Virtual Library

Background:
According to WHO, “approximately one person dies every six seconds due to tobacco, accounting for one in 10 adult deaths. Up to half of current users will eventually die of a tobacco-related disease”, which can be lung cancer (87%), pulmonary disease (61%) and coronary heart disease (32%), considering secondhand smoke exposure too as says the Surgeon General´s Report. To protect the health of the Brazilian population, the government has been applying measures, since the 90 years, to reduce the harm caused by tobacco use. Brazil is also committed to reduce the premature mortality from tobacco use in 30% from 2013 to 2025, to achieve one of the nine voluntary WHO Global NCD´s Targets.

Methods:
Quantitative secondary data analysis confronting the cigarette prevalence rates found in Risk and Protective Factors Surveillance for Chronic Diseases Telephone Survey (VIGITEL) and the National Policy for Tobacco Control measures.

Results:
Before ratifying the WHO Framework Convention on Tobacco Control, in 1996 the government started promoting smoke-free places, banning the advertising, promotion and sponsorship, that were finally regulated in 2014. In 2011, the Secretariat of Federal Revenue developed a new system for cigarette taxation to establish a minimum price for a pack of twenty cigarettes and raise the cigarette´s excise tax gradually. In May,2016 the total taxation represents 76% of the cigarette price and will bring to 81% afther December 2016. This is one of the measures of the Framework Convention for Tobacco Control/WHO more cost-effective in the country: Article 6, which deals with the rising prices and taxes on tobacco products to reduce demand. Several surveys and studies point to a reduction in smoking prevalence. Every year, since 2006, the VIGITEL report has shown prevalence rates collected in the entire adult population of the 27 state capitals. In 2015 the frequency of smokers decreased to 10.4%, compared to 2006 which were 15.7% for both sexes. The report also reiterated the effectiveness of the prices and taxes measure, when you compare the frequency of former smokers with lower education, those representing people with lower income. In 2006 they were 25.6%, and in 2015 they increased to 29.1%.

Conclusion:
The present study shows a prevalence decline as a positive result coming from the National Policy for Tobacco Control implementation between the years of 2006 and 2015. To achieve the WHO Global NCD´s Target we still have too much work to do, specilally protect the National Policy from the tobacco industry interference.

Background:
Mutations or translocations in driver genes of lung cancer such as EGFR or ALK are important treatment targets for advanced lung cancer. These alterations are present mainly in never-smokers. Exposure to environmental tobacco smoke (ETS) might provide some explanation to the presence of such genetic traits. Furthermore, ETS exposure might have a different effect should occur at home in adult life, during childhood, or at work. We aim to know if ETS exposure is associated with EGFR mutations or ALK alterations in a huge sample of never smoking lung cancer cases.

Methods:
We recruited never smoking lung cancer cases diagnosed consecutively in 9 Spanish Hospitals since 2011. We collected extensive information on different lifestyle activities and also measured residential radon exposure. Cases had to be older than 30 years with no upper age limit and with no previous history of cancer. A never smoker was defined as: 1) an individual who smoked less than 1 daily cigarette for no more than 6 months or, 2) no more than 100 cigarettes smoked in lifetime. EGFR mutations and ALK alterations were determined using standard procedures. Logistic regressions were performed to analyze the influence of exposure to ETS in different settings (adult life at home, at work or during childhood). The dependent variables were EGFR mutation (of any type) or not, or ALK translocation (present/absent). Results were adjusted by age, gender and residential radon exposure.

Results:
We included 389 never smoking lung cancer cases. 80.5% were females and the median age was 71 and the interquartilic range 61-78 years. 246 patients had EGFR determined (63.2% of the total) and of them, EGFR was mutated in 43%. ALK status was determined in 97 patients (24.9% of the total), and was positive in 16 patients (16.5%). Living at home with a smoker for more than 20 years was not associated with EGFR mutation or ALK translocation, and the same occurred for being exposed to ETS at work. When exposure to ETS in childhood (before 16) was considered, we observed that those exposed to ETS had an OR of EGFR mutation of 0.57 (95%CI 0.31-1.05; p= 0.07). No association was observed for ALK translocation.

Conclusion:
These results suggest that exposure to environmental tobacco smoke in childhood might reduce the chance of EGFR mutation in never smokers with lung cancer. This observation would add more evidence to avoid exposure to ETS in any time of life. Funding: ISCIII/PI13/01765/Cofinanciado FEDER

Background:
Although the most important risk factor for lung cancer is smoking, lung cancer in never smokers (LCINS) is being increasingly reported. Thus, studies of other risk factors for lung cancer are needed. Recently, radon (Rn), a natural, noble gas, was recognized as the second most common risk factor for lung cancer. OBJECTIVES To identify variations in genes associated with lung cancer in never smokers exposed to radon gas.

Methods:
We conducted an optimized next generation sequencing analysis of lung cancer-related genes in normal and tumor tissues from Korean LCINS patients who had been exposed to radon gas indoors. A total of 926 SNPs showing genome-wide statistical significance were analyzed.

Results:
Several genes commonly associated with lung cancer , EGFR and TP53 in chromosomes 7 and 17, respectively, showed significant correlations with LCINS. Others included ERG in chromosome 21, RIT1 in chromosome 1, and BIRC6 in chromosome 2. Meanwhile, several additional loci showed novel associations with LCINS as a result of exposure to radon gas, including PDK1, VHL, WHSC1L1, CHD4, MBD2, ATRX, CCND1, and PTPRD.

Conclusion:
Using next generation sequencing, we found several lung cancer-related genes to be associated with tumors in never smokers exposed to radon. Most of the noted loci have not been shown to be associated with lung cancer, and provide new insights into the development of LCINS. Our findings may serve as a reference for replication and validation studies on the prevention and treatment of LCINS as a result of exposure to radon gas. ACKNOWLEDGMENTS This study was supported by the Korean Ministry of Environment as part of the “Environmental Health Action Program” (grant number 2015001350002).

Background:
Radon gas is the first cause of lung cancer in non-smoking population. The World Health Organization (WHO) recommends radon concentration lower than 100 Bq/m3. In recent years, most of the advances in personalized therapy in NSCLC patients also occurred in non-smokers. Furthermore, limited information is available about the clinical and pathological characteristics in patients exposed to radon gas. We hypothesized that residential radon could be associated to some specific pathological and molecular alterations in NSCLC patients.

Methods:
Prospective study of a cohort of NSCLC patients harbouring molecular alterations (EGFR, BRAF mutations (m), ALK and ROS1 rearrangements (r)) in our centre, between September 2014 and October 2015. A radon detector alpha-track was given to each patient to measure residential radon concentration for 3 months; it was analysed using optical microscopy. We collected demographic information, smoking history, environmental exposure and clinical characteristics. The pathologic characteristics were prospectively revised by a lung cancer pathologist, including histology pattern, grade and inflammatory infiltrate. EGFR and BRAF mutation (m) were analyzed using quantitative real-time polymerase chain reaction (PCR) and ALK and ROS1 rearrangement by fluorescence in situ hybridization (FISH). Data was analyzed using IBM SPSS v.20.

Results:
60 detectors were delivered (10% missing), 48 patients were evaluated (89.6% living in Madrid). Median age 66.5 (29- 82); 33 (68.8%) females; 33 non-smokers (31.3% passive smokers and 35.4% childhood exposure) and 3 (6.3%) light smokers. 100% adenocarcinoma (35.4% mixte, 18.8% acinar, 10.4% solid, 8.3% papillary, 8.3% micropapilllary, 8.4% others and 10.4% unknown); EGFRm 36 patients, ALKr 10 patients and BRAFm 2 patients. Home characteristics measured: 79.2% flat (89.1% measurement at bedroom); building material: 89.6% bricks. Median length of stay was 28 years (2-55). Median height of house 2 floors (0-15). Median of radon concentration: 104 Bq/m3 (42- 915); 60.42% over WHO recommendation. By molecular alteration: EGFRm median 96 Bq/m3 (42-915), ALKr median 116 (64-852) and BRAFm median 125 (125). A significant association was observed between non-EGFR mutation and concentration over the WHO recommendation (p=0.044). In univariant analysis, radon concentration was associated with non-mucinous histology and low tumoral grade (p=0.033 and p=0.023, respectively).

Conclusion:
Our final results have shown no consistent association between residential radon and molecular alterations in NSCLC patients, but a trend has been suggested in ALKr and BRAFm. Large multicenter studies are needed to confirm this hypothesis.

Background:
India with 11.2%(111.9 million) of world’s smokers has 2[nd ]largest population at elevated risk of lung cancer. Almost twice, 206 million(GATS, 2010), are Smokeless tobacco(ST) users in India, highest globally. Supreme Court of India observed that gutka and pan-masala are food products. Beginning in 2012, almost all state governments in India banned gutka and pan-masala containing tobacco. APPREHENSION was raised that ban on ST products will cause switching to smoking by huge ST user population vastly increasing risk of lung-cancer in India. This ban provided natural experiment on which this observational research studied how ban on popular ST products alters pattern of tobacco-use, especially smoking. Findings are expected to be strategically significant to inform future policies.

Methods:
Questionnaire of Global Adult Tobacco Survey-India(2010), developed by WHO,CDC and Govt. of India was modified to answer research questions and accommodate retrospective-cohort study design. Through 2-step randomization process, 500 households were sampled from Delhi. Participants were adults and interviewed during March-June,2016 comprehensively, including tobacco-use currently and before gutka-ban. Inbuilt mechanisms in standardized questionnaire cross-validated self-report and minimised recall bias. Data was entered into SPSS and statistically analysed.

Results:
94% of 500 households visited agreed to participate. 73.4%of pre-ban gutka-users switched to twin-sachet (pan-masala and chewing-tobacco sold separately by gutka-manufacturers to circumvent law). Delhi’s order bans all ST products. But, except premixed gutka, remaining ST products are freely available and consumed. 21.8%switched to khaini or other ST products. A large fraction switched from singledose sachets to multidose sachet. Interestingly, 96.2%respondents believed tobacco as very harmful(84.6%) or somewhat harmful(11.6%). However, only 18.6%gutka users attempted quitting after ban. 4.8%successfully quitted. In our sample, we DIDNOT find anyone switching to smoking due to gutka unavailability. On an opposite thought, one may expect, ban on an ST product(Gutka) will increase awareness and motivate smokers to quit as spillover effect. But it wasn’t observed either.

Conclusion:
In absence of strong quitting promotion campaign, ban on selective tobacco products has limited role in changing prevalence of tobacco use. If selective ST products are banned, ST users preferably switch to other available ST products, BUT NOT to smoking. As majority ST users switched instead of quitting (after gutka-ban without simultaneous quitting campaign), we may logically conclude that effective ban on all ST products may lead ST users to switch to less favourable option of smoking. This is, however, subject to verification by similar study if there is ever effective ban on all ST products.

Background:
Diffuse pulmonary fibrosis may progress into lung cancer through continuous accumulation and rapid proliferation of fibroblasts and repeated epithelial injury. Repetitive injury and repair can lead to multiple genetic alterations affecting cellular growth, differentiation, and survival, which may elicit malignant potential in the injured area. Diffuse pulmonary fibrosis appears on chest images through expression of bilateral reticular or reticulonodular opacities, called interstitial lung diseases. The clinical significance of these diseases remains poorly understood. To investigate whether interstitial lung diseases increase lung cancer incidence in a cohort of patients from a national population.

Background:
Diffuse pulmonary fibrosis may progress into lung cancer through continuous accumulation and rapid proliferation of fibroblasts and repeated epithelial injury. Repetitive injury and repair can lead to multiple genetic alterations affecting cellular growth, differentiation, and survival, which may elicit malignant potential in the injured area. Diffuse pulmonary fibrosis appears on chest images through expression of bilateral reticular or reticulonodular opacities, called interstitial lung diseases. The clinical significance of these diseases remains poorly understood. To investigate whether interstitial lung diseases increase lung cancer incidence in a cohort of patients from a national population.

Results:
A nationwide retrospective cohort study using Korean Health Insurance Review and Assessment Service data, including 13,666 patients with interstitial lung disease (6.4% with concomitant idiopathic pulmonary fibrosis) diagnosed January–December 2009. The end of follow-up was June 31, 2014. Up to four matching chronic obstructive pulmonary disease controls with and without concomitant interstitial lung disease (8,012 cases) were selected to compare the lung cancer high-risk group. Lung cancer was counted after diagnosis of interstitial lung disease, idiopathic pulmonary fibrosis, or chronic obstructive pulmonary disease.

Conclusion:
The incidence of lung cancer was 126.9 cases per 10,000 person-years (2,732 cancers) in the chronic obstructive pulmonary disease group, 156.6 (809 cancers) in the interstitial lung disease group, and 370.3 (967 cancers) in the chronic obstructive pulmonary disease with interstitial lung disease group. Among various interstitial lung disease definitions, idiopathic pulmonary fibrosis showed the highest lung cancer incidence. A total of 112 of the 879 patients with idiopathic pulmonary fibrosis developed lung cancer, an incidence of 381 cases per 10,000 person-years. Interstitial lung diseases have high potential to develop into lung cancer even when combined with chronic obstructive pulmonary disease.

Background:
Tobacco use is a major preventable cause of premature death and diseases, currently leading to five million deaths worldwide which are expected to raise over eight million deaths worldwide by 2030. India is the second largest consumer of tobacco in the world. Tobacco use is a leading cause of deaths and disabilities in India as well, killing about 1.2 lakh people in 2010. About 29% of adults use tobacco on a daily basis and an additional 5% use it occasionally. This study is contemplated with an aim to assess the prevalence of tobacco consumption and the associated factors involved in its consumption, as this group of the population is under constant pressure and account for the workforce of the country. So through this study we could be able to know * The reasons of consumption. * Amount of consumption *Awareness of ill effect of tobacco consumption* Out of Pocket expenditure.

Methods:
ACross sectional descriptive study was conducted among Auto Rickshaw Drivers in Chennai City.Auto drivers who were working for more than two years and present on the day of examination and who were willing to participate in the study were included.Cluster random sampling technique was used. 400 samples were selected from 40 auto stands of various parts of Chennai City.Data was collected using a Survey Proforma which comprised of a Questionnairewhich can assess the frequency of consumption, age of initiation, the amount of consumption, mental stress, economic factors, any past history of disease and most importantly the awareness towards oral cancer.The data recorded was transferred and analysed using SPSS version 20.Chi- square test was used to test the significance between groups.

Results:
Prevalence among auto rickshaw drivers for consumption of tobacco products was very high (87%). Auto rickshaw drivers were mostly used tobacco in the form of Gutkha (72%) and bidi (40%) in comparison to other products. In the opinion of auto rickshaw drivers increase in tax may reduce it consumption and the majority of drivers (70%) think that tobacco must be banned.

Conclusion:
Prevalence of tobacco use among auto rickshaw drivers was very high. Mostly they use tobacco products to reduce stress, to be awake or to remove nervousness but a large number of participants also use them without any reason. Almost one half of the study population was suffering from tobacco related diseases like cough, ulcer on mouth, lung disorder. They are in definite need of tobacco cessation activities.

Background:
Tobacco use continues to be the leading cause of preventable disease and it is responsible for more than 5 million deaths each year worldwide. Despite this, there are still 650 million smokers in the world. The prevalence of smoking among adults accounts for approximately 25% deaths annually. smoking remains the main cause of mortality and morbidity in the developing nations. Healthcare professionals have an important role to play both as advisers influencing smoking cessation and as role models. However, many of them continue to smoke. Several studies have demonstrated the efficacy of smoking cessation programs and the importance of physician’s advice to their patients. The aims of the present study are as follows: (i) to evaluate smoking prevalence, knowledge and attitudes, and tobacco cessation training (ii) to examine the difference between smokers and nonsmokers;

Methods:
A structured questionnaire consisting of 14 questions related to tobacco/smoking habits, cessation training and role of health professionals in tobacco control were asked to the study population and their response was recorded. Random sampling method was used and data was collected from a cross-sectional survey. The surveywas conducted between January and February 2015. Statistical analysis was done using SPSS version 17 and Logistic regression model was used to identify possible associations with tobacco smoking status. The level of significance was

Results:
A total of 259answered the questionnaire of which 29% declared to be smokers. About 53% of the males have smoked at least once in their life and the age of cigarette initiation was 16-17 years for 28% of the sample.76%considered health professionals as behavioural models for patients, and 96% affirmed that health professionals have a role in giving advice or information about smoking cessation. Although 87% heard about smoking related issues during undergraduate courses, only 17% received specific smoking cessation training during specialization. 93% of the sample agreed that health professionals should receive specific training on smoking cessation according to while 6% were of the opposite opinion.

Conclusion:
The present study highlights the importance of focusing attention on smoking cessation training, given the high prevalence of smokers among physicians specializing in medicine and dentistry, their key role both as advisers and behavioural models, and the limited tobacco training offered in the curriculum. In the field of public health, tobacco screening, and intervention is one of the most effective clinical preventive services.

Background:
Albania is a country with a high prevalence of smoking but a national cancer registry has not been initiated yet and data on lung cancer are scarce.

Methods:
Aim - Methods: In 2010-2014, 1254 patients presented to our hospital with either symptoms or an abnormal finding in their chest X-ray and were diagnosed with lung cancer. This is a descriptive retrospective study, reporting data on the histological type of cancer and smoking history

Results:
Results: Of the 1254 patients, 79% (n= 1001) were men and 21% (n=253) women . Age range was (16-89), with mean age in men 62.4 ±8,5 and in women 58±10,2. Diagnosis was confirmed by histology [table 1] : Regarding NSCLC, 78% of patients had an advanced stage (III and IV). Only 268 patients were non-smokers, 126 were ex-smokers and the remaining 67 % (n=860) were current smokers with high exposure (92 pack/years). Day hospital avarage is 7 day,and day range was(1-21) with SD± 6.4. Performance status was:60.2% improved,35.2% idem,3.3% dead in hospital.

	Squamous cell carcinoma	Adenocarcinoma	Small cell	others	Total
men	56%(n=560)	22%(n=220)	15%(n=150)	7%(n=71)	100%(n=1001)
women	11%(n=27)	72%(n=183)	5%(n=13)	12%(n=30)	100%(n=253)

Conclusion:
In Albania,lung cancer is an increasing pathology (p<0.005) and there is a high prevalence of squamous cell carcinoma especially in men,probably associated with the heavy history of smoking and most patients are diagnosed at a late stage.Policies for smoking cessation should be strengthened and a lung cancer screening program should be initiated.

Background:
Tobacco consumption, and more specifically active smoking, remains the main risk factor for lung cancer (LC) and continues to be the target of awareness campaigns worldwide. However, in recent decades, other risk factors have been identified, including passive smoking, atmospheric pollution and occupational exposure. This analysis focuses on awareness of LC risk factors among the lay population and physicians.

Methods:
The 4th French nationwide observational survey, EDIFICE 4, was conducted by phone interviews of a representative sample of 1602 subjects, aged between 40 and 75 years, from June 12 to July 10, 2014. A mirror survey was also conducted by phone among physicians between July 9 and August 8, 2014. Both surveys were conducted using the quota method on representative samples of 1602 lay persons and 301 physicians. The following analyzes were conducted amongst 1463 lays persons with no history of cancer and 301 physicians. Interviewees were asked to cite the five main risk factors for LC.

Results:
LC risk factors associated with tobacco in general were widely cited in first position by both physicians and the lay population (100% and 96%, respectively; P≤0.01), with the role of active smoking (100% vs 94%, P≤0.01) and passive smoking (77% vs. 68%, P≤0.01) clearly identified. Twice as many physicians cited asbestos as a risk factor, ranking it in second place, compared with the lay population (77% vs. 30%, P≤0.01). Atmospheric pollution was cited to the same degree by physicians and the lay population (49% vs. 43%, P=0.05), the latter ranking it second. Heredity and family history came fourth (32% vs. 13%, P≤0.01) and alcohol fifth (13% vs. 10%, not statistically significant), in both populations. Infections and other respiratory disorders were cited by less than one person in ten (7%). Poor dietary habits were very rarely cited by either physicians or the lay population (<1% vs 4%, respectively, P≤0.01).

Conclusion:
The awareness of risk factors for lung cancer is broadly consistent with the established risk factors, among both physicians and the lay persons in our survey. As expected, tobacco was ranked first, followed by atmospheric pollution and asbestos, though the latter is less present in the mind of the lay population compared to physicians. It is noteworthy that even among physicians, a history of respiratory disorders was only marginally acknowledged.

Background:
Chronic airway inflammation has been emerging targets for lung cancer chemoprevention as well as treatment of COPD. The aim of the present study was to determine the role of roflumilast and aerosolized budesonide in benzo(a)pyrene-induced lung cancer in mice and to elucidate the possible their mechanisms.

Methods:
Female A/J mice were given a single dose of benzo(a)pyrene. Intraperitoneal administration of roflumilast (1mg/kg, 5mg/kg) began 2 weeks post-carcinogen treatment and continued tri-weekly for 28 weeks. Aerosolized budesonide was administered by aerosol delivery for 2 min/day and 5 days/week. Tumor load was determined by averaging the total tumor volume in each group.

Results:
Benzo(a)pyrene induced an average tumor size of 9.4 ± 1.8 tumors per mouse, with an average tumor load of 19.5 ± 3.8mm[3]. Roflumilast treatment at 1 and 5 mg/kg did not inhibit tumor number, however, reduced tumor load, an average of 8.8 ± 2.0 mm[3] at 5mg/kg treatment, significantly. Aerosolized budesonide administration did not show reductions of tumor number or load. The decreased expressions of cyclic AMP and protein kinase A caused by benzo(a)pyrene were increased by roflumilast treatment. NF-κB expression in tumor tissues was lower in the roflumilast group than the place group.

Conclusion:
In vivo experiments in the benzo(a)pyrene-induced model of lung cancer show that roflumilast significant inhibits tumorigenicity via suppression of inflammation. Possible mechanisms between cAMP pathway and lung cancer development will needed to be determined.

Background:
Kava is extracted from the roots of piper methysticum and is consumed by South Pacific Islanders as a relaxing beverage. Epidemiologic evidence points to a protective effect of kava against tobacco-induced lung cancer. NNK is a potent tobacco-specific carcinogen indisputably linked to lung cancer formation. Kava reduced NNK-induced lung adenoma formation in the A/J mouse model. Data also suggest that enhanced NNAL detoxification may be a potential mechanism by which kava exerts a chemopreventive effect. In humans, urinary NNAL is a validated biomarker of NNK uptake. We conducted a clinical trial in smokers to assess the effect of kava on NNK metabolism. The primary objective was to compare urinary total NNAL before and after kava administration. Secondary objectives included comparing the NNAL-gluc/NNAL-free ratio, determining the safety of kava, and quantifying O[6]-methylguanine adducts. The hypothesis was that kava administration would result in increased levels of NNAL in the urine (and increased NNAL-gluc/NNAL-free ratio), reflecting increased elimination and/or increased detoxification of NNK. Additionally, we hypothesized that kava could reduce O[6]-methylguanine adducts.

Methods:
We conducted a single-arm, open-label clinical trial in adult healthy smokers, in which subjects took a commercial kava supplement three times daily for seven days. Twenty-four hour urine collections were collected at baseline, days 4-5, and days 6-7 of the kava intervention for NNAL quantification. Blood samples were collected at baseline, day 4, and day 7 of the kava intervention for safety monitoring and for DNA adduct analysis. Subjects also completed a detailed tobacco questionnaire, food diary, smoking diary, and cigarette evaluation scale (CES) questionnaire. To date, 17 subjects (goal = 18) have completed the study.

Results:
The results and statistics are being finalized. Short-term kava administration was safe with no evidence of hepatotoxicity. Subjects experienced less of the reinforcing effects of smoking after short-term kava administration as determined by the CES scores. The total CES score decreased on average by 4.47, from 45.53 to 41.06 (p=0.053, 95% CI -0.06-9.01). Notably, the smoking “satisfaction” scores decreased by 0.607 (p=0.024, 95% CI 0.09-1.12).

Conclusion:
This is the first study investigating the effect of kava on NNK metabolism in humans and is the first step to gain a more sophisticated mechanistic understanding of kava’s role in potentially modulating tobacco-related lung cancer risk. Short-term kava administration is safe in healthy adult smokers. Kava holds potential as a possible chemopreventive agent for smokers or tobacco cessation aide.

Background:
Smoking accelerates the incidence of asbestos-related lung cancer. We evaluated emphysematous changes by chest CT and pulmonary function for asbestos-related lung cancer in Japan.

Methods:
Two hundred and twenty-two patients of asbestos-related lung cancer compensated by Japanese compensation law were evaluated as age, gender, smoking index, histology, survival, therapy and occupational history including first asbestos exposed age, asbestos exposing terms and latency from the first asbestos exposure to lung cancer． Radiographic evaluation was done by chest CT using Goddard classification of emphysema. Pulmonary function test was done by spirometry and flow-volume curve.

Results:
Ages range from 49 to 92 years with a median of 75 years. Male occupied 97.7%. Non-smoker is only 13 patients and other 209 are smokers with Brinkman Index ranges from 45 to 3000 of a median of 900. For histology of lung cancer, 60.4% are adenocarcinoma and 22.4% of squamous cell carcinoma, 12.6 %of small cell carcinoma and 1.8 of large cell carcinoma and 2.6 % of pleomorphic carcinoma et al. Eighty seven patients were operated and other 87 patients performed chemotherapy. Best supportive therapy is 34 patients. Median survival was 15.8 months. For asbestos histories, median first exposed age was 23 years, asbestos exposing term was 32 years and the latency of lung cancer was 50 years. For Goddard score of emphysematous changes, 28% showed 0 point and 33% of 1~4 points and more than 21 points occupied only 4%, which means very low percentages of emphysematous changes for these asbestos-related lung cancer, nonetheless of high percentages of heavy smokers. For pulmonary function test, FEV1.0% is 70.5%±11.3% and %FEV1.0 is 85.6±22.2%. More than half patients are normal pulmonary function except more than 1,000 of Brinkman index or more than 15 points of Goddard score. From the classification of GOLD criteria, 54.1% are normal, stage 1 is 20.7%, stage 2 is 22.5 % stage 3 is 1.8% and stage 4 is only 0.9%.

Conclusion:
Almost all of asbestos-related lung cancer in Japan are heavy smoker, but 61% showed none or low grade of emphysematous changes by chest CT and only 2.7% had severe pulmonary dysfunction.

Background:
Lung cancer (LC) is the most common and deadliest cancer in the world. In the Republic of Macedonia, within the period 2002-2012, the LC took the first place according to the frequency of appearing in men, while it was on the fourth place in women. The number of the risk factors is great being connected with the occurrence of LC. The aim of this study was to analyze the role of genetic factor, professional exposure and the habit of cigarette smoking in occurrence of lung cancer.

Methods:
The research was conducted as a case-control study. It included 185 patients diseased of LC (investigated group-IG) and the same number of persons without malignant disease (control group-CG). In the study were included only interviewees with pathohistologically confirmed LC. Through calculating the risks of the Odds ratio (OR), the risk-factors, which had a role in occurrence of the disease, were quantified, while with the Confidence intervals (CI), the statistical significance for the error level less 0,05 (p) was defiend.

Results:
According to the investigation results, malignant disease of two members in one family was found in 13,5% of the IG, 9,4% of the CG, respectively. Current smokers (CS) with present hereditary factor had almost 4 times (OR=3,95; 95%CI, 1,78-8,77), greater risk to become ill compared to the never smokers (NS) without hereditary factor. The risk was greater when the same would be compared to the NS with present hereditary factor (OR=8,76; 95%CI, 1,80-42,68).In the diseased, the professional exposition was present in 68,6% from IG, versus 67% in the CG. The highest risk for LC was found in transport workers (OR=2,50;95%CI, 1,01-6,15) and automehanics (OR=2,31;95%CI, 0,76-7,07). CS represented 67% of diseased individuals versus 40,5% of the CG. The risk for them to develop LC was 5,54 (95%CI, 3,0-10,23), times significantly greater compared to the NS. The risk for the disease was significantly greater in individuals who were smoking >20years (y), >20cigarettes/day (c/day), compared to those, who, in the same time period, smoked <20c/day (OR=3,78;95%CI, 2,04-7,01). The risk to develop LC in former smokers (FS), who >20y smoked >20c/day was 2,40 (95%CI, 0,94-6,14), times greater compared to those, who smoked >20y, <20c/day.

Conclusion:
This disease developed twice more commonly in the examined individuals, exposed to professional carcinogens. The LC is multifactor disease for which development, besides smoking, as a main determinant, in mutual interaction are the genetic and other factors of the surrounding and the way of living.

Background:
Diets rich in polyphenols are well-known to reduce lung cancer risk among high-risk populations. We analyzed the efficacy of polyphenols-rich Haskap (Lonicera caerulea L.) fruit extracts in preventing tobacco specific nitrosamine (TSNA)-induced DNA damage in BEAS-2B lung epithelial cells.

Methods:
Monomeric polyphenols of Haskap fruits were extracted in ethanol and water, and profiled. TSNA, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-[(acetoxymethyl) nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) were used (at sub-lethal concentrations) independently to induce the carcinogenesis process in BEAS-2B cells. Cell viability assay was confirmed that the tested concentrations of Haskap extracts were not cytotoxic to BEAS-2B cells.

Results:
The Haskap extracts contain diversity of polyphenols including phenolic acids and flavonoids, however, cyanidin-3-O-glucoside was the most predominant. Pre-treatment of cells with the Haskap extracts could significantly reduce the NNK- and NNKOAc-induced DNA double strand breaks, DNA fragmentation and intracellular reactive oxygen species, compared to non-treated cells. Immunocytochemistry for H2AX-phosphorylation (Serine 139, red) A. NNKOAc 100 µM, 3 h; B. Haskap ethanol extract (50 µg/mL, 3 h)+NNKOAc (100 µM, 3 h). DNA counter-staining was performed with 4,6-diamino-2-phenylindole (blue). Figure 1Figure 2





Conclusion:
The polyphenols-rich Haskap extracts could prevent TSNA-induced DNA damage in lung epithelial cells in vitro. Protective effects of Haskap polyphenols against DNA damage are being investigated in vivo using A/J mice.

Background:
Eventhough lung cancer remains a disease of a median age at diagnosis of 70y, a proportion of patients are diagnosed at 40y or younger. Patients diagnosed before the age of 40 tend to be never-smokers, are stage IV adenocarcinoma, and tend to have an EGFR activating mutation or a EML4-alk translocation. It is crucial to determine the epidemiological characteristics of patients younger than 40y. Our study groups the largest population of patients less than 40y diagnosed with NSCLC.

Methods:
In this epidemiological retrospective study, 249 patients (Argentina=6, Canada=19, Colombia=29, Costa Rica=9, Mexico=89, Nicaragua=2, Panama=19, and Peru=76) with a histologically confirmed NSCLC aged 40 years or less at diagnosis were included. Data included age, gender, histology, stage, EGFR and alk mutation analysis, and date of death or last follow-up. Progression free survival (PFS) and overall survival (OS) were also recorded.

Results:
NSCLC patients aged 40 years or less accounted around a 4% of the total NSCLC population. Median age was 34.5 years (range 14-40), 137 (55%) were women, and 192 patients (77.1%) were non-smokers. Adenocarcinoma was the most frequent histological subtype with 203 patients (81.6%) and 24 patients (9.6%) were squamous. 214 patients (85.9%) were stage IV and 23 patients (9.2%) were stage III at diagnosis. The site(s) of metastasis was obtained in 203/214 stage IV patients where 39.9% (n=81) had lung, 35.6% (n=72) had SNC, and 31.7% (n=64) had bone metastasis. EGFR mutation (EGFRm) analysis was determined in 103 patients with 40 patients (38.8%) having an EGFRm. EML4-alk analysis was determined in 165 patients with 11 patients having a positive translocation (6.7%). The OS for all patients was 14.4 months (95%CI=11.2-17.6), PFS was 5.7 months (95%CI=4.9-6.5), and there was no significant difference according to histological subtype. OS for EGFRm(+) was 42 months (95%CI=30.8-54.0) and for EGFRm(-) was 19.4 months (95%CI=14.8-24.0) (p=0.002); PFS for EGFRm(+) was 11.9 months (95%CI=6.3-17.5) and for EGFRm (-) was 7.1 months (95%CI=5.3-8.9) (p=0.005). OS for alk(+) was 28.0 months (95%CI=15.4-40.6) and for alk(-) was 10.6 months (95%CI=6.9-14.3) (p=0.065).

Conclusion:
NSCLC patients aged 40 years or less constitute a small but important proportion of patients with this diagnosis. Other risk factors may be involved in the pathogenesis of the disease in this population due to a low smoking history found. SNC metastasis at diagnosis seems to be more frequent in this population. EGFR mutation and EML4-alk translocation frequency is higher than the frequency reported in the general population.

Background:
Although prisoners could be at higher risk for lung cancers, very few studies focused on that particular population. In a previous cohort study (Carbonnaux et al. Oncology 2013;85:370–377), we found an early onset of lung cancer in imprisoned patients. The aim of the CARCAN study was to assess epidemiological characteristics, management, prognosis and incidence of lung cancer among prisoners compared to general population.

Methods:
We designed a multi-centric observational case-control study. Cases were lung cancer diagnosed in prison in 3 penitentiary medical units (PMU) of France from 2005 to 2013 (Lyon / Marseille / Toulouse). Up to 3 controls were selected for each case from hospital databases. Controls were randomly matched to cases for center, sex, and year of diagnosis. Overall and age-specific cumulated incidences were calculated in the penitentiary area covered by the 3 participating PMU and in the French population using national statistics.

Results:
Overall, 170 controls and 72 cases met the inclusion criteria and were analyzed. Cases were mainly men (99%). Mean age at diagnosis was 52.9 (±11.0) in prisoners and 64.3 (±10.1) in controls patients (P<10-4). Most of prisoners were current smokers compared to controls (83% vs 53%; P<10-4). We did not find significant difference in histologic type or TNM stage at diagnosis between the two groups. Also, there was no significant difference in first-line treatment type in both groups; especially there was no difference in the rate of patient undergoing supportive care only. Median time from first symptoms to first treatment was 3.3 months [2.7-3.9] in controls compared to 3.6 months [2.7-4.4] in prisoners (P=0.947). We found no significant difference in progression free and overall survival between the two groups. Cumulated incidence (2008-2013) in men was dramatically increased in prisons in each age category compared to the French incidence. Incidence was 4.5 fold higher in prisons than in the general population among 30-40 years old peoples; 3.4 fold higher in 40-50 yo and 1.4 fold higher in 50-60 and 60+ yo categories.

Conclusion:
There is a dramatic shift of lung cancer toward young peoples in prisons. However, presentation, management and prognosis are similar in prisoners compared to controls. These finding should justify a specific screening policy in that high-risk population.

Background:
Health professionals play an important role in cessation and prevention of tobacco use by providing a brief counseling or even a simple advise to their patients. Smoking habit among health professionals themselves may deter them from providing cessation advice and counseling to their patients. Using GHPSS data, we aim to provide updated global, regional, country-level estimates on prevalence tobacco use among medicine, dentistry, nursing and pharmacy students and describe their attitudes towards tobacco cessation training.

Methods:
The Global Health Professions Student Survey collects data on cigarette smoking and use of other tobacco products, training received to provide patient counselling on cessation techniques etc. We analysed country-wise aggregate data on current cigarette smoking’ (smoking cigarettes ≥1 days during the past 30 days), and ‘current use of tobacco products other than cigarettes’ (chewing tobacco, snuff, bidis, cigars, or pipes ≥1 days during the past 30 days), indicators on ‘health professionals’ role’ and ‘cessation training’. We calculated aggregate rates for each World Health Organization regions using ‘metaprop’ command in Stata-11.

Results:
In 236 surveys from 2005 to 2011 from 70 (medical), 56 (dental), 56 (nursing) and 54 (pharmacy) countries 107,527 students (68,809, girls and 37,886 boys) were surveyed. Overall, in all courses smoking was highest in Europe (20%, medical to 40%, dental students) followed by the Americas (13%, pharmacy to 23%, dental students). Other tobacco use rates were higher in the eastern Mediterranean (10-23%) and Europe (7-13%) countries. Tobaccco use among female students was lowest in Asian and African countries. In countries survyed ≥70% of students agreed that medical professionals are role models and have a role in advicing and information about smoking cessation to their patients and public. In the countries surveyed in all the regions, only about 9.2-36.9% of students (except 80% among dental students in the eastern Mediterranean) reorted that they have received formal training on smoking cessation approaches. and ≥80% of all students agreed they should receive a formal cessation training.

Conclusion:
Health professions students ready to receive cessation training. Tobacco control experts should work with medical educators to discourage tobacco use among health professional students and implement integrated smoking cessation training into their medical curricula. Implications: Our results provide a global snapshot and regional estimates of tobacco use among health professions students and cessation training. Results highlight the need for cessation advice/assistance to health professions students currently using tobacco and the need for introducing cessation training particularly in developing Afro-Asian countries.

Background:
In the lung screening population, the prevalence of lung cancer is typically a small percentage (2.3% (10/440) in our institution’s program). A much more common, treatable, and potentially overlooked condition in the lung screened patient population is nicotine addiction. The National Lung Screening Trial contained 49% current smokers. A review of our lung cancer screening program showed 70.2% (309/440) of patients were active smokers at the time of lung screening.

Methods:
Our lung screening program is designed so that all scheduling would be done by a coordinator who is a Nurse Practitioner and a Certified Tobacco Treatment Specialist. A telephone call to schedule the scan was done by the coordinator and basic tobacco cessation intervention was integrated into every call. Futher follow up as face-to-face counseling was offered. We reviewed institutional data to determine what proportion of active smokers would agree to individualized counseling when it would be conveniently offered at the point of the scan, by the person coordinating the program.

Results:
Over a consecutive 26 month period, 440 patients underwent lung screening. The majority of patients (70.2%, 309/440) were actively smoking. Telephone intervention reached 100% (309/309). The telephone intervention consisted of an Ask, Advise, Refer strategy which offered further resources including: a quitline referral, a weekly group counseling session referral, and an indepth personal counseling session which would be provided at the time and place of the screening scan. The same tobacco treatment specialist who provided telephone intervention, met face-to-face with 80.6% (249/309) of active smokers for in depth counseling and development of a cessation plan.

Conclusion:
Our lung screening program detected lung cancer in a minority of participants (2.3%, 10/440) but encountered nicotine addiction in the majority of participants (70.2%, 309/440). Positioning Certified Tobacco Treatment Specialists as coordinators of lung screening programs ensure that all participants receive at minimum a telephone intervention. The initial telephone intervention coinciding with scheduling the screening scan allowed a relationship to develop between the patient and the coordinator (who is tobacco cessation specialist). Most participants who were smoking (80.6%, 249/309) agreed to in depth counseling which was conveniently provided at the point of service with the screening scan. Without integration of the resources, few patients would have sought out cessation counseling. As lung screening will recur annually, this will provide longitudinal support. Further data about acceptance of counseling and data about long term cessation in a lung screening program will be gathered.

Background:
Lung cancer is a devastating disease with a poor prognosis. Chemoprevention has came out as a very promising protective strategy against cancer and numerous natural compounds in diet have shown their curative potential on lung cancer. Catechin is mainly found in green tea and possess anti-oxidative, anti-inflammatory and antiproliferative activity. The present study was designed to investigate the mechanism-based chemopreventive nature of catechin hydrate (CH) against B(a)P induced lung carcinogenesis in Swiss albino mice and possible role of aldehyde dehydrogenase 1 (ALDH1).

Methods:
B(a)P was administered orally (50 mg/kg body weight) twice a week for four successive weeks to induce lung cancer in mice. CH was supplemented to mice at doses of 20 and 40mg/kg b. wt. The body weight, lung weight, lactate dehydrogenase (LDH), lipid peroxidation (LPO), xanthine oxidase (XO), carcinoembryonic antigen (CEA), antioxidants armory activities (SOD, CAT, QR, GPx, GR, GST and GSH) were estimated. Further, histopathological analysis of lung tissue and Immunohistopathology analysis of ALDH1, VEGF, PCNA, NF-kB, COX-2, caspase-3 and Bcl-2 were also carried out

Results:
Administration of B(a)P resulted in increased XO, LPO, LDH, and CEA with subsequent decrease in activities of tissue anti-oxidant armory. It also resulted in up-regulation of VEGF, PCNA, NF-kB, COX-2, caspase-3 and down regulating Bcl-2. ALDH1 expression was also increased in B(a)P-induced lung cancer group. Pre-treatment with CH at a dose of 20 and 40 mg/kg b. wt. significantly decreased in XO, LDH, LPO, CEA and increased anti-oxidant armory. Moreover, assessment of protein expression revealed that CH pre-treatment effectively regulated hyperproliferation, inflammation and apoptosis in lung of mice. Immunohistochemical analysis also revealed that CH pre-treatment showed significantly reduced in ALDH1 expression. Further, the antiproliferative effect of CH was confirmed by histopathological analysis.

Conclusion:
Overall, Our findings suggest that catechin hydrate inhibits B(a)P-induced lung tumor formation by modulating hyperproliferation, inflammation, apoptosis and ALDH1 expression.

Background:
Surgical resection is the optimal treatment modality for NSCLC, while smoking has been shown to have a negative survival impact. We evaluated smoking’s impact on overall survival within a population-based cohort of patients with surgically-resected NSCLC.

Methods:
We examined all patients who had a curative-intent NSCLC resection from 2009-2016 in 4 contiguous Dartmouth Hospital Referral Regions of the US. We compared patient and clinical characteristics among never, former (stopped >1 year prior), and active smokers using the Chi-square and ANOVA tests. Survival analyses were conducted with the Kaplan-Meier method and Cox Proportional Hazards models.

Results:
Of 2,202 patients, 206 (9%) were never, 846 (38%) were former, and 1,150 (52%) were active smokers. Significant demographic and clinical differences between cohorts included age, sex, race, insurance, comorbidities, pulmonary function, method of detection, ASA status, extent, primary site and length of resection, histology, and histologic grade (all p<0.05). Short-term post-operative mortality (at 30-, 60-, 90-, 120-days) rates for never smokers were 1%, 2%, 4%, 4%; for active smokers, 4%, 6%, 7% and 8%; and for former smokers, 5%, 7%, 9%, and 11%; and differed significantly by smoking status (p=0.0539, p=0.0316, p=0.0187, p=0.0017). At 5 years, overall survival was 69% for never smokers, 55% for active, and 49% for former smokers (p=0.0002) (Figure 1). Controlling for age, sex, race, insurance, histologic grade, extent of resection, and length of surgery, and compared with never smokers, active smokers had 1.3 times (p=0.05) the hazard of death and former smokers had 1.4 times the hazard of death (p=0.04). Figure 1



Conclusion:
In this population-based cohort, smoking is negatively associated with post-operative mortality and long-term overall patient survival; although active smokers had better survival outcomes than former smokers.

Background:
Smoking cessation interventions are often ineffective, although negative health effects of smoking are well established. However, evidence suggests that diagnosis of a severe medical condition or a surgical intervention may force people to quit smoking without any counseling. Aim of this study was to determine the smoking cessation rate among patients undergoing lung resection and factors associated with perioperative smoking cessation.

Methods:
All lung resection patients in one thoracic surgery department in 6 years were included. A phone-interview was conducted with all (accessible) patients aged > 16. Wilcoxon rank-sum test, and chi-squared or Fisher exact test were used for statistical analysis.

Results:
In 6 years 970 patients were operated on; 406 (229 male, 177 female; mean age 56.4 [range 16 to 85] years) were available for the study. At the time of surgery 155 patients (38.2%) were non-smokers, 82 (20.2%) ex-smokers, and 169 (41.6%) current smokers. 56.3% of males and 22.6% of females were smokers (p<0.0001). 145 patients had lung cancer and 261 patients other causes for lung resection, with different smoking distribution in these 2 groups (p<0.0001). Sixty nine patients (40.8%) quit smoking before the operation: 22 due to the planned operation, 23 due to the newly diagnosed disease, and 24 for other reasons. Seventy two patients (42.6%) did not smoke after hospital discharge including 66 (39.1%) also a year later. An additional 40 (23.7%) patients had tried to stop, and 57 (33.7%) continued smoking. The quit rate was higher among lung cancer patients versus others (uncorrected p=0.007), and patients operated through thoracotomy versus VATS (uncorrected p=0.0295); and was not influenced by age, gender or duration of smoking before quitting.

Conclusion:
Almost 40% of patients undergoing lung resection stopped smoking without special counseling, with very few restarting. Smoking cessation rate was higher among patients with lung cancer and patients operated through thoracotomy.

Background:
The association between cigarette smoking and lung cancer mortality is well known. Some studies have shown a decreased overall survival (OS) in early stage non-small cell carcinoma (NSCLC) patients that continue to smoke after diagnosis. It is documented that in patients with metastatic disease, continued smoking increases resistance to systemic therapies but the impact of smoking cessation during treatment on outcomes for these patients is not well defined. Objective: To evaluate the impact of smoking cessation, before initiation of chemotherapy (CT), on survival in advanced NSCLC.

Methods:
Patients referred to our centre, between January 2010 and June 2016, and diagnosed with metastatic NSCLC were analysed. Patients defined as smokers at diagnosis and treated with at least one cycle of chemotherapy were included. Clinical characteristics and survival outcome were reviewed and compared between patients who quit smoke before and after the initiation of chemotherapy.

Results:
A total of 113 patients were included [mean age 59±10 years; 89.4% (n=101)]. The histological type more predominant was adenocarcinoma (70.8%) and the most common sites of metastasis were lung, bone and brain (35.4%, 23.9% and 23%, respectively). The majority of patients had performance status 1 and no weight loss at time of diagnosis (53.1% and 58.4%, respectively) and the comorbidity most prevalent was hypertension (19.5%). The average number of cigarettes smoked was 51±23pack-years and 81.4% of patients smoked >30pack-years. The most used CT regimen was platinum combined with pemetrexed (63.7%). Patients who quit smoking before CT showed a better median OS although not statistical significant (8 vs. 7 months; p=0.478). This was also seen in heavy smokers ≥30 pack-years, with a median OS of 8 vs. 6.5 months (p=0.674). The multivariate analysis only showed an influence of type of CT on survival.

Conclusion:
Although not significant differences in OS between groups were observed in our sample, the median survival was better in patients that quit smoking before the initiation of CT, even in heavy smokers. Continued smoking after CT initiation is known to adversely affect treatment response and quality of life and efforts to encourage smoking cessation even among this population of patients should be made.

Background:
Tobacco use is a leading preventable cause of disease, disability and death worldwide. To expand the fight against the tobacco epidemic, WHO has introduced the MPOWER package of six proven policies:1.- Monitor tobacco use and prevention policies, 2.- Protect people from tobacco smoke, 3.- Offer help to quit tobacco use 4.-Warn about the dangers of tobacco 5.-Enforce bans on tobacco advertising, promotion and sponsorship, and 5.-Raise taxes on tobacco. The aim of this study was to evaluate the student´s perception about the effectiveness of each intervention.

Methods:
Students from public and private universities in Panama city were surveyed. Students were asked to evaluate each policy in a binary answer (less effective or very effective). Chi squared test was used to compare answers between smokers and never smokers

Results:
302 students answered the questionnaire: 157 females (52%) and 145 males (48%). Median age was 21 years. There were 73 smokers (24.2%) and 229 never smokers (75.8%). Median age of start smoking was 16 years (10-25), median of cigarettes per week was 6 (1-48). There were not discrepancies in effectiveness between the two groups in monitoring tobacco use policies (p=0.31). 56% of never smokers and 28% of smokers considered that protect people from tobacco smoke is very effective (p<0.001). Offer help to quite tobacco is considered very effective in 31% of smokers versus 52% of never smokers (p=0.003). To require effective package warning labels is very effective in 24.6% of smokers and 48% of never smokers (p<0.0001). Implement counter-tobacco advertising is equally effective for half of both groups (p=0.06). To obtain free media coverage of anti-tobacco activities is very effective in 53% of never smokers and 32% of smokers (p= 0.002). To enforce bans on tobacco advertising promotion and sponsorship is very effective in 46% of never smokers and 52% of smokers (p=0.34). Increase tax rates for tobacco products and ensure that they are adjusted periodically to keep pace with inflation and rise faster than consumer purchasing power is very effective for 46% of smokers and 57% of never smokers (p=0.09). Strengthen tax administration to reduce the illicit trade in tobacco products did not show difference in effectiveness of both groups (p=0.15).

Conclusion:
MPOWER policies are useful to prevent smoking. The perception of the effectiveness of each intervention varies according tobacco use.

Background:
The tobacco epidemic is one of the biggest public health threats the world has ever faced.Tobacco used is a widespread phenomenon in Bangladesh and that causes numerous deaths and disabilities in a year. The studues conducted elsewhere have strengthened the evidence that mass media campaigns conducted in the context of comprehensive tobacco control programs can promote quitting and reduce smoking as well as smokeless tobacco prevalence.Awareness building campaigns in mass media against tobacco use should be prioritized more and this paper will be an initiative towards enhancing mass media's role in controlling tobacco in Bangladesh.

Methods:
This is a qualitative study and both primary, as well as secondary data were used where information gathered through the Key Informant Interviews (KIIs) and media contents. The employees of media houses (five national papers, two online news portals and six TV channels) were selected as study respondent. Media Content Analysis is used through the broad range of ‘texts’ from transcripts of interviews and discussions along with the materials like reports, footages, advertisements, talk-shows, articles etc.

Results:
The study result documented several opinions of discussants where Mass media was found to play a strong role in support of the amended tobacco control law and its implication that could be created public support against tobacco farming, exposing to companies’ tactics and other tobacco control activities. The study results also revealed that in controlling tobacco supply and demand effectively, media has been assisting the government and anti-tobacco activities productively. Majority of the Key Informants opined spontaneously on tobacco control program publicity, organizational interference, and influence of other activities on media. They also emphasized role of media for activities of anti-tobacco organizations, awareness building actions, popularization of tobacco control law and its amendment.

Conclusion:
The study shows evidence that mass media coverage of tobacco control issues is influencing the context of comprehensive tobacco control programs. To reduce tobacco consumption, along with strict enforcement efforts, media should be used to assist with the implementation of the tobacco control law. A sustained nationwide campaign to educate the masses against the dangers of smoking and smokeless tobacco is needed and media can play an important role in creating further awareness about the dangers associated with tobacco consumption.

Background:
Health is a state of complete wellbeing free from any discomfort and pain. Despite remarkable world-wide progress in the field of diagnostic, curative and preventive medicine, still there are large populations of people living in isolation in natural and unpolluted surroundings far away from civilisation, maintaining their traditional values, customs, beliefs and myths. India has the second largest tribal population of the world next to the African countries. About half of the world’s autochthonous people live in India, thus making India home to many tribes which have an interesting and varied history of origins, customs and social practices. The present study was conducted to assess the tobacco use, awarness and its effect on health among Malayali tribes, Yelagiri Hills, Tamil nadu, India.

Methods:
The inhabitants of the 14 villages of the Yelagiri hills, who have completed 18years and residing for more than 15years present on the day of examination and who were willing to participate in the study were included. Data was collected from a cross-sectional survey, using a Survey Proforma, clinical examinationand a pre-tested questionnaire which included Demographic data, tobacco habits. An intra-oral examination was carried out by a single examiner to assess the Oral Health Status using WHO Oral Health Surveys – Basic Methods Proforma (1997).SPSS version15 was used for statistical analysis.

Results:
Results showed that among 660 study population, 381(57.7%) had no formal education. Among the study population 75%) had the habit of alcohol consumption. Of those who had the habit of smoking, 26% smoked beedi, 10.9% smoked cigarette, 65% chewed raw tobacco, 18% chewed Hans and 28% had a combination of smoking and smokeless tobacco usage. The reason for practicing these habits were as a measure to combat the cold, relieving stress and body pain after work, and the lack of awareness of the hazards of the materials used. Prevalence of oral mucosal lesions in the study population was due to tobacco usage and alcohol consumption and lack of awareness regarding the deleterious effects of the products used.

Conclusion:
From the results of this study it may be concluded that the Malayali tribes were characterized by a lack of awareness about oral health, deep rooted dental beliefs, high prevalence of tobacco use and limited access to health services.

Background:
Background: Screening for lung cancer is recommended among heavy current or former smokers at age 55-80. Transitional Cell Carcinoma of the Bladder (TCC) and lung cancer share same risk factors, however the existence of TCC is not indicated as a reason for screening for lung cancer. Patients with invasive TCC undergo full staging and therefore lung cancer is usually detected if it co-exists. However, in superficial TCC, lung evaluation is not routinely done and may be missed. Here, we have studied the incidence of lung cancer among low stage bladder cancer patients aiming to evaluate if this can be defined as a population at risk.

Methods:
Methods: The SEER (Statistics, Epidemiology and End Results) database was used to determine the Incidence and standardized incidence ratio (SIR), and the average time to discovery of lung cancer in Patients with localized TCC of the bladder (AJCC 6 stages T~0~ through T~1a2~) in years 2000-2013, stratified by age and gender, and compare them to the SIR for all solid tumors.

Results:
Results: based on 89691 patients (F:M ratio 1:3.3), the SIR for all solid tumors was 1.95[CI95%:1.87-2.04] for women and 1.87[1.83-1.9] for men. The SIR for lung cancer in women was significantly higher, 2.40[2.19-2.62], with significance persisting among all age groups >50y. The SIR for men was 1.81[1.73-1.9], not significantly different from the risk for all solid tumors in any age group. The median latency period until discovery of lung cancer was 5.41, 3.54, 2.74 and 0.08 years in women, and 4.41, 3.59, 2.96 and 0.96 in men, for age groups 50-59, 60-69, 70-79 and 80+, respectively.

Conclusion:
Conclusion: Incidence of lung cancer is higher in localized TCC patients than among the general population, and among women it appears to be significantly higher than the general risk of solid tumors. Early stage TCC patients may therefore stand to gain from lung cancer screening, and should be considered as potential screening candidates.

Background:
The US Preventive Services Task Force recommends that smokers aged 55-80 should be screened annually with low dose computed tomography (LDCT). Successful implementation of lung cancer screening depends on being able to reach high-risk individuals. This study identified demographic, smoking history, health risk perceptions, knowledge, and attitude factors of older smokers related to LDCT agreement. Using binary logistic regression we produced a predictive model of factors to explain LDCT agreement.

Methods:
As part of a larger Tobacco Attitudes and Beliefs Study, we conducted a cross-sectional, national, online survey of 549 older (≥ 45 years) current and former smokers. Univariate differences between groups for agreement and non-agreement for LDCT was conducted. Using all variables that demonstrated a significant association with LDCT agreement, a binary logistic regression analysis was conducted to predict agreement to have an LDCT.

Results:
Almost 80% of the sample believed that a person who continues to smoke after the age of 40 has at least a 25% chance of developing lung cancer and if asked, 79.4% would agree to a LDCT. Using Chi Square analyses, nine variables that were significant at the 0.10 level were selected for inclusion in model development. Four of the independent variables made a unique statistically significant contribution to the model: Believes that early detection of lung cancer will result in a good prognosis; Perceives accuracy of LDCT as an important factor in the decision to have a LDCT scan; Believes that they are at high risk for lung cancer; and Believes that a negative LDCT result would decrease worry without encouraging continued smoking.

Conclusion:
Older smokers are aware of the risks of smoking, are interested in smoking cessation, and most are interested in and positive about LDCT. Cognitive aspects of participation in screening are key to increasing the uptake of lung cancer screening and smoking cessation among high-risk smokers.

Background:
Lung cancer represents 13.4% of all newly diagnosed US cancers and 27.1% of all cancer deaths. Early stage lung cancer is generally treated with surgical resection. Many patient- and hospital-level factors influence the selection of appropriate surgical procedures and their outcome. We identified patient- and hospital-level characteristics influencing the type of lung cancer surgical approach utilized in New York State and assessed in-hospital complications and mortality.

Methods:
Patients were selected from the Statewide Planning and Research Cooperative System, SPARCS (1995-2012) based on ICD-9-CM codes of diagnosis (162 and 165) and procedures (32.0-32.9). Surgery was categorized into: limited resection (LR: 32.2-32.3), lobectomy (L: 32.4), and pneumonectomy (P: 32.5-32.6). Statistical analyses were performed in SAS v9.4 and ArcMap v10.3.1.

Results:
There were 36,460 patients (age 60-75 years); 56% underwent L, 37% LR, and 7% P. LR patients were more likely to be older (OR~adj~ 1.01, 95%CI [1.01-1.02]), female (OR~adj~ 1.10 [1.06-1.15]), Black (OR~adj~ 1.24 [1.15-1.34]), with comorbidities (OR~adj~ 1.10 [1.04-1.16]) than L patients. Opposite trends were observed among P patients, except for race. Over time, the odds of P decreased, while those of LR significantly increased (OR~adj~ 1.22 [1.16-1.29] for years 2007-2012 vs 1995-2000). Teaching hospitals were less likely to perform LR over L (OR~adj~ 0.82 [0.75-0.88]), while the opposite was true for hospitals with larger surgery volumes (OR~adj~ 1.07[1.03-1.11]). In-hospital complications were significantly less after LR than L (OR~adj~ 0.66 [0.62-0.69]), while in-hospital mortality was similar (OR~adj~ 0.93 [0.84-1.03]). In-hospital mortality was directly associated with age, length of stay, urgent/emergency admission, and inversely associated with female gender, private insurance, and surgery volumes. Figure 1



Conclusion:
There is a growing trend towards LR, which is still more likely to be performed in older patients with co-morbidities. In-hospital outcomes were affected by patients’ clinical and personal characteristics, and were better after LR than L or P.

Background:
Incomplete resection of NSCLC has a negative impact on survival. We evaluated risk factors associated with positive margins within a comparative observational population-based cohort study.

Methods:
We analyzed curative-intent resections from 2009-2016 from 4 contiguous Dartmouth Hospital Referral Regions in 3 US states. Statistical analyses were preformed using univariate and multiple logistic regression models.

Results:
Among the 2,275 NSCLC-resected patients, 52% were male, 78% white, 45% Medicare insured, and 36% privately insured, with a median age of 67 years. Factors associated with a higher margin positivity rate included male sex, large cell histology, undifferentiated tumor grade, neo-adjuvant therapy, clinical stage IIIA and IIIB, bilobectomy extent of resection, patients with abnormal diffusing capacity of the lungs for carbon monoxide (DLCO), use of bronchoscopic biopsy for diagnosis greater than 1 day before surgery, left lung resection, and tumor size >7cm (all p<0.15, Table 1). American Society of Anesthesiologists (ASA) score, prior lung cancer, smoking status, Charlson score, FEV1, PET/CT, brain scan, bone scan, mediastinoscopy, blood transfusion, and hospital were not associated with positive margins in univariate analyses (all p>0.15). Controlling for sex, histology, tumor grade, tumor size, neo-adjuvant therapy, clinical stage, extent of resection, DLCO, pre-operative bronchoscopic biopsy, and primary resection site in the multiple variable analysis, sex (p=0.0134), clinical stage (p<0.001), extent of resection(p=0.0461), DLCO (p=0.0431), and bronchoscopic biopsy (p=0.0029) were independently associated with risk for positive margins (Table 1).

Conclusion:
This detailed evaluation in a large regional cohort indicates patient-level characteristics are associated with positive surgical resection margins. Our recently published evaluation of the National Cancer Database also identified institutional factors that impact the rates of positive margins. Patient-level, surgeon-level, and institutional-level factors should be considered jointly to fully understand factors impacting margin positivity rates. Figure 1

Background:
MPM is extremely aggressive and has a long-latency period. Hence, detected at advanced stages resulting in an unfavorable prognosis(1-2years). However,MPM prognosis has been improving over the past few years with availability of better diagnostic and treatment regimens. We aim to compare clinico-pathologic characteristics of old-MPM cases referred to National Cancer Institute(NCI)-Cairo university between( 2002-2003)and new MPM cases(2012-2015)

Methods:
Retrospective review of MPM cases presented to NCI..Data regarding demographics, histology, symptoms and signs, tumor staging and CT-findings were obtained from all patients’ records. Pearson's Chi(X2)and Fisher's Exact t-tests were used for statistical analysis.

Results:
1)Old cohort(n=100): 100 patients were encountered. Median age was 46years.Males were 59% of cases. 30% has PS1. Asbestos exposure was documented in 74cases. 44cases were smokers, 25cases were industrial-workers. Family history was positive in 12cases.Dyspnea was the presenting symptom in 92cases,chest pain in 83% and tuberculous pleuritis in 2 cases, effusion in all cases, pleural thickening in 80%,tracheal shift to the opposite-side in 23%, T2 represented 41%. Epithelioid subtype 46.6%. Pathological T2= 34%. 2)New cohort(n=194): 194 patients were encountered.Median age was 53 years.Males and females were nearly equally distributed. Epithelioid subtype was 63.4%. Rt-sided lesions were evident in nearly two-thirds of the cases.Pleural thickening was nodular in 131(69.7%)cases. Inter-lobar fissure was thickened in 29.4%. Mediastinal Pleura was affected in 37.1%. Nearly,half of our cases had effusion.Ossification & calcification were detected in 8(4.1%) cases. Contraction of hemithorax was identified in 77(39.7%)cases. Chest wall invasion(CWI)was present in 18(9.3%) cases. Pulmonary nodules were detected in one-fifth of the cohort. Metastases were detected in 9(4.6%)cases(Figure).Figure 1



Conclusion:
By comparing both groups, we found that more lymph node involvement(N+), less metastasis(M+),older median age,more females, more epithelial subtype, less pleural effusion presentation, more pleural thickening were detected in group 2(new cases) reflecting better staging ( mediastinoscopy& PET-CT),early detection, more incidence in females and better treatment modalities.

Background:
Although lung cancer patients frequently require emergency medical care due to acute unbearable symptoms and life‑threatening conditions, there are limited data on them at the emergency departments (ED). National Emergency Department Information System Database (NEDIS) collects information about ED visits in Korean population. We aimed to determine the frequency and main causes of emergency consultations and the predicting factors for hospital admissions and deaths.

Methods:
In this retrospective observational study, we reviewed all the cases of ED visit for six months, from July 2014 to December 2014, in three university hospitals: Hanyang University Hospital and Chung-Ang University Hospital in Seoul, and Chungbuk National University Hospital in Cheongju. By reviewing all the medical records including NEDIS database, we identified cases with lung cancer and performed descriptive statistics and logistic regressions analysis.

Results:
Of all 62,369 ED visits, there were 292 ED visit (0.5%) by 216 patients with lung cancer. Among them, 76.4% had only one ED visit in study period. The main reasons for consultation were respiratory symptoms (36.8%) and fatigue/alteration of the general state (12.7%), and pain (12.4%). ED visit leads to hospital admission in 74.9% and hospital death in 25.1% of lung cancer patients. In multivariate analysis, the main independent predictor factors of hospitalization are diagnostic phase of lung cancer (odd ratio 9.3) and the transfer from another hospital (odd ratio 4.9). The palliative phase with best supportive care alone (odd ratio 5.2) and abnormal heart rate at the time of ED visit (odd ratio 2.4) are statistically associated with death during hospitalization.

Conclusion:
Our study shows that ED visit is a frequent but clinically important event for patients with lung cancer. We might consider certain risk factors indicating hospitalization and death in lung cancer patients visiting ED to improve delivery of quality cancer care.

Background:
Lung cancer is one of the most common malignancies in the world. In Brazil, it is estimated that 28,200 new cases will be diagnosed in 2016. This cancer affects more men and is usually caused by tobacco exposure. The most common histology is adenocarcinoma and many of these patients have driver mutations which help guide therapeutic choice. The aim of this study was to delineate the epidemiological profile of patients with NSCLC in Brazil and to evaluate the prevalence of testing for ALK translocations and EGFR mutations in patients in the public and private settings in Brazil.

Methods:
Observational, descriptive, retrospective, multicenter study involving 230 public and private institutions in Brazil. We obtained data from a commercial database with 1642 Non Small Cell Lung Cancer (NSCLC) patients treated in the country between January and December 2015. Variables analyzed: age, sex, smoking, presence of EGFR and ALK mutation.

Results:
Out of 1642 patients, 814 were treated in the public service (49.57%) and 828 in private services (50.42%). Most patients were men (58.28% vs. 41.71% female). The mean age at diagnosis was 61.8 years (median 62 years), 32.58% were former smokers, 31.12% current smokers, 19.48% never smoked and data were not available for 16.8% of subjects. Most patients had metastatic disease at diagnosis (65.04%), 23.20% had stage III, 9.31% stage II and 2.43% stage I. 68.57% had adenocarcinoma, 27.52% squamous cell cancers, 1.4% large cell and 2.67% had other histological types. Among the 534 patients with non-squamous histology treated in public settings, 244 patients were tested for EGFR (46,69%) and only and only 36 were tested for ALK (6,74%).In private services, of 656 patients with non-squamous subtypes, 454 were tested for EGFR (69,2%) and 77 for ALK (11,73%).

Conclusion:
Overall, testing for EGFR mutations was below ideal, especially in the public setting. More worryingly, and likely due to the lack of availability of crizotinib in Brazil until 2016, very few patients were tested for ALK translocations in 2015. Much work needs to be done in education and advocacy to improve testing patterns in the country.

Background:
This article reviews ECOG scale values at the first consultation in our Institute, we review demographic and other related variables. ECOG at first consultation is related to treatment options.

Methods:
Between January 2004 and December 2013, all patients diagnosed with a pathology of SCLC and NSCLC at National Institute of Oncology at Paraguay were analyzed retrospectively. ECOG performance status, were recorded. SPSS 20 was used to analyze.

Results:
We studied 478 subjects. At age mean 60,40 [95% CI 59,45 to 61,34] years and ECOG performance status mean 2,13 [95% CI 2,06 to 2,20] points. Frequency of ECOG was to 2: 48.1%, to 3:31.3%, to 1: 19 %, to 4:1.1%, to 0: 0,6% of our population. Place of living predominant ECOG at Rural place: ECOG 2: 50%, ECOG 3: 30.6%. At Urban places ECOG 2:44.9% and ECOG 3:32.4% (P>0.05) ECOG and occupational relation was unemployed ECOG 2: 54.2%, Other professions ECOG 2: 46.9% , Farmers ECOG 2: 43.3,6% , homemakers ECOG 3: 50% (P= 0.008). Most of patient were smokers ECOG 4: 100%, ECOG 2: 86%, ECOG 3: 81%, ECOG 1: 76,1%, (P=0,000). Clinical severity and ECOG relation was predominant at ECOG 0 to Stage IIIB:66.7% and ECOG 1 to Stage IIIB:43.5%. And predominant at ECOG 2 was Stage IV: 54.7%, ECOG 3 and 4 was Stage IV: 60%, both (P=0,000). ECOG 1: 33,8% accept to chemotherapy ,ECOG 2: 46.6% Reject any treatment, ECOG 3: 43.9% Reject any treatment, ECOG 4: 40% accept to radiotherapy (P=0,000). Non-Small cell carcinoma predominant ECOG 2: 48.2% Small cell carcinoma: predominant ECOG 2: 48.1%(P>0.05).

Conclusion:
Our mean ECOG was 2.13 but predominance in our populations is ECOG 2 with 48% and ECOG 3 with 31%. Prevalence of ECOG 2 and 3 at first consultation was found at Rural and Urban Places. Statistical significance was found at work and ECOG performance with prevalence to ECOG 2 except to homemakers who had prevalence to ECOG 3. Association with smoking prevalence was ECOG 4 at first consultation. Is important to conclude that at ECOG 0 and 1 clinical stage IIIB was predominant and to ECOG 2 to 4 was clinical stage IV, which shows relation between clinical severity and ECOG performance, but multivariate analysis will be required. Relation between treatment show a high rejection to treatment at ECOG 2 and 3. With this analysis we need to seek a logistic regression model to search relation with ECOG performance and other variables.

Background:
The prevalence of NSCLC as a second primary tumor has been increasing over the past decades, though very little data are available in the literature. We analyzed the NCDB, an oncology outcomes database administered by the American College of Surgeons and the American Cancer Society, to study the outcomes and patterns of treatment of patients (pts) diagnosed with NSCLC as a second or subsequent primary (SP).

Methods:
The NCDB was queried from 2004 to 2012 for NSCLC pts. Pts diagnosed with NSCLC as SP were compared with pts with de novo (DN) NSCLC as defined by sequence number in the database. Univariate (UV) and multivariable analyses (MV) with overall survival (OS) were conducted by Cox proportional hazards model. Kaplan-Meier plots were produced to compare the survival curves by subgroups along with log-rank p-values.

Results:
A total of 207,518 pts in SP and 697,709 pts in DN groups were included in the analysis, which accounted for 22% and 74% of all NSCLC pts respectively. Pt characteristics (SP/DN %): median age 72/68, male 53/53, white 89/84, stage IV 28/41, treated at academic centers 33/32, government insured 72/57, mean tumor size (cm) 3.5/4.4. An increasing trend in incidence of SP was observed (19.5% in 2004 to 24% in 2012) vs. a decreasing trend in DN (75.6% in 2004 to 73% in 2012). About 12% in SP and 15% in DN received chemotherapy as part of their treatment. Surgery was performed in 39% of SP group vs. 28% in DN. Radiation was given to 43% of the pts in DN vs. 36% in SP. On UV and MV analysis, SP was associated with better survival than DN (HRs 0.84 and 0.93 respectively; p<0.001). The SP group had higher 5-year OS (23% vs. 19.6%, p<0.001) and a higher median survival (17 vs. 11.5 months) compared to DN. On stratifying by stage, DN had inferior survival in stage IV pts (HR 1.12, p<0.001) compared to SP but better survival in stage I and II pts (HRs 0.86 and 0.93, p<0.001). No difference in OS was seen in stage III pts (HR 1.01, p= 0.4).

Conclusion:
The incidence of second primary has increased over the past decade. Second primary NSCLC is diagnosed at an earlier stage, smaller tumor size, and is associated with a better survival, compared to de novo NSCLC.

Background:
The National Lung Screening Trial (NLST) showed that screening with low-dose computed tomography (CT) as compared with chest radiography reduced lung cancer-mortality. There is no data's on the faisability and cost-effectiveness of CT lung cancer screening program in the French setting.

Methods:
We estimated mena life-years gaineds, costs and incremental cost-effectiveness ratio (ICER) for screening with low-dose CT compare to no screening. Estimations of life-years gained were based on the efficacy of NLST trail applied to the general french population using the same inclusion criteria's that NLST trail (age of 55 to 74 years with a minimum of 30 pack-years of smoking and no more than 15 years since quitting) adjusted to sex, age and smoking status. Costs were limited to directs costs from the payers perspective. We also performed sensitivity analysis based on several asssumptions of program efficacy.

Results:
The target population was 5 551 141 subjects. Compared with no screening, screening with low-dose CT, over a period of 2 years, will have an additional cost of 941 978 €, will provide 52.4 additional year of life with a corresponding ICER of 17 969 € per year gained. Sensititiviy analysis showed that this result is sensitive to program efficacy (number, stage and survival of lung cancer diagnosed by the program) and to subjects compliance rate to the program.

Conclusion:
Cost effectiveness of CT lung cancer screening program in a French population using the same main inclusion criteria and outcomes of NLST trial appears as acceptable from the french health system

Background:
Lung cancer is the most commonly diagnosed cancer in men and the third most common in women. However, detailed analyses of patient newly diagnosed with advanced non-small cell lung cancer (NSCLC) in routine clinical care, compared to the general population, is limited.

Methods:
This non-interventional study is based on existing data from the population-based national Swedish Cancer Registry. All adult patients diagnosed with advanced NSCLC between 2006 and 2013 receiving care in the regions of Skåne and Västra Götaland counties (about 37.6% of NSCLC patients in Sweden) were included and matched (1:4) by age at diagnosis, gender and region of residence to a sample from the general population (controls). In- and outpatient visit data was extracted from regional databases in order to assess prevalence of selected baseline comorbidities detected in the year before diagnosis.

Results:
In total, 4,758 patients with advanced NSCLC were identified and matched to 18,996 controls. At the date of the initial NSCLC diagnosis, the median age was 69.0 (range 22-97; 96.2% above 50), and 52.7% were men. The stage of disease was IIIb in 19.8% (n=944) or IV in 80.2% (n=3,814) of the patients. When specified, adenocarcinoma was the most frequent histology (70.4%) followed by squamous cell carcinomas (25.4%). A total of 50.9% of the NSCLC patients had recorded morbidities in the year preceding the diagnosis compared to 34.8% of the controls (Odds Ratio (OR) 1.94; P<0.001)). Patients with advanced NSCLC had significantly more often (P<0.001) respiratory disorders (16.4% vs. 3.2%; OR 6.02), infections (13.4% vs 6.1%; OR 2.37), anaemia (3.4% vs. 1.7%; OR 2.08), musculoskeletal disorders (3.7% vs. 2.5%; OR 1.51), and cardiovascular disease (27.8% vs. 22.7%; OR 1.31). No significant difference was observed in prevalence of gastro-intestinal disorders, metabolic disorders or skin disorders. Regarding disorders of the central nervous system, while depression was more frequently present among NSCLC patients (3.8% vs. 2.8%; OR 1,38) dementia was more prevalent among controls (0.6% vs. 1.2%; OR 0.48). As expected, brain metastasis was diagnosed overwhelmingly more for NSCLC patients (53 cases, vs. 2 cases; OR 107.0).

Conclusion:
The present study suggests that patients diagnosed with advanced NSCLC have a significantly higher morbidity burden than the general population in these regions in Sweden.

Background:
We studied the relationship between 1-year mortality and weight at diagnostic in 6,965 adult patients followed for primary lung cancer in 104 general hospitals.

Methods:
Patients were classified into 5 groups: Group 1, underweight with recent weight loss; Group 2, underweight without recent weight loss; Group 3, normal weight; Group 4, overweight; Group 5, obese. Kaplan-Meier method (1-year mortality) and Cox multivariate analysis (independent risk-factors) were used.

Results:
Respectively, 11%, 4%, 45%, 29%, and 12% of patients belonged to Groups 1, 2, 3, 4, and 5. One-year survival was lower in Group 1 (27% [24%-30%]) and higher in Group 4 (50% [48%-52%]) or 5 (53% [50%-57%]) than in Group 2 (47% [41%-53%]) or 3 (43% [42%-45%]) (Fig. 1). As compared with normal weight, overweight was an independent protective factor. Independent protective/risk factors are presented in Table 1. Interaction analyses showed that overweight was a significant independent protective factor for stage IIIA and IIIB cancer (HR=0.77 [0.6-0.99], p=0.038; HR=0.75 [0.59-0.97], p=0.029, respectively). Figure 1

Variable	HR	95%CI	P
BMI (group)
3	1
1	1.06	0.96-1.17	0.26
2	1.03	0.85-1.23	0.789
4	0.92	0.85-0.99	0.036
5	0.9	0.81-1.01	0.061
Age (years)
<=40	1
41-50	1.07	0.74-1.54	0.714
51-60	1.02	0.72-1.46	0.899
61-70	1.05	0.74-1.49	0.796
71-80	1.11	0.78-1.59	0.553
>80	1.54	1.07-2.22	0.02
Sex
Men	1
Women	0.81	0.74-0.88	<0.001
Smoking
Never-smoker	1
Former-smoker	1.19	1.06-1.35	0.004
Current-smoker	1.27	1.13-1.44	<0.001
PS
PS0	1
PS1	1.58	1.45-1.73	<0.001
PS2	2.66	2.4-2.95	<0.001
PS3	5.6	4.95-6.34	<0.001
PS4	10.61	8.62-13.05	<0.001
Stage
<=IIB	1
IIIA	1.89	1.61-2.21	<0.001
IIIB	3	2.56-3.52	<0.001
IV	4.71	4.13-5.38	<0.001

Conclusion:
In 2010, in France, in real life conditions, 1-year survival was low in lung cancer patients with low BMI at diagnosis and recent weight loss. Overweight appeared to be a protective factor in particular for stage IIIA and IIIB cancers.

Background:
We studied the impact of the distance between chest and thoracic surgery departments on the outcome of patients followed, for primary lung cancer diagnosed in 2010, in the chest department of 104 French general hospitals participating in KBP-2010-CPHG study.

Methods:
6,083 patients with non-small-cell lung cancer (NSCLC) participated in this study. Univariate and multivariate analyses were performed to identify independent factors for surgery and 1-year mortality. Distance from the usual thoracic surgery department in 2010 was collected for each chest department and included in the model as a 4-class variable: 0 km (same hospital), 1­-34 km, 35­-79 km, and ≥80 km.

Results:
Overall, 23% of hospitals had a thoracic surgery department; otherwise, mean distance between the hospital and the surgical center was 65 km. 1,157 patients (19%) were operated on; vital status was known for 5,876 patients (97%). Distance was not an independent factor for surgery and for mortality. Independent factors for surgery and mortality are presented in Tables 1 and 2. Table 1- Surgery (multivariate analysis: adjusted odd-ratios)

	OR	95% CI	p
Distance (km)
0	1
1-34	0.97	[0.74-1.27]	0.833
35-79	0.88	[0.66-1.18]	0.399
>=80	1.02	[0.78-1.32]	0.91
Age (year)
Continuous	0.95	[0.94-0.96]	<0.001
Stages
IV	1
I	248.18	[172.48-357.11]	<0.001
II	155.78	[107.70-225.32]	<0.001
IIIA	34.23	[24.80-47.25]	<0.001
IIIB	2.33	[1.40-3.89]	0.001
Histology
Adenocarcinoma	1
Squamous-cell carcinoma	0.77	[0.61-0.96]	0.023
PS
PS0	1
PS1	0.58	[0.47-0.71]	<0.001
PS2	0.12	[0.08-0.17]	<0.001
PS3	0.08	[0.04-0.16]	<0.001
PS4	0.07	[0.02-0.32]	<0.001

Table 2- Mortality (multivariate analysis: adjusted hazard-ratios)

	HR	95% CI	p
Distance (km)
0	1
1-34	1.02	[0.94-1.11]	0.661
35-79	1.00	[0.91-1.10]	0.985
>=80	1.01	[0.93-1.09]	0.887
Age (year)
Continuous	1.01	[1.01-1.01]	<0.001
Sex
Men	1
Women	0.86	[0.80-0.94]	<0.001
Stages
IV	1
I	0.15	[0.13-0.18]	<0.001
II	0.29	[0.25-0.34]	<0.001
IIIA	0.41	[0.37-0.46]	<0.001
IIIB	0.65	[0.58-0.72]	<0.001
PS
PS0	1
PS1	1.58	[1.45-1.73]	<0.001
PS2	2.79	[2.52-3.09]	<0.001
PS3	5.75	[5.11-6.48]	<0.001
PS4	10.2	[8.52-12.20]	<0.001
Smoking
Never-smoker	1
Former-smoker	1.18	[1.05-1.33]	0.005
Current-smoker	1.33	[1.18-1.49]	<0.001

Conclusion:
In 2010, the absence of an on-­site thoracic surgery department did not impair outcome in NSCLC patients managed in the chest departments of French general hospitals.

Background:
The introduction of new effective modalities for the treatment of metastatic non-small cell lung cancer (NSCLC), such as targeted therapies and immunotherapy, has resulted in reports of long-term survival in small sub-groups of patients treated with these therapies. It is therefore important to understand the frequency and characteristics of long-term survivors in large cohorts of advanced-stage lung cancer patients who were diagnosed and treated prior to the advent of these new therapies.

Methods:
A survival analysis of data from the Surveillance Epidemiology and End Results database was performed. The cohort was limited to patients diagnosed with stage IV NSCLC, squamous and adenocarcinoma histology only, between 1991 and 2007, with follow-up through 2012. Outcomes and factors associated with extended survival were evaluated in the 10% of patients with longest survival (long-term survivors, ≥21 months) vs. 90% short-survival patients (< 21 months). Patients surviving ≥ 5 years were compared with those surviving <5 years and ≥21 months. Demographic, tumor characteristic, and treatment differences between long-term and short-survival patients were compared using chi-square and Student’s T-test for categorical and continuous variables, respectively. For descriptive analyses unadjusted for confounders, Kaplan Meier curves and log-rank tests were used to compare survival by histology and long-term survival status.

Results:
Among the 44,387 patients diagnosed at stage IV, long-term survivors (4,544) are distinguishable from short-survival patients (39,843) by younger age, female sex, Asian/ Pacific Islander race, lower tumor grade, adenocarcinoma histology, upper lobe site, and treatment with surgery. Among only long-term survivors (≥ 21 months), predictors of longest survival are younger age, lower tumor grade, and treatment by surgery and radiation. Median survival increased over time from 3 to 4 months for short-survival patients versus 30 to 36 months for long-term survivors. Notably, 1.5% of patients survived >5 years even prior to modern combination chemotherapy regimens, targeted therapies, and immunotherapy.

Conclusion:
Despite the poor overall survival of patients diagnosed with stage IV NSCLC, the top 10% of survivors have significantly longer survival than the rest and a sub-population of individuals with extraordinary survival is identifiable. The discrepancy in median survival between long-term survivors and the rest suggests that long-term survivors comprise a disproportionate percentage of clinical trial participants and provides a rationale for more detailed clinical and molecular analyses in order to improve therapeutic targeting and future study design.

Background:
Although immunotherapy has revolutionized the field of cancer treatment, response rates are only ~20% in non-small cell lung cancer (NSCLC) patients and cost of this therapy is high. Predictive biomarkers are needed to identify patients likely to benefit. Converting digital medical images into high-dimensional data (‘Radiomics’) contains information that reflects underlying pathophysiology and that can be revealed via quantitative analyses. We extracted radiomic imaging features from baseline CT scans (prior to initiation of immunotherapy) and identified features that predict response to immunotherapy in NSCLC patients. This work is an initial test of the hypothesis that radiomic data may predict who will respond favorably and who will not.

Methods:
We curated a subset of data and images from 13 different institutional immunotherapy clinical trials. Patients were stage III/IV NSCLC and received PD-1, PD-L1, or doublet checkpoint inhibitors. All target nodules were identified on the CT prior scan prior to initiation of immunotherapy. RECIST guidelines 1.1 were used to measure patient response from baseline to last follow-up scan. Based on last follow-up, 43 patients had progressive disease (PD) and 28 patients with partial response (PR) or complete response (CR). Since we focused on extreme responses, stable disease (SD) patients were not included in the current analyses. We extracted 219 radiomic features including size, shape, location, and texture information from a total of 210 target nodules (lung, lymph nodes, or other). Backward-elimination analyses were utilized to generate parsimonious radiomic models associated with objective responses (PD vs. PR/CR) and post estimation computed performance statistics.

Results:
There were no significant differences for the patient characteristics between patients with PD vs. CR/PR. Analysis of the radiomic features for all target nodules to differentiate PD patients vs. PR/CR patients resulted in a final model containing 2 features that provided an AUROC of 0.64 (95% CI 0.56–0.72). When we analyzed features for only lung target nodules, we identified a final model with 4 features that produced an AUROC of 0.79 (95% CI 0.68–0.89). When we analyzed the imaging features for lymph node target nodules, we found that a final model with 1 feature yielded an AUROC of 0.67 (95% CI 0.51–0.82).

Conclusion:
Radiomic features of lung target nodules have better performance statistics for predicting response to immune therapies compared to target nodules from other organ sites. With this model, cutoffs can be chosen to reduce non-responders with high confidence. Change feature analyses following therapy are underway.

Background:
Expression of programmed death-ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) patients might identify patients who would benefit from PD-L1 blocking antibodies. In a retrospective cohort of NSCLC patients, we characterized PD-L1 expression and other biomarkers to determine if PD-L1 expression is a prognostic biomarker and whether patient characteristics could be identified to determine those associated with high expression.

Methods:
This was a retrospective analysis of 136 NSCLC patients diagnosed between 1997 and 2015 with stage IIIB and IV disease and treated at Moffitt Cancer Center and affiliated institutions. All patients had at least 2 lines of standard of care chemotherapy and sufficient archival tumor tissue for PD-L1 testing by the Ventana SP263 validated assay and mutation status testing by targeted DNA sequencing with the TumorCare Panel. High PD-L1 expression was defined as ≥ 25% of tumor cells with membrane positivity for PD-L1 at any intensity above background staining. Statistical analyses were performed comparing PD-L1 expression by patient characteristics. Survival analyses were performed using Kaplan-Meier survival curves and the log-rank statistic. All statistical tests were two-sided; P-value of less than .05 was considered statistically significant.

Results:
Of the 136 tissues tested for PD‐L1 expression, 116 (85.3%) were collected by surgical resection and 20 (14.7%) were collected by biopsy. Mean sample age was 7.2 years (SD=2.8 years). 82 of the 136 samples also underwent targeted DNA sequencing. In this patient cohort, 51.5% were male, 83.1% were ever smokers, 90.4% were White, 39% were stage IV at time of tissue collection, 71.3% had adenocarcinoma, 28.7% had four or more lines of therapy, and 24.2% had high-expression for PD‐L1. There were no statistically significant differences with respect to PD-L1 expression for patient characteristics, overall survival (OS), or progression-free survival (PFS). Additionally, there were no statistically significant differences with respect to PD-L1 expression by EGFR (WT/PD-L1<25% = 74.4% vs. WT/PD-L1≥ 25% = 88.5%), KRAS (WT/PD-L1<25% = 74.7% vs. WT/PD-L1≥ 25% = 69.2%), and ALK status (Neg/PD-L1<25% = 98.5% vs. Neg/PD-L1≥ 25% = 100%). However, mutation load (total number of non-synonymous mutations) was statistically significantly correlated with PD-L1 expression (correlation coefficient = 0.23; P = 0.03).

Conclusion:
In this study of NSCLC patients treated with 2 or more lines of standard of care chemotherapy, PD-L1 expression (high vs. low and as a continuous covariate) was not statistically significantly associated with OS or PFS. We did observe a novel positive correlation between PD-L1 expression and mutational load.

Background:
In the United States, lung cancer occurs in about 225,000 patients and causes over 158,000 deaths annually. Due to a shift in smoking patterns between the genders that started decades ago, the incidence of lung cancers appeared to have shifted accordingly. Thus, we analyzed our institutional data as described below.

Methods:
This is a retrospective study that compares two populations of lung cancer patients in two different five-year periods a decade apart: one from 1996 to 2000 consisting of 1355 lung cancer patients and the other from 2011 to 2015 consisting of 2220 lung cancer patients from our institutional tumor registry data. We included lung cancers that have been associated with smoking such as adenocarcinoma, squamous cell carcinoma, and small cell lung cancer. The two populations were compared in category of gender; race and marital status were also included to examine any major population shifts during these time periods. Crude survival at two-year follow up period was also examined. The results were analyzed using Excel as well as Matlab. This project is IRB approved.

Results:
From 1996 to 2000, the percentage of male population with lung cancer associated with smoking was 63%. This number decreased significantly to 51% in the period 2011-2015 (p-value < 0.00001). The percentage of African American patients in 1996-2000 was 14% and decreased to 11% in 2011-2015 (p-value 0.0039). The number of divorced patients increased from 8.1% to 11% (p-value 0.0025). The number of widowed patients decreased from 12.3% to 9.8% (p-value 0.0096). For patients with stage IV lung cancer diagnosed from 1996 to 1998, the crude survival rate at 2-year follow up was 17%, which increased to 29% in patients diagnosed from 2011 to 2013 (p-value < 0.00001).

Conclusion:
Our results demonstrate that the proportionate incidence of lung cancer in males has decreased significantly from the late 90s to the early 2010s. The change in race and marital status, while statistically significant, is less dramatic. The percentage of African American population has also decreased significantly. The crude survival rate at 2 year follow up for those with stage IV lung cancer significantly increased. While this could, in part, be due to stage migration, a real prolongation due to improvements in systemic therapy is likely. More attention should be drawn to the fact that nearly twice as many women die from lung cancer compared to breast cancer, for a more proportionate support of research efforts.

Background:
Physical activity is an effective way to positively influence health outcomes among cancer survivors. Few studies have examined physical activity and sedentary behaviour among lung cancer survivors. Further, these studies have used self-report measures of physical activity, which may bias results (e.g., overestimation) and lead to incorrect conclusions. Only one study to date has reported on objectively-assessed sedentary behaviour among lung cancer survivors. The primary aim of this currently ongoing study is to determine the prevalence of objectively-assessed physical activity and sedentary time among lung cancer survivors.

Methods:
Lung cancer survivors in Southern Alberta diagnosed between 1999 and 2014 are currently being recruited to participate. Eligibility criteria include: confirmed non-small cell lung cancer, completed treatment, and not living in hospice/palliative care. Consenting participants wear an Actigraph[®] GT3X+ accelerometer on their hip for seven days. Time spent sedentary, in light and in moderate-to-vigorous intensity physical activity are derived from the accelerometer data and processed using 60-second epochs. Physical activity and sedentary behaviour accumulated in 10 minute and 30 minute continuous bouts will be examined.

Results:
Recruitment began in June, 2016. A total of 660 survivors were invited and 113 have agreed to participate. Of the 374 survivors that did not respond, most indicated they were not interested (n=115). Others denied having lung cancer (n=6) or had invalid contact information (n=27). Six were deceased. Currently the response rate is 18.2%. Of the 113 that consented, eight participants withdrew due to health concerns (n=4), time constraints (n=2), and loss of interest in the study (n=2). Of the 105 participants, the median age at diagnosis was 66 years, and 72 years at recruitment, the majority were female (n=62), and 85 had a smoking history. Adenocarcinoma was the most common diagnosis (n=65). The majority of participants were diagnosed stage I (n=53) with others diagnosed at stage II (n=23), III (n=18), and IV (n=11). Overall, 65 survivors underwent a lung resection while 18 of those received adjuvant therapy. Other treatments included concurrent chemotherapy and radiation (n=11) and radical radiation alone (n=23).

Conclusion:
This study will be the first to report on objectively assessed physical activity and sedentary time among a population-based sample of lung cancer survivors. Despite inherent difficulties of this type of research (e.g., older population), the positive response rate suggests high participation interest. We expect to reach our recruitment target of 140 patients by September, 2016, with data analysis completed in November, 2016.

Background:
Lung cancer represents 13.4% of all newly diagnosed US cancers and 27.1% of all cancer deaths. Health disparties exist in accessing proper care and receving surgical treatment. We examined the role of Patient to Hospital distance (P-H) in access to care.

Methods:
Patients were selected from the New York State Statewide Planning and Research Cooperative System (1995-2012) based on ICD-9-CM diagnosis (162 and 165) and procedures (32.0-32.9). Surgery was categorized into: limited resection (LR: 32.2-32.3), lobectomy (L: 32.4), and pneumonectomy (P: 32.5-32.6). Distance calculations (ArcMap 10.3.1) and linear regressions (SAS v9.4) were performed to determine the factors influencing P-H.

Results:
There were 36,460 patients (age 60-75 years); 56% underwent L, 37% LR, and 7% P; 95% of patients underwent surgery at a hospital < 70 kilometres (km) from their home (mean±SD 20.49±30.24 km; median 11.10 km). P-H was shorter in LR (19.10±27.71 km) than L (21.00±30.96 km) and P (23.87±36.56 km; p < 0.001). At multivariable analysis, P-H was positively associated with teaching hospitals (β: 3.33, p < 0.001), admitted during 1995-2000 (β: 1.08, p < 0.001) and 2001-2006 (β: 1.23, p < 0.001), and P (β: 1.57, p < 0.001), and inversely associated with female gender (β: -0.49, p = 0.016), age at admission (β: -0.17, p < 0.001), black race (β: -8.22, p < 0.001), Medicaid (β: -3.37, p < 0.001), private insurance (β: -0.79, p = 0.004), rural hospitals (β: -2.80, p < 0.001), LR (β -0.81, p < 0.001), and mortality (β -1.05, p = 0.081). Similar associations were found in the L subgroup; among LR patients there was no statistically signficant association between P-H and female gender. Figure 1



Conclusion:
Significant differences exist in P-H and patient/hospital characteristics, which may affect type of surgery and outcome. P-H should be incorporated to improve health disparities in accessing surgical care.

Background:
Lung cancer is the leading cause of death of all tumors in China. But due to imbalanced development of different provinces, the surgical treatments of Non-small cell lung cancer in different areas of China diverse. The Chinese National NSCLC outcome registry was founded in 2013, which covers 17 provinces across China. We analyze the data of 5060 NSCLC patients retrieved from this registry to reveal the imbalanced circumstances.

Methods:
Data of stage I-III patients were obtained from the NSCLC surgical outcome registry, which included 5060 patients who underwent lung resection surgeries from 17 tertiary hospitals nationwide in 2013-2014. Baseline data , surgical treatment pattern parameters, pathology, number of lymph nodes dissected, and total hospital costs. Heterogenity of quantitative data was analyzed using Kruskal-Wallis test.

Results:
Among the 5060 patients, the mean age was 59.7 , while 3204 were male. Mean pre-op forced expiratory volume in 1 second (FEV1) was 2.23L（P<0.01）, FEV1/FVC was 81.8%(P<0.01). 64.6% patients combined with at least one comorbidity. The average diameter of the tumor was 3.28cm(P<0.01). Mean operation time was 181.2 minutes. (P<0.05).The post-operative pathology confirmed 59.8% as adenocarcinoma while 30.2% as squamous carcinoma. Based on the data submitted by different centers, 88.4%(mean,0 to 98.41) patients who were confirmed as stage III patients received adjuvant therapy before surgery(P<0.01). The rate of minimally invasive surgery was 48.1(mean, 8.1 to 94.7)% in different regions(P<0.01). The number of stations of lymph nodes harvested was 5.8(mean, 4.3 to 7.4)(P<0.05). Mean hospital cost was 55070 (mean, 43051 to 69686 ) RMB(P<0.01).

Center	No. of lymph nodes in lobectomy	Cost(CNY)	VATS%	Adjuvant therapy ofStage III%	No. of patientssubmitted
1	6.3	46861	90.2	99.3	838
2	6.4	52891	27.5	98.8	788
3	4.5	47516	59.0	84.8	729
4	5.6	70875	60.3	50.3	676
5	5.3	51742	78.9	98.6	392
6	7.4	65409	26.1	100	387
7	6.8	50696	8.5	100	293
8	4.8	42206	8.3	98.6	265
Total	5.9	57757	50.07	88.45	5060

Conclusion:
The heterogenity of surgical treatment is quite huge in different centers of China. The baseline status before surgery, pre-operative therapy strategy, surgical technique, and health economic data submitted to the registry showed imbalanced development of NSCLC surgical treatment in different regions of China.

Background:
Neuroendocrine lung tumors (NET) are rare and heterogeneous neoplasms. Typical carcinoids (TC) and atypical carcinoids (AC) have better prognosis and their treatment is mainly focused on surgery. Large cell neuroendocrine carcinomas (LCNLC) are commonly widespread at diagnosis. Here we present clinical characteristics and outcome of patients with TC, AC and LCNLC treated at AC Camargo Cancer Center (ACCCC).

Methods:
We selected 114 consecutive patients with TC, AC or LCNLC treated at ACCCC from 2000 to 2016. Demographic variables included individual data, diagnostic and treatment patters. Data was collected and processed to obtain outcomes and survival information. It was intended to obtain not only epidemiologic characteristics, but also to determine and confirm selected variables as prognostic.

Results:
Main demographic results are described in the Table below:

VARIABLES		TC	AC	TOTAL
NUMBER OF PATIENTS		64(56.1%)	24(21.1%)	88(100%)
SEX	MALE	53.1%	41.7%	45.5%
	FEMALE	46.9%	58.3%	54.5%
MEDIAN AGE	YEARS	56.35	53.74	57.48
CLINICAL STAGING	I	83.9%	55.0%	76.8%
	II	4.8%	20.0%	8.5%
	III	6.5%	0.0%	4.9%
	IV	4.8%	25.0%	9.8%
ECOG	0	71.7%	84.6%	76.4%
	≥1	20.3%	15.4%	25.4%
COMORBIDITIES	YES	56.9%	65.2%	59.3%
	NO	75.4%	66.7%	73.1%
SMOKING HISTORY	YES	24.6%	33.3%	26.9%
	NO	75.4%	66.7%	73.1%
OTHER MALIGNANCIES	YES	32.8%	4.2%	25%
	NO	67.2%	95.8%	75%
FAMILY HISTORY	YES	59.4%	66.7%	61.4%
	NO	40.6%	33.3%	38.6%
SURGERY	YES	92.2%	79.2%	88.6%
	NO	7.8%	20.8%	11.4%

For the entire population, median progression free survival (mPFS) was 158.5 months. mPFS was not reached (NR) for TC and AC, with 5-year PFS 81% for TC and 84% for AC and 10-year PFS 69% for TC and 59% for AC; no Statistical Significance (SS) was found between TC and AC in mPFS (p=0,549). mPFS was worst, with SS, for age ≥65 years (158.5x61.5 months; p=0.018), staging ≥2 (NRx75.7 months; p=0,027), ECOG ≥1 (158.5x35.2 months; p=0.001), node positive disease (N0xN≥1: NRx33.9; p=0.001). Same tendencies were observed for TC, also with SS, but not for AC. Median overall survival (mOS) retained the same tendency: 5-year OS was 74% for TC and 55% for AC and 10-year OS was 74% for TC and 47% for AC. mOS was not reached. Age ≥ 65 years (p=0.001), ECOG ≥1 (p=0.001) and staging ≥ 2 (p=0.001) also were predictable for worst mOS.

Conclusion:
This study demonstrates an epidemiologic descriptive outlook of neuroendocrine LC in a Brazilian population. Older age, worst performance and higher staging were prognostic for poor outcomes in survival.

Background:
Lung cancer is the leading cause of cancer related morbidity and mortality in both the sexes in Nepal. It accounts for 15.4 % of total cancer as per hospital based Cancer Registry in Nepal. Majority of patients are diagnosed and treated at advanced stage. This can be partly contributed to long lag period between the onset of symptoms and the initiation of cancer treatment. This study tries to evaluate the factor contributing delays in various steps in lung cancer diagnosis and treatment.

Methods:
This retrospective cross-sectional observational study was conducted at Department of Clinical Oncology, Bir Hospital, National Academy of Medical Sciences (NAMS), Nepal. We reviewed the record of the all registered, histologically diagnosed lung cancer patient during the year 2012 and 2013

Results:
A total of 123 patient’s were diagnosed as Lung cancer and their records were evaluated. Out of these 123 patients, 60% of the cases were males. The mean age was 63.93 years with the youngest being 35 and the eldest was 83 years. Significant number of patient was in stage III (59%) and IV (33%). About 89% of the patients were smokers. Non-small cell lung cancer (NSCLC) accounted 83% and small cell lung cancer was (SCLC) 17%. A total of 17% (21) of patient were on empirical Anti-tubercular treatment (ATT) since the onset of current symptoms. While analyzing delay with independent T test showed mean delay of 25.01 days (-/+ SD 6.17) in patient without ATT and with ATT delay was 57.09 days (-/+ SD 8.05) (p=<0.01). Thirty five percentage (43) of patient received treatment within 1 month from the first hospital visit, 28% (34) within two months and 37%(46) within 3-4 months of the first hospital visit. The delay in specialist visit was shorter in advanced cancer and small cell cancer may be because of the acute presenting symptoms.

Conclusion:
Various factors contributing for the delays are lag time from symptom onset to first visit with primary physician, delay due to investigation and symptomatic treatment under primary physician care, delay further aggravated by empirical but inappropriate ATT, further delay due to diagnostic procedure to establish the cancer diagnosis. Thus proper and timely referral to the specialist from primary physician will reduce these delays and help to avoid situation where curable disease become incurable and significantly alters the prognosis.

Background:
Malignant pleural mesothelioma is an aggressive thoracic malignancy associated with exposure to asbestos, and its incidence is anticipated to increase during the first half of this century. Chemotherapy is the mainstay of treatment, yet sufficiently robust evidence to substantiate the current standard of care has emerged only in the past 5 years.

Methods:
A retrospective cohort study of 100 MPM patients referred to NCI, Cairo University in 3 years. Detailed data, Pearson's Chi (x2) square and Logistic regression model were used for statistical analysis.

Results:
We found a statistical significant relation between age ( 0.005), male gender (0.002), endemic area residence( 0.001), industrial workers ( 0.018), duration of exposure (0.04), smokers (0.009), Simian virus ( 0.019), P53 ( 0.001), RbP (0.001), PS ( 0.0.16) and development of high grade toxicity of platinum based chemotherapy. Median age = 46 years, only 17% of cases developed high grade toxicity complications of platinum based chemotherapy. Males were 59% of cases. PS, residence, smoking, occupation, history of asbestos exposure, family history, simian virus, P53, Rbp, dyspnea, chest pain, cough, expectoration, haemoptysis, weight loss, fatigue, metastatic symptoms, chronic lung infection, Tuberculous pleuritic, effusion, pleural thickening, Tracheal shift, TNM staging, surgical operations, pathological staging, radiotherapy ,cause of death and chemotherapy toxicity are assessed in our patients.

Conclusion:
Many factors predict high grade chemotherapy toxicity. So, search for target therapy and immunotherapy instead of chemotherapy in this selected group can improve both quality of life and response rate.

Background:
Lung cancer still remains as the principal death cause in many regions around the world. Unfortunate, between 60-70% of patients are diagnosed with advanced disease (clinical stage IIIB-IV). We report the overall survival of advanced lung cancer in patients treated at a private institution (Oncosalud – AUNA).

Methods:
We analyzed data of 75 patients with advanced lung cancer and treated at Oncosalud-AUNA between 2013-2014. Overall survival was determinate using Kaplan-Meier method and survival curves comparison were performed using logrank test.

Results:
The median age was 70 years (range: 39-91) and 49% of patients were women. In patients with clinical stage IV, the metastatic sites were generally brain (28%), osseous (18%), cervical and supraclavicular (14%). The 66.7% of patients received chemotherapy with/without radiotherapy, 9% radiotherapy only and 24% non-treatment. In patients previously treated with chemotherapy, 52% received targeted therapy. The 77% of patients hab died, the follow-up median of survivors was 23 months (CI95%: 17-29), survival median was 9.6 months (CI95%. 5.6-13.5) and 1 and 2 years survival rate were 38% and 23%, respectively. The survival rate at 1 and 2 years in those receiving targeted therapy ware 65% and 43%, and those who did not receive were 35% and 10%. The overall survival present a difference regarding to ECOG scale (p = 0.015) and CYFRA 21.2 (p = 0.04).

Conclusion:
Overall survival for our patients is similar to other series. Patients under ECOG scale <2 and CYFRA 21.1 < 3.3ng/ml had a relatively better prognostic.

Background:
We need to understand the living quality in our population, so we review performance status focus on ECOG 3,4,5 that includes a concept of capable of limited self-care, because this increases expenses at families and health system. We need to understand the variables that increases risk of higher ECOG values.

Methods:
Between January 2004 and December 2013, all patients diagnosed with a pathology of SCLC and NSCLC at National Institute of Oncology at Paraguay were analyzed retrospectively. ECOG performance status were recorded and SPSS 20 was used to analyze with logistic Binary regression

Results:
We studied 478 subjects. At age mean 60,40 [95% CI 59,45 to 61,34 ] years and ECOG performance status mean 2,13 [95% CI 2,06 to 2,20] points. Bivariate correlations show no relation with age, gender, living place, work, smoking, alcohol consumption, histopathology of lung cancer only with motive of consultation and clinical severity. In our model of predicting a ECOG 3 to 5 adding first motive of consultation show a Nagelkerke R2: 0.14, Hosmer y Lemeshow P: 0.95. Adding to the model clinical severity Nagelkerke R2: 0.07 Hosmer y Lemeshow P: 1.0. Variables in our predicting model show at clinical severity IIB stage OR:6,62 [95% CI 1,13 to 38,52 P=0.035], clinical severity IIIA stage OR: 3.85 [95% CI 1,18 to 12.51 P=0.025],clinical severity IIIB stage OR:4,49 [95% CI 1,87 to 10,78 P=0.001]. At limited-stage SCLC clinical severity OR: 10,12 [95% CI 1,88 to 54,34 P=0.007]. At first motive of consultation chest paint OR: 3,13 [95% CI 1,38 to 7,11 P=0.006]. Cough OR: 2,30 [95% CI 1,11 to 4,76 P=0.024]. Palpable Tumoral mass OR: 8,35 [95% CI 1,65 to 42,07 P=0.010].

Conclusion:
Regardless our expectations about relation of disability of patient with lung cancer about place of living, work, gender, age this variables show no relation with ECOG at 3 to 5. In Our review we found a prediction model with clinical severity adding 7% to prognostic of limited self-care and by adding to the model first motive of consultation a 14% of prognostic of worst ECOG status. If first consultation motive is chest pain, cough or palpable tumoral mass, this are strongly related with worst ECOG values. As a conclusion most of our patients are diagnostic in advance clinical stages with a bad performance status which will limited our options to treatment. All of these can be related with a late consultation or a late detection of the disease.

Background:
Mortality from lung cancer ranks first in men and third in women in Mexico. We aim to assess the mortality rate from lung cancer in the Mexican population during the period from 1990 through 2014.

Methods:
In this longitudinal study we analyzed the mortality rate of lung cancer, adjusted for age, sex and degree of marginalization. The mortality rate was adjusted applying the direct method. In adittion, we used the 2010 Mexican Population,which was taken from the Population and Housing Census. Deaths were taken from the mortality database of the Ministry of Health of Mexico. The degree of marginalization of the population was made based on the marginality index established by the National Population Council, it includes five categories of marginalization: Very low, low, medium, high and very high. The 33 states of the Mexican Republic are divided into these categories, approximately being six to seven in each. Finally, the annual percentage change was calculated.

Results:
During the study period a decrease we observed a decrease in the lung cancer mortality rate adjusted for age and gender. Thus, the rate in 1990 was higher (110 deaths per 100,000 population), which decreased to 90 per 100,000 in 2014, representing a decrease of more than 15%. In relation to gender we observed a decrease in the mortality rate for both genders. In addition, the mortality rate in women was three times lower from that among men througout the whole period of study. Nonetheless, we observed a slight increasing trend for women in the last years. Regarding the marginalization index we observed that the highest mortality rates occur in the states that comprise the categories with low and very low marginalization. Moreover, decline in mortality was also observed in those categories, unlike the categories of high and very high marginalization, where a slightly increase was observed during the period of study.

Conclusion:
Mortality from lung cancer has declined during the studied period, a situation that may be due to various situations such as diagnosis at earlier clinical stages or treatments more effectively, or under registry of mortality in the states that make up the categories with high or very high marginalization. Such situations must be studied in greater depth to identify the causes for the decline in mortality from lung cancer.

Background:
The objective of this study was to describe the trends of histology and age of patients with non-small cell lung cancer (NSCLC) treated with lung resection according to gender. The histology of lung cancer is changing in developed countries and there is still little information available for developing countries.

Methods:
Retrospective analysis of all patients (N=1030) with resected NSCLC between 1986 and 2015 in a university hospital of Southern Brazil. Differences in histology, stage and type of surgery were analyzed by sex and period (1986-1995, 1996-2005 and 2006-2015).

Results:
Most patients were males (64.5%), main histologic types were adenocarcinoma (44.5%) and squamous cell carcinoma (40.6%). Mean age at surgery was 56.5 years for women and 58.9 years for men in first period, and 62.2 for women and 64.6 for men in the last period (p<0.001), suggesting that it was approximately 2.4 years higher for men (p<0.001), in spite of the period. The proportion of histologic types were different by gender (p<0.001), showing that overall squamous cells carcinoma was more frequent in men (46.9%) than in women (29%), and the opposite occurred for adenocarcinoma (40.4% versus 51.8% for men and women, respectively). Analyses by period showed squamous cells carcinoma declined from around 38.9% in the first period to 23.2% in 2005-2015 for men, virtually equaling the proportion of adenocarcinoma in the last period. The proportion of adenocarcinoma in women increased from 11.9% in the first period to 24% in the last. Considering all NSCLC patients, females with adenocarcinoma represented 11.9% in the first and 24% in the last period. Figure 1



Conclusion:
As seen in developed countries, rates of lung cancer in females are rising over the last three decades, but have not surpassed men rates yet. Adenocarcinoma is consistently the most frequent histological type in women. In men squamous cell rate has decreased.

Background:
Lung is the leading death cause cancer related worldwide when considering both genders. The great effort to reduce smoking and introduce of cigarette changed lung cancer epidemiology. In developed countries the increase of adenocarcinoma and decrease of squamous cell carcinoma are well known. Other characteristic reported is the rising number of with the disease. Better understanding of current lung cancer epidemiology is necessary to design public health strategies for prevention, diagnosis and treatment.

Methods:
Retrospective analysis of all patients with non small cell lung cancer submitted to anatomical lung resection between 1986 and 2015 in an University Hospital of South Brazil. Patients were divided in three periods 1986-1995, 1996-2005 and 2006-2015. The same pathologist group made histology diagnosis and all staging was updated according to the new IASLC 7th edition. All analyses were performed using the SAS program.

Results:
In our Institution 1030 patients underwent anatomical lung resection for lung cancer between 1986 and 2015. 64.5% were males, average age 62.1 years, 40.6% squamous cell carcinoma and 44.5% adenocarcinoma, 23% advanced stage IIIA. The female proportion increased from 26.6% on first period to 44.2% on last period. Mean age at surgery treatment was 56.4 years for women and 58.9 for men on first period, and 62.2 for woman and 64.6 for men on the last period (p<0.001). The proportion of squamous cell changed from initially 49.6%, then 43% to 34.8% on the last period (p<0.001). In comparison in the same sequence we have for adenocarcinoma prevalence starting on 38.1%, 41.2% and most recently 49.5%. Stage IIIA was predominant on all periods with 23%, however early stages IA and IB combined represent 47.1% in the last group. Type of surgery was predominantly lobectomy and was verified decreasing in pneumonectomy rate.

Conclusion:
Analyses including gender showed that lung cancer in women is raising over the years but didn’t surpassed men yet. Women adenocarcinoma has increased the participation on total cases. The significant decrease of pneumonectomy reinforces the changes on surgical management technics and also correlates with more early staging diagnosis. The mean average age on surgery has increased for both men and women.

Background:
Lung Cancer is one of the leading causes of morbidity and mortality worldwide. It is most commonly attributed to smoking; a smaller proportion is attributed to occupational exposure. However, an earlier published study by Krishnamurthy et al 2012 from the authors institute reported the increasing trends of “Nonsmoking associated lung cancers” in the Indian subcontinent. A larger prospective study aimed at validating the initial findings was planned and this formed the basis of the present study.

Methods:
All consecutive histologically confirmed patients with lung cancer who presented to the outpatient department over a year (November 2014 to October 2015) were included in this current prospective study. A comprehensive questionnaire administered by a trained social worker captured all the demographic details including age, sex, occupation, smoking habits including exposure to second hand smoke, type of cooking fuel, histopathology and stage at presentation among others. (And later analyzed by SPSS Version 22.0)

Results:
713 patients presented with clinico-radiologicaly suspicious findings of lung cancer in the said period. A pathological confirmation of lung cancer could be ascertained in 495 patients and this cohort was further analyzed. The median age of presentation was 58 years; the male to female ratio was approximately 2.5:1. 52.12 % patients were nonsmokers. Adenocarcinoma (63 %) was the predominant histology. Nonsmokers, both among men (p=0.02) and women (0.001) presented more frequently with adenocarcinoma histology. Interestingly, 84.9 % (45/53) rural and 76.1 % (19/25) urban women who were nonsmoker reported exposure to indoor air pollution (second hand smoke/fuel used for cooking purposes) which was significantly associated with adenocarcinoma histology. (p=0.01) A majority of the patients (69.1 %) presented with clinical stage IV. (7[th] edition TNM) Nearly 60% of patients presented in ECOG performance status 3-4, nonsmokers incidentally presented in a better performance status than smokers (p = 0.017). 53% of the patients unfortunately were deemed suitable only for best supportive care. Only 97 patients (19.6 %) were offered potentially curative treatment and radical surgery accounted for < 3 % of the overall management.

Conclusion:
This prospective study validated our initial observation of the increasing trends of lung cancers among nonsmokers. Further, this study also reflected the global trend of rise in adenocarcinoma histology. These findings in a larger perspective will help clinicians better understand the magnitude and the direction of the lung cancer epidemic and also aid policymakers in better channelizing the resources for effective public health interventions.

Background:
BACKGROUND: To analyze principal histological types of lung cancer, as well as sex and the age of patients, staging, tumor location, smoking history, Chronic obstructive pulmonary disease (COPD) history, and survival of lung cancer patients in Croatia.

Methods:
METHODS: This was a retrospective study based on the analysis of medical charts of patients treated at the University Hospital „Split“, Split, Croatia, during 2015. and 2016.

Results:
RESULTS: From July 2015 to February 2016, 332 patients with lung cancer, most of whom (75,30%) were male, were treated. Patients were between 36 and 85 years, with median age 65,5 years. There were 273 (84,78%) patients with NSCLC (Non-Small Cell Lung Cancer) and 49 (15,22%) patients with SCLC (Small Cell Lung Cancer). The most common histological type in patients with NSCLC was adenocarcinoma (58%), followed by squamos cell carcinoma (38%), NSCLC NOS (not otherwise specified) (2%), adenosquamos carcinoma (1%) and large cell carcinoma (1%). Among patients with NSCLC 13% are EGFR positive. Patients had mostly lung cancer in right lung (59,35%), most of them were smokers (88,40%), 20,70% patients had COPD and 21,05 % patients had pleural effusion. Concerning staging, 73,80% patients had stage IIIB and IV at the time of diagnosis, and the remaining 26,20% were classified as stage I-IIIA. The principal sites of metastases were lungs (24,45%), bones (23,6%), and liver (12,9%). One-year survival was 23,21% and median overall survival was 8,82 months for patients presented with stage IIIB and IV. Median age at death was 67,5 years.

Conclusion:
CONCLUSION: In accordance with the literature most of the lung cancer patients in Croatia are men, older age (but younger compared to developed countries), the most common histological type is adenocarcinoma. Most of the patients have cancer in the right lung, most of them are smokers and minority had COPD or pleural effusion. Most cases are presented in advanced stages at the moment of diagnosis. This affects on survival rate, which is lower compared to developed countries. To increase survival rate in Croatia smoking cessation should be encouraged, lung cancer screening, diagnosis and therapy should be improved, patients should be included in clinical trials and palliative care for terminal patients should be improved.

Background:
Nonsmall cell lung cancer (NSCLC) has varying epidemiological patterns in different countries and also in different regions of each country. In a country with a high prevalence of lung cancer such as India, regional variations in demography exist.

Methods:
We did a retrospective analysis of histologically confirmed metastatic NSCLC patients who presented to our Department of Medical Oncology between August 2012 and July 2014. The patients were interviewed regarding their history of smoking (never smokers, light smokers, and heavy smokers). Never smokers were defined as those who have smoked <100 bidi/cigarettes in their life until disease onset. Light smokers were defined as those who smoke <10–100 bidi/cigarette pack years. Heavy smokers were defined as those who smoke more than 100 bidi/ cigarette pack years until disease. All lung cancer biopsy specimens were histologically characterized by morphology and immunohistochemistry (IHC). A diagnosis of AC was made if IHC staining was positive for CK7, TTF1, and napsin A; similarly, SCC was diagnosed if CK5/6 and p63 staining was positive. After confirmation of the histology subtype, patients were confirmed to have metastatic disease after assessment with contrast‑enhanced computerized tomography thorax, abdomen scan, and radiolabeled bone scan. Our study did not include the small cell lung cancer patients. The data were analyzed on SPSS version 22 (IBM) software.

Results:
A total of 304 patients were analyzed. About 55.6% of the patients were in the age group of 41–60 years. About 79.6% of the patients were symptomatic for <6 months before presentation. About 63.5% of the patients were smokers presenting with a median age of 59 years whereas nonsmokers formed 36.51% of the patients presenting with a median age of 47 (P < 0.001). About 82.6% of the male patients and 4.1% of female patients were smokers. Equal number of all patients had adenocarcinoma (AC) and squamous cell carcinoma (SCC) histology. AC histology was more common in the nonsmoking group (62% of patients). SCC histology was seen in 54.3% of smokers. Metastasis to the contralateral lung and pleura was seen in 58.2% of patients.

Conclusion:
NSCLC presents at a young age. Smoking is a significant risk factor and it is common in the urban populations as in the rural areas. Both AC and SCC histologies presented in equal proportions

Background:
The clinico-pathological profile of primary lung cancer has changed considerably over the last few decades in India. Available literature suggests that the features of lung cancer in India like prevalence, incidence, aetio-pathogenesis and presentation vary markedly from the west. We performed a prospective evaluation of the unique demographic features of lung cancer with specific emphasis on smoking and histopathological trends.

Methods:
We analysed all pathologically proven lung cancer cases registered over a period of initial 30 months in the department of Pulmonary Medicine of this All India Institute of Medical Sciences, Bhubaneswar. The patients were evaluated for their epidemiological, clinical and pathological profiles. The data were recorded in MS Excel spreadsheets and subjected to appropriate statistical analysis. Data was collected directly from patients’ paper and electronic medical records. All patients of histologically proven lung cancer were included.

Results:
A total of 179 patients were included in the database of which 6 patients were excluded for significant missing data. There were 114 male and 59 women) average age 57.24 with a M:F ratio of 1.93 :1. Over half (56%) of the patients were active or past smokers, while 48% patients had not been exposed to active or passive smoking. Bidi (tobacco flake wrapped in tendu leaf) smoking was more common (37%) than cigarettes (19%) while 9% smoked both. Exclusive chewed tobacco use was seen in 12% while combined use of chewed and smoked tobacco was seen in 4% patients. The proportion of women never-smokers with lung cancer was significantly higher (89%) compared to men (28%). More than two-thirds patients (69.8%) presented with metastatic disease. Amongst patients with a definitive cytohistological diagnosis, the prevalence of adenocarcinomas was highest (56.3%) followed by squamous (30.8%), small cell (8%) and NSCLC NOS (4.9%).

Conclusion:
Adenocarcinoma is the commonest histological subtype in this region. Prevalence of lung cancer among non-smokers is also high in the eastern part of India. The demographic profile of patients with lung cancer in eastern India is unique with a much higher proportion of tobacco chewers and non-smoker especially in women. There is significant epidemiological trends towards a predominant adenocarcinoma histology. Most of the patients present at an advanced stage, probably due to lack of awareness and limited diagnostic resources in this part of the country. Although there are direct association with smoking, there has been an increase in the non-smoking lung cancers worldwide.

Background:
Lung cancer is the fourth most common cancer in the Gulf Cooperation Council countries among males and the third among females. It is the commonest cancer among Bahraini males accounting to 16.9% of all cancers and the third in Bahraini females contributing to 5.8 % of all female cancers. The aim of this study was to describe the epidemiology of lung cancer among the Bahraini population during the period 1998-2011.

Methods:
All Bahraini registered lung cancer cases in the national cancer registry from 1 January 1998 to 31 December 2011 were included in the study. Incidence rates were calculated using the CANREG software, in which the annual crude incidence rates, age specific incidence rates and the age standardized incidence rate (ASR) were computed.

Results:
Six hundred sixty four lung cancer cases (72.4%, males and 27.6% females) were diagnosed during the study period. The annual average number of cases was 47.5 per year. The mean age at diagnosis during the study period was 70.1 years. The average annual ASR was 26.1/ 100,000 among males and 10.0 / 100,000 among females. There was a tendency for a decreased trend of the ASR during 1998-2011 in both sexes. Twenty six percent of lung cancer cases were squamous cell carcinoma and 17.9% adenocarcinoma. The grades of 70.3% were unknown and 13.4% were poorly differentiated. The stage was unknown for 65.0% of the cases, while 18.5% had distant metastasis and 9.8% were localized. The majority (88.9 %) of the lung cancer cases were dead by the end of the study period with a five-year survival rate of 3.0%.

Conclusion:
A welcomed decline in the incidence of lung cancer has been noted over the past 14 years. However, more efforts should be put to reduce the proportion of lung cancer cases with unknown stage and grade. The incidence of histological types, which are strongly dependent on tobacco smoking, notably small cell, squamous cell and large cell carcinomas, accounted for over one third of lung cancer cases. Future research should be directed towards better understanding of the lung cancer risk factors and the effectiveness of tobacco control measures in in the country.

Background:
In the patients with NSCLC were tested for diagnostic EGFR mutation, in addition to activating mutations, such as Exon 19 Deletion & Exon 21 L858R that is occupies a large amount. The mutation is said to be resistant to EGFR TKIs occupies smaller amount. However, this is also a challenge for the care and treatment for patients with NSCLC.

Methods:
Retrospective, cross-sectional descriptive statistics, clinical case series.

Results:
PRIMARY MUTATIONS WITH EGFR TKIs RESISTANCE §Activating EGFR Mutations: Exon 19 Deletion: 356/597 cases = 59,63% and Exon 21 L858R: 176/597 cases = 29,48%. §Primary Mutations with EGFR TKIs Resistance - Single mutations: 46/597 cases = 7,71% (Male: 27 cases + Female: 19 cases), including: Exon 20 Insertion: 25 cases, Exon 18 G719X: 14 cases, Exon 20 S768i: 5 cases, Exon 20 T790M: 2 cases. §Primary Mutations with EGFR TKIs Resistance - Double mutations: 19 / 597 cases = 3,18% (Male: 11 cases + Female: 8 cases), including: Exon 21 L858R + Exon 20 T790M: 6 cases, Exon 19 Deletion + Exon 20 T790M: 5 cases, Exon 18 G719X + Exon 20 Insertion: 2 cases, Exon 21 L858R + Exon 20 G786i: 3 cases, Exon 18 G719X + Exon 20 S768i: 3 cases. ACQUIRED MUTATIONS WITH EGFR TKIs RESISTANCE §Total cases with the gene mutation diagnosis repeating again (Sampling with Histology or Cytology): 113/597 cases (18,93%). Detection rate for new resistance EGFR TKIs mutations: Number of Cases: 56 cases/113 cases (49,56%). §Distribution rate % of acquired resistance mutations in EGFR TKIs: · Exon 20 T790M (Single & Double Mutations): 28 cases (50 %) · MET Amplification: 4 cases (7,14 %) · HER2 Amplification: 13 cases (23,21 %) · Small Cell Carcinoma Transformation: 5 cases (8,93 %) · PIK3CA Mutation: 2 cases (3,58 %) · The Other Mutations: 4 cases (7,14 %) §The expression of EGFR T790M mutation: T790M Single Mutation: 14 cases; T790M + Exon 20 Insertion: 3 cases; T790M + A763V: 4 cases; T790M + L777G: 1 case; T790M + Y801C: 3 cases; T790M + G719X: 2 cases; T790M + S768i: 1 case.

Conclusion:
§ There are the expressed resistance mutations EGFR TKIs in NSCLC patients at Pham Ngoc Thach Hospital, including: Primary EGFR TKIs Resistance & Acquired EGFR TKIs Resistance. § The rate of drug-resistant mutant EGFR TKIs are small but still important issues in the Care and Treatment of lung cancer.

Background:
Threonine 790 is regarded as a “gatekeeper” to inhibit ATP from binding to EGFR Tyrosine kinase domain. T790M mutation could increase the affinity for ATP. About 30% de novo T790M mutation was detected in NSCLC patients and showed spatiotemporal heterogeneity and variation in individual NSCLC patients. We hypothesize that T790M mutation may be a “regulating” mechanism for EGFR signal transduction pathway and is probably cleared quickly by DNA repair system in healthy persons.

Methods:
Peripheral blood samples were taken from 50 healthy volunteers, 12 resectable lung adenocarcinoma (LADC), 100 advanced LADC (11/100 acquired resistance for gefitinib). A novel cSMART assay (circulating single-molecule amplification and resequencing technology, which counts single allelic molecules in plasma base on next generation sequencing) was performed for detection and quantitation of EGFR mutation in ctDNA from plasma. Matching tumor biopsy samples were obtained from 100 advanced LADC, the EGFR gene mutations were determined by amplification refractory mutation system (ARMS) -PCR analysis.

Results:
The sensitivity and specificity of cSMART plasma assay for EGFR L858R, 19del and T790M was showed in Table 1. The sensitivity and specificity for T790M was obviously lower (50.0% and 72.9% respectively). No L858R and 19del but 14.0% T790M DNA copies were found in plasma from 50 healthy volunteers, and only 1 copy T790M was detected. The T790M positive rate of resectable and advanced LADC patients were almost similar (33.3% and 28.0%) in plasma, 1 or 2 T790M copies were found in the resectable patients and varied from 1 to 622 T790M copies with an average of 34.0 in advanced LADC patients. Patients with acquired resistance to gefitinib, 45.4% harbored T790M with an average of 268.2 copies in plasma. All but one advanced patients harboring T790M mutation were accompanied with other EGFR mutations.

Conclusion:
T790M mutation may be of a “regulator” and has physiological function.

Table 1 EGFR mutations detection by plasma cSMART assay and tumor samples ARMS-PCR in advanced LADC patients

EGFR mutations	Sensitivity	Specificity
L858R	91.7%(22/24)	100.0%(76/76)
19Del	79.2%(19/24)	100.0%(76/76)
T790M	50.0%(2/4)	72.9%(70/96)

Background:
ROS1 fusion is of a low frequency genetic alteration in non-small cell lung cancer (NSCLC). The clinical trial showed that NSCLC patients harboring ROS1 fusion could benefit from crizotinib treatment. Several studies reported that immunohistochemistry (IHC) could be employed as a screening method for detecting ROS1 fusion in tumor tissue using Anti-ROS1(D4D6) antibody. Malignant pleural effusion (MPE) is the common sample type in advanced NSCLC, the reliability of ROS1(D4D6) for detecting ROS1 fusion in MPE cell blocks (CBs) should be explored.

Methods:
Anti-ROS1(D4D6) monoclonal antibody (Cell Signaling Technology, Danvers, USA) IHC testing was performed on 227 formalin fixed paraffin embedded (FFPE) MPE CBs from lung adenocarcinoma patients. RT-PCR using ROS1 fusion gene detection kit (AmoyDx) was performed to detected ROS1 gene fusion as a comfirming test. Some other lung cancer driver genes were also detected in both IHC/RT-PCR ROS1 positive samples, among which EGFR, KRAS, BRAF, PIK3CA and HER2 20 exon mutation was tested by amplification refractory mutation system kits(AmoyDx), ALK and RET fusion was tested by RT-PCR kits(AmoyDx).

Results:
4 of 227 MPE samples(1.76%) of lung adenocarcinoma were interpreted as ROS1 fusion-positive cases by IHC staining, and the cytoplasmic and membranous granular positive signals were displayed. Comparison with RT-PCR testing results, 3 of 4 IHC positive cases were verified by RT-PCR, the sensitivity was 100% and specificity of was 99.6%. The clinicopathologic features and other genes status of 3 both IHC/RT-PCR positive patients were showed Table 1. One ROS1 IHC/RT-PCR positive lung adenocarcinoma patient received crizotinib therapy and obtained partial response.

Conclusion:
ROS1 (D4D6) would be a reliable antibody for screening ROS1 fusion-positive lung adenocarcinoma on FFPE MPE CBs by IHC assay and shows high specificity in the FFPE MPE CBs samples .

Table 1 Clinicopathologic features and genes status of ROS1 IHC/RT-PCR positive patients

	Gender	Age	Smoking	TTF1/ P63	E/k/B/ P/H	A/R	Therapy	Efficacy	PFS/Follow -up
p1	Male	55	Smoker	+/-	WT	WT	crizotinib	PR	10m+/alive
p2	Female	60	Never	+/-	WT	WT	No		1m/Dead
P3	Male	62	Never	+/-	WT	WT	PEM+CIS	PR	9m+/alive

E/K/B/P/H: EGFR/KRAS/BRAF/PIK3CA/HER-2 20 exon mutation, A/R: ALK/RET fusion, WT: wild type, PEM+CIS: Pemetrexed plus Cisplatin,PR: partial response, m: months

Background:
Lung carcinoma represents 2.9% of cancers seen at King Faisal Specialist Hospital and Research Centre (KFSH&RC) as per KFSH&RC Tumor Registry (2013), and 4% of cancers in Saudi Arabia as per National Cancer Registry (2010). EML4-ALK re-arrangement play an important oncogenic driver role in lung adenocarcinoma tumorgenesis in 3-5% of cases. ALK gene rearrangement (inversion in chromosome 2) testing can identify patients with adenocarcinoma who are sensitive to ALK tyrosine kinase inhibitors. No data is available on the prevalence of ALK rearrangement changes in Saudi lung cancer patients. The aim of this study is to evaluate the prevalence of ALK gene rearrangement in lung adenocarcinoma of Saudi patients.

Methods:
A total of 172 cases of lung adenocarcinoma diagnosed at KFSH&RC between January 2013 to May 2016 were identified. Formalin-fixed paraffin embedded tissue samples of these patients were analyzed for ALK gene rearrangement using fluorescence in situ hybridization (FISH), utilizing break-apart probes from Vysis (Abott Molecular, II, USA).

Results:
Eleven (11) cases exhibited ALK gene rearrangement (6.4%). Nine out of eleven cases were stage IV and two cases were stage III. Median patients age was 47 years (21-71 year) with male predominance (males 77%, female 25%). All cases were moderately to poorly differentiated adenocarcinoma. None of our cases showed signet ring cells or abundant intracellular mucin.

Conclusion:
Our findings showed the incidence of ALK gene rearrangement in lung adenocarcinoma in Saudi patients is 6.4%. This is slightly higher in comparison to the published data which may be attributed to KFSH&RC being a tertiary referral Center.

Background:
Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors have improved the outcome of patients with EGFR-mutated lung adenocarcinoma (ADC). However, EGFR mutation occurred in about only 10-15% of ADC, but other alterations are emerging as potential target of drugs. We analyzed the frequency of potentially targetable driver alterations in a series of advanced EGFR-wild type (wt) NSCLC patients.

Methods:
724 advanced EGFR-wt NSCLC patients enrolled from the Wide Catchment Area of Romagna (AVR) between January 2013 to December 2014 were included in the study. KRAS, BRAF, ERBB2, PIK3CA, NRAS, ALK, MAP2K1, RET and DDR2 mutations were analyzed by Myriapod[®]Lung Status kit (Diatech Pharmacogenetics) on MassARRAY[®] (SEQUENOM[®] Inc, California). ERBB4 was evaluated by direct sequencing and EML4-ALK and ROS1 rearrangements were assessed by immunohistochemistry or fluorescence in situ hybridization.

Results:
331 (45.7%) patients showed at least one alteration. Of these, 72.2%, 6.3%, 3.6%, 1.8%, 2.1% and 1.2% patients had mutations in KRAS, BRAF, PIK3CA, NRAS, ERBB2 and MAP2K1 genes, respectively. Only one patient showed a mutation in ERBB4 gene. EML4-ALK and ROS1 rearrangements were observed in 4.3% and 1.4% of all patients, respectively. The distribution of mutations in relation to gender and smoking habits is reported in the Table. Overlapping mutations were observed in 7 KRAS-mutated patients: 2 (28.6%) patients were also mutated in PIK3CA, 4 (57.1%) showed also an EML4- ALK translocation and one (14.3%) had a ROS1 rearrangement. One (0.3%) patient showed both BRAF and PIK3CA alterations. Correlation analyses between the different mutations and patient outcome are ongoing.

GENE	Mutated Patients N (%)	Gender		Smoking Habits*
		Female (%)	Male (%)	Smoker (%)	Never Smoker (%)
KRAS	239 (33)	93 (39)	146 (61)	115 (48.1)	9 (3.8)
BRAF	21 (3)	11 (52.4)	10 (47.6)	11 (52.4)	1 (4.8)
NRAS	6 (0.8)	4 (66.7)	2 (33.3)	4 (66.7)	-
PIK3CA	12 (1.6)	4 (33.3)	8 (66.7)	5 (41.7)	-
MAP2K1	4 (0.5)	-	4 (100)	1 (25)	-
ERBB2	7 (0.9)	5 (71.4)	2 (28.6)	-	1 (14.3)
EML4-ALK	31 (4.3)	20 (64.5)	11 (35.5)	12 (38.7)	8 (25.8)
ROS1	10 (1.4)	7 (70)	3 (30)	3 (30)	5 (50)

*: some data are missing

Conclusion:
Driver mutations were detected in about 50% of EGFR wt lung ADC patients. Such alterations could represent potential targets for therapy and could be evaluated in routine multiplexed testing to obtain a wider tumor molecular characterization.

Background:
Adequate testing for molecular changes in non-small cell lung cancer (NSCLC) is necessary to ensure the best possible treatment. However, it is unknown how well molecular testing is performed in daily practice. Therefore we aimed to assess the performance of testing for EGFR, KRAS mutation and ALK translocation in metastatic NSCLC on a nationwide basis.

Methods:
Using the Netherlands Cancer Registry, all stage IV non-squamous NSCLC from 2013 were identified and matched to the Dutch Pathology Registry (PALGA). Data on molecular testing for EGFR, KRAS and ALK were extracted from excerpts of pathology reports. Proportions of tested and positive cases were determined and interlaboratory variation was assessed. Finally, degree of concordance between ALK immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) results was evaluated.

Results:
In total, 3393 stage IV non-squamous NSCLCs were identified, and 3183 (93.8%) were matched to PALGA. Fifty-two tumors were excluded as pathology reports described a lung tumor other than non-squamous NSCLC or a tumor with another origin, leaving 3131 tumors. All 48 laboratories had access to molecular testing, either in house or via outsourcing. The table shows the nationwide proportions of cases tested and positive for EGFR, KRAS and ALK, as well as the interlaboratory variation. EGFR and KRAS mutations occurred together in 8 patients, ALK translocation occurred together with EGFR mutation in 3 patients and with KRAS mutation in 2 patients. In 272 cases, ALK had been tested using both IHC and FISH, and the methods were conclusive in 253 cases. IHC and FISH were concordant in 239 cases (94.5%; Kappa 0.728, p=0.069), 5 discordant cases were IHC+/FISH- and 9 were IHC-/FISH+.

Nationwide proportions of tested and positive cases and interlaboratory variation.

	Total	Tested cases; n (%)	Range in tested cases between Laboratories	Positive cases; n (%)	Range in positive cases between laboratories
EGFR	3131	2237 (71.4)	33.7% to 93.5%	243 (10.9)	6.1% to 21.6%
KRAS	3131	2292 (73.2)	20.9% to 93.6%	845 (36.9)	27.0% to 48.1%
ALK	3131	905 (28.9)	7.0% to 72.6%	51 (5.6)	0% to 13.6%
ALK (in case of EGFR and KRAS wildtype)	1227	685 (55.8)	19.4% to 100%

Conclusion:
These results suggest that in 2013 molecular testing was suboptimal in the Netherlands, especially for ALK. To determine whether molecular testing has improved, 2015 data will be analyzed in the near future as well.

Background:
A translocation in the anaplastic lymphoma kinase gene is found in 3-5% of non-small cell lung cancer (NSCLC). Patients with this mutation (ALK+) have shown marked responses to the tyrosine kinase inhibitor crizotinib. Second line Crizotinib has been available in Scotland since October 2013 for patients with ALK+ NSCLC. Since January 2014, reflex testing at diagnosis of all non-squamous NSCLC has been carried out in the West of Scotland (WoS) regardless of stage. Here we present the demographics of an Alk +ve cohort from an unselected Scottish NSCLC population and their clinical outcomes.

Methods:
Details of patients with Alk+ NSCLC were obtained from the regional molecular genetics laboratory. 60 patients with NSCLC from WoS tested ALK+ between 1st January 2014 and 30[th] June 2016. Patient records were reviewed retrospectively.

Results:
Median laboratory turnaround for alk testing was 21 days in 2014, 14 days in 2015 and 13 days in 2016. 3 (5%) patients were under 50 years, 8 (13%) 50-60 years, 23 (38%) 60-70 years, 17 (28%) 70-80 years and 9 (15%) patients were > 80 years old at time of testing. Median age was 69. 55% were male and 45% female. 67% were current or ex smokers. Only 22% were never-smokers. The majority (82%) had stage 3 or 4 disease at diagnosis. Only 39% (17/38) of patients with stage 4 ALK+ NSCLC were well enough to receive chemotherapy and 4 of these (24%) did not complete all planned cycles. 10 patients have received crizotinib so far. Median number of cycles is 3 overall (range 1-9). Where documented, reasons for discontinuation were disease progression or death from NSCLC (4), sudden death not related to NSCLC (2), intercurrent illness (2) and pneumonitis (1) . 3 patients continue on crizotinib as of June 2016. Of the 13 patients who have received crizotinib, 3 have had a PR, 4 SD 2 PD and 4 NE. Updated outcomes will be presented.

Conclusion:
A high quality reflex ALK testing service is being delivered in WoS with clinically acceptable turnaround times. Our ALK+ patients do not entirely reflect the literature and are frequently elderly, current or ex-smokers with advanced and aggressive disease. This may reflect the unselected nature of this population. Many Alk+ patients were too unwell to receive chemotherapy or tolerated it poorly and did not have the opportunity to access an alk inhibitor. First line Alk inhibitors may improve outcomes for this group of patients.

Background:
Lung adenocarcinoma patients in advanced stage of disease that harbor EGFR sensitizing mutations are eligible for treatment with tyrosine kinase inhibitors (TKI) due to a high likelihood of response. Most patients will ultimately develop resistance at disease progression. The T790M mutation is a dominant resistance mechanism to TKI. EGFR plasma testing enables non-invasive monitoring and detection of T790M. We followed patients with EGFR sensitizing mutations by measuring EGFR mutations in plasma during TKI treatment.

Methods:
We analyzed patients who were diagnosed lung adenocarcinoma stage IV, detected EGFR sensitizing mutations in tumor tissue samples and treated with TKI at University Clinic Golnik. We collected baseline plasma samples prior to TKI treatment and consecutive plasma samples at different time intervals after initiation of therapy. At the beginning, two separate tests, cobas® EGFR Mutation Test for tissue (CE-IVD) and plasma (under development) were used, and since October 2015 one test for tissue and plasma, cobas® EGFR Mutation Test v2 (Roche, Pleasanton, CA, USA) is used. Detected EGFR mutations in plasma samples were expressed as semi-quantitative index (SQI) which reflects a proportion of mutated versus wild-type copies of the EGFR gene.

Results:
During 2-year period we collected 414 peripheral blood samples from 63 patients and performed 619 EGFR plasma tests. There are 25 patients with baseline and serial follow-up EGFR plasma tests, 16 patients with only serial follow-up EGFR tests since they started with TKI treatment before EGFR plasma testing was available, 5 patients are included in adjuvant setting, and 17 patients had no monitoring due to various reasons. Maximum number of EGFR plasma tests done per patient was 27 at 20 time-points. When introducing EGFR plasma testing, we prepared two aliquots of plasma out of 10 ml blood sample in EDTA-tubes and run test for both aliquots. Results of reproducibility study showed 95% concordance rate between both aliquots and thus we modified protocol to run the second aliquot only if the first one was negative. At disease progression, reappearance of EGFR sensitizing mutations with increasing SQI levels was detected. In 14 patients who progressed we detected T790M mutation, in 10 of them during monitoring TKI treatment. We also observed daily variation in EGFR mutation levels in the plasma.

Conclusion:
These data support the value of EGFR plasma testing to monitor the patient`s response to TKI and detect T790M resistance mutation prior to clinical progression in a routine clinical setting.

Background:
Patients with anaplastic lymphoma kinase (ALK) gene rearrangements manifest good responses to ALK inhibitors and thus, accurate and rapid identification of ALK gene rearrangements is essential for the clinical application of ALK-targeted therapies. The aim of this study was to investigate the diagnostic accuracy of the recently developed ALK RNA-in situ hybridization (RNA-ISH) assay, using formalin fixed paraffin embedded samples of lung adenocarcinoma tissue.

Methods:
We first tested whether ALK RNA-ISH could be performed in 11 resected lung adenocarcinomas in which ALK gene rearrangements were confirmed by immunohistochemistry (IHC, D5F3), and/or fluorescence in situ hybridization (FISH), and also clarified the intra-tumor heterogeneity of ALK RNA-ISH by counting 100 tumor cells in 10 different loci (total, 1000 tumor cells) in each tumor. Secondly, we analyzed the diagnostic accuracy of ALK RNA-ISH in tissue microarrays (TMAs) containing 294 lung adenocarcinoma cores (by counting 100 tumor cells in each core) with no information about ALK gene rearrangements and compared these results with those of conventional IHC and FISH tests.

Results:
ALK mRNA expression was observed in all 11 resected lung adenocarcinomas by the ALK RNA-ISH assay and the median of positive tumor cells was 67.7%, whereas ALK mRNA expression was not observed in normal lung cells in the background. Next, 5 ALK positive cases were found by IHC and/or FISH in the 294 cases of lung adenocarcinoma. The median of positive cells by ALK RNA-ISH in these 5 cases was 75.6% (range: 40-94%), whereas the median of positive cells by ALK RNA-ISH in the remaining 289 cases was 0.3% (range: 0-15%). When the cutoff value was set as 15% based on the first test, the ALK RNA-ISH–positive and ALK RNA-ISH–negative cases were readily distinguishable with 100% sensitivity and specificity compared with the results of IHC and/or FISH.

Conclusion:
Our findings suggest that the ALK RNA-ISH assay is useful for detecting ALK positive lung adenocarcinomas with high sensitivity and specificity compared with the conventional IHC and FISH test. Thus, this study provides important and timely insight into the clinical testing of ALK in lung cancer because the RNA-ISH assay detects the target mRNA easily and rapidly.

Background:
EGFR mutations are present in approximately 15% of NSCLC Caucasian patients, with a similar frequency described in Argentina.Exon 19 deletions and exon 21 L858R are consider common mutations (>90 %) that predict better progression free survival with EGFR-TKIs than with chemotherapy treatment. Most relevant uncommon mutations had a frequency ranged from 1.9% to 7.9% between different populations and their outcome, in general, is less favorable.

Methods:
We analyzed, retrospectively, our dataset of EGFR mutational status in the last two years with the objective to describe the frequency and characteristics of the patients with uncommon EGFR mutations in our population of NSCLC patients. The mutational analysis was performed on formalin fixed paraffin-embedded tissue blocks. EGFR exons 18 through 21 were amplified by PCR- based technology.

Results:
A total of 113 patients underwent EGFR testing since January 2014 until June 2016. Among them, 29 cases (25.7 %) harbored EGFR mutations. The exon 19 deletions (n=9; 8%) and L858R point mutation (n=6; 5.3%) accounted for the 51.7 % of EGFR mutated cases (13.3% of the population explored) while 48.3 % were uncommon mutations (12,4%). In the last group, mutations sites were: G719X in exon 18 (n=9; 8%), L861Q in exon 21 (n=2; 1.8%), INS20 in exon 20 (n=2; 1.8%) and S768I in exon 20 (n=1; 0.9%). All the 14 patients carrying EGFR uncommon mutations had adenocarcinoma histology. In addition, they were more frequently observed in men than in women (79% versus 21%) and in smokers than in nonsmokers (65% versus 45%). The mean age was 62.5 years. Most of the patients (n=11; 75.6%) had advanced disease (stage IIIB-IV) at diagnosis. No one had Asian ethnicity. Seven patients (50%) received EGFR-TKIs for first or second line treatment (4 erlotinib, 2 afatinib and 1 gefitinib). None of them showed sustained clinical benefit. At present, 7 out of 12 patients had died.

Conclusion:
Although the clinical characteristics of our cohort are similar to the data published, we noted a higher and unusual frequency of EGFR uncommon mutations especially exon 18 G719X.All cases treated with EGFR-TKIs showed poor sensitivity to therapy. Time to treatment and accessibility to appropriate therapy in this subgroup are important issues to explore in future reports from public institutions of our region.

Background:
In non-small cell lung cancer (NSCLC), circulating tumour DNA (ctDNA) has gained acceptance as a potential alternative to tissue biopsies to identify targetable mutations. Individual ctDNA platforms have varying abilities to detect specific mutations. A prospective, multicenter study was conducted to determine concordance, sensitivity, and specificity of ctDNA genotyping, with archival tissue DNA (atDNA) as the reference standard.

Methods:
Patients with incurable advanced NSCLC at the BC Cancer Agency were enrolled over 14 months. Next-Generation Sequencing (NGS) and high-throughput multiplex amplification of a 27-gene panel (Raindance) was used for atDNA analysis. Four mL of plasma was collected in Streck (Cell Free DNA BCT) tubes for ctDNA genotyping using the Boreal Genomic OnTarget. Analysis of concordance, sensitivity, and specificity was conducted with atDNA used as the standard.

Results:
Seventy-six patients were enrolled, median age 66, 33 (44%) male, 69 (91%) metastatic disease, 47 (62%) with primary disease in-situ. Twenty-six EGFR mutations in 22 atDNA samples, and 12 mutations in 11 ctDNA samples were detected, with a concordance of 78%, sensitivity of 39%, and specificity 98%. One EGFR T790M mutation was positive by ctDNA alone. Twenty-one KRAS mutations in 21 atDNA samples were detected. Within this subgroup, 10 ctDNA samples had KRAS mutations with a concordance of 76%, sensitivity of 50%, and specificity of 80%. Fourteen KRAS mutations were detected by ctDNA only. The interval between archival tissue and ctDNA collection, and time between treatment and ctDNA collection, did not significantly impact the rate of concordance (p> 0.05).

Conclusion:
Although the sensitivity is limited, the Boreal Genomic OnTarget ctDNA analysis is specific in identifying clinically relevant EGFR mutations and has acceptable concordance rates between ctDNA and atDNA testing. Targetable EGFR and KRAS mutations were detected in ctDNA but not atDNA, which may reflect site of biopsy, tumor heterogeneity, or technical limitations of assays used. Given the high specificity and non-invasive nature of this test, positive results in EGFR mutations can be used to direct therapeutic decisions, especially accounting for clonal evolution overtime in detection of resistance mutations.

Background:
2015 new WHO classification lists four rare variants of invasive adenocarcinoma of the lung (VIA): invasive mucinous adenocarcinoma, colloid adenocarcinoma, fetal adenocarcinoma and enteric adenocarcinoma. Very little information is known regarding the molecular alterations and prognostic values for rarity of VIA. The aim of present study was to investigate the common actionable mutations and survival in VIA.

Methods:
Patients who with pathologic confirmed as VIA with completely resected stage I-ⅢA were enrolled from 2010 to 2013. For comparison, we evaluated the gene status and survival from 380 non-VIA lung adenocarcinoma patients in 2012. RT-PCR was utilized for detecting the mutations of EGFR, KRAS, NRAS, PIK3CA, BRAF, HER2 and the fusion of ALK, ROS1 and RET. Survival curves were plotted with Kaplan-Meier method.

Results:
Thirty one patients were recruited from 1120 lung adenocarcinoma,including invasive mucinous adenocarcinoma (n=15), enteric adenocarcinoma(n=9), colloid adenocarcinoma (n=4) and fetal adenocarcinoma(n=3) . The overall frequency of gene abnormality in VIA was 48.4% (15/31). The genes abnormality was as follows: KRAS mutation (n=5), ALK rearrangement (n=4),PIK3CA (n=2), EGFR mutation (n=2), HER2 mutation (n=1) and ROS1 rearrangement (n=1). No mutations of NRAS, BRAF or RET were observed . The frequency of gene abnormality was lower in VIA than non-VIA patients (48.4% vs.74.7%,P=0.0015). No recurrence free survival difference existed in the VIA and non-VIA patients (38.0 vs.47.0 months,P=0.524) . A trend of worse overall survival in VIA than those with non-VIA patients was found(48.0vs.57.0 months, P=0.052).

Conclusion:
VIA is rare in lung adenocarcinoma with lower frequency of common gene abnormality. Invasive mucinous adenocarcinoma was the most frequent subtype and KRAS was a predominant actionable mutation in VIA patients. A trend of worse survival existed in VIA than non-VIA patients.

Background:
ALK, ROS1 and RET rearrangements represent three most frequency of fusion genes in non-small cell lung cancer (NSCLC). Rearrangements of the three genes are predominantly found in lung adenocarcinoma，while,rare in non-adenocarcinoma. The aim of this study was to investigate the frequency, clinicopathological characteristics and survival of ALK, ROS1 and RET arrangements in non-adenocarcinoma NSCLC patients.

Methods:
We screened ALK,ROS1 and RET arrangements in patients with completely resected non-adenocarcinoma NSCLC using reverse transcriptase polymerase chain reaction (PCR). All positive samples were confirmed with fluorescence in situ hybridization (FISH). Survival analysis was performed with Kaplan-Meier method and log-rank for comparison.

Results:
Totally, 385 patients, who underwent complete resection, including 245 with squamous cell carcinoma, 85 with adenosquamous carcinoma and 55 with large cell carcinoma were enrolled. Twelve patients were identified as harboring fusion genes,including seven with ALK, three with ROS1 and two with RET rearrangements. The frequencies of fusions in adenosquamous carcinoma, squamous cell carcinoma, and large cell carcinoma were 8.2%,1.6% and 1.8%, respectively. The median age of 12 patients was 49.5 years and three patients had smoking history. No survival difference existed between fusion genes positive and negative patients (36.7 vs.50.2 months,P=0.21).

Conclusion:
The frequencies of ALK, ROS1 and RET rearrangements are low in non-adenocarcinoma NSCLC patients, and the clinical characteristics are similar with those in lung adenocarcinoma. Fusions of the three genes are not prognostic marker for non-adnocarcinoma NSCLC patients.

Background:
ERBB2 (HER2) is a driver gene identified in non-small cell lung cancer (NSCLC). The prevalence, clinicopathology, genetic variability and treatment of HER2-positive NSCLC in Chinese population are unclear.

Methods:
Eight hundred and fifty-nine patients with pathologically confirmed NSCLC were screened for HER2 mutations using Sanger sequencing. Next-generation sequencing (NGS) was performed in positive cases. HER2 amplification was detected with FISH. Overall survival (OS) was evaluated using Kaplan-Meier methods and compared with log-rank tests.

Results:
Twenty-one cases carrying HER2 mutations were identified with a prevalence of 2.4%. HER2 mutations were more frequently encountered in females, non-smokers and adenocarcinoma. NGS was performed in 19 out of 21 patients, The results showed 16 cases with additional genetic aberrations, most commonly associated with TP53 (n = 6), followed by EGFR (n = 3), NF1 (n = 3), KRAS (n = 2) and other mutations. One patient harbored HER2 amplification(figure 1). Four patients with stage IV received afatinib treatment, and three showed stable disease with a median progression-free survival of 4 months and one patient was diagnosed with progressive disease. No survival difference existed between HER2 positive and negative patients( (49.3 months vs.45.0 months, P = 0.150).

Conclusion:
HER2 mutations represent a distinct subset of NSCLC. NGS showed that HER2 mutations commonly co-existed with other driver genes. Afatinib treatment displayed moderate efficacy in patients with HER2 mutations.

Background:
Currently, multicenter studies involving a large number of patients have not been not undertaken to detect the frequencies of EGFR mutations and ALK rearrangement in malignant pleural effusion (MPE) samples of patients with non-squamous, non‒small-cell lung cancer (NSCLC), we undertook a multicenter, observational study of Asian patients with untreated stage IV NSCLC.

Methods:
Eligible patients had untreated of EGFR and ALK inhibitor stage IV non-squamous NSCLC patients with MPE. The EGFR and ALK status of MPE and partially paired tumor tissue was determined with reverse transcription polymerase chain reaction (RT-PCR).

Results:
Among 210 patients with pleural effusion samples confirmed as malignant, 16 had EML4-ALK fusion gene rearrangements and 89 had EGFR mutations. No ALK/EGFR coaltered gene was found. Tumor tissue of 56 patients were collected. EGFR and ALK concordance rates between MPE samples and matched tumor tissue samples from 56 patients were 87.5% (49/56) and 96.1% (49/51), respectively. There was a tendency for a longer progression free survival in patients with EGFR accordance in comparison with those with EGFR discordance between tumor tissue and MPE samples (9.8 vs 6.2 months, respectively; p = 0.078). A same trend was found in patients with ALK accordance and discordance (10.0 vs 3.2 months, respectively; p = 0.004) .

Conclusion:
These results demonstrate that MPE can be substituted for tumor tissues for EGFR and ALK gene detection. Patients with gene mutations or arrangement discordance between tumor tissue and MPE samples showed a inferior efficacy of targeted therapy than those with accordance.

Background:
EGFR-TKIs show significant therapeutic effects against non-small cell lung cancer (NSCLC) with EGFR-activating mutations, however 20-30% of them have no response to EGFR-TKIs. HER-family receptors play a critical role in tumor progression, differentiation and survival in lung cancer. Recent studies suggest that the overexpression of HER-family receptors have a potential risk of EGFR-TKIs resistance. The aims of this study were to investigate the association between EGFR-mutation and the expression of HER-family receptor in regard to clinical outcomes.

Methods:
We invested EGFR mutation by direct PCR analysis and HER2-4 expression by immunohistochemistry (IHC) of 231 consecutive non-small cell lung cancers, who had undertaken an operation from January 2007 to July 2012. The intensity of HER2-4 was graded (score: 0-3) from negative (score: 0-1) to positive (score: 2-3). The observed protein expression levels were analyzed for correlation to EGFR mutation status, clinicopathological parameters and the responses of EGFR-TKI treatment.

Results:
EGFR mutation was observed in 40% of lung cancer, 61% of p. ［Leu858Arg］and 31% of exon 19 deletion. Positive expression rates of HER2, HER3 and HER4 were 22.9%, 1.2%, 38.5%, respectively. HER4 positive rare of EGFR positive group was significantly higher than that of EGFR negative group (52% vs 30%, P<0.01), however there was no difference in HER2 expression. In histological type, positive rates of HER2 and HER4 in adenocarcinoma were higher than that in squamous cell carcinoma (HER2: 26% vs 13%, P=0.07, HER4: 49% vs 13%, P<0.01). HER4 positive rate of HER2 positive group was significantly lower that of HER2 negative group (43% vs 26%, P=0.03). The response rate of EGFR-TKIs in HER2 positive group was low, but high in HER4 positive group.

Conclusion:
Our data showed that HER4 expression was independent form EGFR mutation and HER2 expression, but related to the sensitivity against EGFR-TKIs. HER4 positive patients may be candidate for pan-HER inhibitor augments.

Background:
The most lethality in NSCLC is due to uncontrolled tumor metastasis. Epidermal growth factor receptor (EGFR) has been confirmed to be an effective biomarker in EGFR-TKIs treatment for advanced NSCLC. Previous studies have demonstrated EGFR mutation abundance could predict benefit from EGFR-TKIs treatment. However, there is no investigation on the correlation between EGFR mutation abundance and tumor metastasis. Here we aimed to explore potential effect of EGFR mutation abundance on tumor metastasis and EGFR-TKIs prognosis.

Methods:
3913 patients from Henan Cancer Hospital diagnosed with NSCLC were enrolled. The EGFR mutation abundance in tumor specimens was quantified by amplification refractory mutation system (ARMS). Above 10% was defined as high abundance, and below 10% was defined as low abundance.

Results:
The ratio of high mutation abundance in age < 60 years group was significantly higher than that in age ≥ 60 years group (55.4% vs. 42.5%, P=0.000), similar distribution was also detected in 19 exon deletion and L858R subgroup (P=0.003 and 0.030, respectively). Whereas the distribution of EGFR mutation abundance was no difference between surgery and biopsy specimens (P=1.000). Additionally, the ratio of high abundance in 19 exon deletion was obviously higher than that in L858R and rare mutations (66.5% vs. 31.6% vs. 51.1%, P=0.000). Meanwhile, the ratio of high abundance in patients with hepar metastasis was significantly higher than that in patients without hepar metastasis (57.2% vs. 47.8%, P=0.036), but that in brain or bone metastasis was demonstrated no significant difference (P=0.897 and P=0.293, respectively). The subgroup analysis among above metastasis patients indicated the ratio of high abundance in 19 exon deletion was significantly higher that in L858R. Furthmore, the difference in median PFS between 19 exon deletion and L858R group was significant (17.5 months vs. 9.2 months, P=0.003).

Conclusion:
EGFR mutation abundance was not associated with the methods of collecting specimens. The younger patients more likely harbor high EGFR mutation abundance. Hepar metastasis status was associated with EGFR mutation abundance. Median PFS in 19 exon deletion patients was notablely longer than that in L858R group for EGFR-TKIs treatment, which may refer to high abundance in 19 exon deletion.

Background:
Histological transformation including small cell carcinoma and epithelial to mesenchymal transition (EMT) is one of the discovered mechanisms of the acquired resistance to EGFR-TKI. We report two cases of Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor(EGFR-TKI) resistant pulmonary adenocarcinoma associated with EMT features that showed osteosarcomatous differentiation in rebiopsy specimens.

Methods:
We identified two patients with primary lung adenocarcinoma that showed osteosarcomatous transformation on second biopsy (n = 60) between 2010 and 2016. Histomorphologic features and EGFR mutation results were compared between initial and second biopsy samples.

Results:
Case 1 A 55-year-old female, non-smoker presenting chronic cough was found to have pulmonary adenocarcinoma harboring EGFR exon 19 deletion mutation with disseminated intrapulmonary metastasis. After 1 year of gefinitib treatment, radiologic evaluation showed left iliac metastases with extraosseous ossification. Case 2 A 58-year-old man who had undergone right upper lobectomy of the lung was diagnosed as adenocarcinoma with pT1N0M0 harboring an EGFR exon 19 deletion mutation. Three years after surgery, metastatic lesions developed in the right lower lobe and pleura. After 15 months of conventional chemotherapy, 2nd biopsy for the pulmonary lesion confirmed T790M mutation and additional metastatic lesions found in T2 and T5 vertebral bodies were removed by surgical curettage. Rebiopsy of the metastatic bone lesions of these two patients showed metastatic adenocarcinoma merging with poorly differentiated sarcomatous components. Remarkably, the spindle shaped sarcomatous tumor cells produced ill-defined eosinophilic lace-like osteoid. These osteoid components were closely associated with the tumor cells and deposited as disorganized features. The sarcomatous neoplastic cells intermingled with osteoid demonstrate unequivocal features of malignancy, which is different from reactive osteoid or callus formation. EGFR mutation status were same from that of primary lung specimen and IHC showed vimentin expression and decreased E-cadherin (EMT feature).

Conclusion:
To the best of our knowledge, this is the first report to describe pulmonary adenocarcinoma with osteosarcomatous differentiation in rebiopsy specimens of EGFR-TKI resistant patients. As the evaluation of metastatic sites for rebiopsy were bone lesions in both patients, the role of the tumor microenvironment may support the transformation from adenocarcinoma to osteosarcomatous phenotype. A few previous studies suggested that a bone environment is essential for osteosarcoma development from transformed mesenchymal stem cells. Our findings suggest that the differences of the intrinsic nature between epithelial and osteosarcomatous mesenchymal cancers may be the cause of the acquired resistance of EGFR-TKI.

Background:
Analysis of the epidermal growth factor receptor (EGFR) gene is currently one of the most important tests in establishment of a treatment strategy for primary lung cancer. Major mutations of exon 19 deletion and exon 20 point mutation (L858R) are particularly well known in adenocarcinomas, and tyrosine kinase inhibitors (TKIs) have provided significant benefits to patients with such mutations. Complex mutation patterns of EGFR have also been reported, but their significance is unknown.

Methods:
Clinicopathological features and response to treatment of non-small lung cancer (NSCLC) with complex mutation of the EGFR gene were investigated in cases treated at Kanagawa Cardiovascular and Respiratory Center from June 2014 to March 2016.

Results:
EGFR gene analysis was performed in 334 cases, of which 108 had EGFR mutations. These cases included 7 (6.5%) with complex mutations: two males (28.6%) and five females (71.4%) with an age of 71.0±6.0 (mean±SD) years. Five of the 7 cases underwent surgery and two were inoperable. The histological diagnoses were adenocarcinomas (n=6) and squamous cell carcinoma (n=1). The pathological stage in the surgical cases (all adenocarcinomas) were IB, IIA, and IIIA in 2, 1 and 2 cases, respectively. Both inoperable cases were clinical stage IV. The EGFR complex mutation patterns were 19 deletion and 20 T790M (n=2, with one case with acquired TKI resistance), 18 G719X and 21 L861Q (n=3), 19 deletion and 21 L858R (n=1), and 20 T790 and 21 L858R (n=1). In the five surgical cases, two (pStage IIA, 19 deletion and 20 T790M; pStage IIIA, 18 G719X and 21 L861Q) received postoperative TKI therapy because of recurrence. Both patients had a poor response to TKIs and both died. The patients in another three surgical cases are alive without receiving TKI therapy. Two inoperable cases (19 deletion and 20 T790M; 18 G719X and 21 L861Q) were treated with standard chemotherapy and TKIs, and also died. The disease-free survival period in the surgical cases was 532±319 days and the overall survival period in the case with postoperative recurrence and the inoperable cases was 810±563 days. The progression-free survival period in patients treated with TKIs was 101.5±90.6 days.

Conclusion:
In patients with EGFR mutation, 6.5% have complex mutations, of which 85.7% are minor-on-minor or minor-on-major mutations. Lung cancer with complex mutation of EGFR tends to have a poorer response to TKIs compared to cases with a major single mutation.

Background:
The vast majority of advanced non-small cell lung cancer (NSCLC) patients with EGFR mutant tumors will develop disease progression following successful treatment with an EGFR tyrosine kinase inhibitor (TKI). Resistance to EGFR-TKIs is due to various mechanisms, such as the secondary mutation (T790M) or the activation of alternative pathways (MET, AXL). What has not been fully appreciated is that EGFR blockade induces an imbalance in favor of survival, increases activity of STAT3 and enriches lung CSCs through Notch3-dependent signaling. EGF-PTI was designed to elicit an antibody response against EGF, in order to reduce EGF receptor signaling and limit tumor growth. We have explored whether EGF-PTI alone or in combination with EGFR TKIs may efficiently inhibit STAT3 and target CSCs.

Methods:
EGF PTI was provided by Bioven (Europe) Ltd. Gefitinib, erlotinib and the recently FDA-approved third-generation EGFR TKI, AZD9291 (osimertinib) were purchased from Selleck chemicals. Western blotting was used to assess the effect of the drugs on ERK, AKT and STAT3 phosphorylation and on Notch and PARP cleavage in EGFR (del19) mutant NSCLC PC9 cells and gefitinib-resistant PC9-GR4 cells. PC9-GR4 cells have been established in our lab and harbor the resistant T790M mutation (T790M+). The protein expression of AXL and CSCs markers such as HES1 (downstream effector of Notch) and Bmi1 was also examined.

Results:
Gefitinib, erlotinib or AZD9291 suppressed EGFR, ERK1/2 and AKT phosphorylation in PC9 cells but increased STAT3 phosphorylation on the tyrosine residue 705 in both PC9 and PC9-GR4 cells. EGF-PTI suppressed STAT3, EGFR and ERK1/2 and the combination of each of the three EGFR TKIs with EGF-PTI lead to more potent inhibition of STAT3, EGFR and ERK1/2. The EGF-PTI induced AKT phosphorylation was reversed when EGF PTI was combined with EGFR TKIs. Interestingly, EGF-PTI blocked Notch cleavage and decreased the expression of HES1. The expression of Bmi1 and AXL were also attenuated with EGF PTI and apoptosis was enhanced through the induction of PARP cleavage.

Conclusion:
EGF PTI may reverse mechanisms of resistance to single EGFR inhibition and the combination of EGF PTI with EGFR TKIs efficiently inhibits downstream signaling pathways in T790M+ cells. Based on these results, the design of a proof-of-concept trial with the combination of EGF PTI with gefitinib for the first line treatment of EGFR mutant NSCLC patients is in progress.

Background:
Molecular testing of lung cancer is currently performed on a relatively restricted set of standard-of-care markers (normally EGFR and ALK). The introduction of next generation sequencing (NGS) in clinical laboratories has permitted the adoption of broader testing using mutation panels. This study compared the number of mutations detected and outcomes generated as a result of replacing standalone EGFR and ALK assays with a combined mutation and gene fusion assay based on NGS in routine clinical practice.

Methods:
Panel testing was implemented using a bioinformatically selected subset of targets from the Thermo-Fisher Oncomine[TM] Focus assay. The results of testing were compared against the incumbent assay platforms (Roche Cobas® EGFR, Abbott Vysis[TM] ALK) to determine differences in mutation detection and resultant alterations to patient treatment.

Results:
Over a 5 month period of testing, 231 lung adenocarcinomas were analysed using the extended mutation and fusion panel. In total 126 of 231 cases had a mutation or fusion identified; EGFR (n=33), KRAS (n=76), BRAF (n=8), ERBB2 (n=2), ALK-fusion (n=5), RET fusion (n=1). Additionally, one off-target fusion was detected during assay QC. Of the above results 31 EGFR and 5 ALK would have been detected using the benchmark Roche Cobas EGFR and Abbott Vysis ALK FISH methodologies. The additional detections can be classified as nonactionable (KRAS, BRAF) or actionable (RET fusion, 2 EGFR mutations not identified by Cobas, 2 ERBB2 mutations and one off-target fusion). Of the 6 actionable genetic lesions, 4 selected for targeted therapies in lung cancer (EGFR, ERBB2). Two fusions detected by this assay (CCDC6-RET and TMPRSS2-ERG) suggested an alternative diagnosis to that of lung cancer when reviewed with morphology, immunohistochemistry and clinicopathological correlation.

Conclusion:
When compared with standalone assay testing, panel testing of lung cancer identified mutations in an additional 39% of patients and identified genetic lesions that altered targeted therapy selection (2%) or diagnosis (1%). Broad mutation panel testing using NGS has shown itself to be superior at the level of clinical decision making by ascribing a molecular subtype to 39% more cancers and identifying an additional 2% of cases where targeted therapies may be of benefit. Crucially, the addition of 'off-target' mutations and fusions prompts re-examination and, where necessary, correction of primary diagnosis at a rate of 1% in our hands. This feature of panel testing has been overlooked and it is critical for the oncology and pathology communities to be aware of its significance.

Background:
Extended genetic testing of NSCLC tumor samples provides a foundation for personalized cancer treatment and use of new targeted medication. Testing with Next Generation Sequencing (NGS), mostly performed at university hospitals, has not been available for all patients due to geographic and economic reasons. Many lung cancer patients carry a heavy burden of disease and extensive travelling can negatively impact quality of life. The ability to perform a modern state-of the art work-up at local hospitals, without compromising on diagnostic quality, will enable equal access to personalized treatment for lung cancer patients.

Methods:
In Gävle County hospital routine diagnostic immunohistochemistry (IHC) on biopsies is performed at the local pathology lab. In the case of NSCLC the formalin-fixed, paraffin embedded (FFPE) tissue samples are sent to Uppsala University hospital for further molecular pathology and NGS testing. A targeted NGS test (18 gene panel) was established for mutation screening of small biopsies and cytology specimens (Moens et al., J Mol Diagn, 2015). Fusion genes - ALK, ROS1 and RET - are analysed by IHC, FISH and nanoString. Structured biobanking of surplus biopsies and blood samples during treatment, for explorative biomarker testing and research, was set up as a regional extension of the UCAN infrastructure, including detailed registration of clinical baseline and real-time follow-up data in a dedicated database.

Results:
Inclusion of patients in the biobanking cohort started gradually during 2015 in Uppsala, and in February 2016 in Gävle. The cumulative inclusion in the UCAN biobank is updated at www.u-can.uu.se (see Statistics). To date (July 2016) 70 patients have been included at Gävle County hospital covering 95% of the newly diagnosed NSCLC patients. So, far 242 patients from the region were tested by NGS yielding 23 EGFR+ (9.5%), 75 KRAS+ (31%), 5 BRAF+ (2.1%, codon 600), 2 MET (0.8%, exon 14 skipping), 1 ERBB2 (0.4%, exon 20 insertion), and 6 PIK3CA (2.5%, exon 9/20) cases. Fusion gene analysis resulted in 5 ALK+ (2.1%), 1 ROS1 and 1 RET patients.

Conclusion:
Decentralised local patient care, tissue/blood sampling and biobanking in combination with centralised molecular testing allows advanced lung cancer diagnostics and clinical research in networks of county hospitals. Survival benefits from modern targeted drugs, for national lung cancer cohorts, can only be achieved and evaluated in population-based settings without bias related to selective referral to major cancer centers.

Background:
Next generation sequencing (NGS) enables us to detect comprehensive genetic aberrations within a tumor sample, which provides potential alternative to well adopted clinical diagnostic approaches such as amplification refractory mutation system PCR (ARMS-PCR) and FISH. However, there is no enough data to illustrate the overall concordance between NGS with traditional clinical diagnostic approaches. This study is aimed to fill in this blank.

Methods:
We have used 20 cell lines from ATCC and 19 FFPE samples to construct molecular standards and there are 50, 34 and 48 samples for SNV and Indel, CNV and fusion, respectively. All the mutations were verified by Sanger sequencing or QuantStudio 3D digital PCR. To assess the performance of NGS, an amplicon-based NGS strategy was used to detect gene mutations in molecular standards. In order to illustrate the overall concordance between NGS with ARMS-PCR and FISH, we further verified NGS in 2500 retrospective FFPE samples from non-small lung cancer (NSCLC) and breast cancer patients.

Results:
So far, we have detected genetic aberrations in 108 FFPE samples. For SNV and Indel, we focused on the mutation profile of EGFR, KRAS, BARF and PIK3CA, which were the most common mutations in NSCLC. In molecular standards, 34 of 50 (68%) were positive for Sanger and 33 of 50 were positive for NGS, thus the sensitivity, specificity and accuracy was 97%, 100% and 98%, respectively. In FFPE samples from 31 lung cancer patients, NGS results were consistent with ARMS-PCR. For CNV, in molecular standards, the copy number of HER2, MET, EGFR and FGFR1 detected by NGS was high consistent with digital PCR and R[2 ]was 0.9673. In FFPE samples from 45 breast cancer patients, 80% of cases (36/45) were HER2 amplification positive and 20% (9/45) were negative for FISH, 34 HER2 positive and 9 HER2 negative for FISH were also classified by NGS. Thus, the overall concordance between NGS and FISH were 95.56%. For ALK and ROS1 gene fusion, the overall concordance were both 100% in 48 molecular standards (NGS versus Sanger sequencing) and 32 FFPE samples (NGS versus FISH).

Conclusion:
Our result reveal that the amplicon-based NGS strategy for detecting genetic aberrations is of high accuracy and comparable with standard clinical diagnostic approaches, and therefore provides a promising diagnosis approach for clinical in the future.

Background:
Chromosomal rearrangements of anaplastic lymphoma kinase (ALK) compose approximately 4-6% of non–small-cell lung cancer (NSCLC) and the patients can be prescribed with targeted therapy. Nevertheless, acquired resistance towards existing ALK-specific inhibitors is inevitable in most cases. This suggests that a detailed molecular characterization of NSCLC with ALK rearrangement and dissection of ALK-specific signaling pathway are strongly needed.

Methods:
Approximately 3000 NSCLC cases with EGFR and KRAS wild types were screened for ALK alterations by immunohistochemistry (IHC). From the 158 ALK IHC-positive samples, we further validated ALK rearrangement through fluorescence in situ hybridization and RNA color-coded probes. We then performed targeted deep sequencing using a customized panel embedded with 81 select set of cancer associated genes in 129 ALK-positive cases for their molecular characterization. Additional clinical analysis and drug viability assays between EML4-ALK long and short forms were assessed as well. We also conducted multiplexed direct mRNA expression profiling using a panel of 770 essential key driver genes that take part in most cancer pathways. Target gene silencing, overexpression, migration assay, and immunoprecipitation were conducted for functional analysis of identified molecules.

Results:
We found that most ALK genomic breaks occurred at intron 19 (92.7%), which conjoined with partner genes through non-homologous end joining repair system. ALK fusion profiling exhibited that 82% of fusions were EML4-ALK (129/158), 2.4% were HIP1-ALK (4/158), 1.8% KIF5B-ALK (3/158), 1 case was KLC1-ALK, and the rest were unrecognized ALK fusions (21/158). From the unknown partners, we identified 3 novel ALK fusion partners: GCC2, LMO7, and PHACTR1. We also identified 4 novel somatic mutations of ALK: T1151R, R1192P, A1280V, and L1535Q. RNA expression profiling further revealed that NSCLC with ALK rearrangement showed higher expression of ITGB3 with statistical significance. Combinatorial treatment of ALK (crizotinib) and ITGB3 (LM609 antibody) decreased cell migration and invasive properties of ALK-positive cell lines. Furthermore, from our new classification system of EML4-ALK variants, the cases with short EML4-ALK form showed more advanced stages and more frequent metastases than the cases with long form in NSCLCs.

Conclusion:
This is the primary mass-scale study of ALK-rearranged NSCLC to our knowledge. Through this integrated analysis, we provide genomic details and clinical insight of NSCLC with ALK rearrangement. Also, we suggest a novel therapeutic approach of treating ALK-rearranged NSCLC patients with ALK and ITGB3 inhibitors.

Background:
The reported prevalence of ALK rearrangement in NSCLC ranges from 2%-7%, depending on population and detection method. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proven to be a reproducible and sensitive technique. Reverse transcriptase-polymerase chain reaction (RT-PCR) has been advocated and most recently the advent of targeted Next-Generation Sequencing (NGS) for ALK and other fusions has become possible. This is one of the first studies comparing all 4 techniques in resected NSCLC from the large ETOP Lungscape cohort.

Methods:
96 cases from the ETOP Lungscape iBiobank (N=2709) selected based on any degree of IHC staining (clone 5A4 antibody, Novocastra, UK) were examined by FISH (Abbott Molecular, Inc.; Blackhall, JCO 2014), central RT-PCR and NGS. H-score 120 is used as cutoff for IHC+. For both RT-PCR and NGS, RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers were used covering the most frequent ALK translocations. For NGS, the Oncomine™ Solid Tumour Fusion Transcript Kit was used, allowing simultaneous sequencing of 70 ALK, RET and ROS1 specific fusion transcripts associated with NSCLC, as well as novel ALK translocations using 5’-3’ ALK gene expression ‘Imbalance Assay’.

Results:
NGS provided results for 90 cases, while RT-PCR for 77. Overall, 70 cases have results for all 4 methods, with fully concordant 60 (85.7%) cases (49 ALK-, 11 ALK+). Before employing the ‘Imbalance Assay', in 5 of the remaining 10 cases, NGS differs from the other methods (3 NGS-, 2 NGS+), while in the other 5, NGS agrees with RT-PCR in all, IHC in 2, and FISH in 1. Using the concordant result of at least two of the three methods as true negative/positive, the specificity and sensitivity of the fourth is 96/94/100/96% and 94/94/89/72% for IHC/FISH/RT-PCR/NGS, respectively (incorporating imbalance: NGS sensitivity=83%). Imbalance scores are presented here for 18 NGS- cases: 9 ‘NGS-/FISH+/IHC+’, 9 ‘NGS-/FISH-/IHC-‘. Among the ‘NGS-/FISH+/IHC+’, there is strong evidence of imbalance in 4 cases (score’s range: 0.0144-0.0555), uncertain in 5 (range: 0.0030-0.0087), and no evidence (scores≤0.0004) in the 9 negative cases.

Conclusion:
NGS is a useful screening tool for ALK rearrangement status, superior to RT-PCR when RNA yield is limited. When using NGS, it is critically important to integrate the 5’-3’ imbalance assay and to confirm with one or more additional methods in the ‘imbalance’ cases. Data further highlight the possibility of missing actionable rearrangements when only one screening methodology is available.

Background:
Detecting mutations is becoming important for both predicting disease progression and drug responses to treatment of cancer patients. Current mutation detection methods for cancer diagnosis are mainly based on the invasive sampling technique such as a tissue biopsy, but some patients may not available for this invasive procedure. Therefore, circulating tumor DNA (ctDNA) would be a good alternative for those patients. However, testing methods for tissue biopsy sample are not applicable to ctDNA samples due to relatively lower sensitivity. A highly sensitive assay method is required for detecting mutations in liquid biopsy samples.

Methods:
We have developed a highly sensitive and simple method to detect somatic mutation from ctDNA in patient’s plasma. This new real-time PCR-based testing method (PANAMutyper™) has maximized unique properties of peptide nucleic acid (PNA). It contains a PNA clamp and PNA detection probes in the each reaction tubes. A optimized PNA clamp can tightly bind to only wild-type DNA sequences, and then suppress amplification during the PCR reaction. Meanwhile, a PNA detection probe that conjugated with a fluorescent dye and a quencher, can detect a specific target mutant-type DNA and each mutation can be genotyped by melting peak analysis. PANAMutyper™ is able to detect 47 different mutations in exon 18, 19, 20 and 21 of EGFR gene with detection limits as low as 0.01%.

Results:
In order to confirm the validity in real condition, we conducted spiking test. Ten of mutant clones DNA were used as standard materials. (G719A, G719S, G719S, S768I, Exon19 deletion, two Exon20 insertion, T790M, L858R, L861Q). Mix the each mutant clone DNA and human plasma to 1, 10, 100, 1000 copies per microliter concentration. And then we extracted ctDNA from prepared sample. As a result, all of ten mutant clone DNA was detected 1 copy/㎕. This result suggest that PANAMutyper™ is applicable to ctDNA derived from patient’s plasma sample.

Conclusion:
The high concordance, specificity, and sensitivity of this test have demonstrated that EGFR mutation status can be accurately assessed by PANAMutyper™ using ctDNA. Therefore, PANAMutyper[TM] can be used in various clinical areas including companion diagnostics and monitoring acquired mutations.

Background:
Determination of ALK gene rearrangements has been traditionally performed in biopsies and/or surgical specimens. However, advanced lung cancer is often diagnosed by FNA cytology obtained through minimally invasive procedures, and frequently cytological specimens are the only samples available, thus emphasizing the necessity to expand ALK analysis to cytologic specimens. We assessed the feasibility of determining ALK gene rearrangements in different types of cytological samples.

Methods:
We studied prospectively 268 cytological samples from 268 NSCLC patients for ALK gene rearrangements by FISH (Vysis LSI ALK Dual Color Break Apart and ZytoLight SPEC ALK Dual Color Break Apart). Tumour samples were obtained by bronchoscopy -FNA in 45 cases (19.79%), EBUS-FNA in 63 (23.50%), EUS-FNA in 56 (20.89%), CT-FNA in 28 (10.44%), Ultrasonography guided-FNA in 24 (8.95%), and direct FNA in 23 cases (8.58%). Two cavity fluids (0.74%), 4 imprints from surgical specimens (1.49%), and 22 cases received for consultation (8.20%) were also studied. ROSE was done in all FNA procedures. FISH was performed on stained smears in 133 cases (49.62%) (114 Papanicolau and 19 Diff-Quick), ThinPrep in 48 (17.91%), SurePath in 12 (4.47%), and cell block in 75 cases (27.98%). All cases were tested for EGFR and KRAS mutations.

Results:
Two hundred thirty five samples (87.68%) were adequate for FISH analysis. Fifteen cases (5.59%) had ALK gene rearrangements. One case had a concurrent EGFR mutation in exon 21 plus the T789M mutation, and two had also KRAS mutations (G12D and G12C respectively). FISH study was unsuccessful in 33 cases (12.31%): 8 from stained smears (6.01%), 12 from ThinPrep (25%), 8 from SurePath (66.66%), and 5 from cell blocks (6.66%). Correlation cytological / paraffin embedded samples was performed in 10 cases with a concordance rate of 100%.

Conclusion:
ALK gene rearrangements may be definitely detected in cytological samples and particularly in direct smears. Both, Papanicolau and Diff-Quick smears are suitable samples for FISH analysis. The nuclei on cytology smears are not truncated, which allows for the detection of the true number of FISH signals in a nucleus. It is mandatory an exquisite management and care of the samples to preserve quality. Coexistence of ALK gene rearrangements and EGFR and KRAS mutations were observed in one and two cases respectively, indicating that such alterations are not necessarily mutually exclusive

Background:
Cytological materials are widely used in diagnosis and staging of lung cancer due to advanced stage of disease at the time of presentation. Mutational oncogenic drivers in pulmonary adenocarcinoma (ADC) include EGFR, Kras and Her-2/neu. We utilized archived FNA smears and pleural effusion samples of ADC for detection of oncogenic molecular drivers.

Methods:
Pleural fluids (36) and May Grunwald stained FNA smears (18) were used for mutation anaysis. Sanger sequencing and ARMS and Scorpions PCR performed. Each tumor sample was evaluated for all classes of genomic alterations, including base-pair substitutions, insertions/deletions, as well as intronic/CDS polymorphisms.

Results:
There were 31 males and 23 females, aged 18 to 82 years with mean age 65.5 years. A total of 9 patients (16.6%) were found positive for EGFR mutations (Table 1, Figure 1). EGFR exon 18 intronic poylmorphism was seen in 4 cases and EGFR exon 20 showed intronic and CDS polymorphism in most cases. However, none of the cases were found to be positive for EGFR, exon 18, 20, Kras and Her-2/neu mutation. Figure 1 Figure 2





Conclusion:
These findings verify the feasibility of analysis of oncogenic drivers in cytological specimens in advanced ADC. Stained aspiration smears can be used after establishing diagnosis and checking adequacy of the specimen.

Background:
AKL gene rearrangements are predictive alterations in non small cell lung carcinomas (NSCLC). Immunohistochemistry (IHC) has become a valuable tool in assessing ALK status, however unusual staining patterns, such as heterogeneous diffuse moderate and focal intense stains, may occur and can make evaluation difficult.

Methods:
We correlated immunohistochemistry unusual staining patterns with ALK status by fluorescence in situ hybridization (FISH). Of 851 cases tested, we found 14 (1.6%) cases with inconclusive staining patterns that can be summarized in: a) diffuse granular cytoplasmic moderate stain, with or without background mucin stain (10 cases) b) focal intense granular cytoplasmic stain in overall negative or weakly positive tumors (4 cases). IHC was performed on an automatized Benchmark staining module using Ventana ALK (D5F3) CDx assay with Optiview amplification kit. FISH was performed using ALK break-apart probe set (Vysis LSI ALK Dual Color, Abbott Molecular). Cases were considered ALK-FISH positive if ≥15% tumor cells showed split red and green signals (separation of 2 diameters or more) and/or single red signals

Results:
Of 10 moderate granular cytoplasmic stain cases, 4 had also abundant mucin backround stain 6 where markedly heterogeneous with areas of weak and moderate cytoplasmic granular stain. Nine were FISH negative, one yielded no signals (uninformative result) and one specimen corresponded to an acid decalcified specimen and was not evaluated by FISH. Focal intense stain was observed in 5 samples, 3 corresponded to surgical specimens and the rest to small needle biopsies, one of the surgical specimens was FISH positive and the rest, negative.

Conclusion:
Since FDA approval of Ventana ALK (D5F3) IHC CDx Assay, IHC has become a widely used tool for assessing ALK status. Guidelines suggest that weak granular stain should be interpreted as negative and focal intense granular stain in any number of cells, as positive. Even though our sample is small, moderate granular stain was consistently negative by FISH analysis, however, focal intense stain shows more discordant results between tests. To date, no suggestions are made on what should be the minimum amount of tumor in a sample to report an IHC assay. Even though some of these patients with IHC positive/FISH negative results have been reported as responders to Crizotinib, further studies are needed. One specimen with moderate cytoplasmic IHC stain was uninformative due to lack of signals. This raises the issue of the need to standardize preanalytical variables, which can be difficult in some areas of Latin America.

Background:
ALK and ROS1 gene rearrangements are distinct molecular subsets of non-small-cell lung cancer (NSCLC), and they are strong predictive biomarkers of response to ALK/ROS1 inhibitors, such as crizotinib. Thus, it is clinically important to detect patients who will benefit from such treatment and develop an effective screening strategy. In this study, we aim to evaluate the diagnostic performance of ALK/ROS1 RUO FISH probes which can concurrently detect ALK and ROS1 rearrangements.

Methods:
The study populations were composed of three patient cohorts with histologically confirmed lung adenocarcinoma (ALK rearrangement, ROS1 rearrangement and both wild type). Patient specimens consisted of 12 ALK-positive, 9 ROS1-positive and 21 ALK/ROS1-wild type formalin-fixed paraffin-embedded samples obtained from surgical resection or excisional biopsy. ALK rearrangement status was determined by Vysis LSI Dual Color Break Apart Rearrangement Probe (Abbott Molecular, Abbott Park, IL, USA) and ROS1 rearrangement status was assessed by ZytoLight SPEC ROS1 dual color break apart probe (Zytovision. Bremerhaven, Germany). All specimens were re-evaluated by ALK/ROS1 Break Apart FISH RUO 4-color kit. FISH images were scanned via the BioView Duet and interpreted remotely via BioView SoloWeb.

Results:
A total of 42 patient samples were evaluated. The concordance of results obtained from ALK/ROS1 Break Apart FISH RUO 4-color kit was evaluated relative to the ALK and ROS1 rearrangement status of the specimen, as previously determined. One ROS1-positive and 2 wild-type samples were excluded from analysis due to high background. Regarding 12 ALK-positive samples, 12 were ALK-positive by ALK/ROS1 RUO FISH, showing 100% (n=12/12) sensitivity to predict ALK rearrangement. Regarding 8 ROS1-positive samples, 6 cases were ROS1-positive by ALK/ROS1 RUO FISH, showing 75% (n=6/8) sensitivity to predict ROS1 rearrangement. Two cases showed weak ROS1 signals that could not be enumerated. Regarding 19 wild type cases, 18 cases were negative by ALK/ROS1 RUO FISH, showing 95% (n=18/19) specificity, while one case showed poor ROS1 signals which could not be properly enumerated.

Conclusion:
ALK/ROS1 RUO FISH can detect ALK and ROS1 rearrangements simultaneously in NSCLC. The fluorescence of ROS1 signal may be weakened by slide shipment and remote scoring.

Background:
The degree and duration of response to epidermal growth factor receptor (EGFR) inhibitors in EGFR mutated lung cancer are heterogeneous. We hypothesized that the concurrent genomic landscape of these tumors, which is currently largely unknown in view of the prevailing single gene assay diagnostic paradigm in clinical practice, could play a role in clinical outcomes and/or mechanisms of resistance.

Methods:
We retrospectively probed our institutional lung cancer patient database for tumors harboring EGFR kinase domain mutations that were also evaluated by more comprehensive molecular profiling, and assessed tumor response to EGFR tyrosine kinase inhibitors (TKIs).

Results:
Out of 171 EGFR mutated tumor-patient cases, 20 were sequenced using at least a limited comprehensive genomic profiling platform. 50% of these harbored concurrent TP53 mutation, 10% PIK3CA mutation, and 5% PTEN mutation, among others. The response rate to EGFR TKIs, the median progression-free survival (PFS) to TKIs, the percentage of EGFR-T790M TKI resistance and survival were higher in EGFR mutant/TP53 wild-type cases when compared to EGFR mutant/TP53 mutant tumors; with a significantly longer median PFS in EGFR-exon 19 deletion mutant/TP53 wild-type cancers treated with 1st generation EGFR TKIs.

Conclusion:
Concurrent mutations, specifically TP53, are common in EGFR mutated lung cancer and may alter clinical outcomes. Additional cohorts will be needed to determine if comprehensive molecular profiling adds clinically relevant information to single gene assay identification in oncogene-driven lung cancers.

Background:
About 15% of western patient population with advanced NSCLC harbor the epidermal growth factor receptor (EGFR) mutation compared to about 50% in the Asian population. These mutations are more frequently found in NSCLC with an adenocarcinoma histology, in women, East Asians and never smokers. EGFR tyrosine kinase inhibitors (TKIs) are the first-line treatment of choice for NSCLC patients harboring EGFR mutation. Nowadays there is only a scares information regarding EGFR epidemiology and response to EGFR TKI among other ethnicities, although there has been limited reports regarding increased rate of EGFR mutation among arabic patients.

Methods:
Single institution retrospective analysis of serial advanced NSCLC patients tested for EGFR mutation in 2011-2015. Information was obtained using the medical records.

Results:
Of 616 patients with advanced NSCLC tested for EGFR in our institutions in 2011-2015, there were a total of 134 Arabic patients, 38 of them harboring EGFR mutation (28%), as opposed to 64 (13%) among the non-arabic population. Twenty patients had exon 19 deletion, 9 patients had L858R and 1 patient had exon 21 mutation. The median age at diagnosis was 58 (39-80), 22 patients were males and 16 females, of them- 13 were never smokers, 5 are previous smokers, and 20 are active smokers. Thirty-six patients had adenocarcinoma histology while 2 patients had carcinosarcoma and squamous cell carcinoma. The median survival was longer than 9 months. Thirty patients were treated with EGFR TKI, 27 of them as 1[ST] line treatment, and 3 as 2[ND] line treatment. Of the 30 patients treated with EGFR TKI, 69% had partial response, 16% had stable disease.

Conclusion:
Among Arabic patients with NSCLC, the frequency of EGFR mutation is higher than in western population, and is more frequent among males and smokers. The response to EGFR TKI matches the reported literature.

Background:
Histomorphologic changes are known to be associated the acquired resistance of the epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) treatment. Rebiopsy is usually used to detect the underlying molecular mechanism of resistance, however meticulous histologic examination is very important to identify the change of cancer phenotype. Here we tried to investigate histomorphologic changes between the initial and rebiopsy specimens.

Methods:
We retrospectively evaluated the initial biopsy and rebiopsy specimens of 60 patients with acquired resistance to EGFR-TKI between 2010 and 2016 in Seoul National University Bundang Hospital, Republic of Korea. EGFR mutation tests were performed in all specimens. Various histologic parameters including spindle cell components, discohesive growth pattern and stromal change, subtype of the tumor and nuclear grade were evaluated. In addition, immunohistochemistry (IHC) for epithelial-mesenchymal transition (EMT) markers (E-cadherin and vimentin) and neuroendocrine markers (CD56 and synaptophysin) was perfomed.

Results:
In rebiopsy specimens, 18 cases (30%) showed changes of cellular morphology including spindle cell components and discohesive growth pattern representing EMT features. On IHC, acquisition of vimentin expression in spindle cell components and decreased expression of E-cadherin in adenocarcinoma of rebiopsy specimens were identified. Furthermore, histologic transformation to small cell carcinoma (2 cases, 3.3%) with expression of neuroendocrine markers and squamous differentiation (2 cases, 3.3%) were observed.

Conclusion:
In this study, histologic transformation to EMT is the most frequent finding in rebiopsy samples of the patients with EGFR-TKI resistance while small cell carcinoma has been known to be the most common in the literaturesAlthough EMT has been reported to be about 5% of EGFR-TKI resistance, we observed it in approximately 30% of our rebipsy cases. These findings suggest that detailed and meticulous pathologic evaluation plays an important role to find delicate histomorphologic changes associated with EGFR-TKI resistance.

Background:
The Danish Lung Cancer Registry (DLCR) has since 2003 reported all cases of lung cancer in Denmark. Since 2012 data on EGFR mutations and ALK translocations have been included. Little is known on the distribution of EGFR mutations and ALK translocations on a national level in a primarily Caucasian population like the Danish lung cancer population.

Methods:
All Danish lung cancer patients are ascertained based on coded information in the National Patient Register. Supplementary information for each patient is obtained from the clinical units as well as from the National Pathology Register (NPR). Based on SNOMED coding by all departments performing lung cancer pathology evaluation and registered in the NPR the subgroups of lung cancer are identified. The patients are tested for EGFR mutations and ALK translocations according to national guidelines and the results are registered in the NPR. It is estimated that 95 % of all Danish lung cancer patients are present or former smokers and that the sex distribution is equal between the sexes.

Results:
4667 patients diagnosed in 2015 are included. Table 1. Distribution of EGFR mutations and ALK translocation in the 2015 lung cancer population: Figure 1 83.3 % of all patients with lung cancer and adenocarcinoma in Denmark are tested for EGFR mutations and 9.4 % are positive. 73.1 % of adenocarcinomas are tested for ALK translocations and 1.4 % is found to be positive. In total only 8.8 % of all tested lung cancer patients are found to be EGFR mutated and 1.3 % has an ALK translocation.



Conclusion:
Data from primarily Asian lung cancer populations have shown significant higher rates of EGFR mutations and ALK translocations that the findings in this Danish population. Based on these data the cost-effectiveness of the chosen strategy for reflex testing lung cancer patients up front should be reconsidered.

Background:
In the practice of precision medicine, understanding tumor characteristics in the individual patient is crucial. The aim of this study was to analyze tumor aggressiveness from two perspectives: actual growth rate calculated from the tumor; and molecular profiles obtained by next-generation sequencing.

Methods:
Participants comprised patients who underwent preoperative CT two or more times. DNA and RNA of 10 lung adenocarcinoma tumor samples were extracted. Whole-exome and -transcriptome data were obtained, and somatic mutations were detected. Preoperative CT scans were retrospectively reviewed and volume doubling time (VDT) of each tumor was calculated using a modified Schwarz equation.

Results:
Median VDT was 104 days (range, 42-653 days). Median number of somatic missense mutations was 20 (range, 7-306). EGFR mutations were present in 6 patients. Patients were divided into two groups by VDT for further analyses: Slow group with VDT ≥104 days (n=5); and Rapid Group with VDT <104 days (n=5). All patients with EGFR mutation without concomitant KRAS mutation were in the Slow Group. In contrast, a patient with concomitant mutations of EGFR and KRAS showed a considerably rapid growing tumor with a VDT of 45 days. A patient with concomitant mutations in EGFR and PIK3CA had a relatively slow-growing tumor, although VDT was the shortest in the Slow Group (120 days). Figure 1



Conclusion:
EGFR mutation was associated with slow growth of the tumor, although the growth rate may be influenced by concomitant mutation of other driver genes. This may be one of the reasons that the clinical response of tyrosine kinase inhibitors are poor in some patients with EGFR mutation. Assessment of tumor aggressiveness by molecular profiling and by sequential CT are both important for the practice of precision medicine.

Background:
Mutations in the tyrosine kinase (TK) domain of EGFR are oncogenic driver in 10-20% of lung adenocarcinoma (AdC) patients in Western countries. Approximately 90% of EGFR-TK inhibitor (TKI) sensitizing mutations occur as small in-frame deletions in exon 19 or L858R point mutations in exon 21. Recently, novel driver mutations in EGFR with oncogenic and TKI sensitizing activity have been reported. We present here an AdC patient-derived xenograft (PDX) model (PDX12) that is highly sensitive to EGFR-TKI, yet failed to demonstrate classical TKI sensitizing mechanisms.

Methods:
Comprehensive genomics profiling was used to characterize the genotype of PDX12, which was established from a resected stage IIIA AdC patient grafted in NGS mouse. The primary human lung cancer cell line (PHLC12) was extracted from its PDX model (PDX12). Aberrant EGFR cell lines used were H3255 (L858R), H2935 (exon 19 deletion), H1975 (L858R and T790M), and H1944 (wild type). Cell viability was assessed after erlotinib treatment at 1nM - 2μM for 72 hours using MTS assay. Levels of EGFR activation in both pre- and post-treatment by Western blot analysis.

Results:
PDX12 model had no known oncogenic mutations (EGFR wild type) on exons 18-21 by next-generation sequencing, RT-qPCR, and SISH, but was highly sensitive to EGFR-TKI. The IC50 to erlotinib treatment at 72 hr was 67.13 ± 7.63 nM for PHLC12, compared to 9.70 ± 2.64 nM for H3255, 64.88 ± 8.49 nM for HCC2935, > 2 μM for H1975, and > 2 μM for H1944 EGFR mutant or wild type cells, respectively. Western blot analysis demonstrated a relatively higher molecular weight band for EGFR protein with high expression level in PHLC12 when compared to other lung cancer cell lines. Using RT-qPCR, relative expression level of each EGFR domain (extracellular, tyrosine kinase, and c-terminal domain) in PHLC12 showed no difference compared to EGFR wild type. Phosphorylation status of EGFR in PHLC12 was similar in activity as compared to erlotinib sensitive cell lines.

Conclusion:
PHLC12 represents an enigmatic EGFR TKI sensitive lung PDX model without classical TKI sensitizing aberrations. Additional potential mechanisms of EGFR dependency including exon duplication, or post-translational modification of EGFR protein are being investigated.

Background:
Mutations of the epidermal growth factor receptor (EGFR) cause increased activation of EGFR and sensitivity of patients with non-small cell lung cancer (NSCLC) to tyrosine kinase inhibitors (TKIs). The effectiveness of TKIs also depends on mutations of the KRAS gene that encodes small GTPase that is activated in response to a signal from EGFR and transmits it to the cascade of tyrosine kinases. Treatment of patients with KRAS mutations by TKIs is inefficient. Therefore, the research of these alterations plays an important role in determining the possibility of additional factors prognosis of NSCLC and adjusting individual tumor therapy. The aim of this study is to identify mutations in the exons 19 and 21 EGFR gene and in the exon 2 KRAS gene in patients with NSCLC.

Methods:
Analysis of mutations in the EGFR and KRAS genes was performed by PCR followed by sequencing DNA, which was extracted from the tumor and non-tumor lung tissues and blood samples of 97 patients with NSCLC (71 men, 26 women). 51 people have an adenocarcinoma (AC) and 46 people - squamous cell carcinoma (SCC).

Results:
Analysis of mutations in the EGFR gene showed that the frequency of classical mutations is 5,2% of deletions in exon 19 (p.E746_A750del and p.L747_P753>S) and 1,1% of p.L858R mutation in exon 21. All mutations were detected only in the tumor tissue of non-smoking women with AC. Thus, 27,3% of women with AK are carriers of mutations in EGFR gene. These types of mutations were not detected among men. Also in the researched group was identified silent mutation c.2508C>T in exon 21 in the tumor, non-tumor tissue and blood among 3,3% of patients. Analysis of mutations in exon 2 KRAS gene detected 3 types of mutations: p.G12D (1,03%), p.G12C (2,06%) и p.G13C (1,03%). The frequency of all mutations was 4,1% in the total group of patients. The mutations were found only in tumor tissues of men. 75% of mutations carriers are smokers. Analysis of KRAS gene mutations in association with the development of a specific histological type of lung cancer showed that mutations are more common in patients with AC (5,9%) than in patients with SCC (2,2%).

Conclusion:
Thus, in the researched group of patients mutations in the EGFR gene were found only among non-smoking women with AC, mutations in the EGFR gene were detected only among men independently of histological type of NSCLC.

Background:
Epidermal growth factor receptor (EGFR) is a cell surface protein that binds to epidermal growth factor. Over expression of EGFR1 in tumor tissue has been observed in upto 65% of advanced non small cell lung cancer, and has shown promising prognostic potential. In this study, we compared the EGFR1 gene expression in serum adenocarcinoma lung with healthy controls.

Methods:
We analyzed 61 newly diagnosed patients of adenocarcinoma lung and 50 healthy controls. RNA was isolated from blood serum of all subjects and real time PCR (RT- PCR) was performed after complementary DNA (cDNA) synthesis. The level of EGFR1 expression in serum was calculated by relative quantification method and expressed as fold-increase compared to compared with controls. Expression levels were also correlated with various clinico- pathological parameters.

Results:
Out of 61 patients, 42 were males. The mean (SD) age of the entire group was 54.5 (11.5) years. Most of the patients (79%) had stage IV disease. 23 (38%) patients were current/ ex- smokers, with median pack years of 10 (range, 0.5- 100). Majority of patients had KPS of 90 (51%) and ECOG 1 (74%) respectively. Activating mutations in EGFR were observed in the tissue of 14 (21.3%) of 61 patients; of these, 9 were exon- 19 deletions and 4 were exon- 21 point mutations. In the patients, a 19.66 mean- fold increase in serum EGFR gene expression was observed compared to healthy controls. No significant association was found between EGFR expression and other variables i.e., sex, age, smoking habit, performance status, stage of disease and EGFR mutation status.

Conclusion:
Serum EGFR1 gene was over expressed by >16 fold in advanced adenocarcinoma lung compared to healthy controls. The association of EGFR expression with other clinical disease characteristics needs further exploration.

Background:
KRAS mutations are detected in approximately 25% of lung adenocarcinomas (LA). Targeted therapies against KRAS are under investigation. The use of tumor biopsy for molecular testing may be challenging due to the invasiveness of the procedure, the limited material for multiple biomarker analyses and tumor heterogeneity. Mutation detection in circulating cell-free tumor DNA (ctDNA) can overcome these caveats and also be used for tracking tumor dynamics. The aim of this study was to evaluate KRAS mutation detection in plasma samples from LA.

Methods:
Plasma samples from 35 patients with histologically confirmed KRAS mutant LA were collected at initiation of chemotherapy. KRAS mutations were assessed using digital PCR technology (QuantStudio3D Digital PCR System, Thermofisher Scientific). Correlation between ctDNA and tumor biopsies in terms of mutation detection was analysed. In 5 cases plasma samples were obtained during the course of the disease to monitor clonal dynamics.

Results:
Most cases were male (71%), with stage IV disease (83%), and showed KRAS mutation on codon12 (94%). KRAS mutation was found in plasma samples in 28/35 cases, showing a concordance with the tumor of 80%. In patients whose disease was limited to thorax (stages II, III, and IVa) KRAS mutation was detected in 7/10 (70%) plasma samples. Plasma/tumor biopsy concordance in cases with extra-thoracic metastases was 84% (21/25). The 4 false negative cases had low burden of extra-thoracic disease, with bone (2 cases), brain (1 case), and abdominal lymph node (1 case) as the only metastatic location outside the thorax. KRAS clonal dynamics in plasma showed a good correlation with treatment responses in some cases (figure 1).Figure 1



Conclusion:
High concordance in the detection of KRAS mutations was found between plasma and tumor tissue using digital PCR technology, particularly in cases with extra-thoracic disease. Digital PCR allows for tracking clonal dynamics in KRAS mutant LA.

Background:
Significant advances on EGFR-targeted therapy have allowed increasing availability of therapeutic options for non small cell lung cancers. For multifocal lung adenocarcinoma patients in clinic, the EGFR gene mutation is generally examined only on the largest tumor or the one containing the most tumor cells, which could omit the tumors harboring the EGFR mutation and thus loss of opportunity for the tyrosine kinase inhibitors therapy.

Methods:
A total of 58 cases of multifocal lung adenocarcinoma, including 129 intrapulmonary tumors resected surgically, was recruited for this study. The genome DNA samples were prepared from formalin-fixed and paraffin-embedded tumor tissues. The EGFR / KRAS mutational status of each tumor was examined by Sanger’s DNA sequencing. The targeted hotspot mutations in EGFR gene included p.G719S/C/A (exon 18), p.T790M (exon 20), p.S768I (exon 20), p.L858R (exon 21), p.L861Q (exon 21) and deletions in exon 19. The hotspot mutations in KRAS gene were within codon 12 including p.G12C/V/S/R/D/A.

Results:
In this group of 58 patients with multifocal lung adenocarcinoma, 38 patients were found EGFR or KRAS mutations in their tumors. Among them, the EGFR mutations were detected in 59 tumors derived from 34 cases; while the KRAS mutations were detected in 7 tumors from 5 cases. One patient (Case 30) was identified EGFR (exon 18, p.G719A) or KRAS (p.G12A or p.G12C) mutation in her 3 tumors, respectively. It is noteworthy that there were 21 (36.2%) in the 58 cases showing the mutational heterogeneity among the multiple intrapulmonary tumors derived from one individual, and that 6 tumors harbored 2 different types of EGFR mutation. However, none of the specimens investigated contained both EGFR and KRAS mutations within a same tumor. Comparing data from the present study along with the molecular pathological diagnosis records from the Department of Pathology, among the 58 cases enrolled, 30 cases accepted routine molecular pathological examination to check the EGFR / KRAS mutation, and 28 cases did not. However, 37 out of 66 tumors from the 30 cases were tested -- only 5 patients had all their tumors examined; whereas in the rest total 92 tumors unchecked, 42 EGFR sensitive mutations were identified in this study.

Conclusion:
Current finding suggests that the EGFR and KRAS mutational heterogeneity is widely existed in multifocal lung adenocarcinomas. Therefore, reliable and exhaustive examination for EGFR / KRAS mutation should be executed in the every tumor, to provide more individualized therapy choices for the patients.

Background:
Over the years, significant progress has been made in the treatment of lung carcinoma. While targeting EGFR mutations in the tyrosine kinase domain and EML4-ALK rearrangements have yielded impressive therapeutic gains in lung adenocarcinoma; the rarity of genetic aberration in squamous cell carcinoma (SCC) has restricted the use of molecular-targeting agents. Next-Generation sequencing analysis has identified a high frequency of c-MET mutation in Asian lung SCC specimens, in particular the MET-N375S mutant. This study aims to characterize the functional significance and therapeutic implications of MET-N375S in lung SCC cells.

Methods:
MET (N375S) mutation in tumour tissues was verified with droplet digital PCR. c-MET mutant expressing clones were generated through site-directed mutagenesis and single cell clonal selection. The impact of MET-N375S in lung SCC cells was characterized by defining the downstream effectors (Proteome profiling), biological functions (anchorage-independent growth, invasion and migration assays), transciptomic regulation (RNAseq) and protein-protein interaction (SILAC). Novel binding partners of MET-N375S was verified using co-immunoprecipitation (co-IP) and proximity ligation assay (PLA). Sensitivity of c-MET mutant to various kinase inhibitors was determined with CellTiter-Glo assay.

Results:
MET (N375S) mutation was confirmed in 9/45 lung SCC specimens (20%). Ectopic expression of MET-N375S in lung SCC cells significantly elevated the activation of downstream Src, p38 and ERK1/2 kinases, increased hsp27 expression, while enhanced migratory, invasive and anchorage-independent colony forming ability in vitro. Despite so, comparative transcriptomic analysis revealed that epithelial-mesenchymal signature genes were not induced in cells expressing mutant c-MET. On the contrary, SILAC and co-IP analyses on the MET-N375S interactome demonstrated an increase in binding affinity towards receptor tyrosine kinases (RTKs) as compared to wild type c-MET, which was confirmed with in situ PLA. Moreover, MET-N375S augmented in vitro sensitivity to selective MET inhibitors.

Conclusion:
These findings suggest the role of MET-N375S as an activating mutation that strongly enhance malignant transformation and metastatic potential in lung SCC cells. Mechanistically, the dysregulation of various oncogenic events could be associated with the formation of heterodimers with RTKs. Clinically, MET-N375S could be utilized as a potential predictive biomarker for patients with advanced SCC of the lung to be treated with selective MET inhibitors.

Background:
There have been very few studies on ALK status in lung adenocarcinomas in Latin American population. No prior reports in Northeastern Brazil have been published.

Methods:
We analyzed ALK protein expression with a specific antibody (D5F3 clone) with the Ventana system in a series of consecutive cases from Northeastern Brazil, a previously untested population, and correlated with histologic subtypes according to the 2015 WHO Classification.

Results:
A total of 94 patients (55% female, mean age 62 years) were tested, including 51 solid, 29 acinar, 6 lepidic, 6 papillary predominant and 2 sarcomatoid carcinomas with adenocarcinoma components. There were 9 positive cases (9.5%), 5 solid predominant, 3 acinar predominant and 1 lepidic. One of the 3 acinar cases had a mucinous component. The positive cases were more often male (p<0.05) with no significant differences in relation to age, smoking history or histologic subtype. The negative cases showed a 32% EGFR mutation rate. Follow-up will be presented.

Conclusion:
We will describe a series of consecutive adenocarciomas from a population with unknown ALK status with a higher than expected rates, and correlate with the 2015 WHO Classification of Tumors.

Background:
Although the use of NGS (next-generation sequencing) is indeed feasible for the study of druggable rearrangements, the sensitivity and specificity of targeted NGS has been challenged on several grounds: use of fixed tissue, poor quality/insufficient sample, RNA-contamination risk or long turn-around time. Our relatively high rate of failures (7.6%) with the RNA workflow of targeted NGS (data not shown), prompted us to investigate possible alternatives. The objective of this study is to demonstrate an efficient solution based on multiplexed fluorescence in situ hybridization (FISH) for the comprehensive characterization of fusions (ALK, ROS1, RET and NTRK1) in lung adenocarcinomas (ACs). We have implemented for the first time in the literature a novel four-colour ALK/ROS1 probe and used an automated scanning system for scoring these four translocations.

Methods:
A total of 64 patients with early stage lung ACs who underwent surgery at HM Sanchinarro University Hospital were considered. All slides were carefully reviewed to identify the different histological patterns. Afterwards we performed FISH to study ALK, ROS1, RET and NTRK1 in each pattern. We used both commercially (RET and NTRK1) and non-commercially (ALK/ROS1 dual) available Vysis break-apart probes (Abbott Molecular, USA). Slides were captured and scored with the BioView (Rehovot, Israel) automated imaging and analysis system, using dedicated applications for each probe. Positive cases were all confirmed with targeted NGS (Oncomine Focus Assay[TM], Life Technologies, USA).

Results:
Seven out of 420 slides did not hybridize (1.6%). We found two ALK (2.8%) and two ROS1 (2.8%) positive tumors, while no translocations were identified in RET or NTRK1. The rearrangements were present in all the different histological patterns within a given tumour, with a similar proportion of positive cells. The two major patterns of positivity were represented. The mean percentage of positive cells was 65% (range 46 to 84%). The NGS results confirmed the FISH findings.

Conclusion:
Simultaneous FISH testing for ALK and ROS1 is feasible on a single slide. The strategy reported herein provided reduced overall scoring time and ensured sensitive counting. This model might be easier to reconcile (lower cost, shorter turn-around time, and less failure rate) with subsequent comprehensive genotyping. Therefore, it could be used prospectively in advanced lung ACs to align the use of several technologies. Acknowledgements This study was partially funded by Abbott Molecular and Instituto de Salud Carlos III (ISCIII), Fondo de Investigaciones Sanitarias (FIS), Fondos FEDER-Plan Estatal de I+D+I 2013-2016 (PI14-01176).

Background:
There is limited Brazilian data on epidermal growth factor receptor (EGFR) gene activating mutations prevalence and their clinicopathologic associations especially in the Northeast, with no previous epidemiological reports. The current study aimed to assess the relationship between EGFR mutations and histologic subtypes according to the 2015 WHO Classification.

Methods:
We assessed the frequencies of EGFR mutations in consecutive pulmonary adenocarcinomas among a population-based sample in Northeastern Brazil. A sample of 351 patients diagnosed from 2014-2016 was analyzed by direct sequencing (45.5%), real time PCR (34.1%) or next generation sequencing (20.4%).

Results:
The overall mutation rate was was 30.5%. The ratio of exon 19 deletions to exon 21 L858R mutations was 1.2:1. Three patients had T790M mutations detected after progression in use of targeted therapy. Female sex (P = 0.002), never smoking status (P = 0.002), and nonsolid subtype of ADC (P = 0.001) were associated with EGFR mutations on univariate analyses. Acinar predominant and lepidic predominant tumors had a higher rate of mutation but with no statistical significance when evaluated solely. Papillary component did not show correlation with mutations. Tumor differentiation correlated significantly with their incidence of EGFR mutations, lower in poorly differentiated tumors (p=0.005). Follow-up will be presented.

Conclusion:
We will describe a series of consecutive adenocarciomas from a population with unknown status with a higher than expected rates (higher than Southern Brazil and similar to other Latin American countries), and correlate with the 2015 WHO classification of tumors.

Background:
Anaplastic lymphoma kinase (ALK ) rearrangement represents a landmark in the targeted therapy of NSCLC. Several reports showed that IHC is sensitive and specific to detect ALK protein expression, possibly alternative to ALK FISH assay. In this study the concordance between the ALK status by FISH and IHC assay has been determined in a series of 95 advanced NSCLCs; discordant cases were evaluated on the basis of the percentage and the rearrangement pattern of ALK.

Methods:
95 lung NSCLC specimens were tested for ALK rearrangements by IHC assay (ALK D5F3 antibody Ventana,CE-IVD system) and by fluorescence in situ hybridization (FISH). Clinical characteristic and response to crizotinib were reviewed.

Results:
Seven cases (7.3%) showed discordant results with ALK FISH-positive and IHC negative. Among these cases the mean number of FISH positive rearranged nuclei was 40.2% (range 20-54%). All cases showed coexistent split signals pattern positivity with mean percentage 19.1% (range 10-32%.), 5' deletions pattern positivity with mean percentage 21.7% (range 12-34%) and one case also had gene amplifications pattern positivity with percentage of 64%. The polysomy was observed in all cases with mean percentage of 45.7% (range of 16-72%). Five patients with discordant ALK FISH and IHC results received crizotinib; of them, four progressed and one has stable disease.

Conclusion:
In most of discordant cases, a coexistent complex pattern of rearrangements (deleted, invertited and amplified/polysomic patterns) of ALK positive cells in FISH analysis was observed; these complex rearranged cases were not detectable by IHC, probably due to the lack of protein expression. Considering that crizotinib inhibits the ALK protein and not specifically ALK rearrangements, it could be speculated that NSCLCs without IHC ALK expression could be not sensitive to crizotinib. The ALK FISH+/IHC negative discordant group showed a loweer percentage of nuclei positive for ALK rearrangement (19.1% in split signals and 21.7% in 5' deletions) as compared with 64% of gene amplification in one case and with polysomy (45.7%) observed in all cases. These preliminary data highlight the role of IHC analysis and of the percentage of the genetic pattern of ALK rearranged cells in FISH analysis to select patients for anti ALK treatment. Further investigation is suggested about the correlation of complex mutational findings and clinical outcome.

Background:
An ALK genetic translocation event occurs in ~2-7% of non-small cell lung carcinoma patients (NSCLC), resulting in the constitutive expression of an active chimeric ALK protein, which leads to tumor proliferation. Ventana has developed a fully automated immunohistochemistry (IHC) assay using the VENTANA anti-ALK (D5F3) Rabbit Monoclonal Primary Antibody (ALK (D5F3)) to detect the ALK protein in formalin fixed paraffin embedded tissue. ALK IHC testing is becoming more widespread in NSCLC; however, information concerning the impact of pre-analytical conditions on the ALK antigen is limited. We used human cell lines expressing ALK, generated as xenografts to evaluate the effect of Fixation Type, Time, and Delay to Fixation (Ischemia) on the ALK antigen as measured by the ALK (D5F3) antibody staining intensity.

Methods:
NCI-H2228 human NSCLC cell lines were used to generate xenograft tumors in SCID mice. In order to assess ischemia (delay to fixation) H2228 xenografts were used to model ischemia in 10% NBF. The impact of fixation on staining performance of the ALK (D5F3) primary antibody was assessed using the H2228 xenografts to model fixation type and fixation time for 10% NBF, Zinc Formalin, 95% Alcohol, Alcoholic Formalin Acid, B5, and Prefer for 1, 6, 12, 24 and 72 hours. Each of the stained slides were evaluated by a pathologist using a qualitative assessment and scored on a 0-3+ intensity scale (0 = no staining detected, 1+= weak staining detected, 2+= moderate staining detected, 3+ = strong staining detected).

Results:
Fixation in all time points in B5, Prefer, and AFA, as well as ethanol, severely compromised staining intensity of ALK (by decreasing staining intensity greater than 0.5 points). Ischemia greater than 6 hours also decreased staining intensity. In contrast, EGFR (5B7) and TTF1 (SP141) antigens were robust across a wide range of fixation times and types, as well as ischemia times.

Conclusion:
The ALK antigen is highly sensitive to fixative time, type and ischemia. The data indicate that the detection of ALK by IHC is impacted by fixation conditions. Strikingly, antigens detected by other lung antibodies (EGFR, TTF1) are more robust in comparison across a range of conditions. Standardized pre-analytical conditions are critical to achieve appropriate staining for ALK IHC and to mitigate the risk of false negative results. The recommendations for pre-analytical conditions for ALK are to fix at least 6 hours in 10% neutral buffered formalin.

Background:
The epithelial to mesenchymal transition (EMT) is associated with acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in certain non-small cell lung cancers that harbor EGFR mutations. Because no currently available drugs specifically kill cancer cells via EMT, novel treatment strategies that overcome or prevent EMT are needed. A recent report suggested that dasatinib (an ABL/Src kinase inhibitor) inhibits EMT induced by transforming growth factor (TGF)-beta in lung cancer cells. In this study, we analyzed effects of dasatinib on the resistance mechanism in HCC4006 cells harboring EGFR exon 19 deletion, which tend to acquire resistance to EGFR-TKIs via EMT in previous reports.The epithelial to mesenchymal transition (EMT) is associated with acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in certain non-small cell lung cancers that harbor EGFR mutations. Because no currently available drugs specifically kill cancer cells via EMT, novel treatment strategies that overcome or prevent EMT are needed. A recent report suggested that dasatinib (an ABL/Src kinase inhibitor) inhibits EMT induced by transforming growth factor (TGF)-beta in lung cancer cells. In this study, we analyzed effects of dasatinib on the resistance mechanism in HCC4006 cells harboring EGFR exon 19 deletion, which tend to acquire resistance to EGFR-TKIs via EMT in previous reports.

Methods:
HCC4006ER cells with an EMT phenotype were previously established by chronic exposure to increasing concentrations of erlotinib. Sensitivity to dasatinib in parental HCC4006 and HCC4006ER cells was analyzed. Subsequently, HCC4006EDR cells were established by chronic treatment with combination of erlotinib and dasatinib. The expression of EMT markers of these cells and the mechanism of acquired resistance to this combination therapy were analyzed.

Results:
Short-term or long-term, ranging OOhours to XXXmonth, treatment with dasatinib did not reverse EMT in HCC4006ER. In contrast, HCC4006EDR cells maintained an epithelial phenotype, and the mechanism underlying resistance to erlotinib plus dasatinib combination therapy was attributable to a T790M secondary mutation. HCC4006EDR cells, but not HCC4006ER cells, were highly sensitive to a third-generation EGFR-TKI, osimertinib.

Conclusion:
Although dasatinib monotherapy did not reverse EMT in HCC4006ER cells, preemptive combination treatment with erlotinib and dasatinib prevented the emergence of acquired resistance via EMT, and led to the emergence of T790M. Our results indicate that preemptive combination therapy may be a promising strategy to prevent the emergence of EMT-mediated resistance.

Background:
Mutations in the MET exon 14 RNA splice acceptor and donor sites, which lead to exon skipping, have been described with responsiveness to crizotinib. Until now, patient selection has been made in view of MET amplification/high polysomy and protein overexpression, with discrepancies in positivity criteria. We investigated the prevalence of abnormal MET mutational status and the subsequent gene copy number alterations (CNAs) in NSCLC.

Methods:
We routinely tested 190 lung adenocarcinomas and adenosquamous carcinomas for MET exon 14 and flanking introns mutations by PCR-direct sequencing, and for MET gene CNAs by FISH (Abbott Molecular). Amplifications were defined as mean gene by mean centromere ratio ≥2.2, and high gains as mean gene ≥5.0. RT-PCR was performed to validate mutations leading to exon 14 skipping. We collected clinical-pathological data together with EGFR and KRAS mutational status and ALK, ROS1 and RET rearrangements.

Results:
MET alterations were found in 34 patients (17.9%): 11 mutated cases (5.8%), eight gene amplifications (4.2%), and 15 high gains (9.1%). Six out of 11 mutations were confirmed to lead to exon 14 skipping (3.2%). Remarkably, none of these exon 14 skipped cases had concurrent MET amplification nor high gains. Although, KRAS p.G12C and EGFR 19 exon mutations were found concomitant with MET mutation in two of these cases. Among the 14 confirmed MET altered cases (6 exon 14 skipped and 8 amplified): 13 patients were male (93%), with a median age of 65.7 years (range: 40-91), nine current smokers (64%) (40 pack-years, range: 20-60), and eight diagnosed in an advanced stage disease (III and IV) (57%). Correlation with c-MET protein expression by IHC is ongoing, and data will be presented at the meeting.

Conclusion:
As no concurrent MET mutated and amplified cases were found, our data support prospective identification of both, MET exon 14 skipping mutations and gene amplifications. These mutations define a new subset of NSCLC patients that should be analyzed independently of the status of MET gene copy number.

Background:
Rates in targetable gene changes varies between different populations of lung cancer patients. Targetable gene changes include changes in EGFR and ALK gene, as well as KRAS gene. Primary aim of this study was to determine mutation status in purely Caucasian Croatian population.

Methods:
Rates in targetable gene changes varies between different populations of lung cancer patients. Targetable gene changes include changes in EGFR and ALK gene, as well as KRAS gene. Primary aim of this study was to determine mutation status in purely Caucasian Croatian population.

Results:
During 6 months period 324 newely diagnosed primary lung adenocarcinoma were tested. Out of 324 tested patients, 194 were males (60%) and 130 females (40%) mean age 64 years (range 35 to 88 years). Vast majority of patients were in stages 3 and 4 (more than 80%). Among males, 87% of patients were ever smokers, and among females 61% of patients were ever smokers. Significantly higher rates of evers smokers were recorded among males. EGFR mutations were present in 15.7% of patients (51 patients). There was a difference in EGFR mutation rates between males and females (5.6 vs 30.8%, p<0.0001). KRAS mutations (codones 12/12 and 61) were present in 35.8% (116) patients, and ALK translocation detected by IHC in 3.7% (12) patients.

Conclusion:
Molecular testing in primary lung adenocarcinoma patients was done in purely Caucasian Croatian population. EGFR mutation and ALK translocation rates were similar to previously published data. However, KRAS mutation rates were higher than previously published. This can be associated with high smoking rates in Croatian population.

Background:
Patients with epidermal growth factor receptor (EGFR)-mutant lung carcinoma eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKI). In 50% of these cases, a secondary EGFR mutation, T790M, underlies the acquired resistance. Other alterations include amplification of MET, PI3K mutations, changes in MAPK1, Her2, AXL and even transformation to small cell lung carcinoma (SCLC). We assessed histological, clinical characteristics and survival outcomes in Hispanic patients with EGFR mutation after disease progression.

Methods:
34 EGFR-mutant lung cancer patients with acquired resistance to EGFR TKIs were identified as part of a prospective registry (active between January 2011 and January 2015) in which post-progression tumor specimens were collected for molecular analysis using SNaPshot tumor genotyping assay to detect mutations in EGFR, KRAS, BRAF, PI3KCA, TP53, MET and Her2, and FISH for MET, ALK and EGFR. Samples also underwent immunohistochemistry analysis for E-cadherin, synaptophysin, CD56 and PDL1. Post-progression interventions, response and survival were assessed and compared to those with and without T790M.

Results:
Mean age was 59.4±13.9 years; 62% were female, 65% were never-smokers and 53% had a performance status ≥80%; main metastatic sites were lung (16/47%), bone (20/58%), brain (18/52%) and liver (13/38%). All patients received erlotinib as first- line treatment and documented mutations were: 60% DelE19 (Del746–750) and 40% L858R. Overall response rate (ORR) with first line TKI was 61.8% and progression free survival (PFS) was 16.8 months (range, 13.7–19.9 m). After progressing to TKI, all patients were re-biopsied, of whom 16 had the T790M mutation (47.1%); 5 had PI3K mutations (14.7), 5 had EGFR amplification (14.7%), 2 had a KRAS mutation (5.9%), 3 had MET amplification (8.8%), 2 had Her2 alterations (5.8%, deletions/insertions in e20), and one had SCLC transformation (2.9%). 79.4% received treatment after progression. ORR for post-TKI treatments was 47.1% (CR 2/PR 14) and median PFS was 8.3 months (CI95% 2.2–36.6). There were no differences in PFS according to gender (p=0.10) or type of acquired alteration (p=0.63). Median survival was 32.9 months (CI95% 30.4–35.3), and only the use of post-progression therapy affected OS in multivariate analysis (p=0.05).

Conclusion:
Hispanic patients with acquired resistance to EGFR TKIs continued to be sensitive to other treatments after progression. Proportion of T790M+ patients appears to be similar to previously reported results in Caucasians.

Background:
Patients having non-small-cell lung cancer (NSCLC) with activating EGFR-mutations benefit from EGFR-Tyrosine Kinase Inhibitor (TKI) treatment. However, other driver mutations may occur simultaneously and other pathways may be amplified/overexpressed, potentially hampering efficacy of EGFR-TKI treatment. The frequency of such alterations at time of diagnosis was examined.

Methods:
All patients referred to Rigshospitalet, Copenhagen University Hospital for pulmonary adenocarcinoma (ADC) from July 2013 to August 2015 were routinely tested for EGFR-mutations by EGFR Cobas RT-PCR technique (Roche Diagnostics) on DNA extracted from diagnostic tumor biopsies or cytological samples. If positive for EGFR-mutations these patients were, prior to any treatment, also tested by targeted Next Generation Sequencing (NGS; Ion Torrent Ampliseq Colon-Lung v.2 panel and PGM NGS-sequenator) for other simultaneous cancer-relevant mutations, by fluorescence in-situ hybridization (FISH) for MET-amplification, and by immunohistochemistry (IHC) for expression of MET receptor as well as ALK fusion-protein.

Results:
Totally 512 ADC patients were tested, among whom 22 out of 282 advanced stage patients (7.8%) had activating EGFR-mutations, distributed as follows: 1 G719C-mutation, 13 exon 19-deletions, 1 T790M-mutation, 1 S768I-mutation and 8 L858R-mutations with 2 of the patients harboring EGFR-double-mutations G719C + S768I and L858R + T790M (i.e., activating and resistance mutation), respectively. Complete concordance between EGFR-mutation-status by EGFR Cobas and NGS was observed for all NGS tested patients. For one of the patients NGS analysis could not be carried out, due to lacking DNA-extract and remaining tumor tissue. NGS-analysis identified several concomitant driver mutations in the 21 analyzed patients, including one 1 with KRAS-mutation (G12V), 12 with TP53-mutations (7 in TP53-exon 5), 1 with FGFR-mutation (S125_E126insS), 2 with CTNNB1-mutations (S33C and S37F), 1 with MET-mutation (T1010I), 1 with SMAD4-mutation (R135stop), and 1 with PIK3CA-mutation (E545K). FISH and IHC for MET were successful in tumor samples from 16 and 19 patients, respectively. Three patients had concomitant MET-amplification (1 with and 1 without corresponding MET-overexpression, 1 with unsuccessful IHC), whereas 2 other patients had increased copy number (ICN; both with corresponding MET-overexpression). Interestingly, 4 of these 5 patients with MET-amplification or -ICN also carried TP53-mutations. Expression of ALK fusion-protein was not detected in any of the 22 patients with activating EGFR mutations.

Conclusion:
At time of diagnosis, concomitant mutations in other cancer driver genes can be detected in advanced EGFR-mutation-positive NSCLC of ADC subtype. These concomitant mutations may impact the response to first-line EGFR-TKI treatment and represent additional therapeutic targets.

Background:
The epidermal growth factor receptor (EGFR) signaling pathway is one of the most frequently deregulated pathways in non-small cell lung cancer. While targeted therapy prolongs survival in patients harbouring EGFR mutations, resistance to treatment eventually develops in all cases. As multiple genetic and epigenetic alterations are known to disrupt signaling pathways, the objective of this study is to perform a multidimensional analysis of signaling pathways to identify alterations essential to tumorigenesis that are overlooked when assessing a single genomic dimension.

Methods:
Multidimensional integrative analysis of copy number, DNA methylation, and gene expression profiles of 77 lung adenocarcinomas and matched non-malignant tissues identified Signal Regulatory Protein A (SIRPA) as a novel candidate tumor suppressor gene. Following validation of genomic findings in multiple external data sets, the tumor suppressive effects of SIRPA were assessed in vitro and in vivo with a panel of lung cancer cell lines.

Results:
SIRPA negatively regulates receptor tyrosine kinase signaling through activation of the protein phosphatases SHP1 and SHP2 and was found to be underexpressed in 70% of lung tumours, ranking it in the 95[th] percentile of altered genes within the EGFR pathway. Immunohistochemistry (IHC) confirmed reduced protein expression in tumors, which was found to correlate with EGFR mutation and adenocarcinoma histology. In vitro, SIRPA knockdown promoted migration while simultaneously inducing a dramatic senescent phenotype, suggesting SIRPA may act as a barrier to tumorigenesis. This phenotype is dependent upon upregulation of the CDK inhibitor p27, which hypophosphorylates RB leading to cell cycle blockade and reduced tumor growth in vivo. Importantly, increased expression of p27 resulted in mis-localization into the cytoplasm where it is known to promote an invasive phenotype. Inhibition of p27 confirmed previous findings and emphasized the importance of this pathway in lung tumorigenesis. Surprisingly, overexpression of SIRPA increased cell growth and migration, suggesting SIRPA may also possess oncogenic properties due to its regulation of multiple signaling pathways. Overexpression of SHP2 following ectopic expression of SIRPA promotes migration through the inhibition of focal adhesions. This phenotype is abrogated upon siRNA knockdown of SHP2.

Conclusion:
SIRPA is an important player in lung tumor biology, capable of acting as both an oncogene and tumor suppressor due to its ability to regulate multiple signaling pathways. Due to the complex nature in its signaling, future work should focus on elucidating how the timing of alterations to SIRPA affects tumorigenesis to design treatment strategy.

Background:
Many patients with NSCLC are diagnosed at advanced stages. A high percentage of those patients have only cytological samples for morphological and molecular analysis. Furthermore ALK analysis by FISH has to be performed using a specific commercially available probe. The aim of this study is to compare two different commercially available probes for analysis of ALK gene rearrangements by FISH using cytological stained smears.

Methods:
We analyzed 37 cytological stained smears from 37 consecutive cases of FNA (14 Diff-Quick and 23 Papanicolau). ROSE was performed in all procedures. The status of EGFR, KRAS and BRAF was kwon in 28 patients. Two probes were used: LSI ALK BREAK APART (Vysis, Ref: 53206N38020, Izasa) was apply in all 37 cases. Additionally, in 25 of these cases we used the ZytoLight SPEC ALK Dual Color Break Apart (ZYTOVISION, Ref: Z-2124-200, Menarini Diagnostics) probe. Protocols were modified from Basel and Zytolight manufacture guidelines. In order to optimize the amount of probe and to evaluate individual nuclei, we delimited an area on each smear with no nuclear overlap.

Results:
All cases were adenocarcinomas. Two cases showed ALK rearrangement (5.4%). Of the 34 negative cases, 27 were negative-polysomic (77.14%). One case was no assessable (2.7%) due to the impossibility of getting hybridization. Concordance between two probes was 100%. No differences were found between Papanicolau and DiffQuick stained smears .

Conclusion:
FISH-ALK in cytological stained smears gives excellent results. Both commercially avialable probes showed identical performance, both are equally valid. No differences between Papanicolau and Diff-Quick stained smears were observed. In our experience, stained cytological smears are the best choice for the analysis of ALK rearrangements in NSCLC patients, keeping paraffin for cases in which adequate cytological smears are not available.

Background:
Background: KRAS is the most frequently mutated oncogene in NSCLC and lacks an effective targeted therapy. Notably, KRAS mutations occur in both never/light-smokers and smokers. However, the relationship between smoking status and a KRAS+ tumor’s genomic complexity (mutation burden, copy number changes, concurrent mutations in key oncogenic pathways) is unclear. Similarly, the relationship between genomic complexity in tumors from never/light smokers but with a KRAS v EGFR activating mutation is also unknown.

Methods:
Methods: Targeted next-generation sequencing (NGS) data at our institution from 7/13-1/16 was reviewed to identify KRAS+ NSCLC tumors. All patients with a <10 pack-year (py) smoking history (NS) and a subset of heavy smokers (HS) (>40 py) were identified, with clinical and genomic analysis. A comparison cohort of 48 patients with EGFR+ NSCLC was also identified. Fisher’s exact test was used to compare frequency of gene mutations.

Results:
Results: 41 NS and 104 HS KRAS patients were evaluated. NS patients were more likely to be female (34/41 v 66/104, p<.01) and diagnosed with Stage I disease (14/41 v 13/104, p<.01). Compared to KRAS NS patients, tumors from KRAS HS patients were also genomically more complex, with increased total nucleotide variants (median=10 v 7, p<.001) and total copy number variations (median=22.5 v 5, p<.01). Intriguingly, in the cohort of EGFR tumors, total nucleotide variants resembled the KRAS NS cohort (median=6.5) but the total copy number variants were more similar to the KRAS HS cohort (median=25). Compared to KRAS NS tumors, KRAS HS tumors were also more likely to have: a) concurrent mutation in TP53 (43/104 v 8/41, p=.012) and b) concurrent mutations/2-copy deletions in >1 tumor suppressor (TS)/tumor (panel of TP53, STK11, APC, CDKN2A/B, RB) (26/104 v 4/41, p=.042). Although the total number of nucleotide variants in the EGFR cohort was most similar to the KRAS NS cohort, TS distribution in these EGFR tumors was closer to the KRAS HS cohort (TP53 variants in 31/48 and multiple TS variants in 14/48). Finally, median OS for KRAS HS patients with Stage IV disease was 9.7m v 28.7m in KRAS NS patients (HR=0.56).

Conclusion:
Conclusions: The genomic landscape of KRAS+ NSCLC from HS patients is distinct from NS patients and includes increased total mutations and frequency of TS loss. EGFR mutant tumors share some similarities with KRAS tumors from both NS and HS patients. Overall, NS KRAS+ tumors may be a genetically distinct cohort within the broader context of KRAS+ NSCLC.

Background:
EGFR mutations are present in ~15% and KRAS mutations in ~30% of lung adenocarcinomas (LACs). Yet, as several observers have noted, no individual LAC appears to carry mutations in both, even in the setting of resistance to EGFR kinase inhibitors. The typical explanation given is that the two genes are entirely functionally redundant, thereby eliminating any selective advantage to mutation of both. In this study we tested an alternative hypothesis for this mutual exclusivity: that co-occurrence of mutations in both KRAS and EGFR is deleterious to the evolving cancer cell. Furthermore, we aimed to characterize the specific cellular effects and signalling pathways induced by mutant KRAS and EGFR co-induction and determine whether this information could be used to design new strategies to inhibit LACs.

Methods:
Next-generation sequence data for over 600 human LACs were acquired from public sources and analyzed for co-incidence of KRAS and EGFR mutation and the relationship to smoking status. Transgenic mice that express both mutant oncogenes specifically in the lung epithelium were generated to test the effects on LAC development. LAC cell lines with endogenous mutations in either KRAS or EGFR were engineered with lentiviral vectors to conditionally express the second oncogene and assessed for cell viability, gene expression and protein phosphorylation changes. Temporal phosphoproteome assessment of cells co-expressing both oncogenes is currently ongoing to determine the specific signalling pathways affected.

Results:
We confirmed the mutual exclusivity of KRAS and EGFR mutations and demonstrated that this relationship is not due to limited power to detect concurrent mutations in either smokers or non-smokers. While no effect on tumor latency was observed in transgenic mice that express both mutant oncogenes, the resulting tumors preferentially expressed only one of the two transgenic oncogenes, indicating negative selection against co-expression. Introduction of the second oncogene into LAC cells expressing either mutant KRAS or EGFR stimulated the loss of cell viability. The most prominent features accompanying loss of cell viability were vacuolization, other changes in cell morphology, and increased macropinocytosis. Activation of ERK, p38 and JNK was observed in cells expressing both mutants suggesting that an overly active MAPK signaling pathway may mediate this synthetic lethal phenotype. Lastly, this effect is dependent on functionally active EGFR, since inhibition of the EGFR tyrosine kinase with erlotinib rescued cells from the detrimental effects of co-expression.

Conclusion:
Together, our findings indicate that mutual exclusivity of oncogenic mutations may reveal unexpected vulnerabilities and therapeutic possibilities.

Background:
Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer related death worldwide. In recent years molecular characterisation of NSCLC has led to the identification of several driver events including EGFR constitutive activation, ALK-rearrangement and ROS1 fusion. Whilst there are several mechanisms of EGFR-mutation stated in the literature, how genomic heterogeneity related with acquired EGFR resistance to second targeted agent affects response to subsequent therapy has not been noted.

Methods:
We studied EGFR-TKI, gefitinib, in an EGFR 19 deleted lung adenocarcinoma patient to assess whether tissue and liquid biopsy could be integrated with radiologic imagings to demonstrate the impact of individual actionable driver mutation on lesion specific response.

Results:
A 60-year old female, with no previous or family history of malignancy initially presented with EGFR 19 deletion mutation, ROS1 fusion negative and ALK rearrangement negative stage IV, T2aN3M1a lung adenocarcinoma detected in primary lung cancer tissue at the first diagnosis. Biopsy of this patient’s metastatic right cervical lymph node following prolonged response to gefitinb led to the loss of detection of EGFR mutation, and the novel mechanism of acquired resistance with EZR-ROS1 fusion where crizotinib was demonstrated to have good efficacy in all lesions, especially the enlarged lymphadenopathy, and also including diminishing efficacy on metastatic brain lesions. In circulating tumor DNA (ctDNA), mutant EGFR levels disappeared followed by gefitinib treatment, and a recognized EZR-ROS1 fusion was meanwhile identified before crizotinib therapy.

Conclusion:
This case displays tumor heterogeneity in action, where targeted therapy selects against primary drivers, allowing for the establishment of sub-colonies with new drivers within the specific lesion. Parallel analysis of tumor biopsies when disease progressed and ctDNA monitoring showed that lesion specific radiographic responses to subsequent targeted therapies could be driven by district resistant mechanism in the separate tumor lesions within the same patient. This demonstrates that the importance of molecular heterogeneity surveillance ensuing from acquired resistance in managing NSCLC, and the usage of liquid biopsies to allow for a holistic viewpoint of the molecular landscape of this heterogeneous disease.

Background:
Advanced NSCLC patients whose tumors harbor ALK fusions benefit from first line treatment with ALK inhibitors (ALKi). However, insufficient tissue for testing (QNS) occurs ~25% of the time. Patients treated with ALKi ultimately progress. Historically, identification of the resistance mechanism/s required repeat tumor biopsy. Circulating tumor DNA (ctDNA) may provide a non-invasive way to identify ALK fusions and actionable resistance mechanisms without a repeat biopsy.

Methods:
The Guardant360 (G360) de-identified database of NSCLC cases was queried to identify 57 patients (2/2015-6/2016) with 58 ctDNA-detected ALK fusions. G360 is a CLIA-laboratory ctDNA test that detects point mutations in 70 genes and select amplifications, fusions and indels. Available records were reviewed to characterize patients at baseline and at progression.

Results:
Identified fusion partners included EML4 (n=51, 88%), STRN (7%), KLC1 (3%), KIF5B (2%). Thirty patients had no history of targeted therapy (new diagnosis or no prior genotyping, “cohort 1”); 23 patients were drawn at ALKi progression (“cohort 2”). In 6 samples, the patients’ clinical status was unknown. Three additional cases had ALK resistance mutations (F1174C, F1269A/I1171T, D1203N) detected in ctDNA but no fusion detected; historical tissue testing was ALK+. Conversely, in cohort 1, 10 (33%) were tissue QNS (7) or tissue ALK negative (3) while 4 (13%) were tissue ALK+ and 16 (54%) had unknown tissue status. As expected, no documented or putative resistance mechanisms were identified in cohort 1, although TP53 mutations were identified in 43%. Among 18 patients progressing on an ALKi, 7 (39%) contained 1 (4 patients), 2 (1 patient) or 3 (2 patients) ALK resistance point mutations (F1174C/V: 3 occurrences; G1202R: 3; L1196M: 3; G1128A: 1; L1189F: 1; I1171T: 1). Additional events co-occurring in the resistance cohort included 1 each of: BRAF[V600E], MET[E14skip], KRAS[G12], KRAS[G13], HRAS[Q61], EGFR[E330K], KIT[amp], BRAF[amp]. 5 EGFR-mutant NSCLC cases at progression harbored ALK fusions (4 STRN, 2 EML4; 1 patient had both) representing 1% of all EGFR-mutant progressing NSCLC cases in the G360 database. Four of these patients also harbored EGFR[T790M], but the presence of an ALK fusion may represent further subclonal evolution following the selective pressure of an EGFR inhibitor.

Conclusion:
These results add to the growing body of literature demonstrating that comprehensive ctDNA assays provide a non-invasive means of detecting targetable alterations in the first line when tissue is QNS as well as detecting known and novel resistance mechanisms that may inform treatment decisions at progression.

Background:
Tumor heterogeneity, which causes different EGFR mutation abundance, is believed to be responsible for varied progression-free survival (PFS) in lung adenocarcinoma (ADC) patients receiving EGFR-TKI treatment. Frequent EGFR amplification and its common affection inEGFR mutant allele promote the hypothesis that EGFR mutant abundance might be determined by EGFR copy number variation and therefore examination of EGFR amplification status in EGFR mutant patients could predict the efficacy of EGFR-TKI treatment.

Methods:
72 lung ADC patients who harbored EGFR activating mutations and received erlotinib as first line treatment, were examined for EGFR amplification by FISH. EGFR mutational and copy number status were compared with response, overall-survival (OS), and progression-free-survival (PFS).

Results:
Median age was 62-yo (r, 20-87 years), 53 patients were females (73%), and 89% have common mutations. Twenty-two (30.6%) samples with EGFR activating mutations were identified with EGFR amplification. EGFR amplification was more frequent in patients with exon 19 deletion (p=0.05) and in those with better performance status (p=0.01). Patients with EGFR gene amplification had a significantly longer PFS than those without [(25.2 months, 95%CI 22.0-38.5) vs. (12.4 months, 95%CI 5.3-19.5); p=0.002] as well as better OS [(EGFR amplified 37.8 months, 95%CI 30.9-44.7) vs. (EGFR non-amplified 27.1 months, 95%CI 12.8-41.3); p=0.009]. EGFR amplification significantly influenced the response to erlotinib (p=0.0001).

Conclusion:
EGFR amplification occurs in one third of patients with lung ADC harboring EGFR activating mutations, and could serve as an indicator for better response and survival from EGFR-TKI treatment.

Background:
The ability to detect tumour mutations from blood and other bodily fluids promises many sample access, convenience, and monitoring benefits. However, the extremely rare levels at which mutations are present in these fluids obliges the use of optimal extraction and detection methods. Here, the performance of two extraction, two droplet PCR (dPCR) and a droplet next generation sequencing (dNGS) method for blood plasma analysis was systematically evaluated.

Methods:
Limits of detection were assessed using 15 blinded healthy donor blood samples spiked with equivolume mixtures of H1975 (containing EGFR L858R and T790M mutations) and H1650 (EGFR exon 19 deletion, e19del) cells at 10%, 1%, 0.1%, 0.01%, 0.001% H1650 cells in triplicate. A series of 32 blinded blood plasma samples from non-small cell lung cancer (NSCLC) patients with known tumour EGFR mutation status was also tested. Samples were processed for plasma, and 1ml plasma each underwent Qiagen Circulating Nucleic Acid and Promega Maxwell Circulation Cell Free DNA extraction processing. The plasma DNA samples were analysed using the Biorad dPCR and Raindance Raindrop dPCR method for L858R and exon 19 deletion mutations, and the 50-gene Raindance Thunderbolts dNGS protocol.

Results:
No significant difference in DNA yield and detection patterns was observed between the two extraction methods. L858R mutations were detected by both dPCR methods at 0.001% in 1/3 replicates and 0.01% in 3/3 replicates. Of 4 cases with L858R tumour mutations, mutations were detected in the same 3 plasma samples by both Biorad and Raindance dPCR (sensitivity 75%). “False positive” L858R mutations were identified in 2 (specificity 92%) and 7 (75%) cases respectively. For 8 tumour e19del mutations, the sensitivities were 38% and 25%, and specificities were 96% and 75% respectively. Of 16 clinical samples analysed by dNGS, an average of 20 mutations per sample were identified after filtering for quality, non-synomyous, and non-germline variant status. The sensitivity and specificity for detecting 2 L858R tumour mutation was 100% and 50%, and 1 e19del tumour mutation was 100% and 55% respectively. The allele frequencies for the majority of “false positives” for dPCR and dNGS were less than 5%, although some “true positives” were also detected at that level.

Conclusion:
dPCR and dNGS methods can enable detection of tumour mutations in blood, albeit imperfectly. Future work to determine optimal detection thresholds will help to maximize sensitivity and specificity.

Background:
Achaete-scute homolog 1 (ASCL1) is a neuroendocrine transcription factor expressed in 10-20 % of lung adenocarcinomas (AD) with neuroendocrine (NE) differentiation. Previously, we demonstrated that ASCL1 functions as an upstream regulator of the RET oncogene in AD with high ASCL1 expression (A[+]AD). In this study, we examined the potential role of wild type RET in influencing the oncogenic properties of A[+]AD. We also screened for drugs that could selectively target RET signaling and examined the role of the two RET isoform separately.

Methods:
The association of the mRNA expression for the long (RET51) and short (RET9) RET isoforms with overall survival (OS) were assessed in a case-control study of stage-1 A[+]AD patients surgically resected at the Mayo Clinic (1994-2007). Cases and controls were defined as patients who survived < 3.5 years after surgery (n= 29) and > 5 years after surgery (n=38), respectively. mRNA was isolated from FFPE tissue and analyzed by a nanostring assay. Associations of each isoform mRNA with the OS was determined by the area under the receiver operative characteristics (AUC). For drug screening, HCC1833 lung AD cells with endogenously high expression of ASCL1 were stably transfected with either empty vector or an ASCL1-shRNA. Differential sensitivities of tyrosine kinase inhibitors (TKIs) in the pair of syngeneic cell lines were measured by Cell-Titer Glo (Promega). Interactions between EGFR and RET was examined by co-immunoprecipitation.

Results:
Expression of RET51 mRNA was associated with poor OS (p=0.005, AUC 0.71). We detected modestly increased sensitivity to sunitinib and vandetanib in A[+]AD compared with A[-]AD cells. However, the EGFR inhibitors gefitinib and the dual EGFR and HER2 inhibitor lapatinib resulted in ≥ 10 fold higher cytotoxicity in A[+]AD cells than in A[-]AD cells. Subsequent experiments demonstrated that EGF stimulation of EGFR mediates the phosphorylation of RET in multiple A[+]AD cells. RET and EGFR were found to interact only in presence of EGF and predominantly through the long RET isoform (RET51).

Conclusion:
Herein we demonstrate that wild type EGFR predominantly interacts with the long isoform of RET (RET51) in A[+]AD cells. In the presence of EGF this results in activation of RET. High RET51 is associated with worse OS. Furthermore, compared to A[-]AD cells, A[+]AD cells appear to be more sensitivity to EGFR inhibitors. In summary, our results suggest that A[+]AD patients may benefit from treatment with EGFR inhibitors even in the absence of an EGFR mutation.

Background:
For the detection of genomic driver alterations in NSCLC, comprehensive genomic profiling(CGP) or focused molecular testing of biopsied tissue is a well-accepted approach for matching targeted therapies in first line treatment. For NSCLC patients where invasive biopsy represents a serious risk, assessment of circulating tumor DNA(ctDNA) is an emerging alternative.

Methods:
In patients with clinically advanced NSCLC, two 10 mL aliquots of peripheral, whole blood were collected and plasma was isolated. ctDNA was extracted to create adapted sequencing libraries prior to hybrid capture and sample-multiplexed sequencing on an Illumina HSQ2500 to >5000x unique coverage. Results were analyzed with a proprietary pipeline to call substitutions, indels, rearrangements and copy number amplifications.

Results:
In 288 NSCLC patients evaluated, 20 (6.9%) harbored kinase fusions. 17/20 (85%) were adenocarcinomas, all stage IV. Median patient age was 61 years (range 41-81), and 59% were female. 13 (4.5%) cases harbored ALK fusions with partners as follows: nine EML4 (one each of novel partners PPFIBP1 and CACNB4), and two with unidentified partners. All but one case had breakpoints in ALK intron 19, the remaining harboring a novel intron 17 breakpoint. Three cases (1%) harbored KIF5B-RET (canonical breakpoint intron 12), three (1%) had CD74-ROS1 (breakpoints: ROS1 intron 33(2) and intron 32(1)), and one had FGFR3-TACC3. ALK, RET, and ROS1 fusions were observed by tissue testing of NSCLC in the FoundationCore database with similar frequencies. Five patients had a biopsy with insufficient tissue for CGP; three had both sufficient tissue and ctDNA available. The remainder had no tissue available. For one patient, EML4-ALK fusion was detected in both ctDNA and tissue, collected six days apart. For another, CGP identified EGFR L858R + EGFR L709K and the patient had a durable response to afatinib/cetuximab. After progression, ctDNA assay identified FGFR-TACC3 as well as EGFR L858R. For a pre-menopausal, therapy naïve never smoker, female of east Asian heritage, both assays detected a CD74-ROS1 fusion, whereas ROS1 rearrangement was not identified by the prior use of another commercially available ctDNA test. The patient had a major radiographic response by the second cycle of crizotinib treatment.

Conclusion:
Hybrid capture based ctDNA assay can identify kinase fusions in NSCLC when CGP of biopsied tissue cannot be performed and can direct rational use of first line TKIs. This series identified a novel mechanism of acquired resistance to EGFR inhibitors, novel fusion partners and intronic breakpoints for ALK, and a case of false negative testing by another ctDNA assay.

Background:
Transcriptional profiling of lung adenocarcinomas has identified numerous gene expression phenotype (GEP) and risk prediction (RP) signatures associated with patient outcome. However, classification agreement between signatures, underlying transcriptional programs, and independent signature validation are less studied.

Methods:
Published transcriptional profiles from 17 cohorts comprising 2395 lung adenocarcinomas with available patient outcome data were collected from authors’ websites or public repositories as described in the original studies. Tumors were classified according to 18 different GEPs or RPs derived from microarray analysis of lung adenocarcinoma or NSCLC cohorts, using reported original classification schemes or consensus clustering of gene signatures on a per cohort basis. To independently assess the connection between classifications by the lung cancer derived signatures and classification by expression of proliferation-related genes only, a 155-gene breast cancer proliferation signature was used to classify all tumors as low-proliferative (average expression of the 155-genes < median) or high-proliferative. Tumors were also scored according to five reported expression metagenes in lung cancer representing different biological processes; proliferation, immune response, basal / squamous, stroma / extra cellular matrix (ECM), and expression of Napsin A / surfactants on a per cohort basis to contrast subtype classifications with biological functions.

Results:
16 out of 18 signatures were associated with patient survival in the total cohort and in multiple individual cohorts. For significant signatures, total cohort hazard ratios were ~2 in univariate analyses (mean=1.95, range=1.4-2.6). Signatures derived in adenocarcinoma generally displayed better classification agreement than signatures derived in mixed NSCLC cohorts. Strong classification agreement between signatures was observed, especially for predicted low-risk patients by adenocarcinoma-derived signatures, despite a generally low gene overlap. Expression of proliferation-related genes correlated strongly with GEP subtype classifications and RP scores, driving the gene signature association with prognosis. A three-group consensus definition of samples across 10 GEP classifiers demonstrated aggregation of samples with specific smoking patterns, gender, and EGFR/KRAS mutations, while survival differences were only significant when patients were divided into low- or high-risk resembling a terminal respiratory (TRU) like and non-TRU division, respectively.

Conclusion:
By providing this consensus of current GEPs and RPs in lung adenocarcinoma we have connected molecular phenotypes, risk predictions, patient outcome and underlying transcriptional programs of the different classifier types. Our results provide a general insight into the nature and agreement of GEP and RP signatures in the disease, and their prognostic value.

Background:
Non-small cell lung cancer (NSCLC) is a heterogeneous disease, with a wide diversity when it comes to molecular variations. In the non-squamous subset a large variety of altered driver genes have been identified.

Methods:
The mutational status was evaluated in a consecutive Swedish NSCLC cohort consisting of 354 patients, who underwent surgical resection between 2006 and 2010. DNA was prepared from either fresh frozen or formalin fixed paraffin embedded tissue (FFPE) and used for library preparation using a Haloplex gene panel and subsequently sequenced on an Illumina Hiseq instrument. The gene panel covers all exons of 82 genes, previously identified in NSCLC. The panel design utilizes two strand capture and reduced target fragment length compatible with degraded FFPE samples (Moens et al., J Mol Diagn, 2015).

Results:
All previously known hotspot alterations in the driver genes KRAS, EGFR, HER2 (exon 20 insertions), NRAS, BRAF, MET (exon 14-skipping) and PIK3CA (exon 9 and 20) were analyzed in the 252 non-squamous cases, see figure. KRAS mutations were found in 98 patients (39%) whereas EGFR alterations were present in 33 (13%). The prevalence of KRAS mutations is higher than normally reported and could be due to the large fraction of smokers included in this cohort. The EGFR prevalence is a bit higher than previously demonstrated (Sandelin et al. Anitcancer Res, 2015). Mutations in the other driver genes were detected at low frequencies (HER2(3%), BRAF(2%), NRAS(1%), MET(1%) and PIK3CA(1%)). Figure 1



Conclusion:
The preliminary analysis of mutational status in this large unselected Swedish NSCLC cohort reveals mutation frequencies in the common driver genes resembling previous reports on western populations with a high smoking rate. Ongoing analysis of the remaining genes will be used for pathway analysis and could provide a more complete picture of the lung cancer pathogenesis.

Background:
Targeting the MAPK pathway by MEK inhibition results in limited activity in patients with KRAS-mutant non-small cell lung cancer (NSCLC). The lack of effectiveness may be associated with activation of other effectors including STAT3, as well as MEK inhibition relief from negative feedback loops. Indeed, in KRAS-mutant colorectal cancer, MEK inhibition decreases the activity of the metalloprotease ADAM17, which normally inhibits MET signaling and STAT3 activation by promoting shedding of MET endogenous antagonist, soluble "decoy" MET. Herein, we explore the MET-dependent activation of STAT3 as a mediator of resistance to MEK inhibitors, and whether MET or STAT3 inhibitors can synergistically increase MEK-inhibitor-induced growth inhibition in KRAS-mutant NSCLC cells in vitro.

Methods:
Cell viability was assessed by MTT (thiazolyl blue) assay after treatment with the allosteric MEK inhibitor, selumetinib, the small-molecule dual inhibitor of the MET and ALK receptor tyrosine kinases, crizotinib, and evodiamine, an alkaloid isolated from the dried, unripe Evodia rutaecarpa (Juss.) Benth fruit, that exerts an anticancer effect by inhibiting STAT3. RNA was isolated from four KRAS cell lines and the STAT3 and MET mRNA expression analysis was performed by TaqMan based qRT-PCR. Western blotting was used to assess the effect of selumetinb on ERK, AKT and STAT3 phosphorylation.

Results:
We first evaluated the efficacy of the MEK inhibitor selumetinib in our KRAS-mutant NSCLC cell line panel using an MTT cell proliferation assay. H460 cells were relatively insensitive to selumetinib. Following 48-hour treatment with selumetinib, ERK1/2 and AKT phosphorylation were suppressed but a rebound activation of STAT3 occurred in H460 cells. We next investigated whether MET expression was related to the feedback activation of STAT3 signaling following MEK inhibitor treatment. We compared gene expression profiles of the H460 cell line before and after treatment with selumetinib. Interestingly, we found significant upregulation of MET and STAT3 mRNA expression after seven days of selumetinib treatment. To further interrogate the relationship between MEK inhibition and MET-mediated STAT3 reactivation, H460 cells were treated with the combination of selumetinib and crizotinib or selumetinib and evodiamine. A 72-hour exposure to both combinations resulted in a clear cell synergism, as measured by the combination index (CI) analysis, with a CI of 0.79 and 0.78 respectively.

Conclusion:
Collectively our results showed that the feedback STAT3 activation induced by MET, mitigates the effect of MEK inhibition, and provides rationale for further assessment of combined MEK and MET or STAT3 inhibition in KRAS-mutant NSCLC.

Background:
PIM kinases are a family of three serine/threonine kinases: PIM1, PIM2 and PIM3 that have been shown to play a role in tumorigenesis. PIM1 is a downstream effector of oncoproteins ABL and JAK/STAT and regulator of BCL2/BAD and CXCR4. PIM activity is synergistic with the PI3K/Akt/mTOR pro-survival pathway and PIM2 has been shown to phosphorylate translational repressor 4E-BP1 and p70S6 independently of the PI3K pathway. Furthermore a synergism between PIM kinases and c-Myc has been reported. Here we investigate the expression of PIM1/PIM2/PIM3 in NSCLC cell lines and patient matched normal/tissue samples. The effect of a novel combined inhibitor of PI3K/mTOR/PIM kinases (IBL-301) on cell signalling, cell death and proliferation is also examined.

Methods:
PIM1/PIM2/PIM3 expression were examined by Western blot analyses in NSCLC cells (H1975 and H1838). Additionally, the frequencies of PIM1/PIM2/PIM3 expression in NSCLC patient tumour and matched normal adjacent samples (n=31) were investigated. The effectiveness of IBL-301 on cell signalling, cell viability and proliferation were examined by Western blot analysis, cell titre blue and BrdU assay respectively.

Results:
All three PIM isoforms were detected in the lung cancer cell lines tested. Similarly, all three PIM isoforms were expressed across the 31 NSCLC patient tumour and match normal adjacent tissue samples. To investigate this further PIM1 staining of FFPE tumour and match normal tissue from this cohort is currently underway. In two lung cancer cell lines, H1975 and H1838, IBL-301 was found to have a dose dependent effect on proliferation/viability with IC~50~ values in the nanomolar range. Additionally, western blot analyses have indicated that these novel drugs can suppress the phosphorylation of key players in cell signalling pathways linked to tumorigenesis including pAkt, p4E-BP1 and peIF4B.

Conclusion:
This is the first study to investigate the expression of all 3 isoforms of PIM in lung cancer specifically. All 3 isoforms were abundantly expressed across cells lines and patient tumour samples. Observed PIM expression in the immune cells of normal adjacent tissue may indicate a role in inflammation. This finding coupled with the promising in vitro data demonstrate the therapeutic potential of targeting PIM in NSCLC.

Background:
Histopathological diagnosis is important for prognostication and choice of treatment in patients with cancer in the lung. Lung metastases are common and need to be distinguished from primary lung cancer. Furthermore, cases with synchronous or metachronous primary lung cancers (although infrequent) are often handled differently than cases with lung cancer with intrapulmonary metastasis or relapse, respectively. In some cases, morphology and immunohistochemical (IHC) staining is not sufficient for certain diagnosis.

Methods:
Five cases were selected where molecular genetic analysis in form of pyrosequencing or targeted next-generation sequencing (NGS) was of value, not only for treatment prediction, but for certain diagnosis of tumours in the lung.

Results:
Two of the included cases were rare metastases to the lung – rectal cancer with IHC profile consistent with primary lung cancer and malignant myoepithelioma of the breast, respectively – where molecular genetic analysis was of aid for proving the relationship with the primary tumour. The other three cases had multiple lung adenocarcinomas with similar morphology where molecular genetic analysis was of aid to distinguish between an intrapulmonary metastasis and a synchronous primary tumour.

Conclusion:
Comparison of molecular genetic profile may be an important tool for determination of relationship between tumours, at least in some situations, and should always be considered in unclear cases. Further studies on concordance and discordance of molecular genetic profiles between spatially or temporally different tumours with common origin may be helpful for improved diagnostics of pulmonary tumours.

Background:
Repeated biopsy in lung cancer may be necessary at diagnosis or after cancer progression on initial therapy to properly target treatments. The objective of this study is to evaluate the feasibility and safety of repeated biopsy for immunohistochemical and/or mutational analysis in patients with non small cell lung cancer.

Methods:
We have retrospectively analyzed repeated biopsies performed in patients with advanced non small cell lung cancer during the last 4 years. The technical success rates for the repeated biopsy and the adequacy rates of specimens were evaluated. Biopsy-related complications were recorded. Clinical details were collected, specially focusing in EGFR mutation data.

Results:
110 repeated biopsies were performed in 74 patients (34 women, 40 men, mean age 63 [36-84]), the histology was: 74% adenocarcinoma, 12% squamous cell carcinoma, 11% NOS, 3% other. The mean number of repeated biopsies per patient was 1 (1-4). The main reasons for repeated biopsy were immunohistochemical +/- mutational analysis (34/110, 31%), mutational analysis (16/110, 14.6%) and EGFR at progression (28/110, 24.4%). The technical success rate for biopsy was 98/110 (89.1%), and postprocedural complications occurred in 3/110 cases: 2 pneumothorax and 1 wound infection. Biopsy specimens came mostly from primary tumor (56/110, 51%), lymph nodes (26/110, 23.6%) and pleura (9/110, 8.2%), the most used technique was bronchoscopy +/- EBUS (45/110, 40%), followed by percutaneous transthoracic lung biopsy (28/110, 26%) and thoracoscopy and/or mediastinoscopy: (19/110, 17%). Results from repeated biopsy were used to select the next line of treatment in 86/110 procedures (78%), and 40/86 of them (46.5%) allowed to include the patient in a clinical trial. 28 repeated biopsies were performed in 21 EGFR mutant lung cancer patients with acquired resistance at disease progression, T790M was detected in 13/28 (46%) of samples, corresponding to 10/21 (47.6%) EGFR mutant lung cancer patients.

Conclusion:
Our data demonstrate that repeated biopsy in non small cell lung cancer is safe and clinically feasible. Findings from repeated biopsy were used to direct subsequent treatment in 78% of patients.

Background:
Epithelial-mesenchymal transition (EMT) and p53 mutations are known to be pivotal for driving metastasis and recurrence in lung cancer, but the nature of these factors is not completely understood. Some papers have previously described the relationship between EMT and TP53 in other carcinomas, however there have been few reports about the impact of TP53 on EMT and prognosis in lung adenocarcinoma harboring driver mutations such as EGFR or K-ras.

Methods:
The aim of the present study is to clarify the impact of TP53 overexpression in lung adenocarcinoma with driver mutations. A total of 282 lung adenocarcinoma specimens were collected from patients who had undergone surgery in our institute from January 2001 to December 2007. Both EMT markers (E-cadherin, vimentin) and TP53 were analyzed through immunostaining of tumor specimens. The association between EMT and TP53 as well as the patients’ clinical information was integrated and statistically analyzed. EGFR and K-ras mutation were determined by single stranded conformational polymorphism and direct sequencing. Correlations were compared using Pearson's chi-square test and overall survival were compared using the log-rank test.

Results:
Both mesenchymal type (E-cadherin negative, vimentin positive) and TP53 overexpression were significantly correlated with poor prognosis (P=0.0001, P=0.0019). A positive correlation was found between EMT activation level and TP53 overexpression (P=0.017). TP53 overexpression was significantly correlated with poor prognosis in the subgroup of lung adenocarcinoma with driver mutation (EGFR or K-ras) (P=0.011, P=0.026), whereas there was no significant correlation between TP53 overexpression and the prognosis in adenocarcinoma without driver mutations (P=0.359).

Conclusion:
TP53 overexpression is supposed to be the key factor that affects EMT and the prognosis, and also might be an additional therapeutic target for lung adenocarcinoma with driver mutations.

Background:
Young patients diagnosed as non-small cell lung cancer (NSCLC) is rare. Little is known for its genomic alterations and survival. This study retrospectively evaluated the genomic alterations,treatment and prognosis with NSCLC in our institution between January 2009 and July 2014 .

Methods:
All patients were examined for EGFR, KRAS, NRAS, PIK3CA, BRAF, HER2 mutations and ALK, ROS1, RET fusion genes based on reverse transcription PCR. The Kaplan-Meier method was used to estimate survival and comparison using the log-rank test.

Results:
Totally, 54 were with age under 40 years old among 640 patients. Among the 640 patients, three hundred and fifty eight patients were with identified genomic alterations with frequency of 55.9 %. The frequencies of genomic alterations in younger and older were 68.5% and 54.8%,respectively (P=0.05). The frequencies difference between younger and older existed in fusions genes ( 22.2% vs.4.1%,P<0.001), but not mutations genes (46.3% vs.45.6%,P=0.92). There was a trend of shorter recurrence free survival in younger than older (35.2 vs.43.8 months,P=0.050), while no survival difference was found between younger and older (50.2 vs 51.4 months,P=0.112).

Conclusion:
We concluded that younger age of NSCLC is associated with a trend of increased of harboring targeted genes and mainly with difference of fusion genes . An inferior overall survival existed in younger than older.

Background:
Based upon the 2015 Lung Cancer Pathologic Classification, squamous cell carcinoma of lung (SQCC) has been classified as three types of keratinized, non-keratinized and basaloid squamous cell carcinoma (BSCC). The spectrum of common driver genes and clinical prognosis were examined for different subtypes in SQCC in present study.

Methods:
From 2009 to 2013, a total of 201 patients with completely resected stages I-ⅢA SQCC were recruited. Reverse transcription-polymerase chain reaction (RT-PCR) was utilized for detecting the mutations of EGFR, KRAS, NRAS, PIK3CA, BRAF, DDR2, HER2 and the fusion genes of ALK, ROS1 and RET. Survival curves were plotted with Kaplan-Meier method. Cox’s proportional hazard model was used for multivariate analysis.

Results:
The pathological types were BSCC (n=16), non-keratinizing (n=83) and keratinizing (n=102). The overall frequency of gene abnormality was 18.4%. The most common driver genes in a decreasing frequency were PIK3CA mutation (n=14), EGFR mutation (n=8), DDR2 mutation (n=8), KRAS mutation (n=3), HER2 mutation (n=2), ALK rearrangement (n=1) and ROS1 rearrangement (n=1). No mutations of NRAS, BRAF or RET were observed . The frequency of gene abnormality was greater in keratinized (19.6%) ,followed with non-keratinized (19.2%) and BSCC types (6.3%). Targeted therapy was offered for 35 patients, including 32 on EGFR-TKIs (EGFR mutation, n=5; EGFR wild-type, n=27), ALK-positive on crizotinib (n=1) and HER2 mutation on afatinib (n=1). The median progression-free survival (PFS) of EGFR-TKIs were 6.0 and 1.87 months in EGFR mutant and wild types respectively (P=0.004). And the PFS for those two with crizotinib and aftatinib treatment were 8.0 and 3.5 months respectively. . The SQCC patients of BSCC subtype had significantly worse overall survival than those with non-BSCC subtypes (29.0 vs.47.0 months, P<0.001).

Conclusion:
The subtypes of SQCC were associated with varying frequency of gene abnormality. BSCC had a lower frequency of common driver gene abnormality and worse survival.

Background:
Recent advantage of next-generation sequencing (NGS) in the field of cancer genome research has revealed extreme levels of genetic heterogeneity, suggesting the major roles of subclonal mutations in cancer relapse and in rapid emergence of acquired resistance. Unlike inherited mutations, somatic variants often occur at low allele frequencies that require sensitive methods for detection. Based on the results using another highly sensitive method, such as digital PCR, the study of cancer subclones requires to detect mutations that are present in <1% frequency, however, such a level of resolution cannot be obtained by conventional NGS approaches. In the current study, we performed molecular-barcode sequencing for primary lung adenocarcinoma cases in order to analyze mutations with <1% low frequency.

Methods:
Fresh frozen tumor samples from 58 primary lung adenocarcinoma patients whose tumors previously tested positive for EGFR by conventional method (the PNA-LNA PCR clamp method) were collected. All samples were obtained from surgically resected specimen. We used HaloPlex HS method, which is a high sensitivity amplicon-based targeted sequencing method incorporating molecular barcodes in the DNA library, allowing for the identification of duplicate reads to significantly improve the base calling accuracy even at low allelic frequencies compared to conventional NGS methods. We used a panel designed for 47 cancer-related genes including EGFR, and sequenced data was obtained by using Illumina Miseq Reagent v3 (600 Cycle). Normal tissue samples were also sequenced for threshold adjustment.

Results:
Out of 58 samples, EGFR ‘major’ mutation (L858R, Exon19 deletion, G719A/S, T790M) profiles of 57 samples by molecular-barcode sequencing corresponded to those by clinical method (98.3%). The mean coverage of EGFR major mutation was 3078 (243-8285), and the minimal detectable frequency was 0.45%. Of note, we could detect the minor frequency of T790M mutation in addition to the major frequency of L858R mutation, at the allele frequency of 1.92% (20/1042) for T790M and 10.62% (155/1459) for L858R mutation, respectively. Minor genetic alterations, except major mutation, in EGFR were detected in 82.8% (48/58) cases, and the mean number was 2.3 (0-11). These results suggest the clinical applicability of this method.

Conclusion:
We could demonstrate good concordance rate between clinical examination and molecular barcode sequencing. Minor genetic alterations in EGFR were detected in 82.8% (48/58) cases. Further investigations are warranted to establish the confident detection of subclonal mutations with low frequency.

Background:
Therapeutic approaches to lung cancer have shifted toward an emphasis on molecularly targeted therapy in genotypic subsets of patients (pts). Recent advances in next-generation sequencing (NGS) technologies have improved the ability to detect potentially targetable mutations. In this study, we aimed to analysis the genomic alterations of lung adenocarcinoma.

Methods:
A retrospective analysis of genomic data obtained from 99 archived formalin-fixed, paraffin-embedded samples from Japanese pts with lung adenocarcinoma were analyzed by NGS panel of 415 genes. Mutations and frequency of variants present in key oncogenic drivers for each pts were quantified. Fisher’s exact and t-tests were used to identify associations between genomic alterations and clinical characteristics.

Results:
Sex: male 66 pts, female 33 pts. Smoking status: pack-year (PY) <30 (light smoker) 61 pts, PY≥30 (heavy smoker) 38 pts. Of all, the median number of mutation burden and actionable mutation were 13.5 (range 5-33) and 4 (range 1-19), respectively. The most frequent genomic alterations were EGFR (48%), TP53 (40%), CDKN2B (32%), RB1 (21%), and CDKN1B (19%). Representative genomic alterations with a FDA approved targeted therapy such as EGFR mutation (exon 19 deletion and exon 21 L858R), ALK fusion, ROS1 fusion, RET fusion, BRAF (V600E) mutation and, MET amplification were detected in 56 pts (R-GAs group) and the others were 43 pts (O-GAs group). In the O-GAs group, 39 pts had genomic alteration with targeted agent available on or off a clinical trial. As for smoking habit, R-GAs group were significantly associated with light smoker than O-GAs group (p<0.01). In R-GAs group, the median number of mutation burden and actionable mutation were 13 (range 5-20) and 4 (range 1-10), respectively. O-GAs group were significantly greater mutation burden and actionable mutation than R-GAs group (16.2 ± 6.8 vs. 12.3 ± 4.3, p<0.01; 6.5 ± 5.0 vs. 4.5 ± 2.2, p = 0.02, respectively). Of the 43 EGFR pts, there were no concurrent genomic alterations of ALK, ROS1, RET, BRAF and, MET. The most frequent concurrent mutations in EGFR were CDKN2B (37%), TP53 (28%), CDKN2A (23%), CDKN1B (21%), ARID1A (19%), RB1 (16%), and STK11 (16%). Among the EGFR cases, 38 (88%) pts had further genomic alteration with targeted agent available on or off a clinical trial excluding EGFR-TKI.

Conclusion:
With help of NGS, we found most pts might be treated by targeted therapies. Further study may emerge whether concurrent mutations, mutation burden and the number of actionable mutation are associated with survival outcome in lung adenocarcinoma.

Background:
Approximately 15 to 25% of lung adenocarcinomas (LAC) arise in never smokers. They develop through mechanisms distinct from those that affect smokers and are associated with unique histological and molecular characteristics. A significant fraction of LACs in never smokers do not have mutations in known oncogenic driver genes such as EGFR/ALK/KRAS. Furthermore, mutations in oncogenic driver genes appear to be insufficient for tumorigenesis, suggesting that additional alterations are required.

Methods:
To address these issues, we used whole-exome sequencing to comprehensively study 15 LACs from never smokers - seven “triple negative” tumors (with normal EGFR/ALK/KRAS) and eight EGFR-mutant tumors - with the goal of identifying novel mutant genes in these subsets. To identify mutated genes that confer a selective advantage a multistep approach was used to filter variants based on gene expression level, background mutation rate and gene size. Targeted sequencing of 180 genes in the original 15 and an extended panel of 85 tumor/normal pairs validated these alterations and indicated their prevalence in LAC. Sequence data was integrated with copy number and gene expression levels to determine mechanisms, and consequences, of gene disruption. Animal and cell models were used to functionally validate identified genes of interest and explore their role in LAC biology.

Results:
32 unique genes demonstrated significant evidence of conferring a selective advantage including known oncogenes (EGFR/ERBB2/MET) and tumor suppressor genes (p53/RB1/ATM). In addition, RNA-seq revealed fusions involving RET or ROS1 in one tumour each. The variations in MET consisted of truncating and splice-site mutations that we are currently investigating in transgenic mouse models. Pathway analysis indicated frequent mutation in genes implicated in PI3-kinase signaling, RNA splicing and histone modification. Importantly, we identified the hemizygous and homozygous loss of multiple genes from chromosome arm 6q - a genetic locus associated with familial lung cancer susceptibility - including a novel candidate tumor suppressor gene, SHPRH, based on its high frequency of biallelic disruption. SHPRH is an evolutionarily conserved E3-ligase that mediates crucial processes related to DNA repair. We found that SHPRH silencing increased transformation of normal lung cells, increased DNA damage and induced cell cycle changes while SHPRH inhibition sensitized LAC cells to topoisomerase II and PARP inhibitors.

Conclusion:
SHPRH inactivation may induce genetic alterations that cooperate with mutations in driver oncogenes to promote LAC development. Together, this work will expand our understanding of LAC initiation and progression in never smokers and may offer new biomarkers for response to therapy.

Background:
Invasive lung adenocarcinomas have been classified into 5 subtypes by the new WHO classification, of which the micropapillary-predominant subtype is known to have a poor prognosis. However, we previously reported that lung adenocarcinomas harboring a micropapillary component (MPC) of more than 5% showed poorer disease-free survival and overall survival in comparison to the other subtypes (JTCVS 152: 64-72; 2016), despite the MPC not being predominant. Specific biomarkers for the MPC are yet to be developed for the initial diagnosis of adenocarcinoma with an MPC.

Methods:
Total RNA was extracted from snap frozen archived lung adenocarcinoma samples that had been obtained by radical thoracotomy. The diagnosis of each subtype was made by independent pathologists using formalin-fixed paraffin-embedded samples. Frozen sections were made using the archived frozen sample; RNA was isolated after the confirmation of the diagnosis of each sample. RNA-sequencing was conducted using 22 adenocarcinomas and 3 normal lung tissue samples. The 22 adenocarcinomas included micropapillary (n=6), papillary (n=5), lepidic (n=5), acinar (n=3) and solid (n=3) subtypes. The reads per kilo base of exon per million mapped reads data derived from RNA-sequencing were calculated to analyze the gene expression profiles of the MPCs. The expression levels of the genes were validated by a real-time PCR.

Results:
A hierarchical clustering analysis revealed a cluster of tumor samples with MPCs. Two hundred four genes were differentially expressed in adenocarcinomas that contain an MPC. A gene-ontology analysis showed the enrichment of signal-related genes, with 50 significantly upregulated genes, including BAMBI, CXCL14 and VSIG1.

Conclusion:
The results of a gene expression analysis using next generation sequencing revealed the specific gene expression profile of adenocarcinomas that contain an MPC, and several genes might be candidate diagnostic biomarkers for the subtype. In addition, these genes may play an important role in the unique characteristics of adenocarcinomas that contain an MPC.

Background:
Pulmonary large cell carcinoma (LCC) was an undifferentiated and marker-null non-small cell lung cancer (NSCLC) according to 2015 WHO classification criteria. However, there are few studies presented molecular analysis and clinical features of this subtype. The aim of this study is to investigate profile of driver mutations and clinicopathological features of marker-null LCC.

Methods:
From January 2008 to February 2015, 327 cases of surgically resected primary large cell carcinoma diagnosed by H&E staining were enrolled from Shanghai pulmonary hospital affiliated to Tongji university (Lymphoepithelioma-like carcinoma and large cell neuroendocrine carcinoma were excluded with typical morphologic features). Large cell carcinoma was reclassificated with a panel of immunophenotypic markers (adenocarcinoma [ADC]-specific, thyroid transcription factor-1, napsin A and anti-diastase PAS staining; squamous cell carcinoma [SQCC]-specific, cytokeratin5/6, and p40; epithelial mesenchymal transition [EMT], cytokeratin, vimentin and ZEB1; neuroendocrine differentiation, synaptophysin and CD56). Molecular analysis (EGFR, KRAS, BRAF, ROS1, ALK and PIK3CA) and immunomarker PD-L1 of marker-null LCC were detected by ARMS method and immunohistochemistry, respectively.

Results:
113 cases (113/327, 34.6%）were rediagnosed as ADC with solid growth pattern, which showed TTF-1 positive rate 59.3% (67/113, 59.3%) , Napsin A positive rate 82.3% (93/113，82.3%)( P<0.001)) and positive PAS staining (5/113, 4.4%) . 47 (47/327, 14.4%) cases were reclassificated as SQCC, which positively expressed p40 (44 cases, 44/47, 93.6% ) and CK5/6 (31 cases, 31/47, 66%)( P<0.01). 5 (5/327, 1.5%) cases of large cell neuroendcrine carcinoma showed both neuroendocrine morphological feature and CD56、Syn expression. 26 (26/327, 8.0%) were redefined as sarcomatoid carcinoma expressed cytokeratin, vimentin and ZEB1. 136 (136/327, 41.6%) cases of marker-null LCC were diagnosed for lack of specific markers expression. 30 (30/136, 22.1%) cases of marker-null LCC showed PD-L1 positive. Driver genes alteration were found in 3 cases from 30 cases marker-null LCC, including KRAS (n=1), BRAF (n=1) and PIK3CA (n=1). Clinicopathological characteristics indicated that LCC patients were older and more likely to be male nonsmokers.

Conclusion:
Large cell carcinoma was differentially diagnosed by combining with undifferentiated NSCLC morphology and marker-null features. Driver genes mutation testing may significantly impact on the choice of targeted therapy.

Background:
Tumor associated fibroblasts (TAFs) are drawing increasing attention as potential therapeutic targets owing to its direct implication in major steps of tumor progression in solid tumors including non-small cell lung cancer. Accordingly, there is growing interest in defining the aberrant molecular differences between normal and TAFs that support tumor progression. For this purpose, we recently conducted a genome-wide DNA methylation profiling of TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer patients, and reported a widespread hypomethylation concomitantly with a focal gain of DNA methylation indicative of a marked epigenetic reprogramming. Of note, the aberrant epigenome of lung TAFs had a global impact in gene expression and a selective impact on the TGF-β pathway, including the hypermethylation of SMAD3, which is an important transcription factor of the TGF-β pathway. However, the functional implications of the aberrant TGF-β pathway in lung TAFs remains undefined.

Methods:
Patient-derived TAFs and paired control fibroblasts from either adenocarcinoma (ADC) or squamous cell carcinoma (SCC) patients were stimulated with TGF-β1 and their responses were examined in terms of activation and contractility. Activation markers included expression of alpha-smooth muscle actin (α-SMA) and collagen-I, which were assessed by western-blotting and qRT-PCR, respectively. The contractility of single fibroblasts was assessed by traction force microscopy.

Results:
We found a larger expression of activation markers including α-SMA and collagen-I in TAFs compared to control fibroblasts. Likewise, TGF-β1 elicited a larger contractility in TAFs than in control fibroblasts as assessed by traction force microscopy. Of note, these results were consistent with previous observations reported on skin fibroblasts from Smad3 knock-out mice.

Conclusion:
Our findings reveal that lung TAFs are hyperresponsive to TGF-β1, even though their expression of SMAD3 is epigenetically down-regulated. This aberrant response to TGF-β1 may underlie the expansion and/or maintenance of the tumor-promoting desmoplastic stroma in lung cancer.

Background:
Pulmonary sarcomatoid carcinomas (PSCs) are rare, poorly differentiated non-small cell lung cancers (NSCLCs) containing sarcoma or sarcoma-like features, with a worse prognosis than other NSCLCs. The World Health Organization (WHO) classifies three histopathologic subtypes: subtype-1 includes pleomorphic, spindle-cell, and giant-cell carcinomas (PSCGC), subtype-2 carcinosarcoma and subtype-3 blastoma. The value of different subtypes for clinical management and their molecular characteristics are unclear. The aim of this study is to get new insights into PSCs carcinogenesis by a high-throughput sequencing of RNA (RNA-seq).

Methods:
A whole-transcriptome targeted-gene quantification analysis was retrospectively performed on RNA from formalin-fixed paraffin-embedded tissues of 13 PSCs (5 pleomorphic, 2 spindle-cell, 2 giant-cell carcinomas, 4 carcinosarcomas). RNA-seq reads were mapped to the amplicon sequences of the panel and quantified by tools based on alignment algorithms. Differentially expressed genes between subtype-1and -2 were determined using a non-parametric Mann-Whitney U-test (p-value < 0.01) with linearity correction. Moreover, within subtype-1 we compared gene expression levels between monophasic (spindle- and giant-cell) and biphasic (pleomorphic) carcinomas.

Results:
216 genes resulted down-regulated and 15 up-regulated in PSCGC compared to carcinosarcomas (Table 1). There were not significant differences between monophasic and biphasic PSCGC. Figure 1



Conclusion:
PSCs are heterogeneous tumours, barely characterized from a molecular point of view. WHO has recently classified PSCGC and carcinosarcoma as two distinct entities, and our results demonstrated that they effectively have different gene expression profiles. Deregulated genes mostly belong to pathways crucial for cancer, like p53-, MAPK- and Wnt-signaling, and future investigation should clarify their specific role in PSCs. Interestingly, we did not find statistically deregulated genes among monophasic and biphasic carcinomas of subtype-1, thus indicating their molecular similarity. Although this is a preliminary and explorative study, needing further validation, it constitutes a starting point to increase our knowledge of these rare tumours.

Background:
The youthful lung cancer may constitute an entity with distinct clinicopathologic characteristics. The serine/threonine kinase LKB1, also known as Serine/Threonine Kinase 11-STK11, is a known tumor suppressor gene involved in cellular responses such as energy metabolism, cell polarity and cell growth, but the role of LKB1 pathway in lung adenocarcinoma (ADC) is barely studied, especially in young patients

Methods:
Fifty lung ADC patients were retrospectively analysed. After RNA purification from formalin fixed and paraffin embedded tumor tissues, we analysed the mRNA expression levels of LKB1 and of genes involved in its pathway, such as cyclin D1 (CCND1), beta catenin (CCNNB1), lysyl oxidase (LOX), yes-associated protein-1 (YAP-1), and survivin, with NanoString technology, a new tool for a more accurate expression profiling. KRAS mutations were investigated by pyrosequencing analysis. Clinicopathologic characteristics and survival analysis were available for all patients.

Results:
Patients under 50 years old (including 50) were defined as the younger group and patients above 50 years old were defined as the older group. Among all the clinicopathologic characteristics, in the younger group there were more women, less solid and more acinar adenocarcinoma prevalent pattern in comparison to the older group. Younger and older groups showed similar survival rates, as well as KRAS mutations frequencies. Also, in the comparison between the gene expression level of the analyzed genes and the two different age subgroups,no statistical difference was found. We then focused on the LKB1 pathway in all series, independently from the age stratification, founding LKB1 low expression associated with low cyclin D1 (CCND1) (p<0.0001), beta catenin (CCNNB1) (p<0.0001), and YAP1 (p=0.0024) levels, suggesting a target regulation by LKB1. We next tested the expression level of LOX, one of its downstream target, and we found that lung ADC with a high LOX mRNA expression showed a significantly worse prognosis, either in terms of disease-free interval or overall post-operative survival.

Conclusion:
Based on our preliminary results young patients seem to show similar LKB1 pathway expression levels to older group, even if further investigations will be necessary. Moreover, our data suggest LKB1 as a key pathway in lung ADC regardless of age, with a relevant sfavorable role of LOX. A robust assessment of LKB1 and of its downstream gene, LOX in particular, may elucidate the role of this pathway deregulation in lung adenocarcinoma in order to identify potential target for lung cancer therapy.

Background:
The aim of this study was to evaluate the expression of CPA4 a member of carboxypeptidase family and Rel B a member of nuclear transcription factor Kappa B family in imprints of resected NSCLC and correlate these expressions with DNA ploidy and classical prognostic factors.

Methods:
A total of 45 smears of patients who underwent surgical treatment for NSCLC were examined immunocytochemicaly for the expression of CPA4 and Rel B. Imprint smears also were stained using the Feulgen procedure in order to evaluate DNA ploidy in the same cases.

Results:
Thirty two (71,1%) of the tumors were classified as aneuploid. CPA4 positive expression was observed in 18 (40%) and Rel B in 21 (46,7%) of the tumors. We found a significant relationship between DNA ploidy and grade (p=0,005). CPA4 and Rel B expression correlated with grade (p<0,0001 for both) and also with nodal status (p=0,004 and p=0,017, respectively). Cox regression multivariable analysis demonstrated that CPA4 and Rel B expression were independent prognostic factors. The mean DNA index was higher for tumors with negative expression of CPA4 and Rel B (P=0,045 and p=0,025 respectively)

Conclusion:
DNA aneuploidy correlates with poor and moderately differentiated tumors. CPA4 and Rel B positive expression is in relation to well differentiated carcinomas and nodal status

Background:
Atypical adenomatous hyperplasia (AAH) is considered the precursor lesion of adenocarcinoma (ADC), and adenocarcinoma in-situ (AIS) the pre-invasive phase of invasive ADC. Finding incidental synchronous AIS/AAH within lung resection specimens of ADC is an opportunity to evaluate and compare the genomic profiles of the lesions. Different mutation profiles would suggest a field effect with the formation of an early second primary. Identical mutations might suggest a closer relationship with the primary tumor such as an early intrapulmonary metastasis from spread through air spaces. In this study we evaluate the genomic profiles of synchronous AIS/AAH lesions and separate primary ADC in the same lobe of resection specimens.

Methods:
We tested the 13 lesions from six patients identified between August 2015 and June 2016 by targeted next generation sequencing (NGS) using the 50 gene AmpliSeq Cancer Hotspot Panel v2 and FISH for ALK rearrangements. All of our patients had a single radiographically evident ADC and one patient had a second smaller lesion which was proven ADC at resection. All six patients had at least one additional incidental focus of AIS/AAH, not detected radiographically. Our patients ranged in age from 51-67. Five patients were female (83%) and all were current or former smokers.

Results:
All cases were successfully tested and somatic alterations were found in all ADC and three AAH/AIS lesions. None of the samples had an ALK rearrangement. Patient 3, with two ADC, had different profiles between all three lesions tested. In Patient 6, the ADC had an activating EGFR p.L858R mutation and the synchronous AAH lesion showed a KRAS p.G12D mutation.

Results

Patient	ADC site	ADC molecular	AIS/AAH site	AIS/AAH molecular
1	RUL, ADC	STK11 p.E342Q	RUL, AAH	WT
2	LUL, ADC	TP53 p.R158L	LUL, AIS	WT
3	LLL, ADC1 LLL, ADC2	KRAS p.G12V KRAS G12V; PIK3CA p.H1047L	LLL, AIS	TP53 p.H168Q; TP53 p.S94fs
4	LUL, ADC	TP53 p.V157F	LUL, AIS	ATM p.F858L
5	RUL, ADC	KRAS p.G12A; FLT3 p.Y599H	RUL, AIS	WT
6	LLL, ADC	EGFR p.L858R	LLL, AAH	KRAS p.G12D

RUL – right upper lobe; LUL/LLL – left upper/lower lobe; WT – wild-type

Conclusion:
All of the synchronous lesions, including the AAH/AIS lesions, showed different genomic profiles supporting the concept of field cancerization, suggesting that these incidental AAH/AIS foci likely represent de novo secondary tumors.

Background:
The genome wide sequence and microarray has performed flourishingly for major cancer specimen in recent day. However, thymic epithelial tumor(TET) was rare tumor comparatively, there are few reports about epigenetic alternation in TET. Cadherin-3, also known as P-cadherin, is a protein encoded by the CDH3 gene. This study was aimed at identifying the relationship DNA methylation and gene expression of CDH3 gene in TET, especially B3 thymoma and thymic cancer.

Methods:
A)DNA methylation 1) DNA were extracted fresh frozen samples of B3 thymoma and thymic cancer, diagnosed as squamous cell carcinoma. These samples collected from the patients, diagnosed as TET, underwent surgery at Tokushima university from 1990 to 2016. 2) DNA was treated by bisulfite conversion. 3) DNA methylation level was measured by infinium methylation assay(Human Methylation 450K DNA Analysis Kit ; Illumina) exhaustively. 7 cases of thymic cancer and 8 cases of B3 thymoma applied this assay. 4) The methylation levels of CpG sites were calculated as β-values (β = Intensity (methylated)/intensity (methylated + unmethylated) by applying default settings of the GenomeStudio Software's DNA methylation module (Illumina). Analysis of this methylation assay used by R package. B) Immunostaining Immunostaining was performed Envision method. It was used Anti-pan-Cadherin　antibody (ab16505）, dilution 1300 times as first antibody. DAKO Chemomate EnVsion kit was used as second antibody.

Results:
CDH3 had 2 CpG islands. 450K methylation assay set 5 CpG sites on first CpG island as promoter region. (CpG sites:B3 thymoma averageβ-values : thymic cancer averageβ-values : p-value by t-test)=(cg0900242 : 0.07±0.08 : 0.45±0.05 : 2.7×10[-7]), (cg07061260 : 0.21±0.21 : 0.55±0.09 : 3.3×10[-3]), (cg00748373 : 0.19±0.20 : 0.45±0.07 : 0.01), (cg06575065 : 0.28±0.18 : 0.50±0.07 : 0.02), (cg27417609 : 0.35±0.21 : 0.59±0.09 : 0.02). Immunostaining specimen were classified by scoring system measured by stain intensity and stain expanding. It was classified 2points;strong, 1point;weak, 0point;none, in stain intensity. It was classified 3points;diffuse(>80%), 2point;moderate(50-80%), 1point;focal(20-50%), 0point;none(<20%) in stain stain expanding. Expression rate(stain intensity point and stain expanding point) defined more than 4point as expression promotion. Expression promotion rate is 0% in B3 thymoma and 75% in thymic cancer.

Conclusion:
DNA methylation of CDH3 was increasing and P-cadherin protein expression was increasing in thymic cancer compared to B3 thymoma.