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Abstract not provided

Abstract:
Lung cancer remains the leading cause of cancer-related mortality in men and women. Since about 85% of all lung cancer care in the United States is provided in the community setting (1), it is imperative to optimize the delivery of accessible, high-quality lung cancer care in this environment. Implementation of a multidisciplinary clinic is not enough to strengthen physician care across large hospital systems. Many systems have an established weekly tumor board to review patient cases. These multidisciplinary conferences are a venue for involved specialties including thoracic surgery, medical oncology, pulmonary medicine, radiology, radiation oncology, and pathology to provide an opinion regarding the management of each presented case. This forum for open dialogue results in reinforced recommendations and streamlines patient care (2). It can be challenging for physicians in a community setting who are managing multiple tumor types to remain abreast of evolving information regarding each subgroup. Expanding multidisciplinary care beyond the case conference provides a more robust collaboration for physicians managing these patients. For physicians, journal club, continuing medical education programs, and standardized guidelines provide direction regarding the latest advances in diagnosis and management. Specifically in lung cancer, launching a screening program builds partnerships with radiologists pursuing early detection and expands relationships with other practitioners monitoring abnormal chest imaging. For patients, access to smoking cessation directives, chemotherapy teaching, palliative medicine, and survivorship programs enhances the care delivered in community lung cancer programs. A nurse navigator is an invaluable resource providing the patient and family support and education to improve the cancer experience. The navigator also serves as a liaison to ensure interdisciplinary coordination of cancer management. Clinical trials, genetic counselors, interpreters and geriatric oncologists should supplement well-integrated lung cancer networks. Even when all of these components are implemented into a community lung cancer program with the goal to provide the best care, if the core physician care is not strengthened as a part of this process, in a large hospital system, the program will fall short. Thus it is just not what should be executed but how. In large hospital systems, clinicians can become engaged through regional councils, designed to establish the aims of a cohesive lung cancer program and to create a model that will service these recommendations (3). Allowing system-wide participation in customizing the organizational structure of a lung cancer program will result in team development. If team is defined as “a group of people with complementary skills who are committed to a common purpose, performance goals, and approach, for which they hold themselves mutually accountable” (4), then it seems that strengthened care is inevitable. In a large health system, physician teams need to have shared goals and values, need to understand and recognize the competencies of other team members, and need to learn from other disciplines and respect their different views and perspectives. Individual team members may need to reassess exclusive claims to specialist knowledge and authority in order to form effective multidisciplinary teams which can provide the best care. By establishing a one-tiered system, physicians of various expertise find a comfortable niche that is not rewarded by self-promotion. Some physicians may maintain a more traditional generalized oncology clinic. Other physicians may adopt a more academic practice with subspecialty care, clinical trial participation, and literature publications. Collegiate collaborations between these two models strengthen physician care because the gap between private practice and academics is bridged within the same system. Because tumor boards provide multidisciplinary meetings but not necessarily multidisciplinary care, tumor boards may validate physician care but not necessarily strengthen it. A physical multidisciplinary clinic is not required to achieve this success. Instead, a solid team of engaged members focused on a specific disease will enrich the program. Routine meetings of a disease-specific section open to all interested provide a forum to review comprehensive needs for lung cancer patients. When a consistent message is issued from the group, physician care is strengthened. Telephone, video, and internet access to all disease-specific section meetings encourage participation. Communication facilitated by technology is the backbone to the success of this linked enterprise. The format of routine section meetings provides the venue to shift the “what” into the “how.” Announcements for lung cancer events are widely distributed. Consensus-driven standard algorithms of care are reviewed and updated. Finally, research options are reassessed. Integration of clinical trials in the community setting is necessary to strengthen care even in larger systems that may otherwise feel that care is already comprehensive. The best academic programs are often built on a reputation of offering research and clinical trial opportunities. Because the majority of patients with lung cancer are never seen in major academic centers, it is imperative that community programs become involved in clinical trials. Common protocol review (again maximizing participation with technology access) broadens interest. A centralized trials unit blends the team with appropriate system-wide delegation of resources. Utilization of a common internal review board allows for trials to open efficiently as well as simultaneously at multiple sites within the larger system. Local access obviates the need for travel, enhances program visibility, and provides ongoing relationships with the larger worldwide research community. Ultimately, a larger hospital system will benefit from restructuring the community. Physician care can be effectively strengthened not under the traditional hub-and-spoke model but instead as a cancer institute “without walls” (5). Regional councils, subspecialty sections, and multi-site clinical trial options under common review are all successful when system-wide participation is encouraged and when access is easily provided via advanced technology. Consider the large hospital organization as a system not a center. Aim to decentralize cancer care facilities by providing as many of the programs of a tertiary referral center throughout the region, limiting patient travel and lost time while still maintaining balanced quality (6). In order strengthen physician care in large hospital systems, growth measured as patient encounters is not as productive as reorganizing the care team. You can have “many” but still be “One.” 1. American College of Surgeons: Commission on cancer national cancer data base. Benchmark Reports v1.1 2. Fischel RJ, Dillman RO: Developing an effective lung cancer program in a community hospital setting. Clin Lung Cancer 10: 239-243, 2009 3. Dahele M, Ung Y, Meharchand J, et al: Integrating regional and community lung cancer services to improve patient care. Curr Oncol 14: 234-237, 2007 4. Carrier JM, Kendall I: Professionalism and interprofessionalism in health and community care; some theoretical issues. Interprofessional Issues in Community and Primary Health Care, 1995 5. Goldberg P: The Raghavan experiment. The Cancer Letter 39: 1-9, 2013 6. Raghavan D: Costs of cancer care: rhetoric, value, and steps forward. Semin in Oncol 40: 659-661, 2013

Abstract:
Minority populations often suffer disproportionately from lung cancer due to 1) lower levels of education 2) jobs with higher occupational hazards 3) housing in areas with higher environmental hazards and 4) economic disadvantage. Lower socioeconomic status leads to higher rates of uninsured or underinsured populations living in neighborhoods with less access to quality health care. The recently published “Neighborhood Deprivation Study of Lung Cancer”, demonstrated living in a social-economic depressed environment, defined as high rates of low education status (< 10 years formal education), low income (less than 50% of individual median income), high unemployment and high rates of social welfare assistance, leads to higher incidence and mortality from lung cancer[1]. The lung cancer mortality was 13 per 1000 in high deprivation neighborhoods vs 8 per 1000 (OR 1.6) in low deprivation neighborhoods. Minority communities also have more barriers to effective cancer care. Barriers include differences in culturally related health beliefs, (values and preferences that are not understood by health care providers and lead to decreased compliance with medical recommendations), language discordance, provider stereotypes that lead to health care disparities, limited clinic hours of service that do not account for community work patterns, etc. Health care disparities in lung cancer have been studied in treatment decisions for early-stage disease. Bach et al. studied the differences in survival of Medicare beneficiaries with stage I or II NSCLC based on race[2]. The five year survival of black patients was significantly less than white patients, 26 % vs. 34.1% (p < 0.001). The difference in survival could be accounted for by the lower number of black patients treated with surgery, 64.0 % vs. 76.7 % for white patients. Although the difference in treatment had an impact on survival, the authors could not determine if this was due to patient held health beliefs regarding surgery and/or black patients being offered surgical resection less often. A similar outcome was seen by Koshy et al. when examining the National Cancer Database for differences in radiation treatment modalities offered for early stage disease[3]. Socioeconomic factors, including insurance type and race/ ethnicity, were significant variables in determining if a patient received no radiation therapy, conventional radiation therapy or stereotactic body radiation therapy. Each of these barriers is important to study and overcome as efforts to improve the treatment of diverse patient populations will increase the lung cancer cure rate. Applying cultural competence to cancer care delivery will improve adherence to screening and prevention measures, improve compliance to medical treatment and necessary follow-up, and reduce health care disparities. Effectively treating diverse populations of lung cancer patients requires change on multiple levels within healthcare delivery. On an organizational level, the leadership and workforce must allow for greater minority representation, to remain connected to the communities they serve. For minority patients, racial concordance between patient and physician is associated with greater patient satisfaction and higher self-reported quality of care. Specific quality measures for diverse patient populations must be developed. For example, patient-reported health care quality surveys can be adapted to better evaluate culturally diverse populations. Ultimately, health care organizations benefit from establishing ongoing links for consultation with representatives from diverse communities. On a structural level, work-processes can be adapted to aid diverse patient populations. Often the intake process is difficult or cumbersome for minority patients to navigate. Lack of interpreter services or inappropriate health care education materials can limit the effectiveness of the clinic visit. Ngo-Metzger et al. studied the effects of language discordance between patient and provider within a Chinese and Vietnamese population in the US. Patients with language discordant providers reported receiving less health education compared to those with language concordant providers. This effect was mitigated with the use of a clinic interpreter[4]. This language barrier includes key signage and patient information documents. The Joint Commission has published guidance in establishing effective communication, both written and verbal, for diverse patient populations[5]. Finally the clinical (patient- provider encounter) level must be addressed. When cultural differences between provider and patient are not fully understood it becomes a barrier to effective care. Diverse patient populations have specific health beliefs; such as use of home remedies, attitude toward medical care and medical practices, level of trust in doctors and the health system. Each of these differences, if not understood, can interfere with effective care. The Witness Program is an example of a successful, culturally competent approach to health care delivery[6]. Although African-American (AA) women have high rates of breast cancer, screening with mammography was low in this population. To better understand the cultural barriers associated with breast cancer screening among this group, investigators performed multiple focus groups. By directly interviewing AA women in the community barriers to breast cancer screening were identified. The Witness Program® turned cultural barriers into culturally based interventions. Key to this project are Witness Role Models – African American breast and cervical cancer survivors who talk about their experiences with other AA women in a community setting. This approach has led to improved rates of breast cancer screening among the women who participated in this educational program. Training of medical providers in cultural competency is necessary to effectively treat diverse patient communities. Efforts to improve the care of diverse patient populations will increase the lung cancer cure rate. This begins by having a clear understanding of the community that is served, including health care values and beliefs, predominate language used, and any barriers to health care that are present. Training for staff in cultural competency and the ability to evaluate the perceived quality of health care of diverse populations is needed to provide the best care. References 1. Li et al. Journal of Thoracic Oncology, 2015; 10:256-263. 2. Bach PB et al. N Engl J Med. 1999; 341:1198-1205. 3. Koshy et al. Journal of Thoracic Oncology. 2015; 10:264-271. 4. Ngo-Metzger et al. J Gen Intern Med. 22(suppl 2):324-30. 5. The Joint Commission: Advancing Effective Communication, Cultural Competence, and Patient- and Family-Centered Care: A Roadmap for Hospitals. Oakbrook Terrace, IL: The Joint Commission, 2010. 6. Bailey et al. J Natl Med Assoc. 2000; 92:136-142.

Abstract:
The rapid changes in lung cancer treatment,with the development of new treatment modalities such as immunotherapy, coupled with the emergence of low-dose CT screening for lung cancer have made mutli-modality thoracic oncology programs even more vital than previously. Howerver, almost all of these programs are in urban areas, despite a great need in rural communities. Epidemiologic studies show that rural areas have higher lung cancer and all cancer mortality than urban areas (Singh et al J Ca Epidemiol 2011) with even greater disparity in minority and socio-economically disadvantaged rural populations. An analysis of the SEER database found that rural residence had no impact on stage-specific lung cancer survival with the exception of Stage I (Atkins et al, Am J Crit Care Med 191:2015;A3595). Tobacco use is more prevalent in rural communities and may account for much of the higher lung cancer mortality seen in these communities (ALS monograph, Cutting Tobacco's Rural Roots: Tobacco Use in Rural Communities, 2014). It does appear that rural residents are more likely to be diagnosed with an advanced stage of lung cancer than their urban peers (Wen et al, Ann Pub Health and Res 2015 2:1011 ; Johnson et al, Lung Cancer 2014 83:401-7). Survival was lower in rural areas with greater poverty and less educated residents. In addition, rural residents were less likely to receive radiotherapy and chemotherapy, and those in less educated areas were less likely to undergo surgery or chemotherapy (Johnson et al). Another study found poorer outcomes in Stage I lung cancer in rural areas, perhaps due to less access to surgical care (Atkins, op cit). Kim et al compared barriers to clinical trial participation in rural and urban areas of South Carolina (Kim et al, J Comm Health 2013). They found no significant difference between rural and urban residents in willingness to participate in clinical trials but found that rural residents perceived less access to less access to, and awareness and knowledge of, clinical trials. A study funded by the Rand Corporation found that physicians who participated in tumor boards weekly were more likely to enroll lung cancer patients in clinical trials (Kiehl et al, J Onc Practice 2015 11:E267-78). This suggests that rural thoracic oncology programs are likely to improve clinical trial participation by rural residents by improving access and physician particiapation. It is not just access to therapeutic modalities in lung cancer that differs between rural and urban areas; supportive/ palliative care is also different. An analysis of the SEER database found disparities in end of life care as well. Medicare beneficiaries in rural areas had more ER visits in the last 90 days of life than urban residents. Urban residents had more ICU days in the last 90 days of life and were more likely to be enrolled in hospice programs. Minority and lower socioeconomic patients were less likely to use hospice and had more ICU days, inpatient days, and ER visits in the last 90 days of life (Nayar et al, J Comm Health 2014 39:1012-9). These disparities are of particular note since several studies have shown that early palliative care improves survival in lung and other cancers. Greater prevalence of rural thoracic oncology programs may improve access to potentially curative modalities, particularly for early stage disease. Given the higher prevalence of tobacco use in rural populations, these programs should ideally include smoking cessation efforts. Since rural residents are more likely to be diagnosed with advanced lung cancer, the implementation of low-dose CT screening is likely to be particularly beneficial in these communities. It seems likely that rural areas with thoracic oncology programs will have better rates of clinical trial access and participation. Incorparation of early effective palliative care into these programs may improve outcomes and help reduce the disparities seen in end of life care and survival in rural areas. However, resources are often limited in small rural hospitals, so it may be difficult to provide all of these services.

Abstract:
Introduction and Rationale: Assessment and treatment of the cancer patient continues to increase in complexity. Some oncologists have subspecialized in their disease discipline, allowing for a greater depth of knowledge in that particular cancer but not having the breadth of expertise over numerous cancers. The majority of cancer treatment is delivered in the community-based setting where most oncologists practice general oncology. This has created a need for treatment guidelines which help oncologists manage patients in a standard approach. The evolution of treatment guidelines or pathways has several purposes. There has been much research in justifying the implementation of treatment pathways as they lead to consistent care, tend to lower healthcare costs overall, and influence outcomes of care[1-3]. However, each individual system, hospital, or physician will have their own purpose for utilizing pathways, ranging from education of assessment and treatment recommendations to providing evidence to payers for treatment. The development of clinical pathways requires a predetermined strategy. Reports have stressed the need for transparency, inclusiveness, disclosure and frequency of meetings[4][,][5]. This then requires a team to translate the results of these meetings into disseminated information for the clinical teams. The Institute of Medicine published recommendations in 2011 on how to develop these clinical pathways[4]. Various other organizations have published guidelines, such as the National Comprehensive Cancer Network (NCCN), American Society of Clinical Oncology (ASCO), European Society of Medical Oncology (ESMO), Cancer Care Ontario, Cancer Council Australia, and Via Oncology. National Comprehensive Cancer Network (http://www.nccn.org/about/default.aspx) Clinical Practice Guidelines in Oncology The NCCN is the most comprehensive set of guidelines in the United States, covering 97% of all cancers. Guidelines cover the entire cancer spectrum, from prevention to survivorship issues, and are evidence-based and continually updated. Guideline content is consensus based and developed by one of 47 panels consisting of multidisciplinary and disease-specific oncologists and researchers. Within the guidelines, a variety of content can be utilized, including algorithms or decision pathways and discussion text summarizing historical and current data. American Society of Clinical Oncology (http://www.instituteforquality.org/practice-guidelines) Clinical Practice Guidelines ASCO currently has 11 topic areas in which clinical practice guidelines are available for both solid and hematologic malignancies (e.g. use of diagnostic testing and predictive assays, disease-specific treatment, supportive care and survivorship). Each year, ASCO solicits guideline proposals from its members. Expert panels, consisting of oncologists, nurses, pharmacists, and practice managers approve proposal topics and develop the guidelines. Anyone has the opportunity to comment on or provide new evidence for use in the guidelines through the ASCO Guideline Wiki page (https://pilotguidelines.atlassian.net/wiki/display/GW/ASCO+Guidelines). European Society of Medical Oncology (http://www.esmo.org/Guidelines) Clinical Practice Guidelines The ESMO Clinical Practice Guidelines consist of 60 guidelines on cancers of the breast, lung, gastrointestinal tract, head and neck, and more, as well as supportive care and bone health. The ESMO Guidelines Committee is comprised of a Subject Editor and other leading experts and they are charged with authoring, publishing, and disseminating the full clinical practices guidelines and Pocket Guides. Cancer Care Ontario (https://www.cancercare.on.ca/cms/One.aspx?portalId=1377&pageId=7582) Program in Evidence-Based Care (PEBC) The Cancer Care Ontario’s PEBC is a program of the Ontario provincial cancer system, with support from the Ontario Ministry of Health and Long Term Care. Guidelines focus on all stages of cancer, including prevention, screening, diagnostic assessment, treatment, palliative care and survivorship. The PEBC consists of multidiscipline panels (disease-specific and modality-specific guideline development groups) consisting of 200+ physicians, other healthcare providers, and methodologists. Cancer Council Australia (http://www.cancer.org.au/health-professionals/clinical-guidelines/) Clinical Guidelines Network Cancer Council Australia (CCA) is Australia’s national non-government cancer organizations. CCA has published full and condensed guidelines on cancer screening and treatment of lung, esophageal, endometrial, sarcoma, and prostate cancers. The CCA is currently working to transform these guidelines in to a web-based format Via Oncology, LLC (http://viaoncology.com/) Via Oncology Pathways Via Oncology is affiliated with the University of Pittsburgh Medical Center (UPMC) and University of Pittsburgh Cancer Institute (UPCI). The Via Oncology Pathways were developed in 2005 to ensure high quality and standardized care in medical and radiation oncology across UPMC/UPCI facilities and now has grown to include practices from 20 states. Disease committees, consisting of physicians from all participating practices, have developed guidelines covering more than 90% of cancers to demonstrate value to patients, payers and referring physicians. Guidelines are available to participating providers only. Overcoming Barriers and Resistance Many reasons for why physicians do not follow guidelines have been noted in the literature, including: lack of clarity; length of guidelines; guideline format; lack of awareness; lack of familiarity; lack of agreement with the evidence; lack of outcome expectancy; lack of self-efficacy; inertia of previous practice; organizational constraints; excessive frequency of revision; and external barriers[5-8]. Kaster et al recently published data on key domains (stakeholder involvement, evidence synthesis, considered judgment, implementation feasibility, message, and format) related to positive implementation of pathways[5]. These domains largely are nested within two, broad categories: content creation and communication of content[5]. Clinical Trials One aspect that is not as inclusive in all clinical pathways is access or information to clinical trials. This content is an entirely different aspect to clinical pathways as different groups value clinical trials access differently. National accrual rates to clinical trials are low[9][,][10]. Barriers to clinical trial participation are noted on the patient, physician and system level. Clinical pathways that incorporate clinical trials may benefit the patient by increasing physician knowledge of available trials and may benefit the overall clinical trial by increasing accrual rates. EAPathways, Levine Cancer Institute, Carolinas HealthCare System We have developed in-house clinical pathways which include not only treatment pathways to assist clinicians, but also have a number of additional features. These include access to documents, educational resources, clinical trials information and communication to colleagues. The pathways on this proprietary system are developed by our disease-specific sections and housed on our system intranet. Conclusions Clinical pathways are an integral part of patient management. Their utilization is increasing and additional groups are developing these. Functionality and adaptability will be key, especially in the oncology realm, as changes in molecular testing and treatment options are occurring at a faster than ever rate. Educating our practitioners and empowering their ability to accurately assess and treat patients with cancer will enable consistent and efficient care. References 1. Hall SF, Irish JC, Gregg RW, Groome PA, Rohland S. Adherence to and uptake of clinical practice guidelines: lessons learned from a clinical practice guideline on chemotherapy concomitant with radiotherapy in head-and-neck cancer. Current oncology (Toronto, Ont.). Apr 2015;22(2):e61-68. 2. Sullivan WJ. Demystifying pathways in oncology. Managed care (Langhorne, Pa.). Jun 2012;21(6):34-38. 3. Gesme DH, Wiseman M. Strategic use of clinical pathways. Journal of oncology practice / American Society of Clinical Oncology. Jan 2011;7(1):54-56. 4. Graham R, Mancher M, Miller Wolman D, Greenfield S, Steinberg E, eds. Clinical Practice Guidelines We Can Trust. Washington DC: 2011 by the National Academy of Sciences; 2011. 5. Kastner M, Bhattacharyya O, Hayden L, et al. Guideline uptake is influenced by six implementability domains for creating and communicating guidelines: a realist review. Journal of clinical epidemiology. May 2015;68(5):498-509. 6. Kastner M, Estey E, Bhattacharyya O. Better guidelines for better care: enhancing the implementability of clinical practice guidelines. Expert review of pharmacoeconomics & outcomes research. Jun 2011;11(3):315-324. 7. Cabana MD, Rand CS, Powe NR, et al. Why don't physicians follow clinical practice guidelines? A framework for improvement. Jama. Oct 20 1999;282(15):1458-1465. 8. Collins IM, Breathnach O, Felle P. Electronic clinical decision support systems attitudes and barriers to use in the oncology setting. Irish journal of medical science. Dec 2012;181(4):521-525. 9. Go RS, Frisby KA, Lee JA, et al. Clinical trial accrual among new cancer patients at a community-based cancer center. Cancer. Jan 15 2006;106(2):426-433. 10. Comis RL, Miller JD, Colaizzi DD, Kimmel LG. Physician-related factors involved in patient decisions to enroll onto cancer clinical trials. Journal of oncology practice / American Society of Clinical Oncology. Mar 2009;5(2):50-56.

Abstract not provided

Abstract not provided

Background:
As epidermal growth factor receptor (EGFR) mutation a strong predictor of EGFR tyrosine kinase inhibitor (TKI) responsiveness, there are still around 10% TKI-naïve patients early refractory to first line TKIs. We aimed to find clinical predictors of TKIs responsiveness in EGFR-mutant non-small cell lung cancer (NSCLC) patients and create a scoring system as progression free survival (PFS) prediction.

Methods:
This retrospective study evaluated 262 patients harboring EGFR mutation received TKIs as first line therapy for NSCLC between January 2011 and December 2013. Patients were assigned to test (N=131) and validation (N=131) by time sequence. Patients with age ≤ 40, uncommon EGFR mutation, poor performance status, more sites of distal metastasis, and lymphocyte to monocyte ratio ≤3 were independently associated with poor progression free survival. These five factors were included in the scoring system and 3 predictive groups were formed by total score. Table. 1 Univariate and Cox regression analysis of progression free survival

		Univariate analysis	Multivariate analysis
	PFS (M)	P value	P value
Age >40 ≤40	11.6 3.3	0.001	0.002
BMI >24 ≤24	14.9 9.1	0.027	0.928
Gender Male Female	9.3 12.0	0.292
DM YES NO	9.1 11.5	0.500
Smoking Never Former / current	11.5 7.6	0.413
Performance status ECOG 0-2 ECOG 3-4	11.5 2.7	0.009	0.012
Mutation Common Uncommon	11.5 4.1	<0.001	<0.001
Tumor type Adenocarcinoma Non-adenocarcinoma	11.1 9.8	0.789
No. of distal metastasis 0 1-2 >2	21.4 11.3 6.1	<0.001 <0.001	0.015 <0.001
Malignant effusion Yes No	9.1 11.6	0.031	0.946
Lymphocyte to monocyte ratio >3 ≤3	13.4 7.4	<0.001	0.047

Results:
Progression free survival in the test group were 15.7 months(m) for 0-1 points, 9.3 m for 2 points, 4.0 m for 3-6 points (p <0.001). In the validation test, Progression free survival in there predictive groups each were 13.7 m, 9.5 m, 4.8 m (p <0.001). Between the test and validation groups, no significant differences were found in each one of the three predictive groups. Figure 1



Conclusion:
The score appears valid and reproducible. It can stratify NSCLC patients harboring EGFR mutation using first line EGFR-TKIs into long, intermediate and short PFS groups.

Background:
ATP binding cassette superfamily G member 2 (ABCG2) has been demonstrated to be associated with the effect of chemotherapy/targeted therapy in non-small-cell lung cancer (NSCLC) and the single nucleotide polymorphisms (SNPs) of ABCG2 gene are supposed to affect the expression of ABCG2 protein. The purpose of this study was to investigate the correlation between SNPs of ABCG2 and outcome of tyrosine kinase inhibitions (TKIs) therapy in Chinese advanced NSCLC patients.

Methods:
SNP genotyping(34 G/A, 421 C/A, 1143 C/T and -15622 C/T)of ABCG2 gene in 100 patients was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The clinical characteristics of 100 patients were collected. A total of 70 patients were treated with TKIs(gefitinib, erlotinib and icotinib). The association between ABCG2 polymorphisms and clinical characteristics was evaluated. Kaplan-Meier survival curves were plotted for overall survival (OS) and analyzed with the log-rank test. Cox proportional hazards model was applied to evaluate the association between OS and clinical or genomic characteristics and estimated the adjusted HR and its 95 %CI.

Results:
The three polymorphisms of the ABCG2 34 G/A, 421 C/A and 1143 C/T occurred more frequently compared with -15622 C/T in Chinese advanced NSCLC patients. The allele A of 421C>A happened frequently in EGFR mutation positive patients (33.3% vs 9.1%, P=0.038). There was no association between ABCG2 polymorphisms and other clinical characteristics (p> 0.05).The median OS of patients with 34G>A mutant type (GA+AA) was 31.0 (95%CI: 22.9-39.1) months , which was significantly longer than those with wild type (GG) , 18.0 (95%CI: 14.9-21.1) months (p=0.005). No significant difference of OS was found in 421 C/A and 1143 C/T polymorphisms (p> 0.05).

Conclusion:
Our findings demonstrate a strong association between the ABCG2 34G>A polymorphism and the overall survival of NSCLC patients treated with TKI. It may be a possible predictor of the clinical outcome of TKIs therapy in NSCLC patients.

Background:
p53 mutations are common in lung cancer, and have also been described in EGFR mutated patients. The impact of p53 mutations in EGFR M+ patients is controversial, especially if classified as “disruptive” and “non-disruptive” according to their functional effect on the p53 protein as proposed by Poeta and colleagues. The aim of the study was therefore to systematically analyze EGFR and p53 mutations within a cohort of patients with lung cancer stage IV (UICC 7), to correlate alterations with clinical characteristics and to investigate a potential impact of p53 mutations on treatment outcome.

Methods:
267 patients from a single center diagnosed with lung cancer stage IV were studied for the presence of EGFR as well as inactivating p53 mutations. Methods for the detection of EGFR mutations included Sanger Sequencing and hybridization based COBAS testing. P53 mutations were detected by Sanger Sequencing. Clinical characteristics including smoking status were available for all patients.

Results:
267 consecutive patients at the lung cancer center of the Pius-Hospital Oldenburg were studied. The overall EGFR mutation rate was 19% (51/267) in all patients, 80% (41/51) showing common mutations of exon 19 or 21. P53 disruptive mutation showed in 16% (8/51) and p53 nondisruptive mutation occurred in 11% (22/51) whereas p53 WT was found in 47% (24/51). In 8/51 (16%) patients p53 analysis was not successful. OS was 37 months in p53 disruptive mutation and p53 WT patients compared to 19 months in p53 nondisruptive mutation (p<0,05). PFS on 1st line TKI therapy was 18 months in p53 nondisruptive mutation and p53 WT patients and 6 months in p53 disruptive mutation (p<0,024). Similar results could be shown in the EGFR common mutation subgroup but not in the uncommon mutation subgroup.

Conclusion:
Significant differences in PFS and OS in EGFR M+ patients were observed depending on p53 mutation status. P53 mutational status is only predictive when disruptive and non-disruptive P53 mutations are differentiated. P53 should be tested prospectively in EGFR M+ patients as management of patients on 1st line TKI may be different.

Background:
Mutations in the epidermal growth factor receptor (EGFR) are associated with a marked therapeutic response to EGFR-tyrosine kinase inhibitors (TKIs) in patients with advanced non-small cell lung cancer (NSCLC). However, clinical predictors of the survival benefit of EGFR-TKI treatment in NSCLC with EGFR activating mutations have not been well elucidated. Therefore, this study evaluated clinical predictors of survival outcome in patients with EGFR mutant NSCLC who were treated with EGFR-TKIs. Mutations in the epidermal growth factor receptor (EGFR) are associated with a marked therapeutic response to EGFR-tyrosine kinase inhibitors (TKIs) in patients with advanced non-small cell lung cancer (NSCLC). However, clinical predictors of the survival benefit of EGFR-TKI treatment in NSCLC with EGFR activating mutations have not been well elucidated. Therefore, this study evaluated clinical predictors of survival outcome in patients with EGFR mutant NSCLC who were treated with EGFR-TKIs.

Methods:
A total of 224 patients with EGFR-mutant lung adenocarcinomas that were treated with EGFR-TKIs were retrospectively reviewed. Treatment outcomes were evaluated based on clinical factors, number of metastasis site and progression patterns.

Results:
The clinical factors associated with reduced progression-free survival (PFS) and overall survival (OS) by univariate analysis were ECOG performance status (PS) ≥ 2, intra- and extrathoracic metastasis, presence of extrathoracic metastasis, high number of metastasis sites, metastasis to liver or adrenal gland at baseline, and rapid progression of primary tumor at the time of progressive disease (PD). In multivariate analysis, factors that remained significantly associated with shorter PFS were ECOG PS ≥ 2 (Odds ratio [OR] 2.189 [95% CI, 1.374 – 3.437]; P < 0.001) and rapid progression of primary tumor at PD (OR 1.800 [95% CI, 1.059 – 3.058]; P = 0.030).

Conclusion:
Thus, tumor burden, expressed as the number of metastasis sites at the time of EGFR-TKI treatment, and rapid progression of primary tumor at PD are predictive of inferior survival in patients with lung adenocarcinoma with activating EGFR mutations.

Abstract not provided

Background:
Afatinib (A) is a potent irreversible EGFR TKI and nimotuzumab (N) is a humanized anti-EGFR mAb. In this phase Ib/II study, we aimed to assess the safety and activity of A plus N in advanced NSCLC patients with acquired resistance to gefitinib or erlotinib.

Methods:
Major inclusion criteria were advanced NSCLC with activating EGFR mutation or disease control for at least six months with previous gefitinib or erlotinib therapy. In the phase Ib study using classic 3+3 dose escalation method, patients were treated with A 40mg/d or 30mg/d in combination with N 100mg/w or 200mg/w. One cycle was composed of 4 weeks of treatment. In the phase II study, patients were treated with A plus N in the level of RP2D defined in the phase Ib study.

Results:
Overall, fifty pts were enrolled and treated: 13 in phase Ib and 37 in phase II. At the starting dose level (A 40mg/d + N 100mg/w), one out of 6 pts experienced end-of-cycle 1 DLT (G3 diarrhea), and the dose was up to the next level of A 40mg/d + N 200mg/w. Out of 6 pts at this level, 2 pts experienced DLTs (G3 diarrhea and G3 neutropenia, respectively), and RP2D was accordingly determined as A 40mg/d + N 100mg/w. In the whole treatment duration of the phase II, there was no treatment related death and 10 pts (20%) experienced any grade 3 adverse event, including diarrhea and skin rash. Out of evaluable 50 pts in the phase Ib/II study, the response rate was 36% (18 achieved partial response out of 50) and the median PFS was 4.4 months (95% CI:3.2-5.5 months).

Conclusion:
A and N showed an acceptable safety profile and promising antitumor activity in advanced NSCLC patients with acquired resistance to gefitinib or erlotinib.

Background:
Targeted therapies have considerably improved the prognosis of patients with non-small cell lung cancer (NSCLC).Although not precision enough, RESIST criteria was still the most often used response assessment method to reflecting the clinical benefits. We propose a non-invasive, folate receptor (FR)–based circulating tumor cell (CTC) detection approach to interpret treatment response of targeted therapy between baseline and follow-up CTC values in EGFR mutation/ALK translocation advanced NSCLC.

Methods:
One hundred and thirty eight patients were enrolled in our study. Peripheral blood was analyzed for CTCs enumeration on negative enrichment by immunomagnetic beads. Changes of CTCs levels were correlated with radiological response. Sequential analyses were conducted to monitor CTC signals during therapy and correlate radiological effects with treatment outcome.

Results:
CTCs were detected (≥8.7CTC) in 84.8% of patients. Pretreatment and pro-treatment blood samples from all 118 EGFR-mutant (19deltion:56, L858R:57, G719x:3, L861Q:1, 19 deletion + L858R:1), 14 ALK translocation lung cancer patients and 6 EGFR wild type patients were collected. Of 89 eligible and evaluable patients, baseline CTC counts were not associated with response to treatment by RECIST (P=0.353). There is no difference between exon 19 deletion and L858R of baseline CTC values. (19deletion:19.4 CTCs, L858R:20.9 CTCs,P=0.222) The change of CTCs values increased correlation with radiological response (P=0.042) after treatment of targeted therapy. There is no significant difference between exon 19 deletion and L858R of CTCs values pre and pro EGFR-TKI treatment.(3.32 vs.12.1, P=0.783)

Conclusion:
This study confirms the predictive significance of CTCs in patients with EGFR mutation/ALK translocation NSCLC receiving targeted therapy. The change of CTCs value correlated significantly with radiological response. This strategy may enable non-invasive, specific biomarker assessment method for using CTC decreases as an early indication of response to targeted therapy and monitoring in patients undergoing targeted cancer therapies.

Background:
To date there have been limited randomized comparisons of EGFR tyrosine kinase inhibitors (TKI) in EGFR mutant NSCLC. Dacomitinib is a potent, irreversible EGFR inhibitor that demonstrated robust activity in a phase 2 study for patients with common activating EGFR mutations. Additionally, preclinical data suggests greater activity in patients with common EGFR activating mutations in exon 19 or 21. ARCHER 1009 (NCT01360554) and A7471028 (NCT00769067) each compared the clinical activity of dacomitinib (D) versus erlotinib (E) in advanced NSCLC including patients with common activating EGFR mutations; pooled results are presented.

Methods:
Patients (pts) with locally advanced/metastatic NSCLC were randomized following progression with 1 or 2 prior chemotherapy regimens to treatment with dacomitinib (D) (45 mg PO QD) or erlotinib (E) (150 mg PO QD). The Phase 2 study (A7471028) was open label while the Phase 3 ARCHER 1009 study was double-blind and double dummy. Archived tumor tissue, ECOG performance status (PS) of 0-2, adequate organ function and informed consent were required. Results of the two studies were previously reported individually. Analyses were performed by pooling patients with common EGFR activating mutations from both studies to compare efficacy of D versus E.

Results:
121 patients with any EGFR mutation were enrolled into the two studies with 1 patient randomized but not treated; 101 (53 on D) pts had activating mutations in exon 19 or 21. For patients with exon19/21 mutations, the median PFS was 14.6 months (95%CI 9.0–18.2) for D and 9.6 months (95%CI 7.4–12.7) for E and unstratified HR 0.717 (95%CI 0.458–1.124) with 1-sided p=0.073. The median OS was 26.6 months (95%CI 21.6–41.5) for D and 23.2 months (95%CI 16.0–31.8) for E and unstratified HR 0.737 (95%CI 0.431–1.259) with 1-sided p=0.132. The corresponding pooled analyses were conducted separately in exon 19 and exon 21. The adverse-event profile did not differ between the activating mutation subset and the overall population. Figure 1



Conclusion:
Dacomitinib may be associated with an improved PFS and OS compared to Erlotinib in patients with exon 19/21 EGFR mutations. A prospective P3 study comparing D to another EGFR TKI in 1L EGFR mutated NSCLC is ongoing to verify these observations (NCT01774721).

Abstract not provided

Background:
Radiotherapy is the principal treatment modality for patients with brain metastases (BM), however, tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR) shows therapeutic efficacy for brain metastases in patients with EGFR-mutant lung adenocarcinoma. This study was conducted to compare the outcome of TKI alone with TKI plus concomitant radiotherapy in treatment of BM from EGFR-mutated lung adenocarcinoma patients.

Methods:
The inclusion criteria were as following: patients newly diagnosed with EGFR-mutant lung adenocarcinoma, presented with BM, TKI as first-line therapy, and ECOG PS 0-2.

Results:
From January 1, 2009 to September 1, 2014 at Zhengzhou University Affiliated Cancer Hospital, 516 lung adenocarcinoma patients with EGFR gene mutations were reviewed, and 132 cases (25.6%) with newly diagnosed BM were enrolled for the analysis. Among the 132 patients, more than half of them (n = 72; 54.5%) harbored a deletion in exon 19, 97 patients (73.5%) showed multiple intracranial lesions, and 50.8% (n = 67) had asymptomatic BM. 79 patients (59.8%) were treated with TKI alone, 53 with TKI plus concomitant radiotherapy (45 with whole brain radiotherapy, and 8 with stereotactic radiosurgery). The objective response rate of BM was significantly higher in TKI plus radiotherapy group (67.9%) compared with TKI alone group (27.8%, P<0.001). The median time to intracranial progression was 22.3 months. The median intracranial progression-free survival in patients who received TKI plus radiotherapy was 24.7 months, much longer than those treated with TKI alone which was 19.0 months, P = 0.005. Multivariate analysis showed brain radiotherapy (P = 0.012) and intracranial lesion number (P = 0.070) as important prognostic factors for intracranial progression-free survival. In addition, the data of overall survival will be presented at the conference.

Conclusion:
For EGFR-mutated lung adenocarcinoma patients with BM, TKI plus concomitant radiotherapy achieved higher response rate of BM and significant improvement in intracranial progression-free survival compared with TKI alone. Figure 1

Background:
Approximately 20-40% of non–small–cell lung cancer (NSCLC) patients develop brain metastasis (BM) and the survival is very poor with a median overall survival of 4-6 months following whole brain radiotherapy treatment. Recent studies have shown that oral epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) were effective for the treatment of BM from NSCLC with EGFR mutation. However, the relationship between EGFR mutations and prognosis of NSCLC patients with BM remains to be determined. In this study, we investigated the impact of EGFR mutation status on the survival of BM patients from NSCLC

Methods:
730 NSCLC patients were retrospectively reviewed. 136 patients had developed BM during their course of disease. 33 of these 136 BM patients (24.3%) were confirmed to have exon 19 deletions, while 33 had exon 21 point mutation (L858R) (24.3%). Overall survival was evaluated by Kaplan-Meier method. Log-rank test and Cox proportional hazards model were used to analyze the impact of pretreatment and treatment variables on survival.

Results:
The median survival of NSCLC with BM was 8 months. Log-rank test analysis showed that ECOG PS at BM (p=0.000), control of primary tumor (p=0.005), pathology (p=0.01), EGFR mutations (p=0.045) and 19 exon deletion (p=0.007) were associated with a longer survival. In Cox proportional hazards model, EGFR exon 19 deletion (HR=0.558, 95%CI=0.325-0.957, p=0.034), control of primary tumor (HR=2.033, 95%CI=1.098-3.766, p=0.024), and ECOG PS at BM (HR=2.033,95%CI=1.145-1.287, p=0.006) were found to be independent prognostic factors. Moreover, there were significantly differences in the survival between different groups according to RTOG recursive partitioning analysis (RPA) classification system in this cohort of patients (p=0.000)

Conclusion:
Exon 19 deletion is an independent prognostic factor in BM from NSCLC. Our findings suggest that the status of exon 19 deletion may be integrated into the prognostic scoring classification system for NSCLC.

Background:
Erlotinib has a synergistic effect with pemetrexed when treating non-squamous non-small cell lung cancer. The aim of our study was to confirm the efficacy and safety of erlotinib (E) in combination with pemetrexed/cisplatin (E-P) in Chinese lung adenocarcinoma with brain metastases.

Methods:
This study is a prospecive, non-randomized cocurrent controlled study. Lung adenocarcinoma patients with brain metastases, who were erlotinib or pemetrexed treatment-naive and had adequate organ functions, were assigned in parallel to receive either erlotinib 150 mg/day or erlotinib on days 4-21 plus pemetrexed 500 mg/m[2] on day 1 and cisplatin 20 mg/m[2] on day 1-3 every 21 days up to 6 cycles and subsequent oral erlotinib, until progressive disease or unacceptable toxicity. The primary endpoint was intracranial overall response rate (ORRi). Previous data showed that about 56% of the patients treated with E and 78% of the patients treated with E-P, achieved an ORRi. We estimated the minimum sample size of 65 with 70% power (two-sided alpha 0.05).

Results:
69 lung adenocarcinoma patients with brain metastases had received E (n=35) or E-P (n=34) from Jan 2012 through Nov 2014. Demographics and patient characteristics were well balanced between two groups, including EGFR status, gender, age, smoking status, ECOG performance status, brain metastases and number of prior treatments. ORRi, in the E-P arm was superior to that in the E arm (79% vs. 48%, P=0.008) (Table S). Especially in the patients with EGFR wild type or treated as first-line treatment could achieve much better ORRi. Patients treated with E-P arm, compared with E arm as first-line treatment, were associated with better intracranial PFS (PFSi) (median PFSi, 9 months vs. 2 months, P=0.02) and systemic PFS (median PFS, 8 months vs. 2 months, P=0.006).The most frequent adverse events related with erlotinib were higher in the combination arm. No new safety signals were detected. The side effects were tolerable and no-drug related deaths. Table S The ORRi between the E-P and E arm

	group (n)	ORRi (n,%）
Total patients	E (35)	17,48.6%
	E-P (34)	27, 79.4%
P value		0.008
EGFR mutation	E (18)	10,55.6%
	E-P (14)	12,85.7%
P value		0.124
EGFR negative	E (7)	1, 14.3%
	E-P (11)	7, 63.6%
P value		0.066
EGFR unknown	E (10)	6,60.0%
	E-P (9)	8,88.9%
P value		0.303
First-line treatment	E (16)	7,43.7%
	E-P (18)	14 ,77.7%
P value		0.08

Conclusion:
The combination of erlotinib and pemetrexed/cisplatin is effective and improved PFS as first-line treatment in Chinese lung adenocarcinoma with brain metastases. Toxicities are tolerable and the erlotinib-related side-effects were higher.

Background:
Brain metastases (mets) in EGFR/ALK-driven NSCLC are common, and frequently pose treatment dilemmas. Effective systemic therapy with tyrosine kinase inhibitors (TKIs) controls extracranial disease in up to 70% of patients, but often radiotherapy is required for intracranial control. As whole brain radiation (WBRT) may be associated with neurocognitive toxicity, we aimed to evaluate the impact of molecularly targeted therapy and stereotactic radiotherapy (SRS) for EGFR/ALK-driven NSCLC on intracranial disease control with and without WBRT.

Methods:
This retrospective analysis included patients treated with EGFR/ALK-positive NSCLC at Princess Margaret Cancer Centre from 1998-2015, with brain mets at lung cancer diagnosis or during treatment/follow-up. Demographic data were collected from electronic patient records. Time to intracranial progression (TTIP) and overall survival (OS) were calculated from date of diagnosis of brain mets, using the cumulative incidence function and Kaplan-Meier methods respectively; differences between groups were tested with Gray’s or log-rank test.

Results:
From 1998-2015, 162 patients with brain mets from EGFR/ALK-driven NSCLC were identified: 138 in the EGFR cohort, 23 in the ALK cohort and one included in both cohorts for analysis, whose tumour carries both an EGFR mutation and ALK rearrangement. Table 1 contains clinical characteristics and treatment details. In the EGFR cohort, initial brain mets treatment consisted of systemic therapy alone in 19 patients (17 TKI, 2 chemotherapy), SRS +/- surgery in 27 patients and WBRT +/- SRS/surgery in 88 patients. 1-year intracranial progression rates were 26%, 32% and 12%, respectively, and median TTIP was 18, 16 and 40 months [p=0.12]. Median OS was 26, 27 and 34 months respectively [p=0.49]. In the ALK cohort, initial brain mets treatment consisted of systemic therapy alone in 4 patients (1 TKI, 3 chemotherapy), SRS/surgery alone for 4 patients and WBRT +/- SRS/surgery for 15 patients. 1-year intracranial progression rates were 50%, 50% and 13%, respectively, and median TTIP was 18, 14 and 69 months [p=0.028]. Median OS was 35 months, not reached and 51 months, respectively [p=0.75]. Multivariable analysis for the whole group showed that age [p=0.021], number of brain mets [p=0.012] and extracranial control [p=0.008] were significantly associated with OS, but not WBRT [p=0.61].

Conclusion:
In this cohort of patients with brain mets from EGFR/ALK-driven NSCLC, patients treated with WBRT trended to longer TTIP. Although not statistically significant, our data also show a trend towards longer survival in patients who received WBRT. These observations require further validation in this patient population.

	EGFR (N=139)	ALK (N=24)
Median Age (Range)	59(29-86)	53(31-77)
Female Sex	93(67%)	15(62%)
Ethnicity Asian Caucasian Other	58(42%) 63(45%) 18(13%)	7(29%) 13(54%) 4(17%)
Smoking Never Smoker Former/Current Smoker Unknown	108(77%) 30(22%) 1(1%)	19(79%) 5(21%) 0
ECOG PS (Diagnosis) 0 1 2-4	66(48%) 67(48%) 6(4%)	7(29%) 14(58%) 3(13%)
Brain Mets at Stage IV diagnosis	93(67%)	13(52%)
Number of Brain Mets 1 2-4 5+ N/A	32(23%) 39(28%) 62(45%) 6(4%)	9(38%) 6(24%) 9(38%) 0
Symptomatic Brain Mets No Yes	78(56%) 61(44%)	16(67%) 8(33%)
Initial Brain Mets treatment WBRT WBRT+SRS/Surgery SRS+/-Surgery Systemic Therapy None	71(51%) 17(12%) 27(19%) 19(14%) 5(4%)	13(54%) 3(12%) 4(17%) 4(17%) 0

Abstract not provided

Background:
Purines are well known as intracellular sources for energy but also act as extracellular signaling molecules. In the last decade there has been a growing interest in the therapeutic potential of purinergic signaling for cancer treatment. The effects carried out depend on the concentration, expressed pattern of purinergic receptors and general dynamics of synthesis and degradation. In this study we analyze different purines and purinergic receptors in bronchoalveolar lavage (BAL) of patients with non-small-cell lung cancer (NSCLC) to provide further insight on their relevance in the tumor microenvironment.

Methods:
In this prospective clinical trial we enrolled 27 patients with NSCLC and 16 patients with chronic obstructive pulmonary disease (COPD) without signs of malignancy. The study was approved by our local ethics committee and registered as a clinical trial in the German Registry for Clinical Trials (DRKS-ID: DRKS00005415). BAL was performed using flexible bronchoscopes. The bronchoscope was wedged into a subsegment were the tumor was present and a total of 300 ml sterile saline was instilled. The BAL-fluid (BALF) was recovered by gentle aspiration. Purines (ATP, ADP, AMP, Adenosine and Inosine) were analyzed using fluorescence/luminescence based assays. Expression of purinergic receptors and Ectonucleotidases in NSCLC (P2X1, P2X4, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, P2Y13, P2Y14, CD39, CD73) were analyzed using qPCR.

Results:
Patients with NSCLC have significantly lower ATP and ADP concentrations in BALF than patients with COPD without signs of malignancy (p=0.006 and p=0.009). Inosine concentrations however are higher in patients with malignant disease (p=0.01). In the subgroup-analysis of metastasized versus non-metastasized tumors receptor-analysis revealed a higher expression of P2X4 (p=0.07), P2X7 (p=0.0008) and P2Y1 (p=0.009) as well as of the ectonucleotidase CD39 (p=0.007). Analysis of the purine metabolites in the respective groups showed no statistically significant differences. Furthermore there is a positive correlation of the proportion of macrophages in differential cell count in BAL with the expression of P2X7 (r=0.53, p=0.02).

Conclusion:
Previous data suggests pro-inflammatory, zytotoxic and thus anti-neoplastic effects of Adenosine-Triphosphate (ATP) and ADP. Also it has been shown that low ATP concentrations in the tumor microenvironment can lead to enhanced proliferation of tumor cells. In this first in vivo study on purinergic signaling in lung cancer we find lower concentrations of ATP and ADP in samples from NSCLC patients compared to COPD without signs of malignancy in accordance with these findings. Furthermore in aggressive, metastasized NSCLC we find a higher expression of the ectonucleotidase CD39. This enzyme degrades ATP and ADP to Adenosin and has previously been shown to hence induce immune escape in malignant disease. Furthermore we demonstrate elevated expression of P2X4, P2X7 and P2Y1 in the tumor microenvironment of metastasized NSCLC compared to non-metastasized tumors. This suggests a role of these receptors in tumor metastasis, however the exact mechanisms remain unclear. To further illustrate these interactions we are currently initiating a study to identify purinergic receptors in NSCLC tumor cells from pathologic specimen. With this knowledge future translational studies can be conducted to potentially provide new therapeutic targets.

Background:
Lung cancer growth was significantly repressed by the first generation CDK2/9/7 inhibitor seliciclib (R-roscovitine, CYC202, Cyclacel Ltd). This induced anaphase catastrophe and apoptosis to occur. Anaphase catastrophe happens when supernumerary centrosomes attempt mitosis by clustering extra centrosomes. If this clustering is inhibited, cells segregate chromosomes inappropriately and anaphase catastrophe occurs and leads to death of daughter cells. This study explored antineoplastic effects of a next generation CDK2/9 inhibitor: CCT68127 (Cyclacel) against lung cancer cells. CCT68127 inhibits CDK2/9 more potently and selectively than seliciclib (IC50s for CDK2 and CDK9 are 30nM and 110nM, respectively).

Methods:
Antineoplastic CCT68127 effects in murine (transgenic mouse-derived) and human lung cancer cells were compared to seliciclib using luminescent cell viability assays. Cell cycle arrest and apoptosis induction by CCT68127 were detected using fluorescence-based cell imaging after staining with propidium iodide (PI) and double-staining with Annexin V and PI. Multipolar anaphase cells were scored after a tubulin and DNA staining. RPPA (Reverse Phase Protein Assay) analyses were performed in CCT68127 and vehicle-treated lung cancer cells to uncover mechanisms engaged by CDK2/9 antagonism. Expression levels of nearly 200 key growth-regulatory proteins were examined before and after 6, 24, and 48 hours of CCT68127 versus vehicle treatments of murine: ED1 (wild-type KRAS) and LKR13 (mutant KRAS) and human lung cancer cells: H522 (wild-type KRAS) and Hop62 (mutant KRAS).

Results:
IC50s of CCT68127 in murine lung cancer cells (ED1, LKR13, and 393P) were <1µM while IC50 of seliciclib was >25µM. KRAS mutant murine lung cancer cells (LKR13 and 393P) were more sensitive to CCT68127 than the KRAS wild-type line (ED1). In contrast, growth inhibition in C10 immortalized murine pulmonary epithelial cells was negligible. IC50s in human lung cancer cell lines (Hop62, A549, H2122, H522, and H1703) were comparable to murine lung cancer cell lines. KRAS mutant lung cancer cells (Hop62, A549, and H2122) were more sensitive than KRAS wild-type lung cancer cell lines (H522 and H1703). Immortalized human bronchial epithelial cells (BEAS-2B) were resistant to CCT68127 treatment. CCT68127 triggered apoptosis in a dose-dependent manner in murine lung cancer cell lines and at much lower concentrations than seliciclib. CCT68127 caused G1 arrest. Its growth inhibition was partially reversed in washout experiments. CCT68127 also induced apoptosis in human lung cancer cells (Hop62, A549, H522, and H1703). A mechanism responsible for these effects was found. Anaphase catastrophe was triggered by CCT68127 treatment of murine and human lung cancer cell lines and was independent of KRAS mutation status. RPPA analyses uncovered distinct protein profiles after CCT68127 treatment. These included DNA repair, Hippo and Rab GTPase pathway members that were each markedly down-regulated.

Conclusion:
CCT68127 is a next generation CDK2/9 inhibitor that has more potent antineoplastic activity against KRAS mutant and wild-type lung cancer cells than the prior inhibitor, seliciclib. This occurred via induced anaphase catastrophe and was linked to changes in expressed growth regulatory proteins. Taken together, these findings implicate use of a next generation CDK2/9 inhibitor for human lung cancer cases.

Background:
Tumor initiating cells (TICs) are a small subpopulation within cancer that is thought to be resistant to conventional therapies and capable of reinitiating tumors. However, only a few biomarkers of TICs have been well elucidated.

Methods:
By the methods of QPCR, FACS, western blot, colony formation and statistic analysis, we have investigated whether CACNA2D1 (α2δ1) to enrich TICs of non-small cell lung cancer (NSCLC) and and tharget therapy of its antibody.

Results:
In comparison to α2δ1[-], α2δ1[+] cells demonstrated greater TICs properties with higher potential of self-renewal, differentiation and reconstituting tumors. Following treatment, these cells were enriched in clinical samples. We verified a monoclonal antibody of α2δ1 mAb which targets to α2δ1 had therapeutic treatment to TICs of NSCLC and further α2δ1 mAb combined with the common anti-cancer drug of carboplatin was obtained to suppress the established xenograft tumors. Importantly, the disease free survival and overall survival of NSCLC patients with increased α2δ1 expression was significantly shorter than that of patients with decreased expression. Mechanically, our results showed that a role of α2δ1 in up-regulating stemness of NSCLC cells was associated with NOTCH signaling.

Conclusion:
Collectively, our data indicate that α2δ1 could be used as a marker for identifying TICs of NSCLC and targeting these cells might provide a way to treat this disease.

Background:
Heparan sulfate D-glucosamine 3-O-sulfotransferase 3B1 (HS3ST3B1) participates in the biosynthetic steps of heparan sulfate (HS) and found to target VEGF in acute myeloid leukemia(AML) cells thus contributing the angiogenesis and proliferation of AML cells . However, the role of HS3ST3B1 in NSCLC has never been reported. In this study, we aim to investigate the role of HS3ST3B1 in NSCLC epithelial-to-mesenchymal transition.

Methods:
Expression of HS3ST3B1 was investigated by qRT-PCR in specimens of tumor and matched normal tissues of NSCLC patients and also in epithelial and mesenchymal NSCLC cell lines. A549 and HCC827 cell lines was induced to mesenchymal phenotype by TGF-β, and expression of HS3ST3B1, CDH1, and VIM were studied by PCR. HS3ST3B1 was knockdown by siRNA to analyze the effect of HS3ST3B1 on EMT. Computational predicting software was used to predict potential regulators of HS3ST3B1 and dual luciferase report system demonstrated that miR-218 may target HS3ST3B1 in cells. MiR-218 was tranfected into cells to analyze the association of miR-218 and HS3ST3B1 in cells.

Results:
HS3ST3B1 was significantly up-regulated in tumors compared with matched normal tissues(P=0.002). Its expression was also up-regulated in mesenchymal phenotype NSCLC cells lines compared with epithelial phenotype(P＜0.05). When epithelial phenotype NSCLC cells transformed to mesenchymal phenotype induced by TGF-β, HS3ST3B1 was also significantly up-regulated. Moreover, when HS3ST3B1 was knockdown by siRNA in mesenchymal phenotype NSCLC cell lines, cells were reversed to epithelial phenotype morphologically. With Targetscan, we found that HS3ST3B1 was one potential targets of miR-218 and dual luciferase report system demonstrated that HS3ST3B1 was direct target of miR-218 in cells. When miR-218 was transfected into cells, we found that HS3ST3B1 was down-regulated. Figure 1 Figure 2





Conclusion:
HS3ST3B1 may regulate EMT and it can be regulated by miR-218 in NSCLC.

Abstract not provided

Background:
Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer including lung cancer, which is attributed to a greater number of deaths than prostate, breast and colorectal cancers combined. Paxillin is a 68 kDa protein that is an integral part of the focal adhesion and acts as an adaptor molecule. We initially cloned the gene for paxillin, and localized it to chromosome 12q24. We have previously reported that paxillin can be mutated (approximately 8%), amplified (5-7%), and/or overexpressed in almost 80% of lung cancer patient samples. Paxillin protein is upregulated in more advanced stages of lung cancer compared with earlier stages and is a prognostic factor for non-small cell lung cancer (NSCLC). Paxillin gene is amplified in some pre-neoplastic lung lesions as well as neoplastic lesions. We identified 22 different variants of paxillin mutation in our initial investigation especially between the LD and the LIM domains (Jagadeeswaran et al. 2008). There are mutations that have been validated in the TCGA set. We selected six mutants to perform further studies ((P52L, A127T, P233L, T255I, D399N, and P487L as well as wild-type as control). Our investigations focused on an effort to understand the contribution of molecular abnormalities found in paxillin and their relationship to mitochondrial functionality.

Methods:
HEK293 cells as well as a paxillin null NSCLC cell line H522 was used to overexpress the above paxillin mutants and wild-type paxillin. Live cell confocal microscopy was performed to evaluate cell motility, immunoprecipitation to determine interaction with other proteins, and gene expression analysis was performed to evaluate effects on gene expression.

Results:
Among the mutations we investigated, we found that the most common paxillin mutant A127T in lung cancer cells enhanced cell proliferation, focal adhesion formation and co-localized with the anti-apoptotic protein B cell CLL/Lymphoma 2 (BCL-2), which among other sites also localizes to the mitochondria. We further found that when these variant clones of activating mutations were expressed in HEK293 cells, they conferred phenotypic changes resembling neoplastic cells. In gene chip microarrays assay investigating gene expression modulation conferred by these mutations in these same HEK293 cells, we found that P52L, A127T, T255I, P233L and D399N mutations, compared to wild-type paxillin, indeed modulated the expression of a significant number of genes. In particular, there were a number of mitochondrial signature proteins that were altered in the various mutants. Analyzing mitochondrial functions by measuring the interaction of these mutants with mitochondrial proteins MFN2, and DRP1, we identified that they alter mitochondrial dynamics, with significant fission rather than fusion. Paxillin also translocated from the focal adhesion to the mitochondrial membrane. In relationship to cisplatin responsiveness, PXN and mutant overexpression lead to cisplatin resistance.

Conclusion:
These data suggest that wild-type and mutant paxillin variants play a prominent role in neoplastic changes with direct implications in lung cancer progression and hence, its potential as a therapeutic target needs to be explored further.

Background:
The methyltransferase enhancer of zeste homolog 2 (EZH2) belongs to the polycomb repressive 2 complex (PRC2). EZH2 is upregulated in several malignancies including prostate, breast and lung cancer. The EZH2 protein forms one of the critical protein complexes of PRC2 by partnering with EED (embryonic ectoderm development) protein. This EED/EZH2 complex has been shown to interact with histone deacytelase (HDAC). This interaction is highly specific and HDAC does not interact with any other PRC2 protein complexes. In the present study, we investigated the link between EZH2 and HDAC in lung cancer cell lines and in human tumor tissue microarrays (TMAs). We also further investigated EZH2 as a marker for response to HDAC inhibitors.

Methods:
We analyzed EZH2 and HDAC1 mRNA expression in two lung adenocarcinoma datasets (MDACC n=152, and TCGA n=308), and correlated the gene expression with tumors’ clinico-pathological characteristics and patients’ outcome. To study the association of EZH2 and HDAC1 expression with response to the HDAC1 inhibitor suberanilohydroxamic acid (SAHA), we examined mRNA and protein expression by RT-PCR and Western blot, respectively, in twelve lung adenocarcinoma (LUAD) cell lines at baseline and after overexpression or knock-down of EZH2 or HDAC1 gene expression using siRNA. Response to (SAHA) in cell lines was measured by MTT assay and correlated with protein and mRNA expression levels of EZH2 and HDAC1.

Results:
Direct and positive correlation was found between EZH2 and HDAC1 expression NSCLC cell lines (P <0.0001). This correlation was confirmed in NSCLC specimens from MDACC (Spearman’s correlation r=0.416; p < 0.0001) and TCGA datasets (r=0.221; p <0.0001).Patients with high EZH2 and high HDAC1 expression in stage I NSCLC specimens of MDACC and TCGA datasets had lowest survival compared to the patients who had either or both low expressions. Overall survival in the univariate analysis (MDACC dataset; Hazard Ratio (HR)=2.97; p=0.031 and TCGA dataset; HR=2.6 and p=0.041) and multivariate analysis (MDACC; HR=2.92 and p=0.034 and TCGA; HR=3.17 p=0.016). When EZH2 expression was knock down, there was a significant reduction in HDAC1 expression; conversely, when HDAC1 was knocked down EZH2 expression was also decreased. These concordant change in expression was seen both at the protein and mRNA level. Importantly, while all 8 cell lines with high EZH2 protein expression responded to SAHA treatment with average inhibition rate reaching 73.1%, three out of four cell lines with low EZH2 expression had a significantly lower response rate to SAHA inhibition with average inhibition rate 43.2% (P<0.0001). Additionally, altering the expression of EZH2 concordantly altered the sensitivity to SAHA i. e. forced increased expression of EZH2 increased the response to SAHA and vice versa.

Conclusion:
Our data suggest that EZH2 and HDAC expression are correlated in LUAD cell lines in human tissue microarrays and overexpression of both is a negative prognostic indicator. Additionally we show that increased EZH2 expression predicts for response to HDAC inhibitors and thus could serve as a biomarker for selecting LUAD patients with HDAC inhibitors.

Background:
Human bombesin receptors (BnR) are one of the most frequently over-expressed receptor families by human lung-cancers. It is known the activation of the classical members of the BnR family (GRPR, NMBR), causes a marked effect on cell-signaling and growth, often autocrine in nature, on lung-cancer cells. In addition, it has been discovered that the orphan receptor related to the BnR family, BRS-3, is widely distributed in central/peripheral normal tissues, and numerous tumors include lung-cancer cells. However, in contrast to the classical BnRs (GRPR, NMBR), BRS-3 has received little attention in lung-cancer in large part due to the fact its natural ligand is still unknown, and also because, until recently, the lack of specific pharmacological tools to study it. To address this, in this study, we examined the frequency of hBRS-3 expression in lung-cancer cells, and the effects of specific hBRS-3 activation on cell-signaling and cell-function (growth) in different lung-cancer cell lines.

Methods:
17 human lung-cancer (LC) cell lines were studied (9 NSCLC, 8 SCLC), as well as hBRS3 transfected H1299 and Balb 3T3 cells. The BRS-3 selective agonist, MK-5046 and selective BRS-3 antagonist, Bantag-1 were used. BnR expression was assessed by PCR using specific primers for hBRS3, hGRPR or hNMBR. Receptor activation was determined by assessing PLC and MAPK cascade activation using Western Blotting, and cytosolic Ca[2+] release. Proliferation was studied by clonogenic and [[3]H]-Thymidine assays, and EGFR transactivation was assessed using Western blotting.

Results:
Of the 17 LC cell lines, 9 (53%) express hBRS3 [H358, H460, H520, H720, H727, H69, H82, N417, H510], 14 (82%) express hGRPR [H28, H157, H358, H520, A549, H838, H1299, SK-LU-1, H720, H727, H69, H82, H345, H510] and 13 (77%) express hNMBR [H28, H157, H358, A549, H838, H1299, SK-LU-1, H720, H727, H82, H345, N417, H510]. MK-5046 stimulated PLC activation in 6/9 cells which express hBRS3 (H358, H720, H727, H69, H82, N417), and MAPK activation in all 9 hBRS3 cell lines. Cytosolic Ca[2+] increased with MK-5046 addition in all hBRS-3-containing cells, included Balb and H1299 transfected cells, except in H358 cells. Similarly, MK-5046 increased [3]H-Thymidine uptake in 5/9 cells (H460, H520, H720, H727, H82, H510), as well as in Balb and H1299 hBRS-3 transfected cells, and this increase was in a dose-response manner in H727, H69 and N417 cells. MK-5046 stimulated the clonal growth of N417 and H727 cells. MK-5046 addition to H358, H460, H727 (NSCLC) and H69, N417, H510 (SCLC) caused Tyr[1068] phosphorylation of the EGFR.

Conclusion:
These results show the orphan human BnR, hBRS-3 is present in more than one-half of human lung cancer cells. Furthermore, these receptors are functional with their activation effecting cell signaling (MAPK, PLC, Ca[2+]) and cell growth. Transactivation of EGFR is likely an important transduction cascade. These results suggest this orphan BnR, similar to classical BNRs, will be important to assess for growth effects and expression in human lung tumors, and its pharmacological inhibition may be a useful therapeutic approach.

Abstract not provided

Background:
In non-small cell lung cancer (NSCLC), activation of the phosphoinositide-3-kinase (PI3K)/mTOR pathway is common in tumors resistant to Epidermal Growth Factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). CC-115 (Celgene Corporation), an mTOR kinase inhibitor that targets both mTORC1 and mTORC2 as well as DNA-dependent protein kinase (DNA-PK), is currently under early clinical development. We evaluated CC-115 in combination with Erlotinib to overcome resistance to EGFR tyrosine kinase inhibition in non-small cell lung cancer (NSCLC) cell lines and xenografts in nude mice.

Methods:
In the present study we investigated whether CC-115 is able to increase the therapeutic effect of the EGFR TKI Erlotinib in several different NSCLC cell lines which exhibit intermediate or high resistance to EGFR TKIs: A549, H1975, H1650, HCC95, H2122 and H23. Mechanisms of inhibition were analyzed with assays for proliferation, apoptosis, and cell cycle progression. Cell signaling activity was analyzed using phospho-specific antibodies in Western blotting. Xenograft mice studies were performed to confirm the results in vivo.

Results:
CC-115 demonstrated anti-proliferative activity in NSCLC cell lines with various degrees of sensitivity as reflected in different IC50 values, ranging from 0.07 up to 6.9 mM. The anti-proliferative efficacy of Erlotinib was increased in the NSCLC cells synergistically by combination treatment with CC-115 with combination indices down to 0.04-0.2, indicating strong synergy. The synergistic, anti-proliferative effect of the combination treatment could be explained by increased cell cycle arrest and inhibition of signaling components in the mTOR pathway, especially 4E-BP1. In vivo studies in mice xenografts demonstrated a strong synergistic effect of the combination treatment of Erlotinib and CC-115.

Conclusion:
We demonstrate that the therapeutic effect of the EGFR tyrosine kinase inhibitor Erlotinib can be increased by simultaneous treatment with the mTOR kinase/DNA-PK inhibitor CC-115, justifying further clinical studies in lung cancer patients with primary or acquired resistance to EGFR TKIs.

Background:
In-frame insertions in exon 20 of EGFR are infrequent activating mutations in the tyrosine kinase domain that have decreased sensitivity to EGFR inhibitors and currently have no available targeted therapies. In vitro studies ectopically expressing some of the common insertions (3 to 21 bp between codons 762 and 770) show reduced sensitivity to EGFR tyrosine kinase inhibitors (TKIs). Non-small cell lung cancer (NSCLC) patients whose tumors harbor these mutations do not respond to EGFR kinase inhibitors. To date, there are no known patient-derived cell lines that harbor the EGFR exon 20 insertions that recapitulate patient insensitivity to EGFR TKIs. Here we report the isolation and characterization of a patient derived cell line with an EGFR exon20 insertion.

Methods:
The CUTO-14 cell line was derived from a malignant pleural effusion of a lung adenocarcinoma patient harboring the EGFR exon 20 insertion p.A767_V767dupASV after obtaining IRB-approved informed consent. PCR amplification of EGFR exon 20 and subsequent Sanger sequencing was performed on genomic DNA isolated from CUTO-14. H3255 (L858R) and HCC827 (exon 19 del) cell lines were used as controls because they harbor sensitizing EGFR mutations. Cell viability was evaluated by MTS proliferation assay. Phosphorylation status and signaling was analyzed by western blot and an EGFR phosphorylation array. For tumor xenograft studies, nude mice were injected with 1.5 x 10[6] cells in matrigel and evaluated weekly for tumor growth.

Results:
Genomic sequencing of CUTO-14 demonstrated that the cell line maintains the pA767_V767dupASV EGFR exon 20 insertion. CUTO-14 showed relative resistance to gefitinib inhibition compared to HCC827 and H3255 in ERK1/2 phosphorylation assays. CUTO-14 also demonstrated reduced sensitivity to gefitinib compared to HCC827 and H3255 in cell proliferation assays. Tumor formation was observed in mice after injection in nude mice.

Conclusion:
CUTO-14 cells represent a novel model for the investigation of therapeutic strategies for EGRF exon 20 insertions mutations. The cell line has the ability to develop tumors in vivo and importantly shows reduced sensitivity to EGFR TKIs mimicking the lack of response in patients with these mutations.

Background:
Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. We previously demonstrated that the basic helix-loop-helix transcription factor, Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in mouse models, and that inhibition of Twist1 in murine models and KRAS mutant NSCLC cell lines led to oncogene-induced senescence (OIS) and in some cases, apoptosis. Therefore, targeting the TWIST1 pathway represents an exciting and novel therapeutic strategy which may have a significant clinical impact.

Methods:
We used gene expression profiles from KRAS mutant human NSCLC cell lines following shRNA-mediated TWIST1 knockdown to perform connectivity map (CMAP) analysis to identify pharmacologic inhibitors of TWIST1. Growth inhibition was determined through the colony formation and MTS assays. Apoptosis (cl-PARP, active anti-C3) and OIS (SA-β-Gal) was assessed. Genetic (shRNA) and pharmacologic inhibition of the TWIST1-E2A pathway was performed. Lung tumor burden as well as levels of TWIST1 protein, apoptosis and proliferation were measured after treatment with harmine in the CCSP-rtTA/tetO-KrasG12D/Twist1-tetO7-luc(CRT) mice.

Results:
We found that several of our CMAP compounds had significant growth inhibitory effects in NSCLC cell lines. Interestingly, a family of related harmala alkaloids including harmine ranked highly in our CMAP analysis. We observed that harmine could inhibit growth in KRAS mutant NSCLC cell lines through the induction of OIS or apoptosis and phenocopied genetic inhibition of TWIST1. Remarkably, harmine treatment led to TWIST1 protein degradation as well as degradation of its binding partners, the E2A proteins, E12/E47. Furthermore, the growth inhibitory effects of the harmala alkaloids correlated with the ability to degrade TWIST1 and were independent of its ability to inhibit the DYRK kinases. In addition, we demonstrated that heterodimer formation of TWIST1/E12/E47 resulted in a reciprocal stabilization of each binding partner and that E12/E47 are required for TWIST1 mediated suppression of OIS and apoptosis. Importantly, we found that harmine preferential targets the TWIST1-E12 heterodimer for degradation and the growth inhibitory effects of harmine are in due in at least part to the ability to inhibit the TWIST1/E12/E47 heterodimer as overexpression of the E2A proteins can suppress harmine induced cytotoxicity. Finally, we have demonstrated that harmine treatment lead to Twist1 protein degradation and tumor growth inhibition in our Kras[G12D]/Twist1 murine model of lung adenocarcinoma. We are currently testing and designing structure analogs of the initial candidate agents to develop more specific and potent inhibitors of TWIST1.

Conclusion:
We have identified a novel TWIST1 inhibitor harmine that induces degradation of TWIST1 and its binding partners, E12/E47 and inhibits the growth of KRAS mutant NSCLC both in vitro and in vivo. Therefore, we believe that targeting the TWIST1-E2A pathway would be an effective therapeutic strategy. Since TWIST1 is essential not only for KRAS mutant NSCLC but more broadly for oncogene driven NSCLC, the development of this novel class of TWIST1 inhibitors could have a significant clinical impact.

Abstract not provided

Background:
Lung squamous cell carcinoma (SCC) is the second most common histological sub type of lung cancer and accounts for about 30% of all non-small cell lung cancers (NSCLC). We analyzed the NCDB, an oncology outcomes database administered by the American College of Surgeons and the American Cancer Society, to study the epidemiology, patterns of care, outcomes and temporal changes in incidence of SCC.

Methods:
The NCDB was queried from 1998 to 2011 for SCC using ICD-O-3 codes. Temporal changes in incidence were estimated in intervals (1998-1999, 2000-2003, 2004-2007, 2008-2011). The univariate association with covariates between SCC and other subtypes of NSCLC was assessed using Chi-square test or ANOVA. The univariate (UV) and multivariable analysis (MV) with OS were conducted by Cox proportional hazards model and log-rank tests. All statistical analyses were conducted using SAS Version 9.3.

Results:
A total of 435,358 pts with SCC were included in the analysis and accounted for 28% of all NSCLC pts in NCDB. Pt characteristics: median age 70 (18-90 yrs); males 64%; whites 87%; academic centers 27%; metro locations 78%; government insured 72%; Charlson/Deyo comorbidity score (CDS) 0 in 55% and ≥2 in 15%, and stage III/IV- 34/31%. Chemotherapy was used in 39% of pts, radiation in 46% and surgery in 32%. Approximately 19% of the pts did not receive any of the three treatments. Incidence of SCC decreased over time (35%, 28%, 26%, 27%) vs. increasing trend in non-SCC (65%, 72%, 75%, 72%); p<0.001). The trend was similar across all races and sex. SCC was associated with a higher co-comorbidity burden than non-SCC across all stages (CDS 0: 55% vs. 62%; CDS 1: 31% vs. 27%; CDS ≥2: 15% vs. 11%; p<0.001). SCC was associated with inferior 5 yr survival vs. non-SCC in all stages (stage I- 30% vs. 41%, stage II- 16% vs. 21%, stage III- 8.5% vs.10%, stage IV- 1.9% vs. 2.5% respectively; p<0.0001). The 1 yr survival in stage IV SCC is 19.6% vs. 22.2% in non-SCC (p<0.0001). Males had worse survival (HR 1.11 (1.09-1.13; p<0.001). Pts at community centers had worse survival vs. academic centers (HR 1.27 (1.23-1.30; p<0.001). An increasing trend in chemotherapy use was observed (31% in 1998 to 43% in 2011) vs. a decreasing trend in use of radiation (52% in 1998 to 46% in 2011) and surgery (32% in 1998 to 27% in 2011). Chemotherapy was received by 48% of patients with stage IV SCC. Chemotherapy use across other stages: 0/I- 18%, II- 46%, III- 60%. Males were more likely to receive any treatment (OR 1.12 (1.08-1.15); p<0.001). Pts that received any treatment had significantly better 5 year survival than those who did not receive any (20.3% vs. 3.3%, p<0.0001)

Conclusion:
SCC accounted for 28% of all cases of NSCLC in the United States, was associated with higher comorbidities and a significantly worse survival compared to non-SCC of the lung. Chemotherapy was used in only 48% of pts with stage IV SCC.

Background:
Lung cancer is the number one cause of cancer-related death worldwide; with the incidence of non-small cell lung cancer (NSCLC) risen dramatically, the importance of understanding the influence of comorbidities and their treatments takes a great importance. The aim of this study was to investigate the effect of statins and metformin on survival in patients with NSCLC.

Methods:
We reviewed the records of all patients diagnosed with NSCLC at our institution from 2011 to 2013. Demographics, tumor characteristics, comorbidities, statin/ metformin use and survival were analyzed. Cox regression was used for multivariate analysis.

Results:
A total 205 patients were studied. Median age at diagnosis was 65 years (40-91), there were more males than females (56% vs. 44%, p<0.01. 74% (152) were current or former smokers with average 40 pack years (5-60). The Median BMI was 26.5kg/m2 (18-44), ECOG status was 1 (0-4) and serum creatinine of 1.0 (0.4-3.5). Regarding comorbidities: 48% (98) had hypertension, 45% (93) hyperlipidemia, 22% (44) COPD and 19% (39) diabetes mellitus. At diagnosis, 66% (136) of the patients had stage III/IV disease vs. 25% (69) with stage I/II, p<0.0001. Adenocarcinoma was the most common histologic subtype (61%), followed by squamous cell carcinoma (27%). 42% received surgery, 80% systemic chemotherapy and 32% radiation. Median survival was 1520 days (95% CI: 1310-1765). 42% (87) of the patients were taking statins and 13% (27) metformin. About the statin use, 58% of the patients were using statins for 25-48 months, 12% for >48 months, 11% for 13-24 months and 18% for less than 12 months. Female gender (OR: 1.98, p<0.001), age<65 years (OR: 0.89, p<0.01) and statin use (OR: 0.78, p<0.02) were independent predictors of survival by multivariate analysis. Metformin use was not a predictor of survival by univariate or multivariate analysis in this group of patients.

Conclusion:
In our cohort, we observed that almost half of the patients had hypertension and hyperlipidemia with statin use been a significant predictor of survival. Statins are one of the mostly widely prescribed drugs in the US; their proven anti-inflammatory effects may play a role in inhibiting cancer growth. However, the exact mechanisms by which statins inhibit cancer proliferation remains unclear. While other observational studies have looked at statins and risk of developing cancer, we specifically looked at the effect of statins on survival in patients on statin therapy prior to cancer diagnosis. More studies are needed to enhance our understanding of the effect of statins on lung cancer.

Background:
Alterations in glucose metabolism and appetite stimulating hormones have been correlated with inflammation but there is little information on frequency and prognosis in newly diagnosed stage IV non-small cell lung cancer (NSCLC) This study objective was to identify associations of circulating biomarkers of glucose metabolism and inflammation with prognosis in pre-treatment sera from stage IV NSCLC patients selected for platinum doublet based chemotherapy.

Methods:
Pretreatment serum from 118 Pts with frontline stage IV NSCLC were evaluated with the Bio-Plex Pro Human Diabetes Assay panel (adiponectin, adipsin, c-peptide, ghrelin, gastrin inhibitory peptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon, IL-6, insulin, leptin, Plasminogen activator inhibitor-1, resistin, TNFα, vistatin) and HSTCMAG-28SK | MILLIPLEX MAP Human High Sensitivity T Cell Panel - Immunology Multiplex Assay (Fractalkin, GM-CSF, IFNγ, IL-1 β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17A, IL-21, IL-23, ITAC, macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-3α, TNFα) on a FlexMAP 3D system (Luminex Corp.). Pts were treated with standard platinum doublets based chemotherapy. Associations of biomarkers with progression free and overall survival (PFS,OS) outcomes were assessed using multivariate Cox PH analyses.

Results:
Most patients had metabolic levels below the prognostic threshold. However, high levels of insulin, GIP, glucagon, visfatin, ghrelin, GLP-1 were significantly associated (p<0.05) with shorter PFS. Low levels of adipisin (deficiency of which is associated with obesity) was associated with shorter PFS (p=.0185). High levels of pro-inflammatory markers: ITAC, GM-CSF, Fratalkine, INF-ϒ, IL-12p70, IL-13, IL17A, IL-4, IL-23, IL8.4, MIP-α, MIP-1 were also associated with poor PFS (p<0.05) (See Table I for more details on select biomarkers) High levels of these endocrine markers (except insulin and GIP) were associated with shorter OS as were ITAC, GMCSF, IL12p70, IL-13, IL4, IL23, IL5 (p<0.05). Table I. Biomarker correlation with progression free survival

Marker	Cutoff-pg/mL	N <	N >	Median PFS <	Median PFS>	Logrank p
Insulin	1004.9	82	36	6.08	4.04	0.026161
Glucagon	361.2	110	8	5.46	1.71	0.010219
Visfatin	8298.3	109	9	5.65	1.45	8.77E-06
Ghrelin	2897.2	104	14	6.02	2.12	0.009423
GLP.1	268.8	109	9	5.65	1.97	0.000618
ITAC	104.7	99	19	5.82	2.96	0.012529
Fractalkine	271.7	97	21	6.08	3.16	0.0067
IL.12.p70.	17.0	109	9	5.65	3.16	0.010631
IL.13	14.9	105	13	5.82	2.76	0.001533
IL.17A	49.4	102	16	5.82	3.65	0.004862
IL.4	66.1	104	14	6.02	2.96	0.000917
IL.8.4	3.0	25	93	12.8	4.8	0.008985

Conclusion:
Imbalances in the glucose metabolism pathway and increased levels of pro-inflammatory circulating markers were uncommon but consistently associated with a poor prognosis in stage IV NSCLC patients early in their treatment cycle. Alterations in these systems have been associated with cancer cachexia and may be targets for intervention in improving prognosis for select patients with NSCLC.

Background:
Previous population-based studies have shown an association between metformin use and improved survival among diabetic patients with lung cancer. We sought to analyze the effect of diabetes and its treatment in terms of survival in Mexican patients with lung cancer treated at a single institution

Methods:
1106 patients were included. Outcomes were compared between patients with (n=186) and without diabetes (n=920). Characteristics associated with antidiabetic treatment and with proper glycemic control (defined as mean plasma glucose <130mg/dL) were examined. Overall survival (OS) among the different patient populations was analyzed using Kaplan-Meier curves and multivariate analysis was used to determine the influence of patient and tumor characteristics on survival

Results:
OS for the entire population was 18.3 months (95% CI 16.1-20.4). There was no difference in OS between diabetic and non-diabetic patients (18.5 vs 16.4 months, p = 0.62). Diabetic patients taking metformin had a superior OS than those taking other antidiabetic treatment (25.6 vs 13.2 months, p = 0.001), and those with proper glycemic control had a better OS than those without proper glycemic control (40.5 vs 13.2 months, p<0.001). Both the use of metformin (HR 0.57 p = 0.017) and proper glycemic control (HR 0.40, p =0.002) were significant protective factors in the diabetic patient population.

Conclusion:
Proper glycemic control and metformin use have a beneficial effect on the survival of patients with diabetes and lung cancer. Studies using metformin in lung cancer should include measures of proper glycemic control as fundamental variables.

Abstract not provided

Background:
Modern systemic treatment options for advanced NSCLC have largely been established from clinical trials (CTs). It is estimated that less than 10% of cancer patients enter a CT, but this subgroup drives oncology practice and impacts treatment decisions for other cancer patients. The advantage of CTs comes from solid internal validity and stringent methodology. Nonetheless, the generalizability of CTs could be questioned due to the high selectivity of eligibility criteria. We investigated clinical trial eligibility in an unselected NSCLC population

Methods:
With ethics approval, a retrospective chart review was performed of patients with de novo advanced NSCLC assessed by medical oncologists at a large academic cancer centre, serving a mixed urban and rural population, between September 2009 and September 2012. Data collected included patient demographics, stage, performance status, histology, treatment details and outcome. Two sets (A and B) of arbitrary eligibility criteria were created using common criteria from phase 3 CTs. These criteria were applied to this cohort to identify the proportions of patient who would hypothetically qualify for CT enrollment. Criteria A required: ECOG 0 or 1, absence of brain metastases, Creatinine < 120 and the absence of second malignancy. Criteria B, allowing broader inclusion, only required ECOG 0-2 and Creatinine < 120. We investigated survival among eligible/ineligible and treated/untreated patients.

Results:
528 patients were included: 55% male; 50% ECOG 0-1; 58% adenocarcinoma, 22% squamous cell; 7% stage IIIB and 93% stage IV. Using the strict CT criteria (A), only 144 (27%) patients were considered eligible. Of those, 79% actually received systemic therapy. From 384 patients who would have been ineligible for the CT, 178 patients (46%) still received systemic therapy. There was a trend to longer median overall survival (mOS) in the eligible treated compared to eligible non-treated patients (11.6 vs 8.1 months p=0.12). mOS was significantly longer in the non-treated eligible cohort compared to the non-treated ineligible cohort (8.1 vs 3.8 months p=0.003). The eligible treated and non-eligible treated had similar mOS ( 11.6 vs 10.2 months, p= 0.10). When less strict eligibility criteria (B) were applied, 343 patients (65%) would have been eligible, of whom 240 patients (70%) actually received systemic therapy. From the remaining ineligible 185 patients, only 51 (28%) received treatment. The mOS was similar in the treated patient whether eligible or ineligible (10.9 vs 10.1 months, p=0.57). As seen in criteria A, significantly longer mOS was observed in the eligible untreated compared to the ineligible untreated ( 4.9 vs 3.5 months p<0.001).

Conclusion:
While clinical trial criteria restrict study entry to the fittest patients, these results suggest that they do not reflect the broader patient population, as many ‘ineligible’ patients received therapy. Extrapolation of treatment paradigms to non-trial eligible populations is common, and may be reasonable based on these results. We observed similar survival among treated patients, whether trial eligible or not. This suggests that clinical judgement is more important than trial eligibility. In order to broaden trial participation, we could hypothesize that trial eligibility criteria could be relaxed.

Background:
CNS metastases are common in NSCLC, yet clinical trials of new drugs in NSCLC have widely varying inclusion and exclusion criteria in relation to CNS disease. CNS disease that has received local therapy may be dormant, confounding any subsequent drug benefit, whereas untreated CNS disease may reduce PFS if CNS and systemic drug exposure differs. Recently, RANO guidelines propose explicitly explored activity in CNS disease within solid tumor drug trials. The true extent of variation in CNS related enrollment criteria in NSCLC clinical trials has not been documented before.

Methods:
ClinicalTrials.gov was interrogated on September 11, 2014 looking for interventional drug trials including advanced NSCLC. The following characteristics were extracted: 1) trial phase; 2) experimental arm therapy (chemotherapy, targeted therapy, immunotherapy, anti-angiogenic); 3) location (US, International only, US + International); 4) sponsor (Industry, University/IIT, Cooperative Group, NCI); 5) CNS disease allowance (strict exclusion, allowed after local treatment (surgery/radiation), unrestricted/untreated disease allowed). Industry sponsorship was divided into ‘large pharmaceutical’, (top decile by number of sponsored trials) and ‘small pharmaceutical’ (lower 9 deciles). Exclusion of CNS metastasis was treated as a binary variable and grouped as ‘strict exclusion’ vs. ‘allowed CNS metastasis’ (‘allowed with treatment’ and ‘allowed untreated’). Univariable and multivariable logistic regression models were fit to test the association between exclusion of CNS metastasis and trial characteristics. Statistical significance was set at 0.05 with no adjustment for multiple testing.

Results:
Of 735 trials involving NSCLC, 325 (44%) were excluded from analysis mostly because of allowance of early stage NSCLC (50%, n=164), or no active therapy inclusion (45%, n=146). In the remaining 406 trials, patients with CNS metastases were excluded in 58 (14%), allowed after local treatment in 165 (41%), and allowed with no prior treatment in 104 (26%). CNS criteria were not referenced in the available information in 79 (19%) trials which were excluded from further analysis. On univariable analysis, the odds of CNS metastasis exclusion on trial were significantly lower in trials with vs. without targeted therapy (OR 0.44, 95% CI: 0.25-0.78, p=0.005) and significantly higher in trials with vs. without immunotherapy (OR 2.13, 95% CI: 1.06-4.28, p=0.04). No other univariable associations were significant. In multivariable analysis, after adjustment for all other factors, only trials located at international only vs. US only sites had greater odds of exclusion of CNS metastasis (OR 1.64, 95% CI 0.84-3.22; p=0.03).

Conclusion:
Although univariable analysis suggests class of agent may influence trial design, in multivariable analysis trial location was the only variable associated with strict exclusion of CNS metastases. This raises the possibility of exclusion based on historical/cultural rather than scientific factors. With 18% of trials (58/327) excluding all CNS disease and 50% (165/327) only allowing CNS disease if previously treated, less than a third of NSCLC trials permit unequivocal assessment of CNS activity (104/327). Given the high frequency of CNS disease in NSCLC, sponsors should consider consciously tailoring trial designs to more explicitly explore efficacy in this patient population.

Background:
The American Society of Clinical Oncology (ASCO) recently developed a set of recommended targets for clinically meaningful outcomes to encourage patients, advocates and investigators to expect more of clinical trials in the future and to encourage investigators to design trials and select agents that have the most promise. A systematic literature review was conducted to identify where and how research has successfully met the bar for meaningful improvements in NSCLC patient survival.

Methods:
A systematic search strategy was implemented in MedLine and EMBASE to identify randomized phase III trials reporting overall survival (OS) outcomes from 1974-present. Eligible studies were those that reported OS data in terms of a hazard ratio (HR) or median OS. Patients in the eligible randomized trials were NSCLC patients treated in the first (1L) or post-first line (2L+) setting. Data were extracted related to study population, histology, interventions, and survival from trials of patients with advanced or metastatic disease. All eligibility determinations and extracted data were reviewed by two researchers for accuracy.

Results:
The search strategy identified 2051 articles that were reviewed for eligibility. Of these, 245 were eligible for inclusion. 198 (80.8%) studied 1L, 45 (18.4%) 2L+, and 2 investigated both lines. Of all articles, 51 (20.8%) found significant improvement in survival (Figure 1). Of these, 45 (88.2%) were 1L (41 studies). 14/41 (34.1%) were terminated early or had major protocol changes during study enrollment, and 5/41 (12.2%) reported inconsistent outcomes. In significant 1L studies, the median OS improvement ranged from 0.3-7.0m and HRs ranged from 0.35 to 0.87. The 4 2L+ positive studies, OS improvement ranged from 1.4-2.8m and HRs from 0.70-0.86. Of all 198 1L publications, 12 studies met the significant HR <0.80 bar and an additional 12 studies reported statistically significant gains of ≥2.5m OS. Figure 1. Publications reporting significant improvement in survival Figure 1



Conclusion:
There is a need to improve survival outcomes for patients in NSCLC. In 1L, few studies would have met the ASCO target. Only four 2L+ trials were identified with significant improvements in NSCLC. The results of this systematic literature review inform the interpretation of research and the new bar recommended for meaningful outcomes.

Background:
There are general standards for common clinical trial data elements submitted to US Food and Drug Administration (FDA) in a new drug application (NDA) or biologic license agreement (BLA). There is, however, considerable heterogeneity in the structure of data elements that are unique to a particular therapeutic area. As part of an effort organized by Coalition for Accelerating Standards and Therapies (CFAST), we developed specifications for efficacy data standardization in lung cancer.

Methods:
Using FDA guidance documents, NDA and BLA reviews, sample case report forms, and clinical trial datasets, we identified data elements in lung cancer clinical trials that are essential in evaluating the efficacy of new drugs and biologics. We constructed a concept map outlining the data elements and specifying their relational structure.

Results:
Data elements were captured under two main categories: efficacy endpoints (Figure 1) and covariates (Figure 2). Efficacy endpoints consist of overall survival, progression-free survival, objective response rate, duration of response, and disease-free survival. All lesions are assigned an organ-specific code. Data on tumor kinetics are captured as continuous variables supporting precise estimation of response while capturing all the requirements of Response Evaluation Criteria in Solid Tumors, version 1.1. Covariates were divided into disease and patient characteristics. Disease characteristics include specifications on molecular, immunohistochemical, and histological classification of tumors and detailed staging variables. Molecular definitions follow established nomenclature and include information on mutation subtypes as indicated. Patient characteristics include information on prior therapy, race and ethnicity. Figure 1 Figure 2





Conclusion:
The data elements we have identified (Figures 1 and 2) include concepts not adequately captured in current data standards and highlight important regulatory needs for the efficacy evaluation of new drugs and biologics in lung cancer. These specifications will be integrated with CFAST’s efforts to promote the development of lung cancer-specific data standards.

Abstract not provided

Background:
In collaboration with the FDA, the Patient-Reported Outcome (PRO) Consortium’s Non-Small Cell Lung Cancer (NSCLC) Working Group (WG) has completed the initial development of a new PRO measure to assess symptom-related treatment benefit in clinical trials of advanced NSCLC to support labeling claims.

Methods:
Symptoms relevant to NSCLC patients were identified from the literature. This was followed by concept elicitation interviews conducted with patients at six US sites. Interview transcripts were coded using Atlas.ti software and concepts were grouped by similar content and subsequently reviewed by the NSCLC WG members and an expert panel in order to identify the symptoms most relevant for assessing treatment benefit. Preliminary items were generated and combined into a draft measure for cognitive testing with NSCLC patients for both item refinement and for assessing measurement equivalence between the original paper format and an electronic PRO (ePRO) data collection format (i.e., tablet).

Results:
For concept elicitation, the 51 patients interviewed had a mean age of 64.9 years [range 46-86], 51% were female, and 75% were white (non-Hispanic). Current NSCLC staging was: Stage I (12%), III (37%), and IV (51%). A total of 19 (37%) were treatment-naïve, 18 (35%) had received first-line treatment only, and 14 (27%) had received second- or third-line treatment. The most commonly expressed symptom was fatigue, described by patients as tiredness, lack of energy, tiring easily, and weakness. Other symptom concepts expressed included general pain, chest pain, cough, shortness of breath, difficulty breathing, appetite change, and coughing up blood. Items were drafted to assess either symptom frequency or severity for nine distinct symptoms using a 7-day recall period. Cognitive interviews were conducted in 3 waves to support iterative refinement. 20 additional NSCLC patients participated [mean age 65.2 years (range 44-83); 40% female; 75% white (non-Hispanic); 50% Stage III and 50% Stage IV]. During cognitive interviews, an 11-point numeric rating scale (NRS) and a 5-point verbal rating scale (VRS) were tested. Results indicated the 5-point VRS was better understood than the 11-point NRS. Further item refinement resulted in a 7-item measure covering pain (2 items: pain in chest, pain elsewhere), cough, shortness of breath, fatigue (2 items: energy, tiredness), and appetite. Patients reported no differences between tablet and paper data collection formats in regard to how they would interpret and respond to the items.

Conclusion:
NSCLC symptoms elicited from patients, across varied disease stages and treatments, were concordant with the pathophysiology of NSCLC. A new symptom-based PRO measure is being developed in accordance with the FDA’s PRO Guidance. The content is supported by existing literature, patient-reported experiences, and expert opinion. The draft measure (i.e., Non-Small Cell Lung Cancer Symptom Assessment Questionnaire [NSCLC-SAQ]) has been programmed onto an ePRO tablet for quantitative testing. Once complete, the NSCLC-SAQ and supporting evidence will be submitted to the FDA for its qualification as an efficacy endpoint measure to quantify treatment benefit for product evaluation and labeling.

Background:
The evolution of targeted therapy in non-small cell lung cancer (NSCLC) has led to growing complexity of clinical research and a heightened expectation of clinical benefit for participants. Clinical trials in NSCLC increasingly require mandatory tumour samples or research biopsies, both potential barriers for trial participation. We assessed the impact of performing research biopsies in advanced NSCLC on clinical trial enrollment.

Methods:
We conducted a retrospective chart review of patients with advanced NSCLC evaluated for systemic therapy clinical trials at the Princess Margaret Cancer Center from January 2007 to March 2015.

Results:
Of 55 clinical trials reviewed, 38 required tumor samples for enrolment. Six mandated fresh tumor biopsies, whereas archival samples were permitted for 32 trials. All studies were linked to investigational therapy except one trial of molecular profiling not linked to an investigational treatment. Confirmation of a pre-specified biomarker was required in 23 trials in order to receive investigational treatment. Trial participation was offered to 640 patients at 940 unique study encounters, with some patients enrolling in multiple trials. Of 549 encounters where study treatment was offered, 60% proceeded to receive study treatment. Those considering trials without mandatory tissue requirements were more likely to proceed to study enrolment than those considering trials with these requirements (83% vs. 55%, p<0.0001). Those considering trials permitting use of archival tissue were more likely to begin study treatment than those considering trials mandating fresh research biopsies (59% vs. 38%, p=0.0007). For trials requiring current tumour samples, 127 research biopsies were performed. Participants proceeded to study treatment in 51% of these encounters. Study treatment was not offered for the remaining encounters due to lack of the pre-specified biomarker (28%), insufficient biopsy tissue (6%) or non-biopsy related exclusion criteria (15%). Among all 549 trial encounters, the most common barriers to trial enrollment included lack of the pre-specified biomarker (35%), withdrawal of consent (20%), other study exclusion criteria (16%), insufficient biopsy tissue (10%), deteriorating clinical status (10%) and death (5%). Of 391 encounters for the molecular profiling trial, 72% successfully completed molecular profiling. Twenty-two percent had insufficient tissue for analysis and 3% died prior to completion of molecular profiling.

Conclusion:
With the evolution of personalized medicine, a growing number of NSCLC trials require tumour tissue for treatment eligibility. This has emerged as a significant barrier to clinical trial enrollment. Potential solutions include routine tissue banking at diagnosis, facilitating use of available diagnostic samples (e.g. fine needle aspirates) for trials, development of circulating DNA assays for trials, and more resources for timely tissue acquisition.

Background:
Clinical management of lung cancer and personalized cancer therapy have undergone major advances and paradigm shifts in recent years. Repeat tumor biopsy (RTB) at disease progression is not only used for diagnostic confirmation in lung cancer but also has recently been increasingly adopted to profile tumor biomarkers and identify drug resistance mechanisms. However, information on safety and clinical consensus on the use of RTB remain lacking.

Methods:
The aim of this study is to review RTB patterns and safety in advanced lung cancer patients at Cleveland Clinic and its impact on treatment (Rx) decisions. Patients who were diagnosed and underwent RTB for suspected progressive disease between 2007-2013 were included in this retrospective study. Statistical analysis is primarily descriptive.

Results:
The study involved a total of 184 (56% male) patients. Median age at diagnosis was 65Y (21-87). 100 (54%) were treated initially with single modality (Surgery = 41; Chemo = 33; Radiation = 17; targeted therapy = 9) and 83 (45%) with multimodality Rx (2-modality = 57, 3-modality = 26), 1 (1%) unknown. Number of RTB per patient: 1 in 66.3% (n=122), 2 in 20.1% (n=37), 3 in 11.4% (n=21), and 4 in 2.2% (n = 4). The most common procedure employed at 1st RTB was bronchoscopy (44.6%, n = 82), followed by CT-guided biopsy (bx) (20.7 %, n=38), surgery (10.3%, n=19), excision bx (8.2%, n=15), fine needle aspiration of skin & lymph node (LN) (7.6%, n=14), ultrasound guided bx (5.9%, n=11) & others (2.7%, n=5). Lung was the most commonly rebiopsied site (46%) followed by LN (15%). Procedure-related complications were reported in 13 of 181 (7.2%) pts at 1st RTB (data missing in 3 pts), 3 of 61 (4.9%) at 2nd RTB, 1 of 25(4%) at 3rd RTB, and 0 of 4 (0%) at 4th RTB. The 17 (6.2%) complications are shown in the table below. Histologic change was seen in 13 cases, including adeno-to-squamous carcinoma (at erlotinib resistance) and vice-versa, and non-small cell to small cell histology. The T790M-EGFR mutation was noted in 6 cases, the PIK3CA mutation in 1, and a change in ALK translocation status in 3. Medical decision-making was impacted in 16% of cases. Further molecular and genomic analysis of selected cases is in progress.

RTB Complications

Complications	N=17 (%)
Bleeding without hemodynamic compromise	6 (35)
Bleeding requiring transfusion	1 (6)
Pneumothorax	5 (29)
Hemodynamic instability after premedication	1 (6)
Cerebral salt wasting	1 (6)
Tracheaoesophageal fistula	1 (6)
Severe cough	1 (6)
Incomplete procedure	1 (6)
Deaths	0 (0)

Conclusion:
Our study results show that RTB can be safely performed in lung cancer patients using standard techniques and can impact lung cancer diagnosis and Rx decision-making.

Background:
The National Lung Matrix Trial is a flagship trial in the United Kingdom being the first to combine the development of a technology platform that screens for multiple genetic aberrations in tumours with testing of multiple novel genetic-marker-directed drugs. The trial is focused on patients with advanced non-small cell lung cancer and currently includes 8 different drugs and 23 different drug-biomarker combinations. The aim of statistical analysis is to determine whether there is sufficient signal of activity in any drug-biomarker combination to warrant further investigation and this paper evaluates the novel statistical design that has been implemented.

Methods:
The primary outcome measure representing signal of activity is best objective response rate (ORR) in most cases and progression-free survival (PFS) in others. Each drug-biomarker combination could be considered as a single arm phase II trial and could have been designed using standard statistical approaches such as Simon’s two stage design. However, a Bayesian adaptive design was chosen because it gives a more realistic approach to decision-making in this complex setting. It has flexibility to make conclusions without fixing the exact sample size which will be important with the uncertainty around the prevalence of each biomarker. The design allows early stopping of recruitment to any drug-biomarker combinations that do not show sufficient promise at an interim analysis to warrant continuation. It also allows, when appropriate, for information about the primary outcome measure to be shared across different biomarker cohorts within any single drug to aid decision-making. Decision-making is based on the posterior probability distribution for the primary outcome measure, given the observed data and any prior knowledge, calculated using bayesian conjugate analysis. For ORR, the critical threshold which defines a signal of activity is 30% for single drugs and 40% for combinations and for PFS the critical threshold is median 3 months. The interim and final sample sizes were selected to ensure the design achieved pre-defined desirable operating characteristics.

Results:
For both ORR and PFS as primary outcomes, sample sizes of 15 and 30 patients per drug-biomarker cohort for interim and final analyses respectively were selected as giving the required operating characteristics. Simulations of trials for single drugs with ORR as the primary outcome showed that this would ensure a high probability (82%) of correctly stopping early when the true ORR was only 10% and a high probability (90%) of correctly recommending further investigation when the true ORR was as high as 40%. Operating characteristics for the combination drug arms were similar. Operating characteristics for trial arms with PFS as the primary outcome are dependent on the recruitment rate and give high probabilities (>95%) of stopping early when the true median PFS is only 1 month and high probabilities (>80%) of recommending further investigation when the true median PFS is as high as 4 months.

Conclusion:
The Bayesian adaptive design with 15 and 30 patients per drug-biomarker cohort for interim and final analyses respectively gives a flexible, efficient design with good operating characteristics to investigate multiple genetic-marker-directed drugs within a single phase II trial.

Abstract not provided

Abstract:
DNA methylation changes in lung cancer: Defining functional events and use of cancer specific changes for early detection. Epigenetic alterations in lung cancer represent early changes which are associated with tumor initiation and progression. Alterations in DNA methylation include the global loss of DNA methylation in non-promoter region and selective CpG island promoter region methylation leading to gene silencing. Previous studies have focused on individual loci identified through candidate gene approaches. However, recent improvements in technology allow the assessment of genome wide patterns of DNA methylation. The comprehensive genome wide analysis of molecular changes in cancer completed by The Cancer Genome Atlas (TCGA) includes determination of DNA methylation using the Illumina Infinium 450K array. Initial analyses have primarily focused upon defining methylation subtypes. However, this data can be used to determine novel cancer specific events which are associated with transcriptional silencing to identify candidate driver epigenetic alterations. New promoter region DNA methylation changes leading to transcriptional silencing are found in multiple signaling pathways critical for lung cancer development. In addition, a search for common tumor specific DNA methylation provides new markers for early detection strategies. These novel biomarkers can be combined with novel methods developed with extremely sensitive assays for the detection of hypermethylated DNA sequences. By combining these more sensitive methods of detection with highly prevalent methylation changes in lung cancer, utrasensitive detection of tumor specific changes in DNA methylation in blood and sputum samples is possible. This molecular detection can complement CT screening to address the important issue of early detection of lung cancer.

Abstract:
I will discuss opportunities and future directions in profiling lung cancer using mass spectrometry based proteomic technologies. This includes a proposal to perform deep integrated proteo-genomics studies on cancer subtypes to produce more complete views of the tumor architecture, allow contextual understanding of major drug targets, and discover new lung cancer subtypes. Alterations in the genomes of cancers ultimately get integrated and produce a cancer proteome that can be analyzed using modern state of the art mass spectrometry proteomic tools. For example, signaling pathways and networks involved in cancers are built using a ‘parts list’ of the cancer genome, such as through integrating mutated genes, genes altered through differential expression (i.e. copy number gain or loss), and through regulation by micro-RNA molecules. DNA sequencing-based atlases exist for major tumors allowing ‘part lists’ for cancers; however, these atlases lack integration with expressed proteomes and signaling architectures. By taking into account all these alterations in the cancer genome, cancer proteomics can annotate and prioritize proteins and pathways important for cancer growth and survival. Furthermore, microenvironmental influences, known to be important in drug response, are lacking from these DNA based studies. Proteomics can inform about active pathways driving cancers and lead to novel combination therapy approaches for targeting complex oncogenic networks. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly used to study cancer proteomes. This includes examining the ‘expressed proteome’ through shotgun proteomics, global signaling by annotating key post-translational events (phosphorylation, acetylation, ubiquination) events in cancers or assembling protein-protein interaction data that yield network views of cancer. This allows unbiased and global views of signaling events in cancer thus offering complementary views of cancer biology that are not considered by sequencing of genes or gene expression. By integrating DNA-RNA-proteome-network type data, the co-existing driver processes instilled by the genome that either surround or act in parallel to drug targets can be mapped directly onto cancer molecular machines that drive cancer progression and response to therapy. Discovery proteomics has become a widely used tool in our laboratory. This approach provides an unbiased view of the components in a sample, supporting the testing of multiple hypotheses and generating new leads. I will discuss examples integrating complementary mass spectrometry approaches to build molecular snapshots of cancer proteomes, including phosphoproteomics in tumors related to drug resistance (1, 2), drug affinity selection of proteins and identification of drug targets using mass spectrometry (3-6), and protein-protein interaction mapping(7-9). Literature Cited: 1. Yoshida T, Zhang G, Smith MA, Lopez AS, Bai Y, Li J, Fang B, Koomen JM, Rawal B, Fisher KJ, Chen YA, Kitano M, Morita Y, Yamaguchi H, Shibata K, Okabe T, Okamoto I, Nakagawa K, Haura EB. Tyrosine phosphoproteomics identified both co-drivers and co-targeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer. Clin Cancer Res. 2014. doi: 10.1158/1078-0432.CCR-13-1559. PubMed PMID: 24919575. 2. Bai Y, Kim JY, Watters JM, Fang B, Kinose F, Song L, Koomen JM, Teer JK, Fisher K, Chen YA, Rix U, Haura EB. Adaptive Responses to Dasatinib-Treated Lung Squamous Cell Cancer Cells Harboring DDR2 Mutations. Cancer Res. 2014;74(24):7217-28. doi: 10.1158/0008-5472.CAN-14-0505. PubMed PMID: 25348954. 3. Remsing Rix LL, Kuenzi BM, Luo Y, Remily-Wood E, Kinose F, Wright G, Li J, Koomen JM, Haura EB, Lawrence HR, Rix U. GSK3 alpha and beta are new functionally relevant targets of tivantinib in lung cancer cells. ACS Chem Biol. 2014;9(2):353-8. doi: 10.1021/cb400660a. PubMed PMID: 24215125; PubMed Central PMCID: PMC3944088. 4. Gridling M, Ficarro SB, Breitwieser FP, Song L, Parapatics K, Colinge J, Haura EB, Marto JA, Superti-Furga G, Bennett KL, Rix U. Identification of kinase inhibitor targets in the lung cancer microenvironment by chemical and phosphoproteomics. Mol Cancer Ther. 2014;13(11):2751-62. doi: 10.1158/1535-7163.MCT-14-0152. PubMed PMID: 25189542; PubMed Central PMCID: PMC4221415. 5. Li J, Rix U, Fang B, Bai Y, Edwards A, Colinge J, Bennett KL, Gao J, Song L, Eschrich S, Superti-Furga G, Koomen J, Haura EB. A chemical and phosphoproteomic characterization of dasatinib action in lung cancer. Nat Chem Biol. 2010;6(4):291-9. doi: 10.1038/nchembio.332. PubMed PMID: 20190765; PubMed Central PMCID: PMC2842457. 6. Chamrad I, Rix U, Stukalov A, Gridling M, Parapatics K, Muller AC, Altiok S, Colinge J, Superti-Furga G, Haura EB, Bennett KL. A miniaturized chemical proteomic approach for target profiling of clinical kinase inhibitors in tumor biopsies. J Proteome Res. 2013;12(9):4005-17. doi: 10.1021/pr400309p. PubMed PMID: 23901793; PubMed Central PMCID: PMC4127982. 7. Smith MA, Hall R, Fisher K, Haake SM, Khalil F, Schabath MB, Vuaroqueaux V, Fiebig HH, Altiok S, Chen YA, Haura EB. Annotation of human cancers with EGFR signaling-associated protein complexes using proximity ligation assays. Sci Signal. 2015;8(359):ra4. doi: 10.1126/scisignal.2005906. PubMed PMID: 25587191. 8. Li J, Bennett K, Stukalov A, Fang B, Zhang G, Yoshida T, Okamoto I, Kim JY, Song L, Bai Y, Qian X, Rawal B, Schell M, Grebien F, Winter G, Rix U, Eschrich S, Colinge J, Koomen J, Superti-Furga G, Haura EB. Perturbation of the mutated EGFR interactome identifies vulnerabilities and resistance mechanisms. Mol Syst Biol. 2013;9:705. doi: 10.1038/msb.2013.61. PubMed PMID: 24189400; PubMed Central PMCID: PMC4039310. 9. Haura EB, Muller A, Breitwieser FP, Li J, Grebien F, Colinge J, Bennett KL. Using iTRAQ combined with tandem affinity purification to enhance low-abundance proteins associated with somatically mutated EGFR core complexes in lung cancer. Journal of Proteome Research. 2011;10(1):182-90. Epub 2010/10/16. doi: 10.1021/pr100863f. PubMed PMID: 20945942; PubMed Central PMCID: PMC3017669.

Abstract not provided

Abstract:
Study of genomics, epigenomics and proteomics may contribute to an understanding aetiology, prevention, early diagnosis, classification, treatment selection, and novel trial design in lung cancer. The clinical material available for analysis ranges from tumour biopsy to pleural fluid, bronchoalveolar lavage, saliva and even urine. The available techniques are in many cases sensitive (including PCR and mass spectrometry (MS)), and specificity can be optimised especially with reference to normal material such as germline DNA. The omic landscape of lung cancer has been extensively characterised (The Cancer Genome Atlas Research Network, 2014). This provides insight into disease biology via SNP/exome/whole genome sequencing, CpG DNA methylation, mRNA sequencing and protein expression profiling. Epigenomics is a key component since promoter hypermethylation occurs an early event in lung tumourigenesis (Belinsky, S. et al. 2015), targeting tumour suppressor genes. Indeed epigenomics and genomics are intimately linked, with CpG methylation leading to base substitution through 5-methylcytosine deamination, and enhancing the effect of exogenous carcinogens. Although the contribution of smoking to lung cancer aetiology has long been recognised, genomics is now providing insight into somatic mutagenesis as the mechanism of this causal interaction, as well as into tumourigenesis in non-smokers. However, this wealth of genetic and epigenetic information requires further analysis to establish which of these events really drive the phenotype, and which can be biologically validated as targets for therapy. Both genetic and epigenetic targets for therapy of lung cancer have been identified, in the form of both activated oncogenes and loss of tumour suppressor gene function. In some cases tumour genotype proves valuable as a predictive biomarker for patient selection. Several current biomarker-directed trials (such as Lung-MAP and MATRIX) are seeking to identify further successful genotype/therapy pairings. Despite impressive response rates in genomically stratified populations, regulators seem still to require validation of omics-driven treatment selection in a strategy-testing design, randomising to standard of care or personalised therapy. A further therapeutic application of genomics is characterisation of resistance mechanisms, an understanding of which has already led directly to next generation drugs in several drug classes including inhibitors of EGFR and ALK. It is genetic events that are at the origin of the hallmarks of cancer, but proteins, as the effectors of cellular processes, are key to a full understanding of the cancer phenotype. Some have argued that proteomic markers, as a surrogate for the genetic drivers, may be inferior to genomics. Certainly proteomic biomarkers are the less dynamic because their half life is measured in weeks, compared with a few hours for nucleic acids. Beyond the small number of actionable mutations already described in non-small cell lung cancer, the diagnostic, prognostic, and predictive potential of a large number of omic markers has been studied, and in most cases problems with reproducibility have limited their clinical impact. Indeed the utility of multi-gene predictive markers described to date, most likely to be of clinical value in therapy, is limited. An eight-peak MALDI-MS proteomic profile has been developed as a predictive tool (Taguchi, F. et al. 2007). Long suspected, the contribution of tumour heterogeneity to an analysis of tumour omics is now proven to be potentially problematic (Bedard, P. et al. 2013). The study of circulating genomic (eg circulating free DNA, cfDNA) and proteomic tumour markers provides an opportunity for integration of this heterogeneity. Nevertheless, further questions remain. For example, do primary and metastatic sites release similar amounts of DNA and protein into the circulation? However, potential advantages of these liquid biopsies are obvious, as they can be repeated over time without risk or inconvenience to the patient. Still to be fully clarified is the clinical utility of this approach. Possible applications include early discontinuation of toxic failing therapy, evaluation of an emerging resistance mechanism and selection of next therapy, and prognostication (for example, selection for adjuvant therapy). Earlier liquid biopsy methods required initial analysis of potential biomarkers in a tumour, to identify what to look for, followed by detection of this marker in blood samples. This approach requires personalisation for each patient. Newer techniques allow direct analysis, for example next generation sequencing of cfDNA. It is also possible to study the methylation status of cfDNA, so these liquid biopsies may in addition be relevant to the study of tumour epigenomics. cfDNA may be superior to circulating tumour cells (CTCs) as a biomarker since in some patients cfDNA but not CTC is detectable (Bettegowda, C. et al. 2014) The most prominent recent therapeutic advance in lung cancer is the validation of immunotherapy in the context of checkpoint inhibition. While this approach appears to target the tumour only indirectly, via host immunity, there is already good evidence that the genomic context of the target tumour is critically significant (Gubin, M. et al. 2015) The optimum strategy for selection of patients for clinical omic testing remains to be finalised. Should this be for all patients, from the time of diagnosis, or only after completion of standard care? And what material is ideal for testing (archival or contemporaneous biopsy, for example)? Guidelines on what, when and how to test are available (Lindeman, N. et al. 2013), but this advice quickly becomes out of date given the pace of change in the field. Further practical concerns include access to technology, turnaround time for testing, interpretation of molecular pathology results and bioinformatics, and clinical relevance. Fundamental questions arise about which changes are actionable, and the importance of any findings in the germline sequence (incidental or deleterious). Finally, quality control and regulation of omic technologies is demanding and not necessarily well served by existing approaches and infrastructure (Evans, B. et al. 2015), and these aspects must be developed alongside the emergence of these novel technologies. Progress in development of these techniques has been rapid, but maximum utility to patients is still to be developed. Omics have made major contributions to the understanding of lung cancer biology, and to the identification of a growing spectrum of therapeutic targets, but more work remains to be done. References Bedard, P et al. (2013). Tumour heterogeneity in the clinic. Nature 501; 355-364 Belinsky S, et al. (2015). Gene promoter methylation in plasma and sputum increases with lung cancer risk. Clin Cancer Res 11; 6505-11 Bettegowda, C et al. (2014). Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 6, 224ra24 Evans, B et al. (2015). The FDA and genomic tests - getting regulation right. New Engl J Med 372; 2258-2264 Gubin, M et al. (2015). PD-1 blockade in tumors with mismatch-repair deficiency. New Engl J Med 372; 2509-2520 Lindeman, N et al. (2013). Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors. J Thoracic Oncol 8; 823-59 Pastor, M et al. (2013). Proteomic biomarkers in lung cancer. Clin Transl Oncol 15; 671-682 Taguchi F et al. (2007). Mass spectrometry to classify non-small-cell lung cancer patients for clinical outcome after treatment with epidermal growth factor receptor tyrosine kinase inhibitors: a multicohort cross-institutional study. J Natl Cancer Inst 99; 838-46 The Cancer Genome Atlas Research Network (2014). Comprehensive molecular profiling of lung adenocarcinoma. Nature 511; 543-550

Abstract:
In my talk I will report a prospective surveillance program, longitudinally following patients with pre-invasive disease over a 10 year period. It is the largest study of its kind and demonstrates unexpectedly high rates of both local progression to invasive cancer of high grade lesions and the development of synchronous tumours elsewhere in the lung. Further I will show data identifying both gene expression and epigenetic signatures predicting progression of these lesions. These signatures may provide the biomarker strategy we require to identify those patients with lesions at high risk of progression and therefore requiring treatment. Lung cancer accounts for more deaths than breast, prostate and colon cancers combined. Over three quarters of lung cancer patients are diagnosed at a late stage when curative treatment is not possible. Initiatives are underway to detect lung cancer earlier. CT screening of high risk smokers or ex smokers is proven to save lives through increased detection of largely early stage adenocarcinomas (1, 2). Meanwhile sputum cytometry and autofluorescence bronchoscopy of high risk individuals are under investigation as screening tools for the early detection of major airway squamous cell carcinomas in several studies. Squamous carcinogenesis is initiated by pre-invasive dysplastic lesions in the central airways and therefore lends itself to bronchoscopic evaluation. Bronchial dysplasia represents the earliest stages of what is traditionally thought to be a stepwise progression towards invasive disease commencing with squamous hyperplasia and metaplasia followed by mild, moderate, severe dysplasia (SD) and carcinoma-in-situ (CIS) with lesions possessing a greater mutational burden at each stage (WHO classification) (Figure 1). With progression of the lesion there are characteristic morphological changes and increasing cytological disarray. Initial changes affect only the basal epithelium, whilst ‘full thickness’ change is seen in the more advanced CIS. Once the basement membrane has been breached, invasive squamous cell carcinoma has developed. Figure 1 Our early findings, and those of others, have challenged this traditional stepwise model. With longitudinal follow up, few low grade dysplasia lesions (LGD: hyperplasia, metaplasia mild and moderate dysplasia) are seen to progress and largely remain indolent or often regress. High grade dysplasia lesions (HGD: SD and CIS) however, more frequently persist or progress to invasive disease. Bronchial dysplastic lesional destiny is unpredictable and despite research examining the genetic and epigenetic changes that occur, as yet no robust biomarker is able to determine which lesions will continue to progress to invasive disease. Low grade lesions rapidly progressing to cancer have been reported, and these rare lesions have been found to possess a high degree of chromosomal instability including DNA copy number alterations even at a metaplastic stage, seeming to confer a committed course to cancer development. It is likely that close analysis of these rare lesions and other high grade lesions that progress will lead to greater biological insight regarding key lung cancer driver mechanisms. Autofluorescence bronchoscopy (AFB) using blue-violet excitation light has made progress in facilitating not only the detection and delineation of extent of early stage invasive cancers in the airway but also the identification of precancerous central airway lesions that are generally missed on CT. AFB detection of precancerous lesions has been shown to have sensitivity exceeding that of white light bronchoscopy (WLB) alone. The sensitivity of combining AFB with WLB improves detection of bronchial premalignant and malignant lesions up to 96.8% versus 76.3% for WLB alone, whilst corresponding negative predictive values are 97.2% versus 83.1% (3). Treatment of precancerous lesions might be expected to lead to improved survival in those patients harboring them. However our lack of knowledge of the natural history of these lesions, the appearance of new lesions, the regular occurrence of separate lung primaries and the lack of interventional studies in this area leaves the role of early intervention (both surgical and local tissue sparing procedures) under dispute. Due to this poverty of knowledge, our strategy, in keeping with previously published studies, has been the surveillance of all grades of dysplasia. These include our own, initial observations that suggest a low rate of lesion progression but high synchronous invasive cancer occurrence (4, 5). This early experience indicates patients with preinvasive disease are at a globally high risk of developing lung cancer, although not necessarily from the lesion under observation and multiple lesions both centrally and peripherally commonly develop over time. Due to the shared risk factor of tobacco smoke exposure, patients often have significant respiratory and cardiovascular co-morbidity and radical treatment of preinvasive disease may lead to insufficient lung capacity to offer curative intervention to future invasive lung cancer. 1. NCCN Clinical Practise Guidelines in Oncology. Lung Cancer Screening. Version http://www.nccn.org/professionals/physician_gls/pdf/lung_screening.pdf 2. National Lung Screening Trial Research Team, Aberle DR, Berg CD, Black WC, Church TR, Fagerstrom RM, Galen B, Gareen IF, Gatsonis C, Goldin J, Gohagan JK, Hillman B, Jaffe C, Kramer BS, Lynch D, Marcus PM, Schnall M, Sullivan DC, Sullivan D, Zylak CJ. The National Lung Screening Trial: overview and study design.Radiology. 2011 Jan;258(1):243-53. 3. Hanibuchi M, Yano S, Nishioka Y, Miyoshi T, Kondo K, Uehara H, Sone S. Autofluorescence bronchoscopy, a novel modality for the early detection of bronchial premalignant and malignant lesions. J Med Invest. 2007 Aug;54(3-4):261-6. 4. George JP, Banerjee AK, Read CA, O'Sullivan C, Falzon M, Pezzella F, Nicholson AG, Shaw P, Laurent G, Rabbitts PH. Surveillance for the detection of early lung cancer in patients with bronchial dysplasia. Thorax. 2007 Jan;62(1):43-50. 5 Auerbach O, Stout AP, Hammond EC, et al. Changes in bronchial epithelium in relation to cigarette smoking and in relation to lung cancer. N Engl J Med 1961;265:255–67.

Abstract:
Epithelial cancers are thought to arise in a stepwise fashion from premalignant lesions and removal of premalignant lesions in epithelia such as colon and cervix has made a major improvement in survival in these cancers. However, premalignant lesions of the airway epithelium are poorly understood and it is not even known whether they represent a true premalignant state. This is in large part because of the heterogeneity of premalignant lesions of the airway and the fact that most of them will spontaneously resolve, even in high-risk patients. Premalignant lesions are thought to arise because of aberrant repair after injury but our understanding of the biology of normal repair after injury of the airways is limited and thus we do not know what the mechanisms are that drive aberrant repair and even less what the mechanisms are that drive the development of invasive non-small cell lung cancer. In order to increase our understanding of premalignant lesions of the airway, we laser-microdissected representative cell populations along the purported squamous cell lung cancer pathological continuum of patient-matched normal basal cells, premalignant lesions, and tumor cells. We obtained sufficient mRNA to perform high throughput RNA-sequencing. We discovered transcriptomic changes and identified genomic pathways altered with initiation and progression of SCC within individual patients. We used immunofluorescent staining to confirm gene expression changes in premalignant lesions and tumor cells, including increased expression of SLC2A1, CEACAM5, and PTBP3 at the protein level and increased activation of MYC via nuclear translocation. Cytoband enrichment analysis revealed coordinated loss and gain of expression in chromosome 3p and 3q regions, respectively, during carcinogenesis. We also identified several pathways that were upregulated in a stepwise fashion with progression of lesions. One of the pathways found to be upregulated with stepwise progression was redox regulation. Low levels of Reactive Oxygen Species (ROS) are known to be critical for cell regulation, while high levels of ROS are toxic to cells. We found that airway basal stem cells have low levels of ROS at baseline, but injury results in an increase in ROS and this flux from low to higher levels of ROS mediates proliferation of the basal cells via signaling through ROS/Nrf2/Notch1. Perturbation of this pathway at the level of Nrf2 or Notch both in vitro and in vivo results in excessive proliferation of basal cells and the formation of premalignant lesions with hyperplasia and dysplasia of the repairing airway epithelium. Our results provide much needed information about the biology of airway epithelial repair, premalignant lesions and the molecular changes that occur during stepwise carcinogenesis of squamous cell lung cancer, and it highlights a novel approach for identifying some of the earliest molecular changes associated with initiation and progression of lung carcinogenesis within individual patients.

Abstract:
The recently published Fourth Edition of the WHO Classification of Tumours of the Lung[1] recognizes atypical adenomatous hyperplasia (AAH) and adenocarcinoma in situ (AIS) as pre-invasive or premalignant precursor lesions of invasive adenocarcinoma, which arises mostly in the periphery of the lung. In the previous (Third) Edition of WHO classification, AIS was classified as bronchiolo-alveolar carcinoma (BAC), one of the subtypes of malignant adenocarcinoma.[2,3] Reclassification of AIS into the preinvasive category represents a conceptual confirmation of its role in multi-stage pathogenesis of peripheral lung adenocarcinoma.[4-7] This is consistent with the histological hallmark of lack of invasion in AIS, and its association with 100% survival after surgical resection. The neoplastic nature of these lesions are supported at the molecular level with the identification of known genomic aberrations found in invasive lung adenocarcinoma.[8,9] AIS is characterized histologically by the lepidic proliferation of neoplastic epithelium along pre-existing alveolar structures and lacking stromal, vascular or pleural invasion (Figure 1). A majority of AIS is composed of non-mucinous neoplastic cells with Clara cell and/or type-2 pneumocyte phenotype. Mucinous AIS is rare. By definition, AIS is limited to tumors that is ≤ 3 cm in greatest diameter and by TNM classification, is classified a Tis. AIS commonly shows varying degree of stromal thickening by fibrosis and chronic inflammatory cell infiltrate, with some cases showing focal or central area of fibrosis or scar. Around these areas, entrapment of the tumor cells within architecturally distorted and thickened alveolar septa give rise to morphological appearances of invasion. This remains one of the areas of diagnostic difficulty in distinguishing AIS from minimally invasive adenocarcinoma (MIA). However, limited data suggests that MIA is also associated with 100% curability by surgical resection. A majority of AAH are identified incidentally during microscopic examination of non-cancerous lung of surgically resected adenocarcinoma (Figure 2). The reported incidence in lung adenocarcinoma cases may reach up to 30%, and the reported number of lesion can reach up to 40/case, depending on the extent of sampling. They are typically ≤ 5 mm, but size is not a diagnostic criteria for its diagnosis. Histologically it is characterized by slightly thickened alveolar septa that are lined by atypical appearing cuboidal to low columnar epithelial cells with gaps in between them. These cells have similar ultrastructural features as AIS cells, mainly those of type-2 pneumocyte and/or Clara cell. A spectrum of nuclear atypia may be observed but grading has not been recommended, as they have not been demonstrated as reproducible or correlated with neoplastic progression. AAH is considered a precursor of AIS, as they may harbor KRAS or EGFR mutations. In some cases, the histological distinction between AAH and AIS can be very challenging, even though both lesions are considered cured by surgical resection. Further deep genomic analyses of AAH and AIS can provide greater insights into the multistep molecular carcinogenesis of lung adenocarcinoma and potentially novel prevention strategies for this disease. References: 1. WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart. Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG. IARC Press, Lyon, 2015, page 46-50. 2. World Health Organization International Histological Classification of Tumours. Histological Typing of Lung and Pleural Tumours. Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E. Springer Verlag, Berlin, Heidelberg, New York, 1999, page 21-29. 3. WHO Classification of Tumours, Pathology and Genetics. Tumours of the Lung, Pleura, Thymus and Heart. Travis WD, Brambilla E, Muller-Hermelink HK,, Harris CC. IARC Press: Lyon 2004, page 35-44, 73-75. 4. Miller RR, Nelems B, Evans KG, Muller NL, Ostrow DN. Glandular neoplasia of the lung. Cancer 1988;61:1009-1015. 5. Kitamura H, Kameda Y, Ito T, Hayashi H. Atypical adenomatous hyperplasia of the lung. Implications for the pathogenesis of peripheral lung adenocarcinoma. Am J Clin Pathol 1999;111:610-22. 6. Mori M, Rao SK, Popper HH, Cagle PT, Fraire AE. Atypical adenomatous hyperplasia of the lung: A probably forerunner in the development of adenocarcinoma of the lung. Mod Pathol 2001;14:72-84. 7. Chapman AD, Kerr KM. The association between atypical adenomatous hyperplasia and primary lung cancer. Br J Cancer 2000;83:632-36. 8. Westra WH, Baas IO, Hruban RH, Askin FB, Wilson K, Offerhaus GJ, Slebos RJ. K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. Cancer Res 1996;56:2224. 9. Sakamoto H, Shimizu J, Horio Y, Ueda R, Takahashi T, Mitsudomi T, Yatabe Y. Disproportionate representation of KRAS gene mutation in atypical adenomatous hyperplasia, but even distribution of EGFR gene mutation from preinvasive to invasive adenocarcinomas. J Pathol 2007;212:287-94. Figure 1. Adenocarcinoma in situ Figure 1 Figure 2. Atypical Adenomatous Hyperplasia. Figure 2

Abstract:
Adenocarcinoma in situ (AIS) of the lung has an extremely favorable prognosis. However, early but invasive adenocarcinoma (eIA) sometimes has a fatal outcome. We examined epigenetical and genetic abnormalities of very early adenocarcinoma and compared them to early but advanced adenocarcinoma. We had previously compared the expression profiles of AIS with those of eIA showing lymph node metastasis or a fatal outcome, and found that stratifin (SFN, 14-3-3 sigma) was a differentially expressed gene related to cell proliferation. Here, we performed an in vivo study to clarify the role of SFN in progression of lung adenocarcinoma. Suppression of SFN expression in A549 (a human lung adenocarcinoma cell line) by siSFN significantly reduced cell proliferation activity and the S-phase subpopulation. In vivo, tumor development or metastasis to the lung was reduced in shSFN-transfected A549 cells. Moreover, we generated SFN-transgenic mice (Tg-SPC-SFN[+/-]) showing lung-specific expression of human SFN under the control of a tissue-specific enhancer, the SPC promoter. We found that Tg-SPC-SFN[+/-] mice developed lung tumors at a significantly higher rate than control mice after administration of chemical carcinogen, NNK (Fig 1). Interestingly, several Tg-SPC-SFN+/- mice developed tumors without NNK. These tumor cells showed high hSFN expression. These results suggest that SFN facilitates lung tumor development and progression. SFN appears to be a novel oncogene with potential as a therapeutic target. Next, gnetic abnormality in early-stage lung adenocarcinoma was examined. Six in situ lung adenocarcinomas and nine small but invasive adenocarcinomas were examined by array-comparative genomic hybridization (array-CGH), and candidate genes of interest were screened. To examine gene abnormalities, 83 cases of various types of lung carcinoma were examined by quantitative real-time genomic PCR (qPCR) and immunohistochemistry (IHC). The results were then finally verified using another set of early-stage adenocarcinomas. Array-CGH demonstrated frequent amplification at chromosome 3q26, and among the 7 genes located in this region, we focused on the epithelial cell transforming sequence 2 (ECT2) oncogene, as ECT2 amplification was detected only in invasive adenocarcinoma, and not in in situ carcinoma. FISH and IHC analyses also detected amplification and overexpression of ECT2 in invasive adenocarcinoma (Fig 2), and this was correlated with both the Ki-67 labeling index and mitotic index. In addition, it was associated with disease-free survival and overall survival of patients with lung adenocarcinoma. These results were verified using another set of early-stage adenocarcinomas resected at another hospital. Abnormality of the ECT2 gene occurs at a relatively early stage of lung adenocarcinogenesis and would be applicable as a new biomarker for prognostication of patients with lung adenocarcinoma. Figure 1Figure 2

Abstract not provided

Background:
Newly detected nodules after baseline screen are common findings in low-dose computed tomography (LDCT) lung cancer screening, and may complicate management. So far little research focused specifically on nodules newly detected at incidence screening rounds. These nodules develop within a known time-frame and are expected to be fast-growing and potentially malignant. Even so, the majority are benign. The aim of this study was to compare volume and predicted growth rate of benign and malignant new solid nodules detected in the incidence screening rounds of the Dutch-Belgian Randomized Lung Cancer Screening Trial (NELSON).

Methods:
The NELSON trial was approved by the Dutch Ministry of Health. All participants gave written informed consent. In total, 7,557 individuals underwent baseline LDCT screening. After the baseline screening, incidence screening rounds took place after 1, 3 and 5.5 years. This study included participants with solid non-calcified nodules, newly detected after baseline and also in retrospect not present on any previous screening. Nodule volume was obtained semi-automatically by Lungcare software (Siemens, Erlangen, Germany). The growth rate at first detection was estimated by calculating the slowest predicted volume-doubling time (pVDT), according to the formula pVDT=[ln(2)*Δt]/[ln(V2/V1)], using the study’s detection limit of 15mm[3] (V1), the volume of the new nodule at first detection (V2), and the time interval between current and last screen (Δt [days]). The pVDT was calculated for nodules with a predicted volume increase of at least 25% (≥ 18.75mm[3]). Lung cancer diagnosis was based on histology. Benignity was based on histology or a stable size for at least two years. Mann-Whitney U testing was used to evaluate differences in volume and pVDT between malignant and benign nodules.

Results:
During the incidence screening rounds, 1,484 new solid nodules in 949 participants were detected of which 77 (5.2%) turned out to be malignant. At first detection, both the median volume of malignant (373mm[3], IQR 120-974mm[3]) and benign (44mm[3], interquartile-range [IQR] 22-122mm[3]) new nodules, as well as the median pVDT of malignant (144 days, IQR 116-213 days) and benign (288 days, IQR 153-566 days) new nodules differed significantly (P<0.001 for both). The calculated median pVDT of adenocarcinomas (183 days, IQR 138-299 days) and squamous-cell carcinomas (150 days, IQR 117-223 days) was similar to previously published volume doubling-times of fast-growing baseline cancers in the NELSON trial of the same histological type (196 days, IQR 135-250 days and 142 days, IQR 91-178 days, respectively).

Conclusion:
At incidence LDCT lung cancer screening, volume and pVDT can be used to differentiate between malignant and benign nature of newly detected solid nodules. The pVDT is a new measure that can assist in adjusting for time differences in screening intervals.

Background:
Although screening can reduce lung cancer (LC) mortality, the optimal screening strategy (e.g. numbers of screening rounds, screening interval) is unclear. The use of different screening intervals in the NELSON study is unique and makes it possible to investigate how the screening test performances (e.g. lung cancer detection rate, false positive rate) and characteristics of screen-detected lung cancers might change. This study describes the results of a fourth screening round that took place 2.5 years after the third round.

Methods:
The Dutch-Belgian randomized-controlled Lung Cancer Screening Trial (NELSON) aims to investigate whether low-dose CT screening would reduce LC mortality by at least 25% relative to no screening after ten years of follow-up. Therefore, screen group participants were screened four times: at baseline and year 1, 3, and 5.5. Screening test results were classified as negative, indeterminate, or positive based on nodule presence, volume (in case of new nodules) and volume doubling time (in case of previous existing nodules). Participants with an indeterminate test result underwent follow-up screening to classify their final screening test result as positive or negative. Participants with a positive scan result were referred to a pulmonologist for a diagnostic work-up. For this study, we included only participants who had attended all four screening rounds (n=5279). Epidemiological, radiological and clinical characteristics of lung cancers detected in the fourth round were compared with those of the lung cancers detected in the first three rounds. In addition, the risk for lung cancer detection in the fourth round (5.5 year risk) was quantified for subgroups.

Results:
In round four, 46 lung cancers were detected; 58.7% were diagnosed at stage I, 15.2% at stage II and 23.8% at stage III/IV. Adenocarcinomas were correlated with better cancer stage distribution, while small-cell carcinomas (SCLC) were associated with higher stage distribution (p=0.064). False positive rate after positive screening was 59.04% (62/105) and the overall false positive rate of the fourth round was 1.15% (62/5383). Relative to the results of the first three rounds, the LC detection rate was lower (0.80 vs 0.80-1.1) and LC was detected at a more advanced stage (23.8% vs 8.1%). In the fourth round more squamous-cell carcinomas (21.7% vs. 16.3%), SCLC (6.5% vs 3.8%) and bronchioloalveolar carcinomas (8.7% vs 5.3%) were detected. No large-cell carcinomas, large-cell neuroendocrine carcinomas or carcinoids were found in the fourth round. Screening results of the first three rounds led to formation of subgroups with significantly different probability of screening result in the fourth round: participants with previous exclusively negative results had a probability of 97.2% of negative screen compared to participants with ≥1 indeterminate or positive screen (94.6% and 87.1%) in the first three rounds. The risk of detecting LC in the fourth round also differed between these subgroups: exclusively negative results (<1.0%) and any time ≥1 indeterminate or positive result (1.5-1.7%).

Conclusion:
The LC detection rate after the third screening round was slightly lower and the stage distribution of screen-detected lung cancers in the fourth round was slightly less favorable. However, the differences seem limited.

Background:
The Danish Lung Cancer Screening Trial (DLCST) is a European randomized controlled trial comparing annual CT screening with no screening. Inclusion ran from 2004 to 2006, and participants have now been followed for 5 years since last CT screening (approximately 10 years since randomization). The American NLST showed 20% decrease in lung cancer mortality in the screening group, and DLCST is the first European trial to present comparable results regarding effect of screening on mortality, causes of death, lung cancer findings and risk stratification after sufficient follow up.

Methods:
4,104 participants aged 50-70 at time of inclusion and a minimum of 20 pack-years of smoking history were randomized to five annual low-dose CT scans or clinical follow up without CT scanning; thus, participants were younger and had fewer pack-years than participants from NLST. Screening was concluded in 2010. Follow up information regarding date and cause of death as well as lung cancer diagnosis, stage and histology was obtained from national registries, latest follow up date was April 7, 2015. . The effects of age, amount of smoking and COPD on lung cancer mortality in the two randomized groups were explored to evaluate possible effects of risk stratification and selection of high-risk individuals on effect of screening.

Results:
More cancers (100 vs. 53, p<0.001) were found in the screening group, in particular adenocarcinomas (58 vs. 18, p<0.001). Significantly more low-staged cancers (stage I+II: 54 vs. 10, p<0.001) and stage IIIa cancers (15 vs. 3, p=0.009) were found in the screening group. However, stage IV cancers were more frequent in the control group (23 vs. 32, p=0.278), and this was statistically significant for the highest-stage cancers (T4N3M1: 8 vs. 21, p=0.025). No differences in lung cancer mortality or all-cause mortality were observed between the two groups (Log Rank tests: p=0.898 and p=0.885, respectively). However, sub-group analyses including participants with higher age, presence of COPD, and more than 35 pack-years of smoking history showed significantly increased risk of death from lung cancer; the highest-risk group (with COPD and >35 pack-years) showed a 20% reduction in lung cancer mortality when screened. Though this result is not statistically significant due to small numbers, it does show compliance with the results from NLST.

Conclusion:
Although no statistically significant effects of 5 annual CT screening rounds on lung cancer mortality were observed in this small study, results indicate that focus on selection of high-risk individuals may be essential for the effect of CT lung cancer screening. We suggest that balancing benefits with harms—such as false positive findings and overdiagnosis— should bring focus to high-risk profiling of screening participants. Thus, the effects of age, amount of smoking, and COPD on the occurrence and mortality of lung cancer in the two randomization groups seems to indicate that limiting lung cancer screening to a higher-risk group improves the outcome of screening.

Abstract not provided

Background:
Lung-RADS published in 2014 by the American College of Radiology is based on literature review and expert opinion and uses nodule type, size, and growth to recommend nodule management adjusted to malignancy risk. The McWilliams model (N Engl J Med 2013;369:910-9) is a multivariate logistic regression model derived from the Pan-Canadian Early Detection of Lung Cancer Study and provides a nodule malignancy probability based on nodule size, type, morphology and subject characteristics. We compare the performance of both approaches on an independent data set.

Methods:
We selected 60 cancers from the Danish Lung Cancer Screening Trial as presented in the first scan they were visible, and randomly added 120 benign nodules from baseline scans, all from different participants. Data had been acquired using a low-dose (16x0.75mm, 120kVp, 40mAs) protocol, and 1mm section thickness reconstruction. For each nodule, the malignancy probability was calculated using McWilliams model 2b. Parameters were available from the screening database or scored by an expert radiologist. Completely calcified nodules and perifissural nodules were assigned a malignancy probability of 0, in accordance with model guidelines. All nodules were categorized into their Lung-RADS category based on nodule type and diameter. Perifissural nodules were treated as solid nodules, in accordance with Lung-RADS guidelines. For each Lung-RADS category cut-off sensitivity and specificity were calculated. Corresponding sensitivities and specificities using the McWilliams model were determined.

Results:
Defining Lung-RADS category 2/3/4A/4B and higher as a positive screening result, specificities to exclude lung malignancy were 21%/65%/86%/99% and vice versa sensitivities to predict malignancy were 100%/85%/58%/32%. At the same sensitivity levels as Lung-RADS, McWilliams model yielded overall higher specificities with 2%/86%/98%/100%, respectively (red arrows in Figure 1). Similarly, at the same specificities McWilliams’s model achieved higher sensitivities with 100%/95%/85%/48%, respectively (green arrows in Figure 1). Figure 1



Conclusion:
For every cut-off level of Lung-RADS, the McWilliams model yields superior specificity to reduce unnecessary work-up for benign nodules, and higher sensitivity to predict malignancy. The McWilliams model seems to be a better tool than Lung-RADS to provide a malignancy risk, thus reducing unnecessary work-up and helping radiologists determine which subgroup of nodules detected in a screening setting need more invasive work-up.

Background:
Although the National Lung Screening Trial (NLST) demonstrated that 3 annual low-dose CT (LDCT) screens reduced lung cancer specific and overall mortality at 6 years in a defined population of smokers, the decision to implement population-based screening is difficult in the absence of information on factors not evaluated in the NLST including frequency and duration of screening, characteristics of the “at risk” population, program cost and cost-effectiveness. The Canadian Partnership Against Cancer has developed a Cancer Risk Management Model for lung cancer (CRMM-LC) with a screening module informed by data from NLST that can evaluate these factors.

Methods:
CRMM-LC uses longitudinal microsimulation techniques that incorporate Canadian demographic characteristics, risk factors, cancer management approaches and outcomes, resource utilization and other economic factors to assess impacts on population health and costs to the Canadian healthcare system. Data sources include large national population surveys, cancer registries and census data. The diagnostic and therapeutic approaches and outcomes in CRMM-LC are based on input from Canadian lung cancer experts and survival information from medical literature. The simulated mortality reduction from LDCT screening using CRMM-LC is comparable to NLST. The model can projected incident cases, life years and quality adjusted life years over different time periods for populations defined by different age ranges and smoking histories and by screening duration and frequency (annual vs biennial). It can also inform individual provinces of the incremental resources (CT scans, invasive procedures) required for program implementation and project budget impact.

Results:
Based on NLST at risk criteria (55-74 yr old smokers of 30+ pack-years, the base case scenario), 1.4 million or 4% of Canadians would be candidates for LDCT screening in 2014. Annual screening over a 10 year period with a participation rate of 60% and 70% adherence would identify an additional 12,500 (4.7%) incident cases and result in 11,320 life-years saved (undiscounted). Biennial screening would identify 4,620 (1.6%) fewer cases and save 1,454 (12.8%) fewer life-years, but may be more cost-effective than annual screening. Scenarios modeling participation rates of 20, 40 and 80% (linear uptake over 10 years) yield incident cases that vary from 8,380 fewer for the lowest rate to 3,950 more for the highest with life years saved over 10 years ranging from 7,540 fewer to 3,310 more, respectively. The model projects 3,560 more cases would be detected if LDCT was introduced for younger (50 to 69 yr old), 30 pack-year smokers compared to the base case scenario and 1,760 more cases if the threshold number of pack years was decreased to 20 pack-years. The 10 year cumulative incremental cost in Canada of annual and biennial screening would be $1,107 and $709 million, respectively

Conclusion:
CRMM-LC, available at cancerview.ca/cancerriskmanagement, can be used by provincial analysts to estimate the impact of various scenarios on the impact of policy decisions concerning the scope of the LDCT screening program. In the current fiscally constrained healthcare environment, models that can assimilate diverse sources of information and extrapolate beyond clinical trial results can help inform decisions that healthcare administrators confront.

Background:
Screening for lung cancer according to age and smoking history alone could cost billions of dollars of public health expenditure due to the high incidence of potential participants. Risk-adapted lung cancer screening strategies such as participant selection (based on published risk prediction models such as the PLCOm2012 model) and malignancy risk based screening protocols may reduce program costs while improving outcomes among current and former smokers at risk of developing the disease. The Pan-Canadian Early Detection of Lung Cancer Study (PanCan) was designed with the objective of providing economic evidence for an affordable lung cancer-screening program in Canada.

Methods:
Data for 2537 screening participants in the PanCan study (median follow-up time of 4 years) and 25,914 eligible participants from the NLST-CXR arm were included in the analysis. There was adequate power and follow-up to inform the transition probabilities in model and provide the distribution to test all model parameters simultaneously in a probabilistic sensitivity analysis. The cost and health utility inputs are from patient-level trial data with defined ranges of certainty.

Results:
Our results show that risk selection using the PanCan risk prediction model could reduce the need to screen 21,022 (81%) of the NLST population if risk prediction were applied. If risk prediction were applied to Canadians who met the NLST criteria, 2 year program costs could be reduced by 400 million dollars and nearly half a million people could be spared the potential harms from screening that is not likely to result in a Cancer diagnosis. With the economic evidence from the PanCan and NLST trials, we report our initial cost-effectiveness results and will show, for the first time, a definitive description of the uncertainty surrounding our cost-effectiveness ratios.

Conclusion:
Using a model loaded with patient-level screening data has enabled us to predict the likelihood that risk-adapted screening will fall below most commonly referenced thresholds of acceptability for cancer interventions. The initial results and characterization of the parameters affecting cost-effectiveness will be presented. [*]on behalf of the PanCan study team The panCan study is sponsored by the Terry Fox Research Institute and the Canadian Partnership against Cancer, ARCC is funded by the CCSRI The authors thank the National Cancer Institute for access to NCI’s data collected by the National Lung Screening Trial. The statements contained herein are solely those of the authors and do not represent or imply concurrence or endorsement by NCI

Abstract not provided

Background:
BMs are found in up to 30% of patients (pts) with advanced non small cell lung cancer (NSCLC), and are associated with a poor prognosis despite radiotherapy treatment, with a median survival of 6 months (mo). Several data are suggesting the potential brain activity of tyrosin kinase inhibitors (TKIs) alone in NSCLC pts with activating mutations. We retrospectively identified EGFR mutated and ALK rearranged NSCLCs with BMs, to evaluate the efficacy of TKIs and their role in the upfront setting.

Methods:
Out of a cohort of 270 never smoker (NS) NSCLC patients (pts) treated at our Institution from 2/2006 to 2/2015, 89 (32.9%) NSCLCs BMs were identified, synchronous in 27 pts (30.3%). 38 pts (42.7%) harboured an EGFR mutation, 33 pts (37.1%) were ALK rearranged, 18 pts (20.2%) negative for both, were used as a control cohort. Among the EGFR mutated, an in-frame deletion in exon 19 (mostly E746-A750) was found in 26 (68.4%) patients, while a point mutation in exon 21 (L858R) was detected in 10 (26.4%), 1 (2.6%) exon 18 mutation and 1 (2.6%) exon 20 insertion were identified. The majority of EGFR and ALK positive (+) pts with BMs were female (53.9%), median age 52, adenocarcinoma histology, and a good performance status.

Results:
Out of the 71 NSCLCs with BMs EGFR/ALK+, 58 pts (81.7%) received at least one line of chemotherapy, while 13 pts (18.3%) were only treated with TKIs. Of the entire series, 40 pts (56.3%) were treated with standard radiotherapy (WBRT or radiosurgery) prior to TKIs treatment, while 31 (43.7%) received a TKI upfront, distributed as follows: 13 pts (37.9%) were treated with an EGFR inhibitor (gefitinib/erlotinib/afatinib), while 18 pts (62.1%) with an ALK TKI (crizotinib/ceritinib/alectinib). All the pts in the molecular negative cohort, received WBRT and, at least, one line of chemotherapy. Within the entire series, Overall Intracranial Response Rate (OIRR: complete response CR + partial response PR) was evaluated: EGFR+ 31 pts (81.5%), ALK+ 28 pts (84.8%), control cohort 6 pts (33.3%) (p,0.003). Median [95% CI] overall survival (OS) for EGFR mutans, ALK + and EGFR/ALK negative was: 52 months (mo) (32.6-74.4),74 mo (not reached), 25 mo (9.4-40.03) (p,0.003). In the subgroup who received a TKI upfront, all EGFR+ achieved a PR, while all ALK+ obtained an objective response: 4 (22.2%) a CR and 14 (77.8%) a PR. No significant difference in OS between EGFR/ALK+ BMs treated with a TKI upfront versus further line.

Conclusion:
This retrospective study confirms that TKIs are strongly active in patients with BMs from NSCLCs harbouring a sensitive mutation. Brain disease control was achieved in an impressive 81.5% of the EGFR+ pts and 84.8% of the ALK+ subset. Of particular note, is the highest response rate in the TKI upfront arm, with 22.2% attaining a complete remission. We conclude that the use of TKIs in first line setting for BMs treatment may be a reasonable option for asymptomatic subgroup of patients with a long survival expectation, for whom WBRT may be postponed at a later disease stage.

Background:
Venous thromboembolism (VTE) has been demonstrated one of the leading causes of mortality in lung cancer patients. While incidence of VTE in cancer patients varies from 4-20%, at autopsy VTE accounts for as high as 50%. Various strategies of VTE prophylaxis have been proposed, among them low-molecular weight heparins (LMWHs). While different randomized controlled trials (RCTs) showed benefit with LMWHs in regard to VTE, none single RCT was adequately powered for major bleeding. In a meta-analysis of RCTs we aimed to investigate the relation between thromboembolic and bleeding risk associated with LMWHs anticoagulation in lung cancer patients.

Methods:
Established methods were used in compliance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement in healthcare interventions. PubMed, EMBASE, CINAHL, Cochrane, Scopus databases as well as major congress proceedings until April 2015 were screened for RCTs comparing LMWHs with control/placebo. Outcomes assessed were VTE and major bleeding. Odds ratios (OR) and 95% confidence intervals were used as summary statistics. Data were analysed according to Intention-to-treat principle.

Results:
Four RCTs (N=3097) were included in the meta-analysis (Table 1). Average follow-up was 237 days. In a fixed effects model, LMWHs were associated with a significant 50% reduction of the odds of VTE as compared to controls: OR (95% CI): 0.50 (0.35-0.71); p<0.0001; I[2]=0%; (Figure 1A); the number needed to treat =33. A significant, over 2-fold increase in the odds of major bleeding was observed with LMWHs: OR (95% CI): 2.16 (1.16-4.05); p=0.02; I[2]=0%; (Figure 1B); the number needed to harm was 104.

Table 1. Characteristics of included studies

Study	N of pts	LMWH	Dose	NSCLC/SCLC	Follow-up (d)
Agnelli et al. 2009	279	Nadroparin	3800 IU qd	79.9%-20.1%	112
Altinbas et al. 1-2, 2004	84	Dalteparin	5000 IU qd	0%-100%	301
Haas et al. 2012	546	Certoparin	3000 IU qd	100%-0%	168
Woodruff et al. 2013	2202	Dalteparin	5000 IU qd	82.2%-18.8%	365

Figure 1



Conclusion:
Low-molecular weight heparins significantly reduce the risk of venous thromboembolism at a price of increased major bleeding in patients with lung cancer. One episode of major bleeding occurred at every 3 VTEs prevented with LMWHs. Dose-escalation studies are certainly warranted to identify patients who would benefit most from LMWHs.

Background:
In 2011 the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology issued a recommendation to classify patients with advanced NSCLC into specific histological and molecular subtypes and minimize the diagnosis of not otherwise specified (NOS) subtype. The objective of this study was to define the rate of NOS subtype being observed in practice as well as the NSCLC care team’s knowledge and beliefs about a diagnosis of NOS subtype in advanced-stage disease.

Methods:
A series of 5 questions were developed to identify the incidence of NOS subtype being observed in the community as well as relevant care team knowledge gaps and beliefs that may influence the findings. The case vignettes and questions and were based on current standards of care and evidence-base in the treatment of advanced NSCLC. The questions were made available online to healthcare providers either through a survey or as part of 2 certified medical education activities; without monetary compensation or charge. Confidentiality of survey respondents was maintained and responses were de-identified and aggregated prior to analyses. The series of 5 questions was launched in both formats in December 2014 and participant responses were collected over the following 4 months.

Results:
In total, 553 oncologists, pathologists and pulmonologists answered all 5 questions. Oncologists who responded to the questions on average saw about 6-10 patients with suspected or diagnosed NSCLC per month while pathologists and pulmonologists were more likely to see 1-5 per month. Almost 60% of oncologists, pathologists and pulmonologists stated that the incidence of NOS subtype should occur in less than 5% of all cases. Yet, 28% of participating oncologists, 37% of pathologists, and 40% of pulmonologists would find a diagnosis of NSCLC, NOS acceptable. Moreover, 45% of oncologists and 64% of pulmonologists stated that 11% or more of their patients are reported as having a diagnosis of NSCLC, NOS. Reasons for acceptability of NOS subtype differed between clinicians; with more pulmonologists stating it is always acceptable while pathologists and oncologists were more likely to cite age or smoking status, respectively. When asked what contributes to this belief a majority of oncologists and pathologists cited an inability to obtain adequate tissue while pulmonologists were more likely to state that subtyping was unnecessary to prescribe the appropriate therapy (30%) or it was a result of system barriers (25%).

Conclusion:
Despite recommendations from key organizations the incidence of NSCLC, NOS many members of the care team continue to accept a diagnosis of NOS in their patients. Our findings demonstrate a pressing need for additional education of the multidisciplinary care team involved in the diagnosis of advanced NSCLC so as to ensure appropriate diagnosis and treatment.

Abstract not provided

Background:
The majority of patients with epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer respond to first-line EGFR-tyrosine kinase inhibitors (EGFR-TKIs, e.g. gefitinib) but nearly all eventually acquire resistance. The most common mechanism of acquired resistance is a second-site mutation in the EGFR kinase domain, T790M. The phase III, double-blind IMPRESS study evaluated the efficacy and safety of continuing gefitinib plus pemetrexed/cisplatin versus placebo plus pemetrexed/cisplatin in patients with acquired resistance to first-line gefitinib. Study results did not support the continuation of gefitinib after disease progression (by RECIST criteria) when platinum-based doublet chemotherapy is used as second-line therapy. Here we report the results of a retrospective biomarker analysis of plasma circulating free, tumor-derived DNA (ctDNA) from patients in IMPRESS, including T790M profiling, to help understand the IMPRESS clinical trial outcome.

Methods:
Plasma samples for ctDNA isolation were collected at baseline and discontinuation from 151 randomized, non-Chinese patients in IMPRESS (58% of overall IMPRESS population). ctDNA levels of T790M, L858R, and Exon19 deletions were detected using both a quantitative emulsion (BEAMing) digital PCR assay (Sysmex[®]) and a qualitative QIAGEN[®] Therascreen ARMS assay (baseline only). Local EGFR tumor tissue (diagnostic) results were available for 133/151 patients. Mutation concordance rates between tissue and baseline plasma results, and comparisons between the two plasma detection methods, were calculated.

Results:
Baseline ctDNA EGFR mutation results were obtained for >99% (150/151) of patients. Using BEAMing, sensitivity and specificity between baseline plasma EGFR sensitizing mutations and local EGFR tumor tests were 78% (69/89) and 98% (42/43), respectively, for Exon19 deletions, and 82% (31/38) and 97% (91/94) for L858R. The T790M detection rate in baseline plasma samples using BEAMing was 56% (84/150). The Therascreen ARMS assay demonstrated a significantly reduced T790M detection rate of 13% (20/150). Likewise, the sensitivity of the Therascreen ARMS assay with respect to tissue for EGFR sensitizing mutations was also reduced compared with BEAMing: Exon 19: 54% (48/89), L858R: 47% (18/38), though the specificity remained near 100%. In the 97 evaluable plasma samples collected at discontinuation, T790M was detected by BEAMing in 52% (50/97) of patients. When compared with matched baseline plasma, 11 patients had newly acquired T790M mutation at discontinuation while T790M reverted to undetectable in 14 patients. Full plasma profiling data from the complete IMPRESS clinical study population (including 108 patients from China) and correlative analyses of plasma EGFR mutation status with clinical outcome (progression-free survival, overall survival, objective response rate) will be presented.

Conclusion:
In IMPRESS, T790M was detectable with BEAMing digital PCR in the baseline ctDNA samples of 56% of evaluable patients, a rate comparable to similar mutation analyses in this same second-line, EGFR-TKI-failed setting. EGFR mutation detection in plasma using the Therascreen ARMS assay demonstrated comparable specificity to BEAMing but reduced sensitivity. The T790M detection rate afforded by the BEAMing technology will allow for a comprehensive assessment of correlations between clinical outcome in IMPRESS and EGFR mutational status.

Background:
The most important advantage of EGFR mutation analysis in circulating tumor DNA(ctDNA) from plasma is quantitative and dynamic evaluation. Here, we investigated the feasibility of droplet digital PCR(ddPCR) for quantitative and dynamic detection of EGFR mutation in ctDNA and next generation sequencing (NGS) for screening a range of resistance-relevant mutations in plasma DNA in the process of disease progression for patients diagnosed with advanced lung adenocarcinoma.

Methods:
Seventy-three patients were enrolled in this study. Tumor tissues were sampled before treatment, and paired plasma DNA samples were collected pre- and post- EGFR-TKI therapy. Sixty-seven of 73 patients obtained blood samples in the time-point of disease progression. All 73 patients presented EGFR mutation in tumor tissues tested by denaturing high performance liquid chromatography（DHPLC）method. We measured the absolute quantities of plasma EGFR mutant and wild-type alleles by ddPCR. Multi-genes testing was performed using NGS in twenty-seven plasma samples from the twelve patients.

Results:
Taking the EGFR mutation in tumor tissue as the standard, the EGFR mutations detection sensitivity in plasma DNA was 74%（54/73）. According to EGFR mutation status in TKI-naïve patients, all 73 patients were divided into two subgroups that carried mutation in both of specimens (B+/T+,n=54) and mutation only in tissues rather than in plasma ctDNA(T+ /B-,n=19) . The B+/T+ group showed superior progression-free survival (PFS, median, 12.6 vs. 6.7 months, P<0.0001) compared to T+ /B- group. The patients with high EGFR mutated abundance in plasma ctDNA (＞5.15%) showed better PFS (median, 15.4 vs 11.1months; P=0.021) compared with those with low EGFR abundance (≤5.15%). EGFR mutation dynamic alteration during EGFR-TKIs therapy was analyzed and showed patients with decreased quantity of EGFR mutated alleles after disease progression（n=29）showed better PFS compared with non-decreased quantity group(n=38) (median, 12.7 vs 7.1 months; P=0.001). However, NGS results came from 12 patients’ matched plasma DNA showed that 66.6% total mutational copies were elevated and 76.5% mutual mutation frequency increased after disease progression. Besides canonical EGFR pathway, mutated genes in plasma DNA were significantly enriched in cell cycle and TGF-β pathways when disease progressed. Quantification of mutant allele fraction by means of either NGS or ddPCR assay showed excellent agreement.

Conclusion:
Droplet digital PCR is a highly sensitive method for EGFR mutation analysis in plasma DNA of patients with advanced lung adenocarcinoma, while NGS shows good performance in multiple genes testing especially novel and uncommon genes. High EGFR sensitive mutated abundance（>5.15%） in plasma samples of TKI-naïve patients can predict better PFS of EGFR-TKI treatment.

Background:
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) can achieve dramatic response in EGFR activating mutation positive lung cancer patients. However, the duration of treatment is quite different. Some patients experienced longer progression-free survival (PFS) of more than 1 year, whereas some had PFS of shorter than 6 months. Our previous study showed that the relative EGFR mutation abundance in tumor tissues could predict benefit from EGFR-TKIs treatment. However, it still remains controversial whether the intratumor heterogeneity of EGFR activating mutation exists. This study explored the intratumor heterogeneity of EGFR activating mutation at the level of single cancer cell.

Methods:
Single H1975 cells which harbor EGFR exon 21 L858R mutation were isolated by flow cytometry (FCM). The whole DNA extracted from a single cell was submitted to perform nested polymerase chain reaction (PCR) amplification of EGFR exon 21. The amplified products from nested PCR were sequenced to evaluate the feasibility of single-cell analysis for EGFR exon 21. Then, six patients diagnosed with lung adenocarcinoma whose fresh frozen specimens harbored EGFR exon 21 mutation tested by direct sequencing were chosen. All of them received gefitnib treatment and the PFS of three patients was longer than 14 months (Group A) while the PFS of other three patients was shorter than 6 months (Group B). By using the established method based on single H1975 cells, EGFR exon 21 mutational status was analyzed in single tumor cells which were captured from tumor sample by Laser Capture Microdissection (LCM). At least 20 tumor cells were captured from each tumor sample. X[2] test was used to compare the amplification rate of nested PCR and EGFR mutational rate between the two groups.

Results:
A total of 104 individual H1975 cells were obtained to detect EGFR exon 21 mutational status through the application of single-cell nested PCR. The amplification rate and allele drop-out rate were 96.2% and 7.0%. A total of 135 tumor cells from six samples were captured. The amplification rate of nested PCR was 84.3% (59/70) in Group A and 93.8% (61/65) in Group B. There was no statistical difference between the two groups (X[2] =3.119, P=0.077). The mutational rate of EGFR exon 21 L858R was 89.5% (17/19), 89.5% (17/19), and 81.0% (17/21) in the three patients in Group A and 72.2% (13/18), 68.4% (15/22), and 66.7% (14/21) in the three patients in Group B respectively. The total mutational rate was 86.4%（51/59）in Group A, which was significantly higher than the total mutational rate 68.9%（42/61）in Group B (X[2] =5.321, P=0.021).

Conclusion:
It is feasible to perform EGFR mutation detection in single cancer cells. The intratumoral heterogeneity of EGFR activating mutation in lung adenocarcinoma does exist based on the analysis in single cancer cells and the abundance of EGFR activating mutation is relevant to the benefit from EGFR-TKIs treatment.

Abstract not provided

Background:
Plasma genotyping of cell-free DNA (cfDNA) has the potential to allow for noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies. We have developed a quantitative droplet digital PCR (ddPCR)-based plasma genotyping assay capable of detecting common EGFR and KRAS mutations in NSCLC (Oxnard et al., CCR 2014). Although rapid and highly specific, this assay lacks the ability to both multiplex and detect complex genomic alterations such as rearrangements. In this prospective study, we evaluate the test characteristics of ddPCR combined with plasma next-generation gene sequencing (NGS) as a new paradigm for plasma genotyping.

Methods:
Patients with newly diagnosed advanced NSCLC were eligible. All patients were required to have a biopsy available or planned for tissue genotyping which was used for gold standard comparison. Patients underwent an initial blood draw and immediate plasma ddPCR for EGFR exon 19 del/L858R and KRAS G12X. A subset of patients additionally underwent plasma NGS using a unique probe set designed by our group to detect rearrangements and mutations in 12 genes (EGFR, KRAS, ALK, ROS1, BRAF, RET, NRAS, ERBB2, MET, MEK1, PIK3CA and p53). This plasma NGS assay utilized a novel bias corrected NGS which minimizes off-target reads (Resolution Bio) performed on a desktop MiSeq platform. Test turnaround time (TAT) was measured in business days from date of blood draw until test reporting.

Results:
120 patients with newly diagnosed advanced NSCLC have been enrolled and 94 have completed tissue and plasma genotyping. Tumor genotype included 25 EGFR exon 19/L858R mutants, 17 KRAS G12X mutants, 24 rare genotypes and 15 others. Median TAT for plasma ddPCR was 3 days (range 1-5). Specificity of plasma ddPCR was 99% for EGFR exon 19 del/L858R (68/69) and 100% for KRAS (77/77). Sensitivity of plasma ddPCR was 76% for EGFR exon 19 del/L858R (19/25) and 71% for KRAS (12/17). Plasma NGS is ongoing with testing completed on 11 patients with a known tumor genotype. 8 had a genotype detected on plasma NGS: 2 ALK rearrangements, 1 ROS1 rearrangement, 1 RET rearrangement, an EGFR G719A mutation, a KRAS G12C and a combined KRAS G12C/PIK3CA mutation - all matched the tumor genotype. Preliminary plasma NGS turnaround time ranged from 5-10 business days.

Conclusion:
Rapid plasma genotyping using sequential plasma ddPCR (1-5 day TAT) followed by plasma NGS (5-10 day TAT) represents a new paradigm for noninvasive plasma genotyping. This approach capitalizes on the use of rapid ddPCR for common targetable mutations and the ability of plasma NGS using an augmented MiSeq platform to multiplex and detect complex alterations. This new model for plasma genotyping uses testing platforms that can readily be employed in most molecular pathology laboratories allowing for widespread adoption.

Background:
The National Comprehensive Cancer Network (NCCN) non-small cell lung cancer guidelines recommend testing for seven genomic targets amenable to matched therapies, including point mutations and insertions/deletions (indels) in EGFR and ERBB2 (HER2), point mutations in BRAF, fusions in ALK, RET and ROS1, and amplification of the MET gene. Novel digital sequencing technology allows assessment of these biomarkers without an invasive tissue biopsy.

Methods:
We prospectively tested cell free DNA from 43 advanced non-small cell lung cancer patients using a cell-free circulating tumor DNA (ctDNA) next-generation sequencing (NGS) panel of 54 cancer genes (Guardant360). Single nucleotide variants (SNVs) in 54 genes and copy number variants (CNVs) in 3 genes (EGFR, ERBB2 and MET) were reported quantitatively as the mutant allele fraction (MAF) in cell-free DNA and the absolute copy numbers in plasma, respectively.

Results:
79% of patients had at least one ctDNA alteration detected. Five (11.6%) had sensitizing mutations in EGFR: EGFR L858R (n=1), EGFR exon 19 deletions (n=4), which may respond to first line tyrosine kinase inhibitors (TKIs) such as erlotinib and afatinib. Three patients with EGFR exon 19 deletions had concurrent T790M resistance mutations, which develop in over half of patients on early generation TKIs. MET amplification, which may respond to crizotinib, was identified in one patient. Clinical outcomes will be reported at the time of presentation. Of the 43 patients in our series, actionable findings were identified in 35 patients (81.4%), with an approved therapy in 5 (11.4%), off label therapies in 19 (44.2%), and clinical trials in 28 (65.1%).

Conclusion:
In our series of NSCLC patients with advanced disease, digital sequencing of cell-free circulating tumor DNA yielded results in approximately 80% of patients. Of these, over 80% had an actionable alteration, including 5 cases with EGFR alterations that could benefit from an approved therapy. Biopsy-free comprehensive sequencing of a patient’s cancer can empower informed treatment decisions from a simple blood draw, especially when repeat tissue biopsy is not feasible or tissue NGS is uninformative.

Background:
Chromosomal rearrangements that result in transcript fusions have been a focus of attention in cancer as they provide attractive therapeutic targets. Identifying tumors that harbor chromosomal rearrangements by in situ hybridization assays has been a challenge in the clinic because these assays demand large quantities of tissue specimens. Cell-free nucleic acids from patient plasma may provide a non-invasive, alternative tool for detecting transcript fusions. Here, we demonstrate the feasibility of detecting ALK fusions with a qRT-PCR assay using cell-free plasma RNA (cfRNA).

Methods:
We designed a one-tube, four-channel multiplex ALK qRT-PCR assay that incorporates two strategies to detect ALK fusions. One channel employs variant-specific primers to detect >90% of the reported ALK fusions. The remaining three channels measure the expression of the 5’ and 3’ ends of the ALK gene relative to an internal reference and to each other, in theory, permitting the detection of all ALK fusions including those without knowledge of the fusion partner. To assess the performance of the multiplex ALK prototype assay, we made contrived samples blending mutant and wildtype cell line RNAs. In addition, we tested the multiplex ALK assay on NSCLC FFPET specimens (n=209). Moreover, to mimic plasma cfRNA, we made contrived samples by blending mutant cell line conditioned media with normal plasma.

Results:
Data from the cell line RNA blends demonstrate that both the variant-specific and the 5’ and 3’ differential expression successfully detect the EML4-ALK fusion-positive RNA. The variant-specific component of the assay is sensitive enough to detect at least 25pg of fusion-positive cell line RNA at a 1:4000 dilution with wildtype cell line RNA. From the NSCLC FFPET specimens, we identified 4 samples positive for the ALK fusion. These results were validated by a reference method that uses anchored-PCR to enrich ALK targets followed by NGS that employs a novel algorithm to identify potential fusion products. In addition, the multiplex ALK qRT-PCR assay detected transcript fusions in blends composed of plasma and EML4-ALK positive conditioned media at a dilution of 3:1. Lastly, we tested the multiplex ALK assay on confirmed ALK-fusion positive NSCLC plasma specimens, and were able to detect ALK fusions (7 out of 8) from as little as 750 ul of plasma.

Conclusion:
In summary, we have developed a one-tube, multiplex ALK qRT-PCR assay that exhibits performance characteristics suitable for transcript fusion detection in plasma cfRNA. Efforts are underway to further test and optomize the performance of this assay in clinical samples and to apply this multiplex qRT-PCR design concept to other transcript fusions including RET and ROS1.

Abstract not provided

Background:
Circulating tumor DNA can be detected in urine efficiently, serially, and completely non-invasively. Utilizing a PCR enriched NGS detection platform, we sought to demonstrate the feasibility of detecting activating and resistance EGFR mutations in urinary ctDNA to understand mechanisms of resistance to targeted therapies in patients with EGFR-mutated lung adenocarcinoma.

Methods:
In a biomarker study of 46 patients enrolled, urine was collected every 3-6 weeks from patients on first line anti-EGFR TKI therapy and then daily at progression during the first week of 3rd generation anti-EGFR TKI treatment when available. Urinary ctDNA was extracted by a method that preferentially isolates short, fragmented ctDNA. Quantitative analysis of EGFR activating exon19del, L858R, and T790M resistance mutations was performed utilizing wild type blocker probes, PCR enrichment, and NGS detection (MiSeq). Early pharmacodynamic events within the first hours to days of anti-EGFR therapy were further studied by quantitating ctDNA mutations and comparing with the reponse or lack of response by RECIST on CT scans 6 weeks after initiation of second line therapy.

Results:
Interim analysis was conducted on 34 patients receiving first line anti-EGFR therapy with erlotinib. The average quantity of DNA obtained per patient was 830ng/70ml of urine. The sensitivity between tissue and urine for EGFR Exon19del, L858R, and T790M was 94%, 100%, and 100% respectively, and interim specificity was 94%, 100%, and 96% respectively. Analysis of longitudinal samples from patients on erlotinib revealed that the EGFR T790M mutation was detected in the urine of 17 out of 24 (71%) patients 4-15 weeks before radiographic progression on erlotinib. All 10 patients who were positive for T790M mutation by tissue were also positive by urine. Three patients were T790M tissue negative but urine was positive for T790M. Early peaks in EGFR Exon19del, L858R, and T790M ctDNA on days 1-4 of urine collected daily within the first week on next generation anti-EGFR TKI correlated with CT radiographic response or lack of response 6 weeks after first drug dosing. Figure 1



Conclusion:
We demonstrate that EGFR activating and resistance mutations can be detected in ctDNA in urine months before progression on anti-EGFR TKIs. Urinary ctDNA testing identifies additional patients who are potentially eligible for next generation anti-T790M treatment. The size of the peaks in ctDNA upon second line anti-EGFR inhibitors correlate with tumor lysis and CT radiographic response. The clinical utility of daily kinetic monitoring of ctDNA in urine after drug adminstration is being further validated in an expanded cohort.

Background:
To fully implement precision therapy in lung cancer, transition to a re-biopsy policy will be required at baseline and at progression after each line of therapy. The molecular testing paradigm is shifting toward next generation sequencing (NGS). As tissues are limited and repeat invasive biopsy introduces cost and risk, novel technologies sensitive and specific enough for multiplexed assessment in cell-free DNA (cfDNA) isolated from patient blood would represent a significant advance. Preliminary experience from investigators suggest a high degree of correlation between repeat tumor biopsy and plasma NGS. Here, we present the Guardant Health (GH) digital sequencing approach in a consecutive series of NSCLC cases.

Methods:
225 consecutive blood specimens from NSCLC patients, collected February–March 2015, were evaluated for cfDNA tumor alterations by digital sequencing using the GH panel of 68 genes. The test includes all reported fusion partners for ALK, RET, ROS1, and NTRK1 and cfDNA amplification for 16 genes. The mutant allele fraction (MAF) was calculated relative to WT in cfDNA. The test is sensitive to a single fragment of mutated cfDNA in a 10 ml blood sample and analytic specificity is >99.9999%.

Results:
Canonical EGFR activating mutations were detected in 20 cases (14 E19del, 3 L858R, 2 E20ins, 1 G719A). EGFR T790M co-occurred in 7 cases (6 E19del, 1 L858R), with EGFR amplification observed in 6 of the 20. Median age for patients with EGFR mut+ was 62.5; 18 female(90%), compared to nonEGFR-mutant cases. Four cases had driver fusions (2EML4-ALK, 2 KIF5B-RET) and five cases harbored an ERBB2 E20ins. KRAScodon 12/13 mutations were detected in 23 patients, while 3 harbored mutations in HRAS(Q61L) and NRAS(Q61L, G13R), and 6 had BRAF mutations (4 V600E, 2 G466X). All putative drivers were mutually exclusive. Mutations in signal transduction factors with confirmed gain-of-function activity included AKT1(E17K), MEK1(K57N, C121S), PIK3CA(E542K, E545K x2, H1047L, M1043V, R93W) and JAK2(V617F x2); truncating or missense mutations (>3% MAF) were observed in NF1 (6 cases), PTEN(1 case), SMAD4(4 cases) and STK11(4 cases). TP53 mutations were detected in 116/225 (51%). Evidence of gene amplification was seen in 32 cases, with 11 harboring multiple events. By function, amp events were observed for G1 cell cycle factors:11, RTKs: 17, MYC: 2; and signal transduction: 21. MAF ranged from 0.06% to 83.4% (av 5.1%; median: 9.8%), reflecting clinical and biologic diversity of patients. In a clinical subset at UC Davis, 27 patients were evaluated and alterations were detected in 18 (66.7%). Actionable findings were identified in 14 (77.8%) including 2 with EGFRL858R, 1 with EGFR E19del, and 1 interesting case with EGFR E19del at 45% MAF, EGFR amplification, and an emerging EGFR T790M clone at 0.54% MAF.

Conclusion:
In a series of NSCLC cases, high-sensitivity, high-specificity cfDNA analysis demonstrated the ability to identify somatic tumor alterations, including clinically actionable predictors, in a majority of patients via a simple blood draw, suggesting that this approach can be used for guiding therapeutic decision-making when repeat biopsy is high risk or not possible. Assuming validation, plasma cfDNA analysis may supplant invasive tumor biopsy in the near future.

Background:
As DNA analytical methods have become more sensitive, attempts to develop accurate clinical tests to assess tumor mutation status by means of patient plasma samples are now being pursued. The potential to accurately quantify EGFR mutations in plasma from non-small cell lung cancer (NSCLC) patients would enable more rapid and more frequent analyses to assess disease status; however, the utility of such analyses for clinical purposes has only recently started to be explored.

Methods:
Plasma samples were obtained from 69 NSCLC patients with EGFR-mutated tumors and 21 negative control cases. EGFR mutations in plasma were analyzed by a standardized allele-specific polymerase chain reaction (PCR) test and ultra-deep next generation sequencing (NGS). A semi-quantitative index (SQI) was derived from dilutions of known EGFR mutation copy numbers. Clinical responses were evaluated by RECIST 1.1 criteria and expressed as percent tumor shrinkage.

Results:
The sensitivity and specificity of the PCR test and NGS assay in plasma versus tissue were 72% versus 100%, and 74% versus 100%, respectively. Quantitative indices by the PCR test and NGS were significantly correlated (P<0.001). EGFR testing at baseline and serially at 4–60 days during TKI therapy revealed a progressive decrease in SQI , starting from day 4, in 95% of cases. The rate of SQI decrease correlated with percent tumor shrinkage at 2 months (P<0.0001); at 14 days it was more than 50% in 70% of patients (rapid responders) (Fig.1A-B). In 2 patients with slow response (Fig.1B), an early increase in the circulating levels of the T790M mutation was observed. These patients were defined as early resistant (Fig.1C). No early T790M mutations were seen in plasma samples of rapid responders, suggesting that slow responders are more prone to develop early resistance.

Conclusion:
Quantification of EGFR mutations from plasma with a standardized PCR test is feasible. To our knowledge, this is the first study showing a strong correlation between the EGFR SQI during therapy and clinical response with relevant implications for patient management. With the strong correlation between EGFR SQI in plasma and clinical outcome, this study opens the way to prospectively design clinical trials to confirm these data and evaluate the diagnostic value of this test. Figure 1

Abstract not provided