A review of novel biological tools used in screening for the early detection of lung cancer

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1 1 Respiratory Unit, Prine Philip Hospital, Hywel Dda NHS Trust, Llanelli, Wales, UK; 2 Shool of Mediine, University of Swansea, Singleton Park, Swansea, West Glamorgan, Wales, UK Correspondene to: Dr R Ghosal, Respiratory Unit, Prine Philip Hospital, Hywel Dda NHS Trust, Llanelli SA14 8QF, Wales, UK; ghosalr@ hotmail.om Reeived 4 November 2008 Aepted 28 Marh 2009 A review of novel biologial tools used in sreening for the early detetion of lung aner R Ghosal, 1,2 P Kloer, 1 K E Lewis 1,2 ABSTRACT Lung aner is the most ommon aner worldwide and auses more deaths per year than any other aner. It has a very poor 5-year survival rate of 8 16%, partly beause of omorbidity preventing urative treatments but mainly beause of the disease presenting with symptoms only when it is at an advaned and inurable stage. When lung aner is deteted earlier and is amenable to radial treatments suh as potentially urative surgery and radial radiotherapy, 5-year survival rates are muh higher (up to 67%). Therefore reliable detetion of lung aner at this earlier (usually asymptomati) stage of disease should be an important way to improve outomes. This review disusses the priniples of sreening with respet to lung aner, onentrating mainly on the biologial modalities used to detet it. The lak of impat ahieved by early studies using sputum ytology (in onjuntion with hest radiographs) is desribed, and then newer tehnology used to measure other biomarkers in sputum, serum, exhaled breath and bronhial muosa to diagnose (early) lung aner is detailed. Many tehniques show promise, but debate ontinues about whih population to sreen and what is the most (ost) effetive modality to use. Moreover, no single biomarker or ombination of biomarkers in sreening has yet been shown to redue lung aner mortality in large prospetive randomised studies. Lung aner is the most ommon aner worldwide in terms of both inidene and mortality, with 1.3 million new ases being diagnosed eah year and 1.2 million deaths, with Europe and North Ameria having the highest inidene and prevalene rates. 1 2 Compared with other ommon aners suh as prostate and breast aner, lung aner has muh lower survival rates, and, despite advanes in radiologial tests, partiularly CT and positron emission tomography sanning, surgial tehniques and postoperative management, radiotherapy delivery and new hemotherapeuti agents, the long-term survival from lung aner has remained remarkably onstant and has not improved signifiantly over the last 20 years. 3 Apart from lowering its inidene by reduing igarette smoking, the key to improving lung aner survival at present is to diagnose it at an earlier stage. This has led to deades of researh to identify a suitable sreening tehnique in order to redue mortality. The objetives of this artile are to desribe the priniples of sreening and show how they are relevant to lung aner. We will briefly disuss purely radiologial attempts at sreening with lowdose spiral CT (LDCT) and how simple radiology (hest radiographs (CXRs)) has been ombined with sputum ytology, before desribing in more detail how novel biologial tehniques (or biomarkers) have been tried and are still being applied in the detetion of lung aner. We will also explain some of the biologial reasoning for the use of ertain biomarkers and, in partiular, how they are based on a better moleular understanding of lung arinogenesis. PRINCIPLES OF SCREENING Our improved understanding of the ellular and subellular hanges that lead to aner, ombined with advanes in biologial and omputer tehnology, has led to a signifiant widening of sreening tehniques. When onsidering a disease entity for sreening purposes, one has to onsider the priniples of sreening. Sreening is an important proess in mediine, used to identify a disease within a defined population to improve health outomes. In 1968, the World Health Organization published guidelines on the priniples of sreening. 4 For a sreening tool to be effetive, the following priniples must be adhered to. (A) The ondition should be an important health problem. Lung aner is the most ommon aner worldwide with 1.3 million new ases diagnosed eah year. 2 (B) The disease should have signifiant mortality and morbidity. Despite variations worldwide in the 5-year survival rate in lung aner, olletively the figures show how devastating the disease is. The overall 5-year survival rate in Ameria is 16%, although this figure is onfounded by only inluding a quarter of the population and exludes those without histologial onfirmation of diagnosis. 5 In Europe, lung aner has an overall 5- year survival of only 10.9% (ompared with a 5-year survival rate of 79% for breast aner and prostate aner of 78%). 6 (C) There should be a latent phase of the disease. Lung aners usually present when a patient develops symptoms, so in most ases, the aner is already advaned at the time symptoms develop. Sreening aims to detet these aners in the earlier asymptomati/latent phase. (D) Intervention earlier in the disease proess should improve outomes. The 5- year survival rate for patients with stage IA non-small ell lung aner (NSCLC) who have undergone omplete surgial resetion of the tumour is muh higher at 67%. 7 (E) The sreening test itself should have ertain harateristis. It must be sensitive (to avoid false negatives and inappropriate reassurane) and speifi (to avoid too many false positives leading to unneessary worry Postgrad Med J: first published as /pgmj on 5 July Downloaded from on 8 July 2018 by guest. Proteted by opyright. 358 Postgrad Med J 2009;85: doi: /pgmj

2 (F) and further expensive/invasive testing). The test should also involve little risk of harm (or muh less than the disease), and should be aeptable to the majority of the population to be sreened. The ost of finding a ase using the sreening tehnique should be onsidered in relation to medial expenditure as a whole. LDCT AND LUNG CANCER SCREENING There have been many observational studies assessing the feasibility of using LDCT in deteting lung aner at an earlier stage and thus allowing urative treatment. The advantage of using suh tehnology is that it allows detetion of muh smaller nodules (2 3 mm), whih would almost ertainly not be visible on CXR. As a result, a larger number of early aners have indeed been deteted; a large study has found some survival advantage, but this was not a randomised ontrolled trial. 8 Other studies with LDCT sreening have reported no effet in reduing mortality The likely explanation for this is a large number of indolent aners, whih would not have led diretly to death, being deteted. The National Lung Sreening Trial (NLST), a randomised ontrol study looking at LDCT sreening tehniques for lung aner, will be reported in the next 2 5 years. 11 A detailed review of radiologial sreening is not the main fous of this artile and is available elsewhere The rest of this artile reviews several biologial tehniques based on sampling body tissues, whih have been studied as potential sreening tools for lung aner. We desribe ommonly used sputum ytologial tehniques before onentrating on novel biologial tools, whih have been applied to sputum, serum, exhaled breath and bronhosopy. Table 1 summarises these biologial sreening tools. SPUTUM CYTOLOGY AND SCREENING IN LUNG CANCER Sputum is a good biologial speimen for sreening beause it is readily aessible and the tehnique is therefore non-invasive. Sputum ytology (ie, mirosopi examination looking for abnormal ells) in ombination with CXRs has been used as a sreening tool in three large studies sponsored by the National Caner Institute. The Memorial Sloan-Kettering Study, The John Hopkins Study and Mayo Lung Projet have, in total, randomised over male smokers (usually over 45 years), to various ombinations of CXRs with or without sputum ytology sreening, at baseline and repeated at interim periods over 3 5 years. Asymptomati, often operable, arinomas were diagnosed at baseline in % of the partiipants, and also further inident tumours during the study. Early results looked promising, but all three studies onluded that adding regular sputum ytologial sreening offered no advantage to CXRs alone, but most importantly there was no survival advantage to the sreened groups ompared with historial ontrols or the Table 1 Summary of biologial tehniques reviewed Tissue sample/proedure Sreening tool Sputum Serum Breath Bronhosopy Cytology Gene promoter methylation Cirulating DNA Vasular endothelial growth fator Proteomis Volatile organi ompounds Autofluoresent bronhosopy Geneti testing of epithelial ells unsreened population A large study from Czehoslovakia involving 6300 heavy smokers also found no signifiant population survival advantage of performing regular CXRs (with or without regular sputum ytology) over 3 years, even after 15 years of follow-up. An interesting and onsistent finding from the studies is the high frequeny of detetion of squamous ell arinoma in the sputum ytology groups. Together, these studies suggest that adding regular traditional sputum ytology (biologial) sreening to CXRs offers no further long-term survival advantage for the sreened population. Some of the improved early survival rates were probably due to a ombination of overdiagnosis, lead-time bias and length-time bias, therefore alternative sreening approahes need to be onsidered. Sine these early studies, improvements have been made in CXR tehnology, and, as a result, a large randomised ontrol study reassessing the role of annual CXRs is in progress. The Prostate, Lung, Coloretal and Ovarian Caner sreening trial (PLCO study) has been instituted by the National Caner Institute and inludes over men and women aged 55 74, who were alloated to two trial arms. One arm is intervention (sreening) and the other is the ontrol arm, with normal healthare routines ontinued. The long-term survival outomes from this study are pending. 26 THE PRINCIPLE OF BIOMARKERS AND LUNG CANCER: UNDERLYING HISTOLOGICAL AND CELLULAR CHANGES The understanding of the moleular biology of lung aner is rapidly developing, and new tehniques are based on understanding the key ellular hanges that our in lung aner. Moreover, non-ytologial/non-radiologial biomarkers are being evaluated in monitoring and prediting treatment response beause of our understanding of these ellular proesses. Both squamous dysplasia and arinoma in situ are preneoplasti abnormalities, whih often (but not always) lead to squamous ell arinoma. 27 Preneoplasti abnormalities in adenoarinoma and arinoid are different and inlude atypial adenomatous hyperplasia and diffuse idiopathi pulmonary neuroendorine ell hyperplasia. 27 When developing biomarkers, researhers have found hanges in gene expression and hromosome struture within these preneoplasti lesions. These inlude mutations in the p53 and ras genes that are assoiated with hyperproliferation and loss of ell yle ontrol, aberrant gene promoter methylation, inreased vasular growth and altered protein expression Unfortunately, the variability of these hanges and our inability to reliably detet them has meant that no single speifi biomarker has yet been identified with adequate sensitivity, speifiity and reproduibility. Various groups are now developing high-throughput tehniques, to apply panels of biomarkers assoiated with these ellular hanges for deteting and monitoring lung aner. SPUTUM BIOMARKERS AND SCREENING FOR LUNG CANCER Gene promoter methylation This is a very promising modality for lung aner sreening. Early hanges in the development of lung aner inlude methylation of promoter sequenes in different tumour suppressor genes. These are assoiated with the silening of transription and inativation of these tumour suppressor genes, playing a ruial role in triggering malignant transformation and progression. 29 Methylation is deteted by speifi PCR analysis. Hypermethylation was investigated in a small proof-of-onept Postgrad Med J: first published as /pgmj on 5 July Downloaded from on 8 July 2018 by guest. Proteted by opyright. Postgrad Med J 2009;85: doi: /pgmj

3 study looking at methylation of p16 and O 6 -methylguanine DNA methyltransferase (MGMT) gene promoters in a high-risk population. Twenty-one patients with squamous ell arinoma were investigated. The presene of one or both gene promoters was found in all 21 samples irrespetive of whether they were taken up to 3 years before or at the time of diagnosis. This ompares with 15% (methylation of p16) and 25% (methylation of MGMT) in 123 ontrols, deemed high risk beause of a smoking history and/or radon exposure. Interestingly, 48% of the patients with squamous ell aner had methylation of both genes ompared with only 4% of ontrols (p,0.001). On followup, three lung aners were diagnosed in the ontrols between 1 and 3 years after sputum olletion, with the MGMT gene being methylated in two of the subjets. 30 Belinsky et al 31 postulated that a panel of methylated genes would yield greater sensitivity and speifiity. They reruited subjets older than 25 years, with >30 pak-year smoking history and airways obstrution with a fored expiratory volume in 1 s (FEV 1 ) (75% and FEV 1 /FVC (fored vital apaity) (0.75. They exluded subjets with any aner diagnosed in the previous 5 years. A total of 3259 subjets provided pooled sputum for 3 days in one ontainer, and the next 3 days in a seond ontainer. The sputum from the seond ontainer was used as samples for the study. There were 1353 ohort deaths, and 182 subjets were diagnosed as having lung aner. One suffiient quality of DNA was analysed, the study ohort onsisted of 98 subjets (ases), and the non-aner ohort was used to form a group of 92 ontrols mathed for age, gender and month of enrolment. Although there were 26 urrent smokers in the ontrol group ompared with 42 in the ases, the total number of smoking pak-years was similar. Fourteen genes were analysed for promoter methylation, inluding p16 and MGMT. It was found that six of the 14 genes (p16, MGMT, DAPK, RASSFIA, PAX5 b and GATA5) were individually assoiated with a.50% inreased risk of lung aner. Sputum olleted within 18 months of a diagnosis of lung aner had more methylated gene promoters than sputum from the same subjet olleted more than 18 months before the diagnosis. The onomitant methylation of three or more genes was assoiated with a 6.5-fold inreased risk of lung aner, with reeiver operating harateristis of a speifiity of 64% and sensitivity of 64%. This is the first study to show how a panel of genes an be used as potential biomarkers in sreening a high-risk population for lung aner and suggests an aumulative hit effet. The researhers onluded that their level of speifiity is not yet high enough for prospetive sreening studies, but alled for more evaluation of andidate gene panels, espeially as tehnology improves. SERUM BIOMARKERS AND SCREENING FOR LUNG CANCER Serum biomarkers have been used to sreen for and monitor different aners. These inlude prostate-speifi antigen for prostate aner, CEA and CA19-9 in oloni aner and CA125 in ovarian aner. More reently, serum has been analysed as a sreening biofluid for lung aner. Cirulating DNA and geneti hanges Raised onentrations of irulating ell-free DNA in patients with aner were first reported in Although the preise mehanism of DNA release into the blood remains obsure, it appears that muh of this irulating DNA is derived from apoptoti and neroti tumour ells. 32 Patients with lung aner show geneti and epigeneti hanges onsisting of hromosome loss, onogene ativation and tumour suppressor gene methylation, so the onentrations of this irulating DNA may allow development of speifi markers. Conentrations of irulating ell-free DNA are generally higher in patients with aner than in healthy ontrols. k-ras and p53 are well-haraterised ommon mutations in human lung aner and preneoplasia. k-ras mutations have been found in the irulating DNA of 20 30% of patients with lung aner, with only one study deteting the mutation in the ontrol group, although the harateristis of the ontrols were not speified p53 mutations have been found in 10 30% of patients with lung aner ompared with minimal detetion in the healthy ontrol groups. 41 One study found p53 mutations in 41% of lung tumours, with the idential mutation identified in the plasma of 73% of them. 42 Vasular endothelial growth fator (VEGF) Reent preliminary data showed signifiantly (p = 0.033) inreased VEGF onentrations (measured using enzyme immunoanalysis) in seven patients with NSCLC ompared with five healthysmokersandsevenontrols. 43 Proteomis Proteomis is the study of proteins, and two approahes have been applied to lung aner. The first, protein profiling, is where patterns of protein expression are used, and the alternative is identifying individual proteins. Yang et al 44 analysed serum from 158 patients with lung aner and 50 ontrols using a mass spetrometry tehnique. Seventy-four lung aner serum samples and 20 healthy ontrols were used to develop a training set. From this, a speifi pattern onsisting of five protein peaks was hosen as biomarkers that ould diagnose NSCLC, and this pattern was used, in a blinded fashion, in an attempt to disriminate the remaining serum samples of 84 patients with lung aner and 30 healthy ontrols. The pattern of proteins yielded a sensitivity of 87% and speifiity of 80%. These tehniques are also being evaluated to assess for premalignant hanges in high-risk people. 45 BREATH BIOMARKERS AND SCREENING FOR LUNG CANCER Volatile organi ompounds (VOCs) in breath Exhaled breath samples are even more aessible than sputum. More than 200 different measurable hemials are exhaled in the human breath. 46 Gordon et al 47 studied VOCs in exhaled air from 12 patients with histologially onfirmed NSCLC and nine healthy ontrols (two were heavy smokers). Gas hromatography ombined with mass spetrometry identified four peaks representing individual VOCs in more than half of the patients with lung aner, whih were ompletely absent in the ontrol group. Gordon et al suggested that unique VOCs are exhaled in the breath of patients with lung aner, whih may have potential diagnosti importane, and reommended that this tehnique be further tested in a larger population. Phillips et al 48 olleted exhaled breath in 108 fasting patients about to undergo a bronhosopy for an abnormal CXR. Lung aner was onfirmed histologially in 60 patients (50 NSCLC, 10 small ell lung aner) and exluded in 48. Many VOCs were ommon to both sets of breath samples, but a group of 22 of these VOCs ould be used to distinguish between lung aner and ontrol ases aording to risk weighting attahed to the test. For example, the VOCs had 100% sensitivity (ie, no false negatives) and 81% speifiity for stage I lung aner if the Postgrad Med J: first published as /pgmj on 5 July Downloaded from on 8 July 2018 by guest. Proteted by opyright. 360 Postgrad Med J 2009;85: doi: /pgmj

4 reeiver operator aepted a post-test probability of A post-test probability of 0.9 yielded lower (67%) sensitivity but 100% speifiity (no false positives). The abnormal VOC peaks onsisted of mainly alkanes and benzene derivatives. Smoking status was self-reported and did not aount for the benzene derivatives, as these were also present in the breath of nonsmokers and ex-smokers. There were no signifiant differenes in the sensitivity and speifiity of VOCs between early and advaned lung aner. Phillips et al also onluded that patterns of VOCs ould at as a fingerprint for lung aner, but alled for validation in a larger general population. The same researhers ompared VOCs in patients with biopsy-proven primary lung aner (n = 67), non-lung aner metastasising to the lungs (n = 15) or abnormal CXRs but no histologial evidene of lung aner (n = 5), and healthy volunteers from the general population (n = 41). The breath test identified over 80 different alkanes and monomethylated alkanes used to generate a preditive model using a panel of nine VOCs. These nine VOCs, in ombination, yielded a sensitivity of 90% and a speifiity of 83% (using a post-test probability of 0.5) of identifying primary or seondary lung aner. There were only minor differenes when subjets were stratified aording to smoking history, histologial aner type or staging. 49 The authors believed that the test was sensitive, ost-effetive and easy to perform and ould be used to omplement other modalities, but again alled for further evaluation. Italian researhers measured 13 VOCs (seven aliphati and six aromati ompounds) in 36 NSCLC ases, 25 ontrols with stable mild-moderate hroni obstrutive pulmonary disease (COPD), 35 asymptomati smokers and 50 healthy nonsmokers with normal spirometry. 50 Although no single VOC ould distinguish NSCLC from the other groups, the panel of VOCs performed with an overall sensitivity of 72% and speifiity 94%. Twenty-six of the patients with NSCLC agreed to have repeat VOC analysis after surgial resetion, whih revealed a signifiant redution in their exhaled isoprene and deane onentrations. Poli et al 50 still onluded that the use of VOCs alone ould not be reommended at this stage in the early detetion of lung aner. The studies disussed thus far involve mass spetrometry of the VOCs. New tehniques using VOCs have reently been developed. One suh system onsists of a olorimetri sensor array, whih has 36 spots made up of different hemially sensitive ompounds. The olour of the spot hanges depending on the hemial it omes into ontat with. A study to assess this priniple was performed by Mazzone et al. 51 The study inluded 143 subjets: 49 had NSCLC, 73 had various hroni lung diseases suh as COPD, idiopathi pulmonary fibrosis, pulmonary arterial hypertension and saroidosis, and there were 21 ontrols. One a predition model had been developed using 70% of the subjets, it ould be used to predit the presene of lung aner in the remaining 30% with a sensitivity of 73.3% and speifiity of 72.4%. Although this suggests only moderate auray of the diagnosis, the study does prove the potential of this tehnique. Mazzone 52 has reently published a review of VOCs in lung aner. BRONCHOSCOPY AND SCREENING FOR LUNG CANCER Many lung aners are diagnosed by histologial analysis of endobronhial samples obtained by fibre-opti bronhosopy. The role of bronhosopy has expanded over the last few years and now enompasses loalised treatments suh as laser therapy, stent insertion and brahytherapy. As well as obtaining Key learning points Lung aner is the most ommon aner worldwide. Five-year survival rates are signifiantly worse than for breast and prostate aner. Sreening using standard tehniques suh as sputum ytology and plain hest radiographs have not been shown to improve mortality. Sreening studies using CT are ongoing. Newer tehniques measuring biomarkers in easily aessible tissues suh as serum, sputum and exhaled breath, and even in less easily obtained tissue suh as endobronhial samples, show some promise in deteting (early) lung aner. Overall, no single heap and reliable test has yet been identified for effetive sreening of lung aner in large population studies, but work is in progress. a histologial diagnosis and allowing some treatment, bronhosopy may have a role in sreening for lung aner. Autofluoresent bronhosopy The priniple behind autofluoresent bronhosopy is that, when blue light (wavelength nm) is shone on to abnormal muosa, fluoresene is redued (ompared with normal muosa), allowing more lesions to be identified (partiularly premalignant ones) ompared with the use of shining standard white light. A review suggested that autofluoresent bronhosopy (in experiened hands) an diagnose arinoma in situ in 1.6% of ases deemed high risk, and moderate and severe dysplasia in 19% of urrent heavy or former smokers with sputum atypia. 53 Moreover, the preinvasive lesions were small, with 55% being (1.5 mm in greatest diameter. Over 1000 ases omparing white light against autofluoresent bronhosopy found 40% of preinvasive lesions deteted by white light bronhosopy alone, but,80% of lesions were deteted by adding autofluoresent bronhosopy. 54 Loewen et al 55 ombined autofluoresent bronhosopy, sputum ytology and spiral CT for surveillane in a very highrisk group. Two or more of the following riteria had to be fulfilled: >20 pak year smoking history; asbestos-related lung disease on CXR; FEV 1,70% of predited, treated aero-digestive aner with no evidene of disease for.2 years. A total of 186 patients were enrolled, and 169 ompleted the baseline tests; 66% of the patients had squamous metaplasia or worse and 7% had a diagnosis of aner at baseline. Sputum ytology missed 100% of the dysplasia and 68% of the metaplasia deteted by autofluoresent bronhosopy, and failed to detet any ases of arinoma or arinoma in situ. Patients who had pulmonary nodules on spiral CT san were 3.2 times more likely to exhibit premalignant hanges on autofluoresent bronhosopy (p,0.001). The study onluded that autofluoresent bronhosopy should be arried out on high-risk people regardless of the results of onventional sputum ytology. Other studies have found no inreased detetion rates of arinoma in situ or dysplasia using autofluoresent bronhosopy ompared with white light bronhosopy in urrent or former smokers, 56 suggesting that autofluoresent bronhosopy is best used in highly seleted ohorts with sputum atypia or when multiple risk fators for lung aner are present. Postgrad Med J: first published as /pgmj on 5 July Downloaded from on 8 July 2018 by guest. Proteted by opyright. Postgrad Med J 2009;85: doi: /pgmj

5 Current researh questions To identify a biomarker that is ost-effetive and with good enough auray to be used as a sreening and monitoring tool to impat on lung aner survival. To onsider ombinations of biomarkers, possibly using more than one tissue or ombined with low-dose CT sans in sreening for lung aner. Key referenes Wilson JMG, Jungner G. Priniples and pratie of sreening for disease. WHO Chronile 1968;22:473. Bah PB, et al. Sreening for lung aner: a review of the urrent literature. Chest 2003;123(1 Suppl):72S 82S. Aviel-Ronen S, et al. K-ras mutations in non-small-ell lung arinoma: a review. Clin Lung Caner 2006;8:30 8. Phillips M, et al. Detetion of lung aner with volatile markers in the breath. Chest 2003;123: Mazzone PJ. Analysis of volatile organi ompounds in the exhaled breath for the diagnosis of lung aner. J Thora Onol 2008;3: Geneti testing of epithelial ells from endobronhial brushings Spira et al 57 investigated whether gene expression profiling of large airway epithelial ells ould be used as a biomarker in histologially normal bronhial muosa obtained by bronhial brushing. A total of 129 urrent or former smokers who underwent fibre-opti bronhosopy for a linial suspiion of lung aner were followed-up until a final diagnosis was made. Sixty were diagnosed as having lung aner (bronhosopy or subsequent lung biopsy yielded lung tumour ells), and 69 without lung aner (investigations yielded a non-aner pathology or the radiologial abnormality resolved). From a training set (n = 77), an 80-gene biomarker panel was identified and then tested prospetively on the remaining 52 ases, obtaining an auray of 83% (80% sensitivity, 84% speifiity). Among the non-diagnosti bronhosopies (n = 92), the auray of the panel of gene biomarkers was 85% (89% sensitive, 83% speifi). By adding the biomarker panel to the traditional tests, the diagnosti sensitivity was improved to an impressive 95% with a 95% negative preditive value. Spira et al also onluded that gene expression in ytologially normal bronhial epithelial ells an serve as a lung aner biomarker. CONCLUSIONS Lung aner is ommon, and only a small proportion of patients survive more than 5 years. Sreening studies using serial CXRs and sputum ytology examination (and ombinations of them) have not improved group mortality. Sreening with LDCT sanning is still under review. Importantly, a better understanding of the biology of lung arinogenesis and improved tehniques in DNA and hemial analyses are being used to develop ost-effetive, high-throughput tehnologies on samples of readily aessible tissues suh as blood, sputum and even exhaled breath. As yet, none have been proven in large studies, but, as these novel biologial tehniques ontinue to evolve, it is hoped that, in the future, ombinations of biomarkers will lead to non-invasive, ost-effetive sreening tools to improve outomes in this ommon and devastating disease. TRUE/FALSE QUESTIONS (ANSWERS AFTER THE REFERENCES) 1. Lung aner is the most ommon aner worldwide. 2. For a sreening modality to be effetive, it must have a high sensitivity and speifiity. 3. Standard ytology and plain hest radiograph sreening studies have shown an inreased survival advantage in the sreened group ompared with the ontrol non-sreened group. 4. Patients with lung aner may have a different profile of volatile organi ompounds in their breath from those without lung aner. 5. The addition of autofluoresent bronhosopy to traditional white light bronhosopy may double the hanes of diagnosis of a preinvasive lesion. Competing interests: None. REFERENCES 1. Caner Researh UK. 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