Cadm 1's expression levels in peripheral blood: A potential marker for diagnosis, disease progression and clicical outcome for NSCLC.

MARIA TERESA CORTE REAL TELES BRANCO Cadm1’s expression levels in peripheral blood: A potential marker for diagnosis, disease progression and clinical outcome for NSCLC Dissertação de Candidatura ao grau de Mestre em Oncologia – Especialização em Oncologia Molecular submetida ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Porto. Orientador – Professor Doutor Rui Manuel de Medeiros Melo Silva Categoria – Professor Associado Convidado Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto e Grupo de Oncologia Molecular e Patologia Viral, Centro de Investigação do Instituto Português de Oncologia do Porto Co-orientador – Mestre Mónica Patrícia Silva Gomes Afiliação – Grupo de Oncologia Molecular e Patologia Viral, Centro de Investigação do Instituto Português de Oncologia do Porto Informação Técnica TÍTULO: Cadm1’s expression levels in peripheral blood: a potential marker for diagnosis, disease progression and clinical outcome for NSCLC Tese de Candidatura ao Grau de Mestre em Oncologia – Especialização em Oncologia Molecular submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto AUTOR: Maria Teresa Corte Real Teles Branco DATA: outubro 2015 EDITOR: Maria Teresa Corte Real Teles Branco MORADA: Rua da Música nº1 LOCALIDADE: Coruche CÓDIGO POSTAL: 2100-135 Coruche CORREIO ELECTRÓNICO: [email protected] 1ª EDIÇÃO: outubro 2015 “When nothing is sure, everything is possible.” Margaret Drabbler Agradecimentos Ao terminar mais uma etapa da minha vida não podia deixar de agradecer àqueles que foram essenciais para a realização deste projeto. Em primeiro lugar gostava de agradecer à Professora Doutora Berta Silva, Diretora do Mestrado em Oncologia, pela oportunidade de ingressar neste mestrado. Ao Professor Doutor Rui Medeiros, meu orientador, agradeço a oportunidade de fazer parte da sua equipa. Obrigada por toda a ajuda e disponibilidade que me ofereceu, pelo entusiasmo que demonstrou por este projeto, e por me ensinar a pensar além do óbvio. Um grande agradecimento à Doutora Mónica Gomes. Não seria possível ter feito este trabalho sem a sua ajuda. Mostrou-se disponível para me ensinar e ajudar desde o primeiro momento. Motivou-me quando os resultados não foram os que eu esperava. Ajudou-me a ver e a pensar no que pode vir depois deste trabalho e isso foi essencial para que eu não desmotivasse. Obrigada por tudo o que me ensinou, pelos “brainstormings” e pelo exemplo que deu na maneira como trabalha. Foi essencial para a realização desde projeto. A todos os colegas do piso -1, muito obrigada pela recetividade e disponibilidade. Todos, sem exceção, se mostraram disponíveis para me ajudar, ensinar e acompanhar, sempre que precisei. Quero muito agradecer à Andreia, Liliana, Luísa, Joana e Marta por me fazerem sentir que os 300 quilómetros de distância que nos separam não mudam a nossa amizade. Conseguiram estar “presentes” sempre que precisei e foram um pilar essencial nesta jornada. São cinco pessoas fantásticas e cinco profissionais que admiro muito. A toda a família e amigos que me fazem feliz por voltar a casa, e que não me fizeram sentir a distância nos últimos dois anos. Obrigada mãe, por me mostrar todos os dias o que é empenharmo-nos num projeto e levá-lo até ao fim. “Quando acreditamos no que fazemos, queremos sempre fazer melhor” e eu espero um dia conseguir deixar a minha marca nos projetos em que me envolvo tal como a mãe faz. Obrigada pela mãe que é, por acreditar em mim e por me ter apoiado em tudo o que fiz até aqui. Pai, desde pequena que me ensinou a questionar o que me dizem e a pensar por mim em vez de ir atrás dos outros. Esse espírito crítico e “cético” é essencial num investigador e esteve bem presente ao longo deste trabalho. Desde o primeiro dia que mostrou interesse no que eu faço, questionou as minhas escolhas e fez-me querer justificar cada passo do meu trabalho. Obrigada por tudo isto e pelo exemplo que é como pai, como homem e como profissional. À Mariana e ao António, agradeço por me fazerem sentir orgulhosa por ser irmã mais velha. Fazem-me querer ser melhor do que sou para dar o melhor exemplo possível. Ao meu tio Carlos Maria, por me ter feito voltar às raízes, pelas gargalhadas e pelas histórias. Esses fins de semana foram essenciais para renovar energias e preparar uma boa semana. Por último, e mais importante de todos, quero agradecer à tia Lena e à Mafalda. Deram-me muito mais que um teto. Deram-me uma casa, uma nova família. Cresci, com o exemplo que dão todos os dias e nada disto teria sido o mesmo sem as duas. Obrigada pelos miminhos, pela companhia, pelas histórias e por me fazerem sentir em casa e parte da família. Nunca esquecerei o quão importante foram nestes dois anos, especialmente durante a elaboração deste trabalho. A todos, muito, muito obrigado! 5 Abbreviations A A Adenine AJCC American Joint Committee on Cancer ATL Adult T-cell leucemia B BRCA2 Breast Cancer 2 C C Cytosine CADM1 Cell Adhesion Molecule 1 CASK Calcium/Calmodulin-Dependent Serine Protein Kinase cDNA Complementary Deoxyribonucleic Acid cfDNA Circulating Cell free DNA CI Confidence Interval CRTAM class-I MHC-restricted T cell-associated molecule Ct Cycle threshold CTC Circulating Tumor Cells D DNA Deoxyribonucleic Acid E EDTA Ethylenediaminetetraacetic Acid EGFR Epidermal Growth Factor Receptor EML4-ALK Echinoderm Microtubule-associated protein-like 4 - Anaplastic Lymphome Kinase G G Guanine GUSβ glucuronidase, beta H HER-2 Human Epidermal growth factor Receptor 2 I IARC International Agency for Research on Cancer IgCAM immunoglobulin superfamily cell adhesion molecule IGSF4 ImunoGlobulin SuperFamily 4 K K-RAS Kirsten Rat Sarcoma L LOH Loss of Heterozigoty 6 M miRNA microRNA MPP3 Membrane Protein, Palmitoylated 3 mRNA messengerRNA N necl-2 nectin-like molecule 2 NK cells Natural Killer Cells nm nanometers NPV Negative Predictive Value NSCLC Non-Small Cell Lung Cancer O OR Odds Ratio P PPV Positive Predictive Value Q qPCR Real Time Polymerase Chain Reaction S SCLC Small Cell Lung Cancer SD Standard Deviation SgIGSF Spermatogenic Immunoglobulin Superfamily SynCAM Synaptic Cell Adhesion Molecule T T Thymine TSG Tumor Suppressor Genes TSLC1 Tumor Suppressor of Lung Cancer 1 Y YAC Yeast Artificial Chromosome 7 Table of contents Resumo ...........................................................................................................................13 Abstract ...........................................................................................................................17 1. Introduction ..................................................................................................................21 1.1. Cancer: Epidemiology and general concepts ........................................................21 1.2. Lung Cancer .........................................................................................................22 1.3. Non-small cell lung cancer ....................................................................................24 1.4. Biomarkers ............................................................................................................25 1.5. CADM1 .................................................................................................................28 2. Objectives ....................................................................................................................35 2.1. Main Objective ......................................................................................................35 2.2. Specific Objectives ................................................................................................35 3. Material and Methods ..................................................................................................39 3.1. Patient selection and sample collection .................................................................39 3.2. RNA extraction and cDNA synthesis .....................................................................40 3.3. Cadm1 relative quantification ................................................................................40 3.4. Statistical Analysis .................................................................................................41 4. Results .........................................................................................................................45 4.1. Cadm1 as a diagnostic marker for NSCLC ............................................................45 4.1.1. Association between Cadm1 expression levels and NSCLC. .........................45 4.2. Cadm1 influence in tumor progression and NSCLC advanced stages ...................47 4.2.1 Association between Cadm1 expression levels and NSCLC advanced stages 47 4.2.2. Association between Cadm1 expression levels and NSCLC long distance metastasis selecting for possible confounding variables...........................................48 4.3. The influence of Cadm1 expression levels in 1year survival rates .........................50 5. Discussion ...................................................................................................................53 5.1. Association between Cadm1 expression levels from peripheral blood and NSCLC. ....................................................................................................................................54 5.2 Association between circulating Cadm1 expression levels and tumor progression and clinical outcome ....................................................................................................55 14 não metastizados e estadios metastizados). Os níveis de expressão de Cadm1 no sangue periférico parecem influenciar apenas a sobrevivência a um ano dos indivíduos não metastáticos e não dos indivíduos com tumores em estadio IV. Indivíduos com tumores não metastáticos e baixos níveis de expressão de Cadm1 em circulação apresentam uma pior taxa de sobrevivência do que aqueles que têm tumor não-metastático, mas elevados níveis de Cadm1 (p=0,016). Apesar de os níveis de expressão de Cadm1 no sangue periférico aparentemente não representarem um bom marcador de prognóstico, os nossos resultados suportam o seu uso como biomarcador de diagnóstico apresentando uma sensibilidade de 57,3%, especificidade de 61,5%, VPP de 72% e VNP de 54%. Não existindo à data um marcador ideal para diagnóstico de cancro do pulmão e visto que os métodos utilizados apresentam maiores custos, maiores riscos de indução de efeitos secundários e impossibilidade de aplicação em toda a população, concluímos, com base nos nossos resultados, que o Cadm1 poderá ser um potencial biomarcador de diagnóstico de CPNPC através de uma simples amostra de sangue. Abstract 17 Abstract More and more, cancer has become an emerging public health problem worldwide and it is estimated that there will be 19.3 million new cases per year by 2025 due to growth and ageing of the global population. Lung cancer is the leading cause of cancer-related death worldwide. Most lung cancers (80%) are non-small-cell lung cancers (NSCLC) and of these, more than 65% of the patients present with locally advanced or metastatic disease. When diagnosed in an early stage it's operable and the five-year survival rate is approximately 50%–70%. However, the five-year survival rate drops to 2%–5% for patients whose cancers are diagnosed after their tumors have spread distantly. Although early detection can save lives, the current screening tests for high-risk individuals have not resulted in improvements in disease-specific survival. It has become imperial to develop a non-invasive approach based on molecular biomarkers that may significantly improve the assessment of risk and early diagnosis. One candidate marker is cell adhesion molecule 1 (CADM1), a putative tumor suppressor gene located in 11q23.2 region, which encodes a glycoprotein of the immunoglobulin superfamily and that is found to be downregulated in many solid tumors. It has been related both with suppression of tumorigenesis and decreased metastization, especially in non-small cell lung cancer (NSCLC) and in most cases its downregulation appears to be silencing by promoter methylation. The aim of the present study was to evaluate if Cadm1 expression levels can be used as biomarker for diagnosis and/or prognosis for NSCLC. To evaluate the potential of using Cadm1 expression levels in peripheral blood as a diagnostic marker for NSCLC we conducted a hospital-based case-control study using blood samples from 89 NSCLC patients and 52 healthy individuals. In a second phase we performed a follow-up study with 89 NSCLC patients to evaluate the association between Cadm1’s expression levels in peripheral blood and disease progression and clinical outcome. When we compared the expression profiles of healthy individuals against NSCLC patients, we observed clear differences of expression between both groups. Cadm1 presents lower expression levels in NSCLC patients compared to the higher levels measured in healthy individuals (OR=2.147; CI95%=1.068-4.32; p=0.031). Also, we observed that this differences are independent of gender (HR=0.401; CI95%= 0.181-0.887; p=0.024). When studying the association between Cadm1’s expression levels and disease progression we didn’t find any differences between early stages and more advanced stages, or between non metastatic and metastatic tumors. Furthermore, Cadm1’s expression levels in peripheral blood only appears to 18 influence one-year survival in non-metastatic patients and not in those who have stage IV tumors. Patients with non-metastatic tumors and low Cadm1’s expression levels in circulation have lower survival rates that those who have non-metastatic tumor but high Cadm1’s expression levels (p=0.016). Even though it appears that Cadm1’s expression levels in peripheral blood aren’t a good prognostic marker, our results support the potential of using Cadm1 as a diagnostic biomarker with a sensibility of 57.3%, specificity 61.5%, PPV of 72% and NPV of 54%. Since there isn’t an ideal screening method for lung cancer and the methodologies used are either expensive, have side effects, or are not accessible or applicable to everybody we conclude, based on these results, that Cadm1 may become a potential biomarker for NSCLC through a simple blood test. Introduction 21 1. Introduction 1.1. Cancer: Epidemiology and general concepts More and more, cancer has become an emerging public health problem worldwide and it is estimated that there will be 19.3 million new cases per year by 2025 due to growth and ageing of the global population [1]. At present time, it represents the first and the third cause of death in the developed countries and in the less developed countries, respectively [2]. According to GLOBOCAN 2012 - the International Agency for Research on Cancer (IARC) online database - there were 14.1 million new cancer cases, 8.2 million cancer deaths in 2012, compared to 12.7 million and 7.6 million, respectively, registered in 2008 [1]. Estimates prevalence rates for 2012 show that there were 32.6 million people alive (over the age of 15 years) who had had a cancer diagnosed in the previous five years [3]. The term 'cancer' defines over one hundred different diseases that can arise from virtually any tissue or organ in the body. It is a heterogeneous disease, with different etiology and natural history that develops through the interactions between environmental and genetic factors, and involves deregulation of multiple pathways responsible for the fundamental cell processes, such as death, proliferation, differentiation and cell migration [4]. Hanahan and Weinberg proposed that cancer cells acquire six biological capabilities during the multistep development of human tumors that are shared by nearly all cancers and are considered to represent the hallmarks that govern the conversion of normal cells into cancer cells. Those include growth signal autonomy, insensitivity to anti-growth signals, apoptosis evasion, replicative immortality, angiogenesis induction and tissue invasion and metastasis. Conceptual progress in the last decade has added two emerging hallmarks of potential generality to this list - reprogramming of energy metabolism and evading immune destruction. In addition to cancer cells, tumors exhibit another dimension of complexity for they contain a repertoire of recruited, ostensibly normal cells that contribute to the acquisition of hallmark traits by creating the ‘‘tumor microenvironment’’ [5]. The process of carcinogenesis is very complex and involves a progressive accumulation of genetic alterations that lead to the transformation of a normal cell to a neoplastic cell [5]. Carcinogenesis can be divided in three main stages: Initiation, promotion and progression. The initiation process consists on the acquisition of non-lethal genetic lesions by the somatic cells, caused by carcinogens (chemical, physical or biological), by epigenetic alterations or by inherited germline alterations [6]. If this cell is stimulated by promoting agents like chemicals or hormones and isn’t able to repair the damages, there is an 22 accumulation of genetic alterations that provides a selective advantage for these cells over normal cells. This is the promotion stage: there is a selective clonal expansion of initiated cells which results in a larger population of cells that are at risk of further genetic changes and malignant conversion [7]. Tumor progression comprises the expression of the malignant phenotype and the tendency of malignant cells to acquire more aggressive characteristics over time. During this process, and through interaction with environmental factors, there may occur genetic and epigenetic alterations that allow the activation of proto-oncogenes and inactivation of tumor suppressor genes (TSGs). Subsequently, multiple events occur that result in the disruption of processes such as cell proliferation and apoptosis, which in turn lead to the growth and development of malignancies. The mutations acquired during neoplastic development make the cells less sensitive to apoptotic signals, cell growth stop and destruction by the immune system. Cumulatively, the self-sufficiency of these cells in growth factors provides them with greater proliferative capacity, angiogenesis and invasive potential. These factors combined with increase in inflammation, genomic instability and cell energy deregulation, favor cancer development and systemic dissemination, enhancing the metastatic process [8]. Even though there has been new developments and achievements in the field of Oncology, either in the early detection of some neoplasia and in the development of new treatments more effective in disease management, the numbers are still increasing and the acquired resistance to these treatments has become a difficult obstacle to overcome. 1.2. Lung Cancer Lung cancer has been the most common cancer in the world for several decades. There were estimated to be 1.8 million new cases in 2012 (12.9% of the total). The disease remains as the most common cancer in men worldwide (1.2 million, 16.7% of the total) with the highest estimated age-standardized incidence rates in Central and Eastern Europe (53.5 per 100,000) and Eastern Asia (50.4 per 100,000), in 2012. In women, the incidence rates are generally lower (approximately 0.6 million, 8.8% of the total) and the geographical pattern is a little different, mainly reflecting different historical exposure to tobacco smoking. Furthermore, lung cancer is the most common cause of death from cancer worldwide, estimated to be responsible for nearly one in five deaths (1.59 million deaths, 19.4% of the total) which is more than breast, colon and prostate cancer combined [1, 2]. In Portugal, lung cancer is the fourth most common neoplasia (4192 cases, 8.5% of total), but has the second highest mortality rate (3441 deaths, 14.3% of the total) following closely the 15.7% death rate caused by colorectal cancer [9]. According to American Cancer Society estimates, the 1and 5-year relative survival 23 rates for lung cancer cases diagnosed during 2003-2009 were 43% and 17%, respectively [10]. The majority of lung cancer cases in men (~85%) and approximately 47% of lung cancer cases in women are estimated as being the consequence of tobacco smoking [11]. Primary prevention should continue to be a major focus of public health campaigns for there is overwhelming evidence that tobacco smoking is the major cause of lung cancer not only based on epidemiological studies. Lung tumors of smokers frequently contain a typical, though not specific, molecular footprint in the form of G:C>T:A mutation in TP53 gene which are probably caused by benzo[a]pyrene one of the many carcinogens in tobacco smoke [11]. However, there are other factors that may play a role in susceptibility to lung cancer or modulate the development of the neoplasia. Occupational exposure to carcinogens like arsenic, asbestos and other chemicals, pulmonary chronic disease, environmental pollution, pulmonary tuberculosis or even family history, viruses, and auto-immune diseases are considered to be possible risk factors, but further studies are necessary to clarify their association with lung cancer [11]. One of the biggest problems associated with poor survival in lung cancer cases is that most patients present with metastatic disease at time of diagnosis (~65%) [12, 13]. Metastasis is a multistep process in tumor development that culminates in a systemic spread of cancer cells. For reasons still unclear, some cells from the primary tumor suffer molecular alterations that causes them to separate from the tumor and enter circulation and invade another organ where a new tumor starts developing. Treatment against metastasis is still very poor because of the heterogeneity of the metastasis since the same type of tumor can develop metastasis in different sites, and even in the same tumor, different cells have a potential to metastasize. A correct evaluation of the histologic subtype and staging at the time of diagnosis is necessary for treatment and patient’s prognosis [14]. Staging for lung tumors is according to the TNM system, proposed by the American Joint Committee on Cancer (AJCC), that classifies the tumor according to its size and invasive potential (T), with the involvement of regional lymph nodes (N) and the absence or presence of long distance metastasis (M) [1416]. Even though 50% of lung carcinomas exhibit more than one major histological type, two main categories are distinguished: small cell lung cancer (SCLC) accounting for about 20% of lung cancer cases, and non-small cell lung cancer (NSCLC) that comprises a number of histological subtypes representing near 80% of lung cancers [11]. 30 apoptosis in A549 cells [56]. Studies in nude mice have demonstrated that re-expression of CADM1 suppresses in vivo tumorigenicity of NSCLC and nasopharyngeal carcinoma cell lines [56, 57]. Apoptosis induction, as well as the formation or maintenance of an epithelial cell structure through homophilic interaction, is one of the possible molecular mechanisms of CADM1/TSLC1 in the tumor suppression of carcinomas. In contrast, CADM1/TSLC1 has been found to be ectopically expressed in adult Tcell leukemia (ATL), and could be implicated in tumor invasion through interactions of the leukemia cells with the endothelial cells [58]. Another interesting study challenging CADM1's role as a tumor suppressor is one by Kikushi et al. They studied a splicing variant of CADM1 present in Small Cell Lung Cancer and their results suggest that CADM1 enhances the malignant features of SCLC, as is observed in ATL, and could provide a molecular marker specific to SCLC [59]. It is now known that CADM1/TSLC1 is expressed universally in human tissues and is frequently silenced in a variety of human carcinomas, such as lung, prostate, liver, stomach, pancreatic and breast carcinoma [48, 56]. Besides mutations and deletions, DNA methylation of CpG islands in the promoter region of cancer-related genes is a frequently acquired epigenetic event in the pathogenesis of many human malignancies leading to gene silencing. So far, aberrant methylation (referred to as methylation) has been described for several genes in various malignancies including lung cancer [60]. Promoter methylation of the CADM1/TSLC1 gene was demonstrated in NSCLC, pancreatic cancers, hepatocellular carcinoma, and prostate cancers, cancers from the esophagus, stomach, nasopharynx, breast, and uterine cervix, as well as meningioma, and seems to be the main mechanism underlying the expression silencing [41, 49, 60-62]. Furthermore, methylation or loss of CADM1 expression was observed preferentially in tumors at advanced pathological stages rather than in early stages and found to be associated with tobacco smoking [63, 64]. Fukami et al. demonstrated that CADM1 promoter methylation was significantly associated with tumor progression of NSCLC from pT1 to pT2 and from stage Ia to IIb [61]. And Heller et al. showed that promoter methylation also occurred more frequently in tumors at grade 3, compared with tumors at grade 1 or 2 in breast cancer [65]. In an immunohistochemical study using a specific antibody against CADM1/TSLC1, Uchino et al. demonstrated that CADM1/TSLC1 expression was inversely correlated with advanced disease stage, lymph node involvement, lymphatic permeation, and vascular invasion. 4-year survival and disease-free survival are significantly shorter in patients with lung adenocarcinoma lacking CADM1 expression [66]. 31 For all that’s been previously said, it seems clear that CADM1 plays an important role in cancer development, especially in NSCLC since it is downregulated in most cases and appears to be correlated with tumor progression, but can it be used as a biomarker? Objectives 35 2. Objectives 2.1. Main Objective The aim of the present study is to evaluate if Cadm1 expression levels can be used as biomarker for diagnosis and/or prognosis for NSCLC patients. Thereby, the present study can be divided in two different parts: 2.2. Specific Objectives 1. Assess Cadm1’s expression levels in peripheral blood as a possible diagnostic marker for NSCLC; 2. Evaluate the association between Cadm1’s expression levels in peripheral blood and tumor progression, namely 1-year survival. Material and Methods 39 3. Material and Methods 3.1. Patient selection and sample collection 3.1.1. The first part of this work consisted of a hospital-based case-control study involving 141 individuals (89 NSCLC patients and 52 healthy individuals) all Caucasian, from the north of Portugal. All participants signed an informed consent according to the Helsinki declaration. The case group consisted of 69 males and 20 females all Caucasian with non-small cell lung cancer diagnosed and treated at the Portuguese Institute of Oncology of Porto (mean age = 61.2; SD=15.7). After patients signed an informed consent according to the Helsinki declaration a blood sample was collected before any medical intervention. All the demographic and histopathological data were collected from the clinical files of each patient and the staging of each patient was made according to the AJCC TNM classification system 2010 (7th edition). For the control group, 52 healthy Caucasian individuals with no history of cancer (mean age = 50.22; SD=17), were randomly recruited from the north of Portugal. All demographic data were collected through a written form, authorized by the ethics committee of the Portuguese Institute of Oncology of Porto, and signed by each individual, according to the Helsinki declaration. All the clinicopathological and demographic data are described in table 1. Approximately 8 ml of peripheral blood were collected from all individuals throught a standard method of intravenous collection with EDTA tubes. The blood tubes were centrifuged 5 minutes at 2800 rpm in order to separate the peripheral blood cells and then preserved in Tripure ® (Roche) at 80ºC. 3.1.2. The second part consisted of a follow-up study involving the 89 NSCLC patients from the case group of the first part. All the clinicopathological data are described in table 1. 46 In order to better visualize these differences we created the graphic below where it is clear that the higher percentage of individuals with low Cadm1 expression levels belong to our case group and the higher percentage of individuals with high Cadm1 expression levels belong to the control group. To have a better understanding of our sensitivity and specificity values, we compared our study with other methods currently in use for diagnosing NSCLC. Table 3 Comparison of specificity and sensitivity values for the currently used methods for diagnosing NSCLC Screening method Specificity (%) Sensitivity (%) References Cadm1 expression levels 61.5 57.3 Current work Low Dose CT 92.6 / 98.3 88.9 / 94.6 [69] / [70] Chest x-ray 78.3 92.6 [69] Panel of 6 antibodys agains 6 tumor related antigens 89-91 36-39 [70] These results suggest that assessing Cadm1’s expression levels in peripheral blood may be a potential diagnostic marker for NSCLC. Figure 5 Percentage of participants (cases and controls) corresponding to the different Cadm1’s expression profiles 47 To further evaluate the potential of monitoring Cadm1 expression levels to diagnose NSCLC we studied the association between gender and the gene's expression levels (table 4) to eliminate possible confounding bias. According to our results, Cadm1’s expression levels are independent of gender. Table 4 Logistic Regression for Cadm1 expression HR CI 95% p Cadm1 High/Low 0.401 0.181-0.887 0.024 Gender 7.639 3.459-16.871 <0.001 pp value from X2 test; HRHazard Ratio; CI 95% - Confidence Interval at 95% 4.2. Cadm1 influence in tumor progression and NSCLC advanced stages 4.2.1 Association between Cadm1 expression levels and NSCLC advanced stages To study whether there was an association between Cadm1 expression levels and the evolution of the tumor we analyzed the expression in early stages (I and II) versus advanced stages (III and IV) as described in table 5. We couldn’t find any differences with statistical significance between the groups (OR=2.30; CI 95%=0.231-23.147; p=0.464). Table 5 Association between Cadm1 expression levels and tumor stages Stage I + II Stage III + IV Total OR CI 95% p Cadm1 mRNA n (%) n (%) n (%) High 1 (25.0) 37 (43.5) 38 (42.7) - - Low 3 (75.0) 48 (56.5) 51 (57.3) 2.30 0.231-23.147 0.464 pp value from X2 test; OROdds Ratio; CI 95% - Confidence Interval at 95% 48 4.2.2. Association between Cadm1 expression levels and NSCLC long distance metastasis selecting for possible confounding variables 4.2.2.1. Selecting for Gender In order to eliminate gender as a possible confounding variable we analyzed the differences in Cadm1’s expression levels between non-metastatic stages and metastatic stages regarding gender. We couldn’t find any differences with statistical significance between the groups in neither female (OR=2.667; CI 95%=0.237-30.066; p=0.417) or male (OR=1.200; CI 95%=0.466-3.093; p=0.706). Table 6 Association between Cadm1 expression levels, metastization and gender Gender Cadm1 Stage I, II and III Stage IV OR CI 95% p n (%) n (%) Female High 1 (14.3) 4 (30.8) - Low 6 (85.7) 9 (69.2) 2.667 0.237-30.066 0.417 Male High 15 (45.5) 18 (50.0) - Low 18 (54.5) 18 (50.0) 1.200 0.466-3.093 0.706 pp value from X2 test; OROdds Ratio; CI 95% - Confidence Interval at 95% 4.2.2.2. Selecting for Smoking status In order to eliminate the smoking status as a possible confounding variable we analyzed the differences in Cadm1’s expression levels between non-metastatic stages and metastatic stages regarding smoking status. We couldn’t find any differences with statistical significance between the groups in neither Smoking (OR=1.466; CI 95%=0.554-3.882; p=0.441) or non-smoking (OR=1.200; CI 95%=0.166-8.659; p=0.856). 49 Table 7 Association between Cadm1 expression levels, metastization and smoking status Smoking status Cadm1 Stage I, II and III Stage IV OR CI 95% p n (%) n (%) Smoking High 13 (41.9) 18 (51.4) Low 18 (58.1) 17 (48.6) 1.466 0.554-3.882 0.441 Nonsmoking High 2 (25.0) 4 (28.6) Low 6 (75.0) 10 (71.4) 1.200 0.166-8.659 0.856 pp value from X2 test; OROdds Ratio; CI 95% - Confidence Interval at 95% 4.2.2.3. Selecting for Histology In order to eliminate histology as a possible confounding variable we analyzed the differences in Cadm1’s expression levels between non-metastatic stages and metastatic stages regarding the different histologic subtypes. We couldn’t find any differences with statistical significance between the groups in neither Squamous Cell Carcinoma (OR=1.469; CI 95%=0.350-6.168; p=0.598) or Non-Squamous Cell Carcinoma (OR=1.067; CI 95%=0.360-3.159; p=0.907). Table 8 Association between Cadm1 expression levels, metastization and histologic subtype Histologic subtype Cadm1 Stage I, II and III Stage IV OR CI 95% p n (%) n (%) SCC High 7 (36.8) 6 (46.2) Low 12 (63.2) 7 (53.8) 1.469 0.350-6.168 0.598 Non-SCC High 9 (42.9) 16 (44.4) Low 12 (57.1) 20 (55.6) 1.067 0.360-3.159 0.907 pp value from X2 test; OROdds Ratio; CI 95% - Confidence Interval at 95% 50 Figure 6 One-year survival by Kaplan-Meier for non-metastatic (A) and metastatic (B) NSCLC according to Cadm1’s expression levels in the blood. 4.3. The influence of Cadm1 expression levels in 1year survival rates When analyzing the association between Cadm1’s expression levels with one-year survival, distinguishing non-metastatic (stages I, II and III) and metastatic (stage IV) we observe different results. According to figure 6A (non-metastatic) there is a difference with statistical significance in 1-year survival rates of patients with Low Cadm1 versus patients with High Cadm1 (p=0.016). In figure 6B (metastatic) the difference between the two groups (High Cadm1 and Low Cadm1) doesn’t have statistical significance (p=0.751). Discussion 53 5. Discussion In the past years it has become clear that the key to lower cancer mortality rates is in early diagnosis and personalized therapy. Because cancer is a very complex disease, finding a specific marker for early diagnosis is complicated and finding a specific target for treatment has proven difficult for most tumors. With the consistent increase in lung cancer incidence and low survival rates it has become imperial to find tools that allow early diagnosis. It is clear that people with tumors diagnosed in early stages have far better survival rates than those whose tumors were in more advanced stages. For lung cancer, one of the obstacles to early diagnosis is the fact that symptoms only appear in more advanced stages and a major hurdle in the attempts to improve the survival of these patients has been the lack of a simple, non-invasive and effective test for early prediction and for monitoring therapeutic efficacy [21, 29]. One potential marker for NSCLC development is CADM1. Even though Cadm1 is expressed in a variety of normal tissues, nowadays, CADM1 is well established as a tumor suppressor, not only in lung cancer, but in different tumors and appears to be downregulated in more advanced stages of the disease [48-53]. This downregulation is mostly due to promoter methylation but can also be the result of LOH. Because CADM1 is an adhesion molecule, it seems logic that its expression is lower when tumor has metastasized, since this process requires cell detachment from the primary tumor. The use of a biomarker for early detection and for monitoring disease progression implies that with a simple test we can differentiate an affected patient from a person without the disease. This means diagnosing tumors in earlier stages that consequently are easier to treat and also, adjust therapy strategies according to each patient’s response. To the best of our knowledge, our study is the first to measure Cadm1 levels in peripheral blood. All the others report the use of tissue samples obtained directly from the tumor to quantify the molecule. When measuring Cadm1’s levels in circulation we’re referring to its expression in blood cells and not plasma or serum. With this in mind, we established two main goals in order to evaluate the potential of using this method for early diagnosis, monitoring disease progression and evaluate clinical outcome: the first was to determine if there was an association between circulating mRNA levels of the CADM1 gene and non-small cell lung cancer in order to validate Cadm1 as a good diagnostic marker; the second was to study the association of circulating CADM1’s expression levels and tumor progression and clinical outcome. 54 5.1. Association between Cadm1 expression levels from peripheral blood and NSCLC. In the beginning of the process of carcinogenesis the cell must undergo a change, genetic and / or epigenetic, which may lead to changes in gene activity resulting in the rupture of the homeostatic balance [71]. Some of these alterations lead to inactivation of tumor suppressor genes (TSGs) and the effect of their inactivation plays a major role in cancer and has been described for several genes in various malignancies. CADM1 is a TSG that has been reported to be functionally involved in pulmonary carcinogenesis and that is highly methylated in most NSCLC cases and inactivation of this gene by promoter methylation has been proposed as a candidate marker for early lung cancer detection [40, 72]. Even though the process through which Cadm1 acts to suppress tumorigenesis is still not clear, being a tumor suppressor it seems logic to find higher levels of Cadm1 in healthy individuals compared with patients with cancer. According to our results, there is a difference with statistical significance between our case and control groups: individuals with Low Cadm1 expression levels, have a higher probability of being diagnosed with NSCLC (OR=2.147; CI95%=1.068-4.32; p=0.031) when compared with individuals with High Cadm1 expression levels. The majority of our control group had High Cadm1 expression levels (61.5%) while in NSCLC group, the majority presented with Low Cadm1 expression levels (57.3%). If CADM1 is a TSG, healthy individuals will have High Cadm1 expression levels. Patients with NSCLC, however, will have lower Cadm1’s expression levels since the disruption of the balance between oncogenes and tumor suppressor genes is a crucial step for carcinogenesis. With CADM1 downregulated, there is a reduction of the signals to stop cell growth and a decreasing in the level of cell adhesion which allows the tumor to develop [73]. The diminished levels of Cadm1 in the blood cells of patients with NSCLC compared to healthy individuals may be due to different factors. It has been proposed that Cadm1 interacts with the immune system [49, 74], so it is logical to suppose that low Cadm1’s expression in the blood cells of NSCLC patients is the result of the interaction between tumor cells and blood cells. Tumor cells signal the cells in circulation to lower their Cadm1 expression levels. If these blood cells signal other cells to lower their Cadm1 expression, the result could be the establishment of a microenvironment with a reduced tumor suppressor activity consequently promoting tumor development, invasion and metastization. How this interaction works, and the pathways leading to Cadm1 decrease in the blood cells is still unknown. 55 On the other hand, we can also suggest that the difference in Cadm1 expression in circulating blood cells between healthy individuals and NSCLC patients is the cause for tumor formation instead of a consequence of the tumor. It is possible to assume that because individuals from our case group had a polymorphism that resulted in lower Cadm1 expression, they were more susceptible to develop NSCLC than our control group. If this was the case, it wouldn’t be the tumor modulating Cadm1’s expression levels in circulation. The fact that those people had less Cadm1 by default would be the reason to why they are more susceptible to develop NSCLC. Weyden et al. showed that Cadm1-null mice died significantly faster than their wildtype littermates due to the spontaneous development of tumors at an earlier age, which supports our suggestion [75]. Whatever the cause for the difference of expression levels, our results support the potential of using Cadm1 as a diagnostic biomarker with a sensibility of 57.3%, specificity 61.5%, PPV of 72% and NPV of 54%. At the present time, there isn’t an ideal screening method for lung cancer and the methodologies used are either expensive, or have side effects, or are not accessible or applicable to everybody. When compared to the current methods used, Cadm1 expression levels in peripheral blood seems to have low specificity and sensitivity (table 3). However, it is important to note that this is a non-invasive method, with no side effects (contrary to those of CT scans and X-Ray), it’s less expensive, faster to obtain results and applicable to everyone. Having a PPV of 72% means that an individual who has Low Cadm1 expression levels in blood cells has a 72% chance of already having NSCLC and should therefore be recommended for further diagnostic tests. Based on these results Cadm1 has a potential to be a remarkable biomarker for NSCLC through a simple blood test. 5.2 Association between circulating Cadm1 expression levels and tumor progression and clinical outcome 5.2.1. Circulating levels of Cadm1 and tumor progression There is still a lot to understand about the role CADM1 plays in tumor development and the pathways it is involved in, but many different studies have reported a correlation between loss of Cadm1 expression and advanced stages (poor prognosis) and metastasis [50, 63, 66]. Inhibition of Cadm1 in the tumor was proven to be important in metastasis formation in different studies and its re-expression found to prevent metastasis formation. Our results, however, didn’t show an association between Cadm1 circulating levels and tumor progression. We compared Cadm1’s expression levels for early stages (stages I and II) against advanced stages (III and IV) and found no statistical association (p=0.464; 62 To validate the hypothesis that our data is the result of tumor interaction with blood cells it would be interesting to analyze both tumor tissue and blood samples from each individual in order to evaluate a possible correlation between them. Also, knowing more about what triggers Cadm1 silencing in tumor initiation may prove essential to understand it’s role in tumor formation and development. Another interesting idea would be testing Cadm1’s expression levels in patients before and after treatment to understand treatment efficacy and the possibility of using Cadm1 as a marker for monitoring tumor development and treatment efficacy. There is a lot we don’t know about this molecule and its role in lung cancer development, but according to our study, the assessment of Cadm1’s expression levels in the blood seems to potentially be a good diagnostic marker for NSCLC. It’s a simple, fast and accessible method that allows to differentiate healthy individuals from NSCLC patients. Continuing this work is essential to understand Cadm1 and its role in NSCLC. References 65 7. References 1. IARC, Latest world cancer statistics, in Global cancer burden rises to 14.1 million new cases in 2012: Marked increase in breast cancers must be addressed. IARC: Lyon, 2013: p. 1. 2. Jemal, A., et al., Global cancer statistics. CA: A Cancer Journal for Clinicians, 2011. 2(61): p. 69-90. 3. Bray, F., et al., Global estimates of cancer prevalence for 27 sites in the adult population in 2008. International Journal of Cancer, 2013. 5(132): p. 1133-1145. 4. DeVita, V.T.L., Theodore, S., Rosenberg, Steven, A. , Devita, Hellman & Rosenberg's Cancer: Principles & Practice of Oncology. Lippincott Williams & Wilkins: Philadelphia, 2008: p. 4-8. 5. Hanahan, D., Weinberg, R.A., Hallmarks of Cancer: The Next Generation. Cell, 2011. 144: p. 646-674. 6. Abbas, K., Mitchell, F., Robbins Basic Pathology 8th edition. Saunders Elsevier, 2007. 7. Cairns, J., Mutation selection and the natural history of cancer. Nature, 1975. 255: p. 197-200. 8. Hanahan, D., Weinberg, R.A., The hallmarks of cancer. Cell, 2000. 100: p. 57-70. 9. Globocan. http://globocan.iarc.fr/Pages/fact_sheets_population.aspx?country=620. (Accessed on February 2015). 10. Society, A.C., Cancer Facts & Figures 2014. Atlanta: American Cancer Society, 2014. 11. Travis, W.D., Brambilla, E., Muller-Hermelink, H.K., Harris, C.C. , World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart. . IARC Press: Lyon 2004. 12. Morgensztern, D., Ng, S.H., Gao, F., Govindan, R., Trends in stage distribution for patients with non-small cell lung cancer: a National Cancer Database survey. Journal of thoracic oncology 2010. 5: p. 29-33. 13. Rami-Porta, R., Crowley, J.J., Goldstraw, P., The revised TNM staging system for lung cancer. Annals of Thoracic Cardiovascular Surgery, 2009. 15: p. 4-9. 14. Mountain, C.F., Revisions in the International System for Staging Lung Cancer. Chest, 1997. 111: p. 1710-1717. 15. Brambilla, E., Travis, W.D., Colby, T.V., Corrin, B., Shimosato, Y., The new World Health Organization classification of lung tumours. European Respiratory Journal, 2001. 18: p. 1059-1068. 16. Serke, M., Schonfeld, N., Diagnosis and staging of lung cancer. Deutsche medizinische Wochenschrift, 2007. 132: p. 1165-1169. 17. Goldstraw, P., Crowley, J., Chansky, K., et al., The IASLC Lung Cancer Staging 66 Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours. Journal of thoracic oncology, 2007. 2(8): p. 706-714. 18. Reck, M.e.a., Management of non-small-cell lung cancer: recent developments. The Lancet, 2013. Volume 382(9893): p. 709 - 719. 19. Melamed, M.R., Flehinger, B.J., Screening for lung cancer. Chest, 1984. 86(1): p. 23. 20. Marcus, P.M., Bergstralh, E.J., Zweig, M.H., Harris, A., Offord, K.P., Fontana, R.S. , Extended lung cancer incidence follow-up in the Mayo Lung Project and overdiagnosis. Journal of the National Cancer Institute, 2006. 98(11): p. 748-756. 21. Manser, R., Lethaby, A., Irving, L.B., Stone, C., Byrnes, G., Abramson, M.J., Campbell, D., Screening for lung cancer. Cochrane Database of Systematic Reviews, 2013(6). 22. Pusztai, L., Chips to bedside: incorporation of microarray data into clinical practice. Clinical Cancer Research 2006. 12: p. 7209-7214. 23. Azzoli, C.G., Temin, S., Qliff, T., Baker, S. Jr., Brahmer, J., Johnson, D.H., et al., 2011 Focused update of 2009 American society of clinical oncology clinical practice guideline update on chemotherapy for stage IV non-small-cell lung cancer. Journal of Clinical Oncology, 2010. 29(28): p. 3825-3831. 24. Jantus-Lewintre, Update on biomarkers for the detection of lung cancer. Lung Cancer: Targets and Therapy, 2012. 3 p. 21-29. 25. Nalejska, E., Maczwynska, E., Prognostic and predictive biomarkers: tools in personalized oncology. Molecular diagnosis & therapy, 2014. 18(3): p. 273-284. 26. Ludwig, J.A., Weinstein, J.N., Biomarkers in cancer staging, prognosis and treatment selection. Nature Reviews: Cancer 2005. 5: p. 845-856. 27. Schilsky, R.L., Personalized medicine in oncology: the future is now. Nature Reviews: Drug Discovery, 2010. 9: p. 363-366. 28. Fine, B.M., Amler, L. , Predictive biomarkers in the development of oncology drugs: a therapeutic industry perspective. Clinical pharmacology and therapeutics, 2009. 85: p. 535-538. 29. Duffy, M.J., Crown, J., A personalized approach to cancer treatment: how biomarkers can help. Clinical Chemistry, 2008. 54: p. 1770-1779. 30. Synowiec, E.M.A., Krupa, R., Wozniak, K. , Gene's polymorphism of DNA doublestranded repair in breast cancer. Post. Biol. Kom., 2010. 37(2): p. 433-438. 31. Stoklosa, T., Golab, J., Prospects for p53-based cancer therapy. Acta biochimica Polonica, 2005. 52: p. 321-328. 32. Korpanty, G.J., Graham, D.M., Vincent, M.D., Leighl, N.B., Biomarkers that currently 67 affect clinical practice in lung cancer: EGFR, ALK, MET, ROS-1, and KRAS. Frontiers in Oncology: Thoracic Oncology, 2014. 4(204). 33. Santos, E., Martin-Zanca, D., Reddy, E., Pierotti, M., Della Porta, G., Barbacid, M., Malignant activation of a K-ras oncogene in lung carcinoma but not in normal tissue of the same patient. Science, 1984. 223(4637): p. 661-664. 34. Suzuki, Y., Orita, M., Shiraishi, M., Hayashi, K., Sekiya, T. , Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene, 1990. 5(7): p. 1037-1043. 35. Schiller, J.H., Adak, S., Feins, R.H., et al. , Lack of prognostic significance of p53 and K-ras Mutations in primary resected non-small-cell lung cancer on E4592: a laboratory ancillary study on an Eastern Cooperative Oncology Group prospective randomized trial of postoperative adjuvant therapy. Journal Clinical Oncology, 2001. 19(2): p. 448-457. 36. Riely, G.J., Pao, W., Pham, D., Li, A.R., Rizvi, N., Venkatraman, E.S., Zakowski, M.F., Kris, M.G., Ladanyi, M., Miller, V.A., Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib. Clinical Cancer Research, 2006. 12(3): p. 839-844. 37. Ciardiello, F., Tortora, G. , EGFR antagonists in cancer treatment. New England Journal of Medicine, 2008. 358: p. 1160-1174. 38. Soda, M., Choi, Y.L., Enomoto, M., Takada, S., Yamashita, Y., Ishikawa, S., Fujiwara, S., Watanabe, H., Kurashina, K., Hatanaka, H., Bando, M., Ohno, S., Ishikawa, Y., Aburatani, H., Niki, T., Sohara, Y., Sugiyama, Y., Mano, H., Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature, 2007. 448(7153): p. 561-566. 39. Gomyo, H., Arai, Y., Tanigami, A., Murakami, Y., Hattori, M., Hosoda, F., Arai, K., Aikawa, Y., Tsuda, H., Hirohashi, S., Asakawa, S., Shimizu, N., Soeda, E., Sakaki, Y., Ohki, M., A 2-Mb sequence-ready contig map and a novel immunoglobulin superfamily gene IGSF4 in the LOH region of chromosome 11q23.2. Genomics, 1999. 62: p. 139-146. 40. Kuramochi, M., Fukuhara, H., Nobukuni, T., Kanbe, T., Maruyama, T.,Ghosh, H.P., Pletcher, M., Isomura, M., Onizuka, M., Kitamura, T.,Sekiya, T., Reeves, R.H., Murakami, Y., TSLC1 is a tumorsuppressor gene in human non-small-cell lung cancer. Nature Genetics, 2001. 27: p. 427-430. 41. Murakami, Y., Functional cloning of a tumor suppressor gene, TSLC1, in human cell lung cancer. Journal National Cancer Institute 2002. 21: p. 6936-6948. 42. Wakayama, T., Ohashi, K., Mizuno, K., Iseki, S., Cloning and characterization of a novel mouse immunoglobulin superfamily gene expressed in early spermatogenic 68 cells. Molecular reproduction and development, 2001. 60: p. 158-164. 43. Biederer, T., Sara, Y., Mozhayeva, M., Atasoy, D., Liu, X., Kavalali, E.T., Sudhof, T.C. , SynCAM, a synaptic adhesion molecule that drives synapse assembly. Science, 2002. 297: p. 1525-1531. 44. Murakami, Y., Nobukuni, T., Tamura, K., Maruyama, T., Sekiya, T., Arai, Y., Gomyou, H., Tanigami, A., Ohki, M., Cabin, D., Frischmeyer, P., Hunt, P., et al., Localization of tumor suppressor activity important in nonsmall cell lung carcinoma on chromosome 11q. Proceedings of National Academy of Sciences USA, 1998. 95: p. 8153-8158. 45. Wakayama T., O.K., Mizuno K. and Iseki S., Cloning and characterization of a novel mouse immunoglobulin superfamily gene expressed in early spermatogenic cells. Mol. Reprod. Dev., 2001. 60: p. 158-164. 46. Ito, A., Jippo, T., Wakayama, T., Morii, E., Koma, Y., Onda, H., Nojima, H., Iseki, S. and Kitamura, Y., SgIGSF: a new mast-cell adhesion molecule used for attachment to fibroblasts and transcriptionally regulated by MITF. Blood 2003. 101: p. 2601-2608. 47. Yamada, D., Yoshida, M., Williams, Y.N., Fukami, T., Kikuchi, S., Masuda, M., Maruyama, T., Ohta, T., Nakae, D., Maekawa, A., Kitamura, T., Murakami, Y., Disruption of spermatogenic cell adhesion and male infertility in mice lacking TSLC1/IGSF, an immunoglobulin superfamily cell adhesion molecule. Molecular Cell Biology 2006. 26: p. 3610-3624. 48. Shingai, T., Ikeda, W., Kakunaga, S., Morimoto, K., Takekuni, K., Itoh, S., Satoh, K., Takeuchi, M., Imai, T., Monden, M., Takai, Y., Implications of nectin-like molecule-2/ IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 in cell-cell adhesion and transmembrane protein localization in epithelial cells. Journal Biological Chemistry, 2003. 278: p. 35421-35427. 49. Murakami, Y., Involvement of a cell adhesion molecule. TSLC1/IGSF4, in human oncogenesis. Cancer Science 2005. 96: p. 543-552. 50. Liang, Q.L., Chen, G.Q., Li, Z.Y., Wang, B.R., Function and histopathology of a cell adhesion molecule TSLC1 in cancer. Cancer investigation, 2011. 29: p. 107-112. 51. Masuda, M., Yageta, M., Fukuhara, H., Kuramochi, M., Maruyama, T., Nomoto, A.,Murakami, Y., The tumor suppressor protein TSLC1 is involved in cell-cell adhesion. Journal of Biological Chemistry, 2002. 277: p. 31014-31019. 52. Watabe, K., Ito, A., Koma, Y. and Kitamura, Y., IGSF4: a new intercellular adhesion molecule that is called by three names, TSLC1, SgIGSF and SynCAM, by virtue of its diverse function. Histology and histopathology, 2003 18: p. 1321-1329. 53. Yageta, M., Kuramochi, M., Masuda, M., Fukami, T., Fukuhara, H., Maruyama, T., Shibuya, M. and Murakami, Y., Direct association of TSLC1 and DAL-1, two distinct tumor suppressor proteins in lung cancer. Cancer Research, 2002. 62: p. 5129-5133. 69 54. Galibert, L., Diemer, G.S., Liu, Z., et al., Nectin-like protein 2 defines a subset of Tcell zone dendritic cells and is a ligand for class-I restricted T-cell associated molecule. The Journal of biological chemistry, 2005. 280(21): p. 955-964. 55. Boles, K.S., Barchet, W., Diacovo, T., Cella, M., Colonna, M., The tumor suppressor TSLC1/NECL-2 triggers NK cell and CD8+ T cell responses through the cell surface receptor CRTAM. Blood, 2005. 106: p. 779-786. 56. Mao, X., Seidlitz, E., Truant, R., Hitt, M., Ghosh, H.P., Re-expression of TSLC1 in a non-small-cell lung cancer cell line induces apoptosis and inhibits tumor growth. Oncogene, 2004. 23: p. 5632-5642. 57. Lung, H.L., Cheung, A.K., Xie, D., Cheng, Y., Kwong, F.M., Murakami, Y., Guan, X.Y., Sham, J.S., Chua, D., Protopopov, AI., Zabarovsky, E.R., Tsao, S.W., Stanbridge, E.J., Lung, M.L., TSLC1 is a tumor suppressor gene associated with metastasis in nasopharyngeal carcinoma. Cancer Research, 2006. 66: p. 9385-9392. 58. Sasaki, H., Nishikata, I., Shiraga, T., et al. , Overexpression of a cell adhesion molecule, TSLC1, as a possible molecular marker for acute type of adult T-cell leukemia. Blood, 2005. 105: p. 1204-1213. 59. Kikuchi, S., Iwai, M., Sakurai-Yageta, M., Tsuboi, Y.,Ito, T.,Maruyama, T.,Tsuda, H., Kanai, Y., Onizuka, M., Sato, Y., Murakami, Y., Expression of a splicing variant of the CADM1 specific to small cell lung cancer. Cancer Science, 2012. 103: p. 1051-1057. 60. Heller, G., Fong, K.M., Girard, L., Seidl, S., End-Pfutzenreuter, A., Lang, G., Gazdar, A.F., Minna, J.D., Zielinski, C.C., Zochbauer-Muller, S., Expression and methylation pattern of TSLC1 cascade genes in lung carcinomas. Oncogene, 2006. 25: p. 959968. 61. Fukami, F., Fukuhara, H., Kuramochi, M., et al., Promoter methylation of the TSLC1 gene in advanced lung tumors and various cancer cell lines. Interantional Journal of Cancer 2003. 107: p. 53-59. 62. Fukuhara, H., Kuramochi, M., Fukami, T., et al., Promoter methylation of the TSLC1 and tumor suppression by its gene product in human prostate cancer. Japanese journal of cancer research : Gann, 2002. 93: p. 605-609. 63. Kikuchi, S., Yamada, D., Fukami, T., Maruyama, T., Ito, A., Asamura, H., et al., Hypermethylation of the TSLC1/IGSF4 promoter is associated with tobacco smoking and a poor prognosis in primary nonsmall cell lung carcinoma. Cancer, 2006. 64. Ito, T., Shimada, Y., Hashimoto, Y., et al., Involvement of TSLC1 in progression of esophageal squamous cell carcinoma. Cancer Research, 2003. 63: p. 6320-6326. 65. Heller, G., Geradts, J., Ziegler, B., Newsham, I., Filipits, M., Markis-Ritzinger, E.M., Kandioler, D., Berger, W., Stiglbauer, W., Depisch, D., Pirker, R., Zielinski, C.C., et al., Downregulation of TSLC1 and DAL-1 expression occurs frequently in breast 70 cancer. Breast Cancer Research Treatment, 2007. 103: p. 283-291. 66. Uchino, K., Ito, A., Wakayama, T., et al., Clinical implication and prognostic significance of the tumor suppressor TSLC1 gene detected in adenocarcinoma of the lung. Cancer 2003. 98: p. 1002-1007. 67. Szumilas, M., Explaining odds ratios. Journal of the Canadian Academy of Child and Adolescent Psychiatry, 2010. 19: p. 227. 68. Glas, A.S., Lijmer, J.G., Prins, M.H., Bonsel, G.J., Bossuyt, P.M., The diagnostic odds ratio: a single indicator of test performance. Journal of clinical epidemiology, 2003. 56: p. 1129-1135. 69. Toyoda, Y., Sensitivity and specificity of lung cancer screening using chest low-dose computed tomography. British Journal of Cancer 2008. 98: p. 1602 - 1607. 70. Vansteenkiste, Screening and early detection of lung cancer. Annals of Oncology, 2012. 23. 71. Strahm, B., Capra, M. , Insights into the molecular basis of cancer development. Current Paediatrics, 2005. 15(4): p. 333-338. 72. Anglim, P.P., Alonzo, T.A., Laird-Offringa, I.A., DNA methylation-based biomarkers for early detection of non-small cell lung cancer: an update. Molecular Cancer, 2008. 7(81). 73. Yageta, M.S., Masuda, M., Tsuboi, Y., Ito, A., Murakami, Y., Tumor suppressor CADM1 is involved in epithelial cell structure. Biochemical and Biophysical Research Communications 2009. 390: p. 977-982. 74. Faraji, F., Pang, Y., Walker, RC., Borges, R.N., Yang, L., Hunter, K.W., Cadm1 Is a Metastasis Susceptibility Gene That Suppresses Metastasis by Modifying Tumor Interaction with the Cell-Mediated Immunity. PLoS Genetic, 2012. 8(9). 75. Weyden, Increased tumorigenesis associated with loss of the tumor suppressor gene Cadm1. Molecular Cancer, 2012. 11(29). 76. Socinski, M.A., Morris, D.E., Masters, G.A., et al. , Chemotherapeutic management of stage IV non-small cell lung cancer. Chest, 2003. 123: p. 226S-243S. 77. Ma, M., Shen, J., Jiang, L., Han, B., Bai, H., Ji, H., Zhao, Y., Jin, B., Yu, Y., Pei, J., Zhang, W., Prognostic factors in patients with stage IV non-small cell lung cancer. The Chinese-German Journal of Clinical Oncology, 2006. 5(5): p. 319-323.