Microsatellite markers; Molecular diagnosis
20.  Smoking and high-risk mammographic parenchymal patterns: a case-control study 
Breast Cancer Research  1999;2(1):59-63.
Current smoking was strongly and inversely associated with high-risk patterns, after adjustment for concomitant risk factors. Relative to never smokers, current smokers were significantly less likely to have a high-risk pattern. Similar results were obtained when the analysis was confined to postmenopausal women. Past smoking was not related to the mammographic parenchymal patterns. The overall effect in postmenopausal women lost its significance when adjusted for other risk factors for P2/DY patterns that were found to be significant in the present study, although the results are still strongly suggestive. The present data indicate that adjustment for current smoking status is important when evaluating the relationship between mammographic parenchymal pattern and breast cancer risk. They also indicate that smoking is a prominent potential confounder when analyzing effects of other risk factors such as obesity-related variables. It appears that parenchymal patterns may act as an informative biomarker of the effect of cigarette smoking on breast cancer risk.
Introduction:
Overall, epidemiological studies [1,2,3,4] have reported no substantial association between cigarette smoking and the risk of breast cancer. Some studies [5,6,7] reported a significant increase of breast cancer risk among smokers. In recent studies that addressed the association between breast cancer and cigarette smoking, however, there was some suggestion of a decreased risk [8,9,10], especially among current smokers, ranging from approximately 10 to 30% [9,10]. Brunet et al [11] reported that smoking might reduce the risk of breast cancer by 44% in carriers of BRCA1 or BRCA2 gene mutations. Wolfe [12] described four different mammographic patterns created by variations in the relative amounts of fat, epithelial and connective tissue in the breast, designated N1, P1, P2 and DY. Women with either P2 or DY pattern are considered at greater risk for breast cancer than those with N1 or P1 pattern [12,13,14,15]. There are no published studies that assessed the relationship between smoking and mammographic parenchymal patterns.
Aims:
To evaluate whether mammographic parenchymal patterns as classified by Wolfe, which have been positively associated with breast cancer risk, are affected by smoking. In this case-control study, nested within the European Prospective Investigation on Cancer in Norfolk (EPIC-Norfolk) cohort [16], the association between smoking habits and mammographic parenchymal patterns are examined. The full results will be published elsewhere.
Methods:
Study subjects were members of the EPIC cohort in Norwich who also attended the prevalence screening round at the Norwich Breast Screening Centre between November 1989 and December 1997, and were free of breast cancer at that screening. Cases were defined as women with a P2/DY Wolfe's mammographic parenchymal pattern on the prevalence screen mammograms. A total of 203 women with P2/DY patterns were identified as cases and were individually matched by date of birth (within 1 year) and date of prevalence screening (within 3 months) with 203 women with N1/P1 patterns who served as control individuals.
Two views, the mediolateral and craniocaudal mammograms, of both breasts were independently reviewed by two of the authors (ES and RW) to determine the Wolfe mammographic parenchymal pattern.
Considerable information on health and lifestyle factors was available from the EPIC Health and Lifestyle Questionnaire [16]. In the present study we examined the subjects' personal history of benign breast diseases, menstrual and reproductive factors, oral contraception and hormone replacement therapy, smoking, and anthropometric information such as body mass index and waist:hip ratio.
Odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated by conditional logistic regression [17], and were adjusted for possible confounding factors.
Results:
The characteristics of the cases and controls are presented in Table 1. Cases were leaner than controls. A larger percentage of cases were nulliparous, premenopausal, current hormone replacement therapy users, had a personal history of benign breast diseases, and had had a hysterectomy. A larger proportion of controls had more than three births and were current smokers.
Table 2 shows the unadjusted and adjusted OR estimates for Wolfe's high-risk mammographic parenchymal patterns and smoking in the total study population and in postmenopausal women separately. Current smoking was strongly and inversely associated with high-risk patterns, after adjustment for concomitant risk factors. Relative to never smokers, current smokers were significantly less likely to have a high-risk pattern (OR 0.37, 95% CI 0.14-0.94). Similar results were obtained when the analysis was confined to postmenopausal women. Past smoking was not related to mammographic parenchymal patterns. The overall effect in postmenopausal women lost its significance when adjusted for other risk factors for P2/DY patterns that were found to be significant in the present study, although the results were still strongly suggestive. There was no interaction between cigarette smoking and body mass index.
Discussion:
In the present study we found a strong inverse relationship between current smoking and high-risk mammographic parenchymal patterns of breast tissue as classified by Wolfe [12]. These findings are not completely unprecedented; Greendale et al [18] found a reduced risk of breast density in association with smoking, although the magnitude of the reduction was unclear. The present findings suggest that this reduction is large.
Recent studies [9,10] have suggested that breast cancer risk may be reduced among current smokers. In a multicentre Italian case-control study, Braga et al [10] found that, relative to nonsmokers, current smokers had a reduced risk of breast cancer (OR 0.84, 95% CI 0.7-1.0). These findings were recently supported by Gammon et al [9], who reported that breast cancer risk in younger women (younger than 45 years) may be reduced among current smokers who began smoking at an early age (OR 0.59, 95% CI 0.41-0.85 for age 15 years or younger) and among long-term smokers (OR 0.70, 95% CI 0.52-0.94 for those who had smoked for 21 years or more).
The possible protective effect of smoking might be due to its anti-oestrogenic effect [1,2,19]. Recently there has been renewed interest in the potential effect of smoking on breast cancer risk, and whether individuals may respond differently on the basis of differences in metabolism of bioproducts of smoking [20,21]. Different relationships between smoking and breast cancer risk have been suggested that are dependent on the rapid or slow status of acetylators of aromatic amines [20,21]. More recent studies [22,23], however, do not support these findings.
The present study design minimized the opportunity for bias to influence the findings. Because subjects were unaware of their own case-control status, the possibility of recall bias in reporting smoking status was minimized. Systematic error in the assessment of mammograms was avoided because reading was done without knowledge of the risk factor data. Furthermore, the associations observed are unlikely to be explained by the confounding effect of other known breast cancer risk factors, because we adjusted for these in the analysis. We did not have information on passive smoking status, however, which has recently been reported to be a possible confounder [5,6,21,24].
The present data indicate that adjustment for current smoking status is important when evaluating the relationship between mammographic parenchymal pattern and breast cancer risk. They also indicate smoking as a prominent potential confounder when analyzing effects of other risk factors such as obesity-related variables. It seems that parenchymal patterns may act as an informative biomarker of the effect of cigarette smoking on breast cancer risk.
PMCID: PMC13911  PMID: 11056684
mammography; screening; smoking; Wolfe's parenchymal patterns
21.  Dysfunctional KEAP1–NRF2 Interaction in Non-Small-Cell Lung Cancer 
PLoS Medicine  2006;3(10):e420.
Background
Nuclear factor erythroid-2 related factor 2 (NRF2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (KEAP1) negatively regulates NRF2 activity by targeting it to proteasomal degradation. Increased expression of cellular antioxidants and xenobiotic detoxification enzymes has been implicated in resistance of tumor cells against chemotherapeutic drugs.
Methods and Findings
Here we report a systematic analysis of the KEAP1 genomic locus in lung cancer patients and cell lines that revealed deletion, insertion, and missense mutations in functionally important domains of KEAP1 and a very high percentage of loss of heterozygosity at 19p13.2, suggesting that biallelic inactivation of KEAP1 in lung cancer is a common event. Sequencing of KEAP1 in 12 cell lines and 54 non-small-cell lung cancer (NSCLC) samples revealed somatic mutations in KEAP1 in a total of six cell lines and ten tumors at a frequency of 50% and 19%, respectively. All the mutations were within highly conserved amino acid residues located in the Kelch or intervening region domain of the KEAP1 protein, suggesting that these mutations would likely abolish KEAP1 repressor activity. Evaluation of loss of heterozygosity at 19p13.2 revealed allelic losses in 61% of the NSCLC cell lines and 41% of the tumor samples. Decreased KEAP1 activity in cancer cells induced greater nuclear accumulation of NRF2, causing enhanced transcriptional induction of antioxidants, xenobiotic metabolism enzymes, and drug efflux pumps.
Conclusions
This is the first study to our knowledge to demonstrate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC. Loss of KEAP1 function leading to constitutive activation of NRF2-mediated gene expression in cancer suggests that tumor cells manipulate the NRF2 pathway for their survival against chemotherapeutic agents.
Biallelic inactivation ofKEAP1, a frequent genetic alteration in NSCLC, is associated with activation of the NRF2 pathway which leads to expression of genes that contribute to resistance against chemotherapeutic drugs.
Editors' Summary
Background.
Lung cancer is the most common cause of cancer-related death worldwide. More than 150,000 people in the US alone die every year from this disease, which can be split into two basic types—small cell lung cancer and non-small-cell lung cancer (NSCLC). Four out of five lung cancers are NSCLCs, but both types are mainly caused by smoking. Exposure to chemicals in smoke produces changes (or mutations) in the genetic material of the cells lining the lungs that cause the cells to grow uncontrollably and to move around the body. In more than half the people who develop NSCLC, the cancer has spread out of the lungs before it is diagnosed, and therefore can't be removed surgically. Stage IV NSCLC, as this is known, is usually treated with chemotherapy—toxic chemicals that kill the fast-growing cancer cells. However, only 2% of people with stage IV NSCLC are still alive two years after their diagnosis, mainly because their cancer cells become resistant to chemotherapy. They do this by making proteins that destroy cancer drugs (detoxification enzymes) or that pump them out of cells (efflux pumps) and by making antioxidants, chemicals that protect cells against the oxidative damage caused by many chemotherapy agents.
Why Was This Study Done?
To improve the outlook for patients with lung cancer, researchers need to discover exactly how cancer cells become resistant to chemotherapy drugs. Detoxification enzymes, efflux pumps, and antioxidants normally protect cells from environmental toxins and from oxidants produced by the chemical processes of life. Their production is regulated by nuclear factor erythroid-2 related factor 2 (NRF2). The activity of this transcription factor (a protein that controls the expression of other proteins) is controlled by the protein Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 holds NRF2 in the cytoplasm of the cell (the cytoplasm surrounds the cell's nucleus, where the genetic material is stored) when no oxidants are present and targets it for destruction. When oxidants are present, KEAP1 no longer interacts with NRF2, which moves into the nucleus and induces the expression of the proteins that protect the cell against oxidants and toxins. In this study, the researchers investigated whether changes in KEAP1 might underlie the drug resistance seen in lung cancer.
What Did the Researchers Do and Find?
The researchers looked carefully at the gene encoding KEAP1 in tissue taken from lung tumors and in several lung cancer cell lines—tumor cells that have been grown in a laboratory. They found mutations in parts of KEAP1 known to be important for its function in half the cell lines and a fifth of the tumor samples. They also found that about half of the samples had lost part of one copy of the KEAP1 gene—cells usually have two copies of each gene. Five of the six tumors with KEAP1 mutations had also lost one copy of KEAP1—geneticists call this biallelic inactivation. This means that these tumors should have no functional KEAP1. When the researchers checked this by staining the tumors for NRF2, they found that the tumor cells had more NRF2 than normal cells and that it accumulated in the nucleus. In addition, the tumor cells made more detoxification enzymes, efflux proteins, and antioxidants than normal cells. Finally, the researchers showed that lung cancer cells with KEAP1 mutations were more resistant to chemotherapy drugs than normal lung cells were.
What Do These Findings Mean?
These results indicate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC and suggest that the loss of KEAP1 activity is one way that lung tumors can increase their NRF2 activity and develop resistance to chemotherapeutic drugs. More lung cancer samples need to be examined to confirm this result, and similar studies need to be done in other cancers to see whether loss of KEAP1 activity is a common mechanism by which tumors become resistant to chemotherapy. If such studies confirm that high NRF2 activity (either through mutation or by some other route) is often associated with a poor tumor response to chemotherapy, then the development of NRF2 inhibitors might help to improve treatment outcomes in patients with chemotherapy-resistant tumors.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030420.
US National Cancer Institute information on lung cancer and on cancer treatment
MedlinePlus entries on small cell lung cancer and NSCLC Cancer Research UK information on lung cancer
Wikipedia entries on lung cancer and chemotherapy (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030420
PMCID: PMC1584412  PMID: 17020408
22.  Frequent loss of the AXIN1 locus but absence of AXIN1 gene mutations in adenocarcinomas of the gastro-oesophageal junction with nuclear β-catenin expression 
British Journal of Cancer  2004;90(4):892-899.
Up to 60% of gastro-oesophageal junction (GEJ) adenocarcinomas show nuclear β-catenin expression, pointing to activated T-cell factor (TCF)/β-catenin-driven gene transcription. We demonstrate in five human GEJ adenocarcinoma cell lines that nuclear β-catenin expression indeed correlates with enhanced TCF-mediated transcription of a reporter gene. In several tumour types, TCF/β-catenin activation is caused by mutations in either adenomatous polyposis coli (APC), β-catenin exon 3, AXIN1, AXIN2 or β-transducin repeat-containing protein (β-TrCP). In GEJ adenocarcinomas, very few APC and β-catenin mutations have been found. Therefore, the mechanism of Wnt pathway activation remains unclear. In the present study, we did not find AXIN1 gene mutations in 17 GEJ tumours with nuclear β-catenin expression (without β-catenin exon 3 mutations). Six intragenic single nucleotide polymorphisms (SNPs) were identified. One of these, the AXIN1 gene T1942C SNP, has a frequency of 21% but is only very recently described despite numerous AXIN1 gene mutational studies. We provide evidence why this SNP was missed in single strand conformation polymorphism analyses. The AXIN1 gene G2063A variation was previously described as a gene mutation but we demonstrate that this is a polymorphism. With these six SNPs loss of heterozygosity (LOH) was found in 11 of 15 (73%) informative tumours. To investigate a possible AXIN1 gene dosage effect in GEJ tumours expressing nuclear β-catenin, AXIN1 locus LOH was determined in 20 tumours expressing membranous and no nuclear β-catenin. LOH was found in 10 of 13 (77%) informative cases. AXIN1 protein immunohistochemistry revealed cytoplasmic expression in all tumours irrespective of the presence of AXIN1 locus LOH. These data indicate that nuclear β-catenin expression is indicative for activated Wnt signalling and that neither AXIN1 gene mutations nor AXIN1 locus LOH are involved in Wnt pathway activation in GEJ adenocarcinomas.
doi:10.1038/sj.bjc.6601589
PMCID: PMC3215949  PMID: 14970870
oesophageal cancer; Wnt signalling pathway; AXIN1; mutation; loss of heterozygosity
23.  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
24.  Abnormal Fhit expression is an independent poor prognostic factor for cervical cancer 
British Journal of Cancer  2003;88(8):1213-1216.
We analysed the expression of the fragile histidine triad (FHIT) gene in cervical cancer to evaluate its clinical relevance in relation to human papillomavirus (HPV) infection. A total of 73 women with cervical cancer of stage Ib or more advanced (67 squamous cell carcionomas, four adenocarcinomas, two adenosquamous carcinomas) were examined for Fhit expression by immunohistochemistry. They were further analysed for the presence of HPV and its subtype. Abnormal expression of Fhit (absent or reduced Fhit expression) was observed in 52 cases (71.2%). The high-risk HPV DNAs for cervical cancer, including type 16, 18, 31, 33, 51, 52, 58, 68, were identified in 63 cases (86%). The abnormal Fhit expression was not related to the clinicopathological factors including histology, tumour stage, and HPV type. Notably, the 5-year survival of patients showing the abnormal Fhit expression was significantly poorer than those showing normal Fhit expression (64 versus 87%, P=0.035). Interestingly, the mean age of the patients with the abnormal Fhit expression was significantly less than those with the normal Fhit expression (51.6 versus 58.7 years of age, P=0.027, student's t-test). These data imply that the aberrant Fhit expression could be a poor prognostic factor independent of HPV. In the light of a high incidence of abnormal Fhit expression in younger patients and HPV as a key player in cervical carcinogenesis, abnormal Fhit expression may accelerate carcinogenesis in concert with HPV.
doi:10.1038/sj.bjc.6600892
PMCID: PMC2747578  PMID: 12698186
cervical cancer; Fhit expression; HPV
25.  Allelic loss on chromosome 10q in human lung cancer: association with tumour progression and metastatic phenotype. 
British Journal of Cancer  1998;77(2):270-276.
We analysed 78 carcinomas of the lung for allelic losses on chromosome 10q. The tumours were of different stage and grade and comprised 22 small-cell lung carcinomas (SCLC), 40 squamous cell carcinomas (SCC), 11 adenocarcinomas, four large-cell carcinomas and one carcinoid. They were investigated by six polymorphic markers located between 10q21 and 10qter. We observed a high incidence of loss of heterozygosity (LOH) in SCLC (91%) and metastatic SCC (56%). Non-metastatic SCC showed deletions in three cases (14%) and no LOH was found in the other types of non-small-cell lung cancer. The statistical analysis indicated that the presence of LOH correlated significantly with advanced tumour stages in the entire collective and in particular within the SCLC and SCC subgroups. For SCC, a positive association was found between LOH and metastases formation, while in SCLC the number of non-metastatic tumours was too small for a final conclusion. Whereas SCLC was frequently characterized by multiple allelic losses, suggesting the deletion of the entire chromosomal arm, SCC showed interstitial imbalances. A high incidence of allelic loss was observed between the markers D10S677 and D10S1223. The analysis of five informative cases suggested the presence of two non-overlapping regions between the loci D10S677/D10S1237 and D10S1213/D10S1223. In SCLC, we did not find mutations in the putative tumour-suppressor gene MXI1. The data indicate that LOH on chromosome 10q is associated with tumour progression in SCC and SCLC. Thus it may become a useful genetic marker in the assessment of the malignant potential of these tumour types.
Images
PMCID: PMC2151238  PMID: 9460998

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