Patent Publication Number: US-2018051344-A1

Title: Embryonic isoforms of gata6 and nkx2-1 for use in lung cancer diagnosis

Description:
Lung cancer (LC) is the leading cause of cancer-related deaths worldwide, accounting for an estimated 1.6 million deaths out of 1.8 million cases in 2012 (Globocan 2012). The incidence pattern of LC closely parallels the mortality rate because of persistently low survival rates. There are two major classes of LC, non-small cell lung cancer (NSCLC, representing 85% of all lung cancers) and small cell lung cancer (SCLC, the remaining 15%) 1 . Histologically, NSCLC is further divided into three major subtypes; squamous cell carcinoma, adenocarcinoma and large cell carcinoma. Adenocarcinoma is the most common form and has approximately 40% prevalence, followed by squamous cell and large cell carcinoma, which represent 25% and 10%, respectively 2 . Clinical manifestations of LC are diverse and patients are mostly asymptomatic at early stages. Symptoms, even when present, are non-specific and unfortunately mimic more common benign etiologies 3 . Traditional diagnostic strategies for LC include imaging tests, such as chest X-ray radiography (CXR) or computed tomography (CT), cytological assessment of sputum or bronchial suctioning and histopathological evaluation of biopsies taken during bronchoscopy, mediastinoscopy, open lung surgery or from metastasis resections 4-6 . In the majority of patients, these procedures are initiated after the development of symptoms, therefore at advanced stages of the disease, when the overall condition of the patient is already impaired and prognosis is poor, as shown by the low five-year patient survival of 1-5% 1 . Strikingly, patient survival is high as 52% if LC is diagnosed early, demonstrating that early diagnosis of LC is pivotal to increase the probability of successful therapy. 
     Accordingly, there is a need for new techniques for diagnosis of specific cancers and their subtypes as well as for further and/or alternative treatment options in cancer therapy. Thus, the technical problem underlying the present invention is the provision of reliable means and methods for the detection of cancer, in particular lung cancer and its subtypes, and for the determination of treatment options. 
     The solution to this technical problem is provided by the embodiments as defined herein and as characterized in the claims. 
     The invention provides a statistical method for assessing whether a subject suffers from cancer or is prone to suffering from cancer. The invention provides an anti-cancer agent and/or radiation therapy, said agent or radiation therapy being selected on basis of the patient group determined by the statistical method provided herein. 
     The object of the invention is solved with the features of the independent claims. Dependent claims refer to preferred embodiments. 
     The invention provides a statistical method of assessing whether a subject suffers from cancer or is prone to suffering from cancer, said method comprising the step of performing at least one statistical algorithm for classification and for regression on measurement data of the subject, wherein the measurement data of the subject comprises at least one of the following: a value of GATA6 Em isoform in at least one sample taken from the subject, a value NKX2-1 Em isoform in said at least one sample, a value of GATA6 Ad isoform in said at least one sample, NKX2-1 Ad isoform in said at least one sample; and wherein at least one of the following is used as at least one classifier or a component of at least one classifier in the statistical method: GATA6 Em isoform, NKX2-1 Em isoform, GATA6 Ad isoform, NKX2-1 Ad isoform, ratio of GATA6 Em isoform/GATA6 Ad isoform, ratio of NKX2-1 Em isoform/NKX2-1 Ad isoform. 
     Statistical algorithms for classification and for regression on measurement data are generally known to the skilled person. Examples of statistical algorithms can be found in the following textbooks:
     “The Elements of Statistical Learning: Data Mining, Inference, and Prediction, Second Edition (Springer Series in Statistics)”, Trevor Hastie et al., Springer, 2011   “Pattern Recognition and Machine Learning”, Christopher M. Bishop, Springer, 2011. B. Schölkopf, A. Smola,  Learning with Kernels—Support Vector Machines, Regularization, Optimization and Beyond , MIT Press, Cambridge, Mass., 2002.   

     Preferably, these algorithms are grossly partitioned into parametric approaches that explicitly model the data by one member of a parametrized family of probability distribuions (e.g., linear discriminant analysis or logit regression), and non-parametric approaches like Neural Networks or Support Vector Machines that do not rely on a distributional assumption. 
     According to an embodiment, said value of the GATA6 Em isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the GATA6 Em isoform comprising the nucleic acid sequence of SEQ ID No: 1 or the GATA6 Em isoform comprising a nucleic acid sequence with up to 55 additions, deletions or substitutions of SEQ ID NO: 1. 
     According to an embodiment, said value of the NKX2-1 Em isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the NKX2-1 Em isoform comprising the nucleic acid sequence of SEQ ID No: 2 or the NKX2-1 Em isoform comprising a nucleic acid sequence with up to 39 additions, deletions or substitutions of SEQ ID NO: 2. 
     According to an embodiment, said value of GATA6 Ad isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the GATA6 Ad isoform comprising the nucleic acid sequence of SEQ ID No: 5 or the GATA6 Ad isoform comprising a nucleic acid sequence with up to 55 additions, deletions or substitutions of SEQ ID NO: 5. 
     According to an embodiment, said value of the NKX2-1 Ad isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the NKX2-1 Ad isoform comprising the nucleic acid sequence of SEQ ID No: 6 or the NKX2-1 Ad isoform comprising a nucleic acid sequence with up to 38 additions, deletions or substitutions of SEQ ID NO: 6. 
     According to an embodiment, the statistical method further comprises the step of processing the measurement data, preferably normalizing, rescaling, dimension reducing, and/or noise reducing. 
     Preferably, the step of processing the measurement data, preferably normalizing, rescaling, dimension reducing, and/or noise reducing is performed before performing the at least one statistical algorithm for classification and for regression on measurement data of the subject. 
     Preferably, the normalizing of the measurement data comprises the normalizing of at least one of the following: microarray or RNA-Seq measurements. 
     Preferably the normalizing of the measurement comprises obtaining abundance estimates and/or detecting outlier and/or removing outlier. 
     Preferably, the reducing of the dimension and/or the reducing of the noise comprises transforming the measurement data into a space where discriminatory methods achieve a higher power. 
     Preferably, reducing the dimension and/or reducing the noise comprises at least one of the following: principal component analysis, non-linear variant principal component analysis, singular value decomposition, non-linear variant singular value decomposition, independent component analysis, non-linear independent component analysis, a kernel principal component analysis. 
     According to an embodiment, the statistical method further comprises the steps of cross-validation and/or bootstrapping. 
     According to an embodiment, the GATA6 Em isoform of said sample is set in relation to a GATA6 Em isoform of at least one control sample and then used as a classifier in the statistical method. 
     Preferably, set in relation comprises at least one of the following: normalizing the value of the GATA6 Em isoform of said sample with respect to the value of the GATA6 Em isoform of the control sample, subtracting the value of the GATA6 Em isoform of at least one control sample from the GATA6 Em isoform of said sample. 
     Preferably, said value of the GATA6 Em isoform of said at least one control sample is obtained by measuring in said sample of said subject the amount of a specific transcription factor isoform wherein said specific transcription isoform is the GATA6 Em isoform comprising the nucleic acid sequence of SEQ ID No: 1 or the GATA6 Em isoform comprising a nucleic acid sequence with up to 55 additions, deletions or substitutions of SEQ ID NO: 1. 
     According to an embodiment, the NKX2-1 Em isoform in said at least one sample is set in relation to a NKX2-1 Em isoform of at least one control sample and then used as a classifier in the statistical method. 
     Preferably, set in relation comprises at least one of the following: normalizing the value of the NKX2-1 Em isoform of said sample with respect to the value of the NKX2-1 Em isoform of the control sample, subtracting the value of the NKX2-1 Em isoform of at least one control sample from the NKX2-1 Em isoform of said sample. 
     Preferably, said value of the NKX2-1 Em isoform in said at least one control sample is obtained by measuring in said at least one sample of said subject the amount of a specific transcription factor isoform wherein said specific transcription isoform is the NKX2-1 Em isoform comprising the nucleic acid sequence of SEQ ID No: 2 or the NKX2-1 Em isoform comprising a nucleic acid sequence with up to 39 additions, deletions or substitutions of SEQ ID NO: 2; 
     According to an embodiment, a ratio of the GATA6 Em isoform and the GATA6 Ad isoform and a ratio of the NKX2-1 Em isoform and the NKX2-1 Ad isoform are used as a classifier. 
     According to an embodiment, the statistical method comprises a linear classifier. 
     Preferably, the statistical method comprises at least one of the following: a linear classifier, preferably a support vector machine and/or a linear discriminant analysis and/or decision trees, a regression method, preferably linear, logistic or probit regression, or a penalized version of the regression, preferably a penalized version of the linear, logistic or probit regression, more preferably a Lasso and/or ridge regression, or a generalized linear model, a neural network, or a regression tree, or ensemble methods built from the above algorithms in a process, preferably boosting. 
     Preferably, the support vector machine is a linear kernel support vector machine. Preferably, the linear kernel support vector machine is the one implemented in the following software: Evgenia Dimitriadou, Kurt Hornik, Friedrich Leisch, David Meyer and Andreas Weingessel (2010). e1071: Misc Functions of the Department of Statistics (e1071), TU Wien. R package version 1.5-24. http://CRAN.Rproject.org/package=e1071. 
     Preferably, the SVM, does not assume that the data from the sample groups are drawn from a Gaussian distribution. The SVM can be considered as the more robust choice in comparison to the linear discrimination analysis. Preferably, the support vector machine finds a separating hyperplane between data from normal and cancerous samples, which is expected to yield a good generalization performance when applied to new, unseen data. Preferably, the distance to this hyperplane is determined by the following function: 
         LC   score =−α·log 2 (ratio of  GATA 6  Em  isoform/ GATA 6  Ad  isoform)−β·log 2 (ratio of  NKX 2-1  Em  isoform/ NKX 2-1  Ad  isoform)−γ,
 
     wherein preferably α=0.607, β=1.431, γ=1.916. 
     Preferably, α=−0.607, β=−1.431, γ=−1.916 
     Preferably, the function comprises a prefactor (−1) such that the distance to the hyperplane is determined by the following function: 
         LC   score =(−1)−(−α·log 2 (ratio of  GATA 6 Em  isoform/ GATA 6 Ad  isoform)−β·log 2 (ratio of  NKX 2-1 Em  isoform/ NKX 2-1 Ad  isoform)−γ),
 
     wherein preferably α=0.607, β=1.431, γ=1.916. 
     The amount of said specific transcription factor isoform(s) can be measured on the mRNA level. 
     The appended example shows that the expression ratio remained stable for both control donor as well as LC EBC samples until 75 ng of RNA starting material. Decreasing the starting material below 75 ng resulted in suboptimal detection of the Em-isoform in the control and the Ad-isoform in the LC group, which led to distorted ratios. If the amount of the transcription factor isoform(s) is determined/measured in accordance with the present invention, it is preferred that the starting material (mRNA/RNA) contains/is more than about 75 ng of RNA. 
     According to an embodiment, the amount of said specific transcription factor isoform(s) is measured via a polymerase chain reaction-based method, According to an embodiment, the amount of said specific transcription factor isoform(s) is measured via a polymerase chain reaction-based method, an in situ hybridization-based method, or a microarray. According to an embodiment, the amount of said specific transcription factor isoform(s) is measured via a polymerase chain reaction-based method. According to an embodiment, said polymerase chain reaction-based method is a quantitative reverse transcriptase polymerase chain reaction. 
     According to an embodiment, the step of measuring in a sample of said subject the amount of a specific transcription factor comprises the contacting of the sample with primers, wherein said primers can be used for amplifying at least one of the specific transcription factor isoforms. According to an embodiment, said primers are selected from the group of primers having a nucleic acid sequence as set forth in SEQ ID NOs 9 to 40, particularly one or more primers/primer pairs having a nucleic acid sequence as set forth in SEQ ID NOs 9 to 24. For example, one or more of the following primers/primer pairs can be used in accordance with the present invention: 
     
       
         
           
               
               
               
             
               
                   
               
               
                   
                 Primers 
                 Primers for Human (5′→3′) (For 
               
               
                 Gene 
                 for Human (5′→3′) 
                 RNA from tissue sections) 
               
               
                   
               
             
            
               
                 Gata6-Em Fwd 
                 SEQ ID NO 9: 
                 SEQ ID NO 10: 
               
               
                   
                 CTCGGCTTCTCTCCGCGCCTG 
                 TTGACTGACGGCGGCTGGTG 
               
               
                   
               
               
                 Gata6-Em Rev 
                 SEQ ID NO 11: 
                 SEQ ID NO 12: 
               
               
                   
                 AGCTGAGGCGTCCCGCAGTTG 
                 CTCCCGCGCTGGAAAGGCTC 
               
               
                   
               
               
                 Gata6-Ad Fwd 
                 SEQ ID NO 13: 
                 SEQ ID NO 14: 
               
               
                   
                 GCGGTTTCGTTTTCGGGGAC 
                 AGGACCCAGACTGCTGCCCC 
               
               
                   
               
               
                 Gata6-Ad Rev 
                 SEQ ID NO 15: 
                 SEQ ID NO 16: 
               
               
                   
                 AAGGGATGCGAAGCGTAGGA 
                 CTGACCAGCCCGAACGCGAG 
               
               
                   
               
               
                 Nkx2-1-Em Fwd 
                 SEQ ID NO 17: 
                 SEQ ID NO 18: 
               
               
                   
                 AAACCTGGCGCCGGGCTAAA 
                 CAGCGAGGCTTCGCCTTCCC 
               
               
                   
               
               
                 Nkx2-1-Em Rev 
                 SEQ ID NO 19: 
                 SEQ ID NO 20: 
               
               
                   
                 GGAGAGGGGGAAGGCGAAGCC 
                 TCGACATGATTCGGCGGCGG 
               
               
                   
               
               
                 Nkx2-1-Ad Fwd 
                 SEQ ID NO 21: 
                 SEQ ID NO 22: 
               
               
                   
                 AGCGAAGCCCGATGTGGTCC 
                 TCCGGAGGCAGTGGGAAGGC 
               
               
                   
               
               
                 Nk2-1-Ad Rev 
                 SEQ ID NO 23: 
                 SEQ ID NO 24: 
               
               
                   
                 CCGCCCTCCATGCCCACTTTC 
                 GACATGATTCGGCGGCGGCT 
               
               
                   
               
               
                 Foxa2-Var1 Fwd 
                 SEQ ID NO 25: 
                 SEQ ID NO 26: 
               
               
                   
                 TGCCATGCACTCGGCTTCCAG 
                 CAGGGAGAGGGAGGGCGAGA 
               
               
                   
               
               
                 Foxa2-Var1 Rev 
                 SEQ ID NO 27: 
                 SEQ ID NO 28: 
               
               
                   
                 TCATGTTGCCCGAGCCGCTG 
                 CCCCCACCCCCACCCTCTTT 
               
               
                   
               
               
                 Foxa2-Var2 Fwd 
                 SEQ ID NO 29: 
                 SEQ ID NO 30: 
               
               
                   
                 CTGCTAGAGGGGCTGCTTGCG 
                 CGCTTCTCCCGAGGCCGTTC 
               
               
                   
               
               
                 Foxa2-Var2 Rev 
                 SEQ ID NO 31: 
                 SEQ ID NO 32: 
               
               
                   
                 ACGGCTCGTGCCCTTCCATC 
                 TAACTCGCCCGCTGCTGCTC 
               
               
                   
               
               
                 Id2-Var1 Fwd 
                 SEQ ID NO 33: 
                 SEQ ID NO 34: 
               
               
                   
                 AACCCCTGTGGACGACCCGA 
                 TGCGGATAAAAGCCGCCCCG 
               
               
                   
               
               
                 Id2-Var1 Rev 
                 SEQ ID NO 35 
                 SEQ ID NO 36: 
               
               
                   
                 GCCCGGGTCTCTGGTGATGC 
                 AGCTAGCTGCGCTTGGCACC 
               
               
                   
               
               
                 Id2-Var2 Fwd 
                 SEQ ID NO 37: 
                 SEQ ID NO 38: 
               
               
                   
                 CTGCGGTGCTGAACTCGCCC 
                 CCCCCTGCGGTGCTGAACTC 
               
               
                   
               
               
                 Id2-Var2 Rev 
                 SEQ ID NO 39: 
                 SEQ ID NO 40: 
               
               
                   
                 GACGAGCGGGCGCTTCCATT 
                 TAACTCGCCCGCTGCTGCTC 
               
               
                   
               
            
           
         
       
     
     According to an embodiment, the amount of said specific transcription factor isoform(s) can be measured on the polypeptide/protein level. According to an embodiment, the amount of said specific transcription factor isoform(s) is measured by an ELISA, a gel- or blot-based method, mass spectrometry, flow cytometry or FACS. 
     According to an embodiment, the cancer is a lung cancer. According to an embodiment, said lung cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). 
     According to an embodiment, the sample comprises tumor cells. According to an embodiment, the sample is a biopsy sample, a breath condensate sample, a blood sample, a bronchoalveolar lavage fluid sample, a mucus sample or a phlegm sample. Preferably, the sample is a breath condensate sample. 
     According to an embodiment, the subject is a human subject. According to an embodiment, said human subject is a subject having an increased risk for developing cancer. A human subject having an increased risk for developing cancer can, for example, be a human subject that is a current or former smoker(s); and/or that was/is exposed to smoke, like environmental smoke, cooking fumes, and/or indoor smoky coal emissions; and/or that was/is exposed to asbestos, some metals (e.g. nickel, arsenic and cadmium), radon, and/or ionizing radiation. A human subject having an increased risk for developing cancer can, for example, be a human subject that has shown cancer-like lesions in a preceding computed tomography scan. 
     According to an embodiment, the method further comprises the detection of one or more additional markers in a sample of said subject. According to an embodiment, said one or more additional markers are one or more markers for classifying cancer. According to an embodiment, said one or more additional markers are one or more markers for classifying lung cancer into subtypes of lung cancer. According to an embodiment, said one or more markers for classifying lung cancer are differentially expressed. 
     According to an embodiment, said one or more markers for classifying lung cancer are one or more markers for classifying non-small cell lung cancer (NSCLC) into subtypes of NSCLC. According to an embodiment, said one or more markers for classifying NSCLC are selected from the group consisting of SFTPA1, SFTPB, NAPSA, hsa-let7-d, VEGFA, VEGFB, VEGFC, VEGFD, PLAUR, TP63, KRT5, KRT6A, KRT7, hsa-miR9, HMGA1 and CDH1. Exemplary nucleic acid sequences and amino acid sequences of these markers are provided in the present application. 
     The specific transcription factor isoform(s) and/or the additional markers (like SFTPA1, SFTPB, NAPSA, VEGFA, VEGFB, VEGFC, VEGFD, PLAUR, TP63, KRT5, KRT6A, KRT7, HMGA1 and/or CDH1) can be measured on the protein/polypeptide or the mRNA level. Additional markers like hsa-let7-d, hsa-miR9, can be measured on the mRNA level. 
     For example, the amount can be measured via a polymerase chain reaction-based method, an in situ hybridization-based method, or a microarray, or a quantitative reverse transcriptase polymerase chain reaction. 
     For example, the amount can be measured on the polypeptide/protein level, for example, by an ELISA, a gel- or blot-based method, mass spectrometry, flow cytometry or FACS. 
     For example, if the specific transcription factor isoform(s) and/or additional marker(s) is/are measured on the protein level, contacting and binding can be performed by taking advantage of immunoagglutination, immunoprecipitation (e.g. immunodiffusion, immunelectrophoresis, immune fixation), western blotting techniques (e.g. (in situ) immuno histochemistry, (in situ) immuno cytochemistry, affinitychromatography, enzyme immunoassays), and the like. These and other suitable methods of contacting proteins are well known in the art and are, for example, also described in Sambrook and Russell (2001, loc. cit.). 
     In case the specific transcription factor isoform(s) and/or additional marker(s) is a protein, quantification can be performed by taking advantage of the techniques referred to above, in particular Western blotting techniques. Generally, the skilled person is aware of methods for the quantitation of polypeptides. Amounts of purified polypeptide in solution can be determined by physical methods, e.g. photometry. Methods of quantifying a particular polypeptide in a mixture rely on specific binding, e.g of antibodies. Specific detection and quantitation methods exploiting the specificity of antibodies comprise for example immunohistochemistry (in situ). Western blotting combines separation of a mixture of proteins by electrophoresis and specific detection with antibodies. Electrophoresis may be multi-dimensional such as 2D electrophoresis. Usually, polypeptides are separated in 2D electrophoresis by their apparent molecular weight along one dimension and by their isoelectric point along the other direction. 
     For example, if the specific transcription factor isoform(s) and/or additional marker(s) is/are measured on the RNA/mRNA level, contacting and binding can be performed by taking advantage of Northern blotting techniques or PCR techniques/via a polymerase chain reaction-based method, like quantitative reverse transcriptase polymerase chain reaction or in-situ PCR, an in situ hybridization-based method, or a microarray. These and other suitable methods for binding (specific) mRNA are well known in the art and are, for example, described in Sambrook and Russell (2001, loc. cit.). 
     If the specific transcription factor isoform(s) and/or additional marker(s) is an mRNA, determination can be performed by taking advantage of northern blotting techniques, hybridization on microarrays or DNA chips equipped with one or more probes or probe sets specific for mRNA transcripts or PCR techniques referred to above, like, for example, quantitative PCR techniques, such as Real time PCR. A skilled person is capable of determining the amount of the component, in particular said gene products, by taking advantage of a correlation, preferably a linear correlation, between the intensity of a Raman signal and the amount of the component to be determined. 
     According to an embodiment, said subtype of NSCLC is classified as adenocarcinoma, if said one or more markers for classifying NSCLC into subtypes of NSCLC are one or more of SFTPA1, SFTPB and NAPSA, and 
     if the level of one or more of SFTPA1, SFTPB and NAPSA is increased compared to a control. Preferably the level of SFTPA1 is the mRNA level or the protein level of SFTPA1. 
     According to an embodiment, said subtype of NSCLC is classified as adenocarcinoma, if said marker for classifying NSCLC into subtypes of NSCLC is hsa-let7-d, and if the level of hsa-let7-d is decreased compared to a control. Preferably the level of hsa-let7-d is the RNA level of hsa-let7-d. 
     According to an embodiment, said subtype of NSCLC is classified as metastatic adenocarcinoma, 
     if said marker for classifying NSCLC into subtypes of NSCLC is VEGFA, VEGFB, VEGFC, VEGFD and/or PLAUR, and
 
if the level of VEGFA, VEGFB, VEGFC, VEGFD and/or PLAUR is increased compared to a control. Preferably the level of VEGFA, VEGFB, VEGFC, VEGFD and/or PLAUR is the mRNA level or the protein level of VEGFA, VEGFB, VEGFC, VEGFD and/or PLAUR.
 
     According to an embodiment, said subtype of NSCLC is classified as squamous cell carcinoma, if said marker for classifying NSCLC into subtypes of NSCLC is one or more of TP63, KRT5, KRT6A, KRT7 and hsa-miR9, and 
     if the level of one or more of one or more of TP63, KRT5, KRT6A, KRT7 and hsa-miR9, is increased compared to a control. Preferably the level of TP63, KRT5, KRT6A and KRT7 is the mRNA level or the protein level of TP63, KRT5, KRT6A and KRT7. Preferably the level of hsa-miR9 is the RNA level of hsa-miR9. 
     According to an embodiment, said subtype of NSCLC is classified as large cell lung carcinoma, if said marker for classifying NSCLC into subtypes of NSCLC is HMGA1, and if the level of HMGA1 is increased compared to a control. Preferably the level of HMGA1 is the mRNA level or the protein level of HMGA1. 
     According to an embodiment, said subtype of NSCLC is classified as large cell lung carcinoma, 
     if said marker for classifying NSCLC into subtypes of NSCLC is CDH1, and
 
if the level of CDH1 is decreased compared to a control. Preferably the level of CDH1 is the mRNA level or the protein level of CDH1.
 
     According to an embodiment, said one or more markers for classifying lung cancer are genomic alterations. A person skilled in the art knows how to determine genomic alterations, a mutation(s) or a polymorphism(s) in a gene by his common general knowledge and the teaching provided herein. Exemplary, non-limiting techniques for determining such genomic alteration(s), mutation(s) and/or polymorphism(s) are described below. 
     Genomic alterations, including mutations and polymorphisms, can be detected by DNA sequencing, including pyrosequencing and Sanger sequencing methods, PCR based methods including restriction fragment length polymorphisms, taqman probes and molecular beacons, or using DNA arrays. Genomic alterations including chromosomal changes, such as translocations or deletions can be identified by conventional cytogenetic stainings, fluorescent in situ hybridization, comparative genomic hybridization and array based comparative genomic hybridization, or PCR based analysis. 
     According to an embodiment, said one or more markers for classifying lung cancer are one or more markers for classifying non-small cell lung cancer (NSCLC) into subtypes of NSCLC. 
     According to an embodiment, said subtype of NSCLC is classified as adenocarcinoma, 
     if said marker for classifying NSCLC into subtypes of NSCLC is KRAS G12D or G12V G--&gt;C/T transversion at codon for Exon 12, and
 
if said marker is present in the sample from the subject.
 
     Preferably, the specific mutations of KRAS found in NSCLC are one or more of: G34T, G35A, G35T and G37T and G38T (the last 2 result in mutations of codon 13 which are also oncogenic) 
     Ref: 21197450. 
     These mutations are negative predictors of response to EGFR therapy in patients. 
     According to an embodiment, said subtype of NSCLC is classified as metastatic adenocarcinoma, 
     if said marker for classifying NSCLC into subtypes of NSCLC is KRAS G12D//TP53 mutations R172H Substitution in p53 (Li-Fraumeni syndrome), and
 
if said marker is present in the sample from the subject.
 
     Preferably, metastatic adenocarcinoma is characterized/classified by a combination of KRAS and TP53 as defined above. 
     According to an embodiment, said subtype of NSCLC is classified as adenocarcinoma in never-smokers, 
     if said marker for classifying NSCLC into subtypes of NSCLC is KRAS G12D G--&gt;G--&gt;A (G35A) transition, and
 
if said marker is present in the sample from the subject.
 
     According to an embodiment, said subtype of NSCLC is classified as adenocarcinoma or squamous cell carcinoma, 
     if said marker for classifying NSCLC into subtypes of NSCLC is TP53 mutations, translocations, and
 
if said marker is present in the sample from the subject.
 
     Preferably, the most frequent mutations in TP53 for Adenocarinoma: G:C247T:A and for Squamous cell carincoma is G:C274T:A and for SCLC is G:C96T:A. 
     According to an embodiment, said subtype of NSCLC is classified as drug resistant adenocarcinoma (patients relapse after tyrosine kinase inhibitors), 
     if said marker for classifying NSCLC into subtypes of NSCLC is EGFR T790M mutation in exon 20, codon 790, and
 
if said marker is present in the sample from the subject.
 
     According to an embodiment, said subtype of lung cancer is classified as small cell lung cancer (SCLC), 
     if said marker for classifying lung cancer into subtypes of lung cancer is/are TP53 mutations combined with mutations in RB1, and
 
if said marker is present in the sample from the subject.
 
     The above mentioned additional markers are suitable markers to classify cancer into subtypes of cancer, and in particular lung cancer into subtypes of lung cancer. This is illustrated by the references below. Accordingly, the one or more additional markers can be suitably be used in accordance with the present invention for a refined analysis using the herein provided statistical method. For example, the expression of one or more of these additional markers can be determined in exhaled breath condensates from patients that are assessed to suffer from cancer or being prone to suffering from cancer in accordance with the statistical method can, in order to classify e.g. cancer subtype (preferably the NSCLC subtype) in the patients. The terms “transition” and “transversion” are used interchangeably herein. 
     For example, the following one or more markers can be used to classify NSCLC into subtypes of NSCLC: 
     Adenocarcinoma: 
     SFTPA, SFTPB and/or NAPSA: (Garber, Troyanskaya et al. 2001, Ye, Findeis-Hosey et al. 2011, Turner, Cagle et al. 2012, Whithaus, Fukuoka et al. 2012, Taguchi, Hanash et al. 2013); and/or hsa-let7-d: (Lee and Dutta 2007, Kumar, Armenteros-Monterroso et al. 2014); and/or KRAS G12D and/or G12V: (Winslow, Dayton et al. 2011); and/or
 
TP53 mutations and/or TP53 translocations: (Kishimoto, Murakami et al. 1992)
 
     The term KRAS G12D or G12V (or more particularly the term “KRAS G12D or G12V G--&gt;C/T transversion at codon for Exon 12”) refers to an amino acid substitution at position 12 of the amino acid sequence of KRAS. The substitution is due to a transversion in the coding sequence of KRAS. Particularly the term “KRAS G12D or G12V G--&gt;C/T transversion at codon for Exon 12”) can refer to a G(35)--&gt;C/T transversion at position 35 of the DNA sequence of KRAS within codon 12. The DNA mutation is G→C/T at position 35 of the coding sequence of KRAS, which is changing codon 12 in the amino acid sequence of KRAS. Coding sequences of KRAS can be derived from databases like NCBI. Exemplary coding sequences of KRAS to be used herein are, for example, shown in the database under accession number GI 575403058 (Transcript variant a) or under GI 575403057 (Transcript variant b). 
     Metastatic Adenocarcinoma: 
     VEGFA, VEGFB, VEGFC, VEGFD, and/or PLAUR: (Shijubo, Uede et al. 1999, Garber, Troyanskaya et al. 2001, Su, Yang et al. 2006) (Han, Silverman et al. 2001, Stacker, Caesar et al. 2001, Li, Hu et al. 2014, Qi, Zhu et al. 2014); and/or
 
KRAS G12D mutations and/or TP53 mutations (such as R172H substitution in TP53 (Li-Fraumeni syndrome)): (Kishimoto, Murakami et al. 1992, Lang, Iwakuma et al. 2004)
 
     The term “KRAS G12D//TP53 mutation(s) R172H Substitution in TP53 (Li-Fraumeni syndrome)” can refer to KRAS G12D mutation(s) and/or TP53 mutation(s) (such as R172H substitution in TP53 (Li-Fraumeni syndrome)). 
     The term KRAS G12D refers to an amino acid substitution at position 12 of the amino acid sequence of KRAS. The substitution is due to a transversion in the coding sequence of KRAS, like a G--&gt;A (G35A) transition. 
     The term “TP53 mutation(s)” (or more particularly the term “TP53 mutation(s) R172H Substitution in TP53”) can refer to an amino acid substitution in the amino acid sequence of TP53. The substitution is due to a transition in the coding sequence of TP53. Particularly the term “TP53 mutation(s) R172H Substitution in TP53” can refer to a G to A transition at position 515 (G515A) of the sequence encoding TP53. Coding sequences of TP53 can be derived from databases like NCBI. An exemplary coding sequence of TP53 to be used herein is, for example, shown in the database under accession number GI 23491728. 
     Adenocarcinoma in Never-Smokers: 
     KRAS G12D G--&gt;A (G35A) transition: (Riely, Kris et al. 2008). The terms “KRAS G12D G--&gt;G--&gt;A (G35A) transition” and “KRAS G12D G--&gt;A (G35A) transition” can be used interchangeably herein. 
     The term “KRAS G12D” or particularly the term “KRAS G12D G--&gt;G--&gt;A (G35A) transition”/“KRAS G12D G--&gt;A (G35A) transition” refers to an amino acid substitution at position 12 of the amino acid sequence of KRAS. The substitution is due to a transition in the coding sequence of KRAS. The terms “KRAS G12D G--&gt;G--&gt;A (G35A) transition”/“KRAS G12D G--&gt;A (G35A) transition” can refer to a KRAS G12D G--&gt;A (G35A) transition. Particularly the term “KRAS G12D G--&gt;G--&gt;A (G35A) transition” refers to an amino acid substitution at position 12 of the amino acid sequence of KRAS which is due to a G--&gt;A (G35A) transition in the coding sequence of KRAS. The amino acid change KRAS G12D results from a change at position 35 in the coding sequence of KRAS, in this case G35 to A. 
     Drug Resistant Adenocarcinoma (for Example Patients Relapse after Therapy with Tyrosine Kinase Inhibitors):
 
EGFR T790M mutation in exon 20, codon 790: (Pao, Miller et al. 2005)
 
     The terms “EGFR T790M mutation in exon 20, codon 790” and “EGFR T790M mutation in codon 790” can be used interchangeably herein. The terms “EGFR T790M mutation in exon 20, codon 790” or “EGFR T790M mutation in codon 790” are also known as “EGFR C2369T mutation”. 
     The term “EGFR T790M mutation”, or particularly the term “EGFR T790M mutation in exon 20, codon 790”, refers to an amino acid substitution at position 790 of the amino acid sequence of EGFR. The amino acid substitution can be due to a transition in the coding sequence of EGFR. Particularly the terms “EGFR T790M mutation in exon 20, codon 790”/“EGFR T790M mutation in codon 790”/“EGFR C2369T mutation” can refer to a C to T transition at position 2369 (i.e. C2369T) of the sequence encoding EGFR. Coding sequences of EGFR can be derived from databases like NCBI. An exemplary coding sequence of EGFR to be used herein is, for example, shown in the database under accession number GI 41327737 (Transcript isoform a), GI 41327731 (Transcript isoform b), GI 41327733 (Transcript isoform c) or 41327735 (Transcript isoform d). 
     Squamous Cell Carcinoma: 
     TP63, KRT5, KRT6 and/or KRT7: (Pelosi, Pasini et al. 2002, Rekhtman, Ang et al. 2011, Whithaus, Fukuoka et al. 2012); and/or
 
hsa-miR9: (White, Neiman et al. 2013)
 
TP53 mutations and/or TP53 translocations: (Kishimoto, Murakami et al. 1992)
 
     Large Cell Lung Cancer/Large Cell Lung Carcinoma: 
     HMGA1: (Hillion, Wood et al. 2009) and/or 
     CDH1: (Kase, Sugio et al. 2000, Garber, Troyanskaya et al. 2001, Asnaghi, Vass et al. 2010) 
     For example, the following one or more markers can be used to classify lung cancer into the subtype small cell lung cancer (SCLC): TP53 mutations in combination with mutations in RB1: (Sutherland, Proost et al. 2011). Mutations in RB1 may refer to mutations in the tumor suppressor gene Retinoblastioma, RB1. The protein is a negative regulator of cell cylce. 
     The invention also provides a computer program product comprising one or more computer readable media having computer executable instructions for performing the steps of one of the aforementioned methods. 
     The present invention relates to a method of treating a subject, said method comprising 
     a) selecting a subject that is assessed to suffer from cancer or is assessed to be prone to suffering from cancer according to the herein provided statistical method;
 
b) administering to said cancer patient an effective amount of an anti-cancer agent and/or radiation therapy.
 
     Preferably, the gene mutations can be used to distinguish patients&#39; response to EGFR therapy as mentioned above. 
     The invention also provides an anti-cancer agent and/or radiation therapy for use in the treatment of a subject, wherein the subject is assessed to suffer from cancer or is assessed to be prone to suffering from cancer according to any of the statistical methods mentioned above. Preferably, the subject/patient is a human subject/patient. In other words, the invention provides an anti-cancer agent and/or radiation therapy, said agent or radiation therapy being selected on basis of the patient group determined by the statistical method provided herein. 
     For example, conventional chemotherapy (like cisplatin based protocols), radiotherapy (like conventional radiotherapy or radiosurgery), and/or more modern approaches employing tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib and/or monoclonal antibodies directed against activating mutations of the tumor (ERGF, ALK or ROS1 mutations) can be used. 
     If the subject is assessed to suffer from non-small cell lung cancer (NSCLC) or is assessed to be prone to suffering from non-small cell lung cancer (NSCLC) according to any of the statistical methods mentioned above, the following treatment options can be used: 
     The treatment options for NSCLC are, for example, based on the stage of the disease. Standard treatments include surgery, platinum-based chemotherapy, radiotherapy, combined chemoradiotherapy and/or targeted therapy. The choice of the course of treatment can depend on the stage of the disease, its spread to the surrounding tissues, patient&#39;s overall medical condition, and/or especially the patient&#39;s pulmonary reserve. 
     If the subtype of NSCLC (like NSCLC stage I, II or III tumors/cancers) is, for example, adenocarcinoma, squamous cell carcinoma or large cell carcinoma, the following treatment options are conceivable: 
     For Stage I tumors, surgery is the most consistent and successful treatment for lung cancer patients. Tumors can be removed by lobectomy, segmental, wedge or sleeve resections or pneumectomy as found appropriate (Molina, Yang et al. 2008, Schuchert, Abbas et al. 2010, 2011, Cagle and Chirieac 2012). Five-year survival rate ranges between 40-67% favoring T1N0 or earlier (Martini, Bains et al. 1995). In the patients with potentially resectable tumors but who are unfit for surgery due to an unacceptably high perioperative risk or for patients with inoperable Stage I tumors, primary radiosurgery or conventional radiation therapy is suggested (Dosoretz, Katin et al. 1992, Gauden, Ramsay et al. 1995). Unfortunately, many patients develop local recurrent or second primary tumors after surgical resection. To prevent this, adjuvant chemo or radiation therapy following surgery is recommended pending on the stage prior to surgery (Martini, Bains et al. 1995). 
     Stage II cancers are routinely treated with surgical resections, however, prognosis is worse than that of Stage I cancers and the 5-year survival rate varies from 25-55% (Martini, Burt et al. 1992). However, patient survival is lower for squamous cell lung cancer. In some cases, neoadjuvant chemotherapy, i.e. preoperative chemotherapy is proposed to be beneficial to reduce tumor size to facilitate surgical resection and eliminate early micrometastases (Burdett, Stewart et al. 2007). In addition, post-operative adjuvant chemotherapy, for instance with cisplatin, may significantly improve prognosis and prevent local recurrences. For inoperable tumors or patients unfit for surgery, radiation therapy is recommended (Pignon, Tribodet et al. 2008). 
     Stage III NSCLC includes both locally and regionally advanced disease. For resectable NSCLC, surgery to remove the complete tumor and the surrounding lymph nodes is recommended, followed by post-operative chemotherapy. Further, neoadjuvant chemotherapy to shrink the tumor and eradicate micrometastases, thus facilitating surgery, is also an approach of choice (Burdett, Stewart et al. 2007). Further, similar to Stage II, patients are shown to benefit with adjuvant chemotherapy using cisplatin. For unresectable Stage III NSCLC, radiation therapy or a concurrent or sequential combination of chemo- with radiation therapy is recommended (Furuse, Fukuoka et al. 1999). 
     If the subtype of NSCLC (like NSCLC stage IV tumors/cancers) is, for example, metastatic NSCLC (such as forms of all NSCLC classes/subtypes, like metastatic adenocarcinoma), adenocarcinoma, squamous cell carcinoma or large cell carcinoma the following treatment options are conceivable: 
     For patients with metastatic NSCLC (Stage IV), treatment is usually aimed to prolong survival and for palliation of disease related symptoms. Standard treatment options include cytotoxic chemotherapy and targeted agents. However, treatment is selected based on comorbidity, performance status, histology, and molecular genetic features of the cancer. First line cytotoxic combination chemotherapy includes a combination of platinum-based chemotherapy (cisplatin or carboplatin) and paclitaxel, gemcitabine, docetaxel, vinorelbine, irinotecan, or pemetrexed (Le Chevalier, Arriagada et al. 1992, Wozniak, Crowley et al. 1998, Mok, Wu et al. 2009). Following the initial response to chemotherapy, maintenance chemotherapy using the initial combination of drugs, or continuing single-agent chemotherapy, or using a new ‘maintenance’ agent is evaluated. (Brodowicz, Krzakowski et al. 2006, Park, Kim et al. 2007, Paz-Ares, de Marinis et al. 2012). Further, based on the molecular analysis of the cancer, patients may benefit from single-agent EGFR tyrosine kinase inhibitors or EML4-ALK inhibitors, as first line treatment (if driver mutations have been encountered) or, even in absence of driver mutations, as second or third line treatment. 
     If the subtype of NSCLC is, for example, adenocarcinoma, the following treatment options are conceivable: 
     Among the currently used combinations, definite recommendations regarding drug dose, schedule or combination cannot be made. However, the exception for this is pemetrexed for lung adenocarcinoma (Scagliotti, Parikh et al. 2008). Adenocarcinoma patients, especially adenocarcinoma in never smokers/never smoker patients, benefit from using EGFR tyrosine kinase inhibitors, such as gefitinib (Mok, Wu et al. 2009). 
     If the subtype of NSCLC is, for example, sqamous cell carcinoma, the following treatment options are conceivable: 
     In contrast, in patients with squamous cell histology (like patients with squamous cell carcinoma), patient response is significantly better using a combination of cisplatin and gemcitabine versus cisplatin and pemetrexed (Scagliotti, Parikh et al. 2008). 
     Lastly, for patients with Stage IV NSCLC, palliative radiotherapy may be used to control vocal cord paralysis, hemoptysis, obstructive symptoms or pain related to bone metastases. Surgical intervention may also be recommended for patients with bronchial obstructions. 
     Standard treatment for recurrent drug resistant NSCLC includes palliative radiation therapy (Sundstrom, Bremnes et al. 2004) and/or combination chemotherapy, for patients who have previously received platinum based chemotherapy. Chemotherapy combinations include Docetaxel, Pemetrexed, Erlotinib after failure of both platinum-based and docetaxel chemotherapies, Gefitinib, Crizotinib for EML4-ALK translocations, EGFR inhibitors in patients with or without EGFR mutations, EML4-ALK inhibitors in patients with EML-ALK translocations (Hanna, Shepherd et al. 2004, Kim, Hirsh et al. 2008, Kwak, Bang et al. 2010, Shaw, Yeap et al. 2011). 
     If the subtype of NSCLC is, for example, large cell lung cancer/large cell carcinoma, the treatment plan depends on the stage and no definite recommendations can be made beforehand. For example, conventional therapy, like chemotherapy/radiotherapy as disclosed herein, can be contemplated. 
     If the subtype of lung cancer is, for example, small-cell lung cancer (SCLC), the following treatment options are conceivable: 
     For treatment purposes, small-cell lung cancer (SCLC) is usually staged as either limited or extensive disease. Limited stage SCLC means that the cancer is only on one side of the chest and includes the lobes and/or lymph nodes on the same side. The tumors are often confined to a small area and can be targeted by a single radiation field. On the other hand, extensive stage represents cancers that have spread to both sides of the chest and may include distant metastases to other organs. 
     Chemotherapy is the mainstay of treatment of SCLC. For limited stage disease, combined modality of chemotherapy and thoracic radiation therapy, called concurrent chemoradiation, is the most widely used treatment. Active drugs usually include a combination of platinum and etoposide. Based on the patient&#39;s health status, radiation therapy may not be recommended and in this case, the patients are treated with chemotherapy alone (Pignon, Arriagada et al. 1992, Warde and Payne 1992, Murray, Coy et al. 1993). Surgical resection for SCLC is limited to management of cases with very limited disease, i.e. small tumors pathologically confined to the lobe of origin. Surgery is generally followed by adjuvant chemotherapy (Osterlind, Hansen et al. 1985, Prasad, Naylor et al. 1989, Smit, Groen et al. 1994). 
     For patients with extensive stage disease, combination chemotherapy, including platinum and etoposide in doses that the least toxic effects is recommended (Okamoto, Watanabe et al. 2007). Further, radiation therapy to the site of distant metastases is also a standard treatment option for patients. This is especially preferred for metastases that are unlikely to be immediately palliated by chemotherapy, such as the brain and bone (Slotman, Faivre-Finn et al. 2007). 
     
       
         
           
               
             
               
                   
               
               
                 Commonly used chemotherapy combinations 
               
               
                 include cisplatin, carboplatin, etoposide, 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Standard  
                 Etoposide + cisplatin 
               
               
                 treatment 
                 Etoposide + carboplatin 
               
               
                 Other  
                 Cisplatin + irinotecan 
               
               
                 regimens 
                 Ifosfamide + cisplatin + etoposide 
               
               
                   
                 Cyclophosphamide + doxorubicin + etoposide 
               
               
                   
                 Cyclophosphamide + doxorubicin + etoposide + vincristine 
               
               
                   
                 Cyclophosphamide + etoposide + vincristine 
               
               
                   
                 Cyclophosphamide + doxorubicin + vincristine 
               
               
                   
               
            
           
         
       
     
     Response rates to chemotherapy are high for SCLC, up to 85-95% in limited disease and 75-80% in extensive disease. However, median survival still remains low, i.e. 14-20 months for limited disease and only 7-10 months for extensive disease. Long term survival is only seen in 5-10% of the patients. (Hoffman, Mauer et al. 2000). 
     In accordance with the present invention the methods, in particular the statistical methods, may comprise the use of FOXA2 Em isoform and/or ID2 Em isoform. 
     For example, the herein provided statistical method of assessing whether a subject suffers from cancer or is prone to suffering from cancer, may (further) comprise the step of 
     performing at least one statistical algorithm for classification and for regression on measurement data of the subject,
 
wherein the measurement data of the subject comprises at least one of the following: a value of FOXA2 Em isoform in at least one sample taken from the subject, a value ID2 Em isoform in said at least one sample, a value of FOXA2 Ad isoform in said at least one sample, ID2 Ad isoform in said at least one sample; and
 
wherein at least one of the following is used as at least one classifier or a component of at least one classifier in the statistical method: FOXA2 Em isoform, ID2 Em isoform, FOXA2 Ad isoform, ID2 Ad isoform, ratio of FOXA2 Em isoform/FOXA2 Ad isoform, ratio of ID2 Em isoform/ID2 Ad isoform.
 
     The term “specific transcription factor Em isoform” according to the present application may relate to FOXA2 (Uniprot-ID: Q9Y261; Gene-ID: 3170) and/or ID2 (Uniprot-ID: Q02363; Gene-ID:3398). If, for example, the amount of a specific transcription factor is measured on mRNA level, the specific transcription factor can be mRNA molecules (or transcript or splice variants). In this context, the transcription factors can be defined as
     i) the FOXA2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 3 or the FOXA2 Em isoform comprising nucleic acid sequence with up to 68 additions, deletions or substitutions of SEQ ID NO: 3;   ii) the ID2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 4 or the ID2 Em isoform comprising nucleic acid sequence with up to 34 additions, deletions or substitutions of SEQ ID NO: 4;   iii) the FOXA2 Ad isoform comprising the nucleic acid sequence of SEQ ID No: 7 or FOXA2 Ad isoform comprising the nucleic acid sequence with up to 74 additions, deletions or substitutions of SEQ ID NO: 7; or   iv) the ID2 Ad isoform consisting of the nucleic acid sequence of SEQ ID No: 8 or ID2 Ad isoform consisting of nucleic acid sequence with up to 30 additions, deletions or substitutions of SEQ ID NO: 8;   

     In a certain aspect, the value of the FOXA2 Em isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the FOXA2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 3 or the FOXA2 Em isoform comprising nucleic acid sequence with up to 68 additions, deletions or substitutions of SEQ ID NO: 3. 
     In a certain aspect, the value of the ID2 Em isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the ID2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 4 or the ID2 Em isoform comprising nucleic acid sequence with up to 34 additions, deletions or substitutions of SEQ ID NO: 4. 
     In a certain aspect, the value of the FOXA2 Ad isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the FOXA2 Ad isoform comprising the nucleic acid sequence of SEQ ID No: 7 or FOXA2 Ad isoform comprising the nucleic acid sequence with up to 74 additions, deletions or substitutions of SEQ ID NO: 7. 
     In a certain aspect, the value of the ID2 Ad isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the ID2 Ad isoform consisting of the nucleic acid sequence of SEQ ID No: 8 or ID2 Ad isoform consisting of nucleic acid sequence with up to 30 additions, deletions or substitutions of SEQ ID NO: 8. 
     In a certain aspect, the FOXA2 Em isoform of said sample is set in relation to a FOXA2 Em isoform of at least one control sample and then used as a classifier in the statistical method; and 
     said value of the FOXA2 Em isoform of said at least one control sample is obtained by measuring in said sample of said subject the amount of a specific transcription factor isoform, wherein said specific transcription isoform is the FOXA2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 3 or the FOXA2 Em isoform comprising nucleic acid sequence with up to 68 additions, deletions or substitutions of SEQ ID NO: 3. 
     In a certain aspect, the FOXA2 Ad isoform of said sample is set in relation to a FOXA2 Ad isoform of at least one control sample and then used as a classifier in the statistical method; and 
     said value of the FOXA2 Ad isoform of said at least one control sample is obtained by measuring in said sample of said subject the amount of a specific transcription factor isoform, wherein said specific transcription isoform is the FOXA2 Ad isoform comprising the nucleic acid sequence of SEQ ID No: 7 or FOXA2 Ad isoform comprising the nucleic acid sequence with up to 74 additions, deletions or substitutions of SEQ ID NO: 7. 
     In a certain aspect, the ID2 Em isoform of said sample is set in relation to a ID2 Em isoform of at least one control sample and then used as a classifier in the statistical method; and 
     said value of the ID2 Em isoform of said at least one control sample is obtained by measuring in said sample of said subject the amount of a specific transcription factor isoform, wherein said specific transcription isoform is the ID2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 4 or the ID2 Em isoform comprising nucleic acid sequence with up to 34 additions, deletions or substitutions of SEQ ID NO: 4. 
     In a certain aspect, the ID2 Ad isoform of said sample is set in relation to a ID2 Ad isoform of at least one control sample and then used as a classifier in the statistical method; and 
     said value of the ID2 Ad isoform of said at least one control sample is obtained by measuring in said sample of said subject the amount of a specific transcription factor isoform,
 
wherein said specific transcription isoform is the ID2 Ad isoform consisting of the nucleic acid sequence of SEQ ID No: 8 or ID2 Ad isoform consisting of nucleic acid sequence with up to 30 additions, deletions or substitutions of SEQ ID NO: 8.
 
     In certain aspects, a ratio of the FOXA2 Em isoform and the FOXA2 Ad isoform and a ratio of the ID2 Em isoform and the ID2 Ad isoform are used as a classifier. 
     The present invention also contemplates the use of obtaining the value of a transcription factor isoform in a sample e.g. by measuring the amount of a transcription factor isoform on the protein level. 
     If, for example, the amount of a specific transcription factor is measured on protein level, the specific transcription factor can be protein molecules. For example, they can be defined as
     i) the FOXA2 Em isoform comprising the polypeptide sequence of SEQ ID No: 52 or the FOXA2 Em isoform comprising polypeptide sequence with up to 43 additions, deletions or substitutions of SEQ ID NO: 52;   ii) the ID2 Em isoform comprising the polypeptide sequence of SEQ ID No: 53 or the ID2 Em isoform comprising polypeptide sequence with up to 13 additions, deletions or substitutions of SEQ ID NO: 53;   iii) the FOXA2 Ad isoform comprising the polypeptide sequence of SEQ ID No: 56 or FOXA2 Ad isoform comprising the polypeptide sequence with up to 43 additions, deletions or substitutions of SEQ ID NO: 56; or   iv) the ID2 Ad isoform consisting of the polypeptide sequence of SEQ ID No: 57 or ID2 Ad isoform consisting of polypeptide sequence with up to 13 additions, deletions or substitutions of SEQ ID NO: 57.   

     In a certain aspect, the value of the FOXA2 Em isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the FOXA2 Em isoform comprising the polypeptide sequence of SEQ ID No: 52 or the FOXA2 Em isoform comprising polypeptide sequence with up to 43 additions, deletions or substitutions of SEQ ID NO: 52. 
     In a certain aspect, the value of the ID2 Em isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the ID2 Em isoform comprising the polypeptide sequence of SEQ ID No: 53 or the ID2 Em isoform comprising polypeptide sequence with up to 13 additions, deletions or substitutions of SEQ ID NO: 53. 
     In a certain aspect, the value of the FOXA2 Ad isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the FOXA2 Ad isoform comprising the polypeptide sequence of SEQ ID No: 56 or FOXA2 Ad isoform comprising the polypeptide sequence with up to 43 additions, deletions or substitutions of SEQ ID NO: 56. 
     In a certain aspect, the value of the ID2 Ad isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the ID2 Ad isoform consisting of the polypeptide sequence of SEQ ID No: 57 or ID2 Ad isoform consisting of polypeptide sequence with up to 13 additions, deletions or substitutions of SEQ ID NO: 57. 
     If, for example, the amount of a specific transcription factor is measured on protein level, the specific transcription factors can be proteins molecules. For example, they can be defined as
     i) the GATA6 Em isoform comprising the polypeptide sequence of SEQ ID No: 50 or the GATA6 Em isoform comprising the polypeptide sequence with up to 30 additions, deletions or substitutions of SEQ ID NO: 50;   ii) the NKX2-1 Em isoform comprising the polypeptide sequence of SEQ ID No: 51 or the NKX2-1 Em isoform comprising the polypeptide sequence with up to 14 additions, deletions or substitutions of SEQ ID NO: 51;   iii) the GATA6 Ad isoform comprising the polypeptide sequence of SEQ ID No: 54 or the GATA6 Ad isoform polypeptide sequence with up to 23 additions, deletions or substitutions of SEQ ID NO: 54;   iv) the NKX2-1 Ad isoform comprising the polypeptide sequence of SEQ ID No: 55 or the NKX2-1 Ad isoform comprising the polypeptide sequence with up to 15 additions, deletions or substitutions of SEQ ID NO: 55.   

     In a certain aspect, the value of the GATA6 Em isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the GATA6 Em isoform comprising the polypeptide sequence of SEQ ID No: 50 or the GATA6 Em isoform comprising the polypeptide sequence with up to 30 additions, deletions or substitutions of SEQ ID NO: 50 
     In a certain aspect, the value of the NKX2-1 Em isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the NKX2-1 Em isoform comprising the polypeptide sequence of SEQ ID No: 51 or the NKX2-1 Em isoform comprising the polypeptide sequence with up to 14 additions, deletions or substitutions of SEQ ID NO: 51 
     In a certain aspect, the value of the GATA6 Ad isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the GATA6 Ad isoform comprising the polypeptide sequence of SEQ ID No: 54 or the GATA6 Ad isoform polypeptide sequence with up to 23 additions, deletions or substitutions of SEQ ID NO: 54 
     In a certain aspect, the value of the NKX2-1 Ad isoform in said at least one sample is obtained by measuring the amount of a specific transcription factor isoform in said at least one sample of said subject, wherein said specific transcription isoform is the NKX2-1 Ad isoform comprising the polypeptide sequence of SEQ ID No: 55 or the NKX2-1 Ad isoform comprising the polypeptide sequence with up to 15 additions, deletions or substitutions of SEQ ID NO: 55. 
     Genes can contain single nucleotide polymorphisms (SNPs). The specific transcription factor Em isoform sequences of the present invention encompass (genetic) variants thereof, for example, variants having SNPs. Without deferring from the gist of the present invention, all naturally occurring sequences of the respective isoform independent of the number and nature of the SNPs in said sequence can be used herein. To relate to currently known SNPs, the transcription factor Em isoforms of the present invention are defined such that they contain up to 55 (in the case of GATA6), up to 39 (in the case of NKX2-1), up to 68 (in the case of FOXA2) or up to 34 (in the case of ID2) additions, deletions or substitutions of the nucleic acid sequences defined by SEQ ID NOs: 1, 2, 3 and 4, respectively. Thus, respective Em transcripts of carriers of different nucleotides at the respective SNPs are covered by the present application. 
     The FOXA2 Em isoform according to the invention is the FOXA2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 3 or the FOXA2 Em isoform comprising a nucleic acid sequence with up to 68; preferably up to 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53 52, 51, 50, 49, 48 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20; even more preferably up to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7. 6, 5, 4, 3 or 2; or even furthermore preferably only 1 addition(s), deletion(s) or substitution(s) of SEQ ID NO: 3. The FOXA2 Em isoform can also be defined as the FOXA2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 3 or the FOXA2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 3 with additions, deletions or substitutions at any of positions 168; 208; 289; 361; 368; 374; 379; 383; 404; 459; 481; 483; 494; 529; 564; 577; 584; 590; 610; 623; 641; 650; 659; 674; 773; 845; 1040; 1075; 1186; 1188; 1240; 1242; 1243; 1304; 1374; 1391; 1408; 1414; 1432; 1458; 1475; 1487; 1522; 1539; 1582; 1583; 1594; 1627; 1631; 1687; 1723; 1737; 1738; 1754; 1812; 1831; 1838; 1940; 1966; 1970; 2070; 2083; 2084; 2093; 2105; 2112; 2200 and 2388. The FOXA2 Em isoform according to the invention can also be defined as the FOXA2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 3 or the FOXA2 Em isoform comprising a nucleic acid sequence with at least 93% homology to SEQ ID No: 3, preferably up to 94%, 95%, 96%, 97% or 98% homology to SEQ ID No: 3; even more preferably up to 99% homology to SEQ ID No: 3. 
     The ID2 Em isoform according to the invention is the ID2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 4 or the ID2 Em isoform comprising a nucleic acid sequence with up to 34; preferably up to 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10; even more preferably up to 9, 8, 7, 6, 5, 4, 3, or 2; or even furthermore preferably only 1 addition(s), deletion(s) or substitution(s) of SEQ ID NO: 4. The ID2 Em isoform can also be defined as the ID2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 4 or the ID2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 4 with additions, deletions or substitutions at any of positions 6; 43; 53; 55; 154; 195; 209; 224; 237; 263; 286; 360; 399; 405; 485; 501; 544; 547; 605; 662; 665; 716; 757; 871; 876; 975; 1085; 1115; 1119; 1149; 1151; 1251; 1333 and 1350. The ID2 Em isoform according to the invention can also be defined as the ID2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 4 or the ID2 Em isoform comprising a nucleic acid sequence with at least 51% homology to SEQ ID No: 4, preferably up to 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% homology to SEQ ID No: 4; even more preferably up to 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID No: 4. 
     Preferably, the above referred “addition(s), deletion(s) or substitution(s)” of the transcription factor isoforms are substitutions. 
     The person skilled in the art understands that a subject which is prone to suffering from cancer is a subject which has an increased likelihood of developing cancer within the next 30 years or preferably within the next 20 or 10 years or even more preferably within the next 9, 8, 7, 6, 5, 4, 3 or 2 years or even furthermore preferably within the next year. An increased likelihood of a subject of developing cancer can be understood as that said subject has an increased likelihood of developing cancer within a given time period as if compared to the average likelihood that a subject of the same age or a subject of the same age and the same gender develops cancer. 
     The term “sample” according to the present invention relates to any kind of sample which can be obtained from a subject, preferably from a human subject. The sample is a biological sample. A sample according to the present invention can be for example, but is not limited to, a blood sample, a breath condensate sample, a bronchoalveolar lavage fluid sample, a mucus sample or a phlegm sample. Preferably, the sample according to the present invention is a biopsy, a blood sample or a breath condensate sample. More preferably, the sample according to the present invention is a biopsy or a breath condensate sample. Particularly preferred is (a) (a) breath condensate sample(s). 
     The term “breath condensate sample” as used herein refers to an “exhaled breath condensate (sample)”. The term “exhaled breath condensate (sample)” can be abbreviated as “EBC”. Accordingly, the terms “breath condensate sample”, “exhaled breath condensate”, “exhaled breath condensate sample” and “EBC” are used interchangeably herein. The use of “breath condensate sample”, in particular “exhaled breath condensate (sample)” allows the non-invasive obtaining of samples from a subject/patient and is therefore advantageous. 
     The herein provided diagnostic method can lead to fast medical intervention for example by means of corresponding anti-cancer therapy, like anti-cancer medication or radiation therapy. Early stage anti-cancer therapies include, but are not limited to, radiation therapy, such as external radiation therapy, photodynamic therapy (PDT) using an endoscope and surgery (i.e. wedge resection or segmental resection for carcinoma in situ and sleeve resection or lobectomy for StageI). In addition, chemotherapy is used alone or after surgery. The chemotherapy drugs may, inter alia, comprise compounds selected from the group consisting of Cisplatin, Carboplatin, Paclitaxel (Taxol®), Albumin-bound paclitaxel (nab-paclitaxel, Abraxane®), Docetaxel (Taxotere®), Gemcitabine (Gemzar®), Vinorelbine (Navelbine®), Irinotecan (Camptosar®, CPT-11), Etoposide (VP-16®), Vinblastine and Pemetrexed (Alimta®). 
     The herein provided methods are primarily useful in the assessment whether a subject suffers from cancer or is prone to suffering from cancer before the subject undergoes therapeutic intervention. In other words, the sample of the subject is obtained from the subject and analyzed prior to therapeutic intervention, like conventional chemotherapy. If the subject is assessed “positive” in accordance with the present invention, i.e. assessed to suffer from cancer or prone to suffering from cancer, the appropriate therapy/therapeutic intervention can be chosen. For example, a subject may be suspected of suffering from cancer and the present methods can be used to assess whether the subject suffers indeed from said cancer in addition or in the alternative to conventional diagnostic methods. 
     Following positive diagnosis with the herein provided inventive method, the diagnosis may be elucidated/further verified with low-dose helical computed tomography and/or Chest X-Ray, by bronchoscopy and/or histological assessment. In early stage or Grade I tumors, surgery to to remove the lobe or the section of the lung that contains the tumor would be the first choice of treatment. It is feasible to supplement the surgery with chemotherapy, known as ‘adjuvant chemotherapy’, to prevent cancer relapse (Howington J A et al. (2013) CHEST Journal 143: e278S-e313S). At later stages, surgery is no longer feasible and a combination of chemotherapy and radiation are advised. Further, for metastatic lesions, chemotherapy and radiation are suggested, mainly for palliation of the symptoms. 
     The term “isoform” according to the present invention encompasses transcript variants (which are mRNA molecules) as well as the corresponding polypeptide variants (which are polypeptides) of a gene. Such transcription variants result, for example, from alternative splicing or from a shifted transcription initiation. Based on the different transcript variants, different polypeptides are generated. It is possible that different transcript variants have different translation initiation sites. A person skilled in the art will appreciate that the amount of an isoform can be measured by adequate techniques for the quantification of mRNA as far as the isoform relates to a transcript variant which is an mRNA. Examples of such techniques are polymerase chain reaction-based methods, in situ hybridization-based methods, microarray-based techniques and whole transcriptome shotgun sequencing. Further, a person skilled in the art will appreciate that the amount of an isoform can be measured by adequate techniques for the quantification of polypeptides as far as the isoform relates to a polypeptide. Non-limiting examples of such techniques for the quantification of polypeptides are ELISA (Enzyme-linked Immunosorbent Assay)-based, gel-based, blot-based, mass spectrometry-based, and flow cytometry-based methods. 
     Genes can contain single nucleotide polymorphisms (SNPs). The specific transcription factor Em isoform sequences of the present invention encompass (genetic) variants thereof, for example, variants having SNPs. Without deferring from the gist of the present invention, all naturally occurring sequences of the respective isoform independent of the number and nature of the SNPs in said sequence can be used herein. To relate to currently known SNPs, the transcription factor Em isoforms of the present invention are defined such that they contain up to 55 (in the case of GATA6), up to 39 (in the case of NKX2-1), additions, deletions or substitutions of the nucleic acid sequences defined by SEQ ID NOs: 1 and 2 respectively. Thus, respective Em transcripts of carriers of different nucleotides at the respective SNPs are covered by the present application. 
     The GATA6 Em isoform according to the invention is the GATA6 Em isoform comprising the nucleic acid sequence of SEQ ID No: 1 or the GATA6 Em isoform comprising a nucleic acid sequence with up to 55; preferably up to 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20; even more preferably up to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7. 6, 5, 4, 3 or 2; or even furthermore preferably only 1 addition(s), deletion(s) or substitution(s) of SEQ ID NO: 1. The GATA6 Em isoform can also be defined as the GATA6 Em isoform comprising the nucleic acid sequence of SEQ ID No: 1 or the GATA6 Em isoform comprising the nucleic acid sequence of SEQ ID No: 1 with additions, deletions or substitutions at any of positions 163; 293; 320; 327; 339; 430; 462; 480; 759; 1128; 1256; 1304; 1589; 1597; 1627; 1651; 1652; 1803; 1844; 1849; 1879; 1882; 1911; 1940; 1949; 1982; 2000; 2002; 2008; 2026; 2031; 2106; 2137; 2142; 2163; 2294; 2390; 2391; 2627; 2691; 3036; 3102; 3240; 3265; 3266; 3290; 3358; 3366; 3578; 3632; 3646; 3670; 3690; 3708 and 3735. The GATA6 Em isoform according to the invention can also be defined as the GATA6 Em isoform comprising the nucleic acid sequence of SEQ ID No: 1 or the GATA6 Em isoform comprising a nucleic acid sequence with at least 85% homology to SEQ ID No: 1, preferably up to 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% homology to SEQ ID No: 1; even more preferably up to 99% homology to SEQ ID No: 1. 
     The NKX2-1 Em isoform according to the invention is the NKX2-1 Em isoform comprising the nucleic acid sequence of SEQ ID No: 2 or the NKX2-1 Em isoform comprising a nucleic acid sequence with up to 39; preferably up to 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10; even more preferably up to 9, 8, 7, 6, 5, 4, 3, or 2; or even furthermore preferably only 1 addition(s), deletion(s) or substitution(s) of SEQ ID NO: 2. The NKX2-1 Em isoform can also be defined as the NKX2-1 Em isoform comprising the nucleic acid sequence of SEQ ID No: 2 or the NKX2-1 Em isoform comprising the nucleic acid sequence of SEQ ID No: 2 with additions, deletions or substitutions at any of positions 269; 281; 305; 304; 420; 425; 439; 441; 450; 486; 781; 785; 825; 950; 1169; 1305; 1344; 1448; 1458; 1467; 1489; 1552; 1633; 1634; 1640; 1641; 1643; 1667; 1673; 1678; 1748; 1750; 1831; 1893; 1916; 1917; 1934; 2099 and 2319. The NKX2-1 Em isoform according to the invention can also be defined as the NKX2-1 Em isoform comprising the nucleic acid sequence of SEQ ID No: 2 or the NKX2-1 Em isoform comprising a nucleic acid sequence with at least 90% homology to SEQ ID No: 2, preferably up to 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% homology to SEQ ID No: 2; even more preferably up to 99% homology to SEQ ID No: 2. 
     Preferably, the above referred “addition(s), deletion(s) or substitution(s)” of the transcription factor isoforms are substitutions. 
     Tables 1, 2, 3, 4, 5, 6, 7 and 8 below provide information on different SNPs of the transcription factors of the present invention. The present invention relates to the respective isoforms independently from the various SNPs which may occur at the different positions of the mRNAs or polypeptides. The SNPs of tables 1, 2, 3, 4, 5, 6, 7 and 8 may occur in the isoforms of the present invention in any combination. For example, a (genetic) variant of the GATA6 Em isoform to be used herein may comprise a nucleic acid sequence of SEQ ID NO:1, whereby the “G” residue at position 293 of SEQ ID NO:1 is substituted by “A”. Further variants of the isoforms to be used herein are apparent from Tables 1 to 8 to the person skilled in the art. The respective SNP information has been retrieved using dbSNP (short genetic variations) database of the NCBI. The SNP information is based on Contig Label GRCh37.p5. A person skilled in the art will understand that also SNPs which are not mentioned in tables 1 to 8 are encompassed by the present invention. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 SNPs of the GATA6 Em isoform 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Contig 
                 Poly- 
                 Codon 
                   
                 Protein 
               
               
                 S. No. 
                 Region 
                 Position 
                 reference 
                 morphism 
                 Position 
                 Function 
                 residue 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 5′ UTR 
                 163 
                 C 
                 G 
                   
                   
                   
               
               
                 2 
                 CCDS 
                 293 
                 G 
                 A 
                 6 
                 Missense 
                 Gly-Ser 
               
               
                 3 
                 CCDS 
                 320 
                 G 
                 C 
                 15 
                 Missense 
                 Gly-Arg 
               
               
                 4 
                 CCDS 
                 327 
                 C 
                 G 
                 17 
                 Missense 
                 Ala-Gly 
               
               
                 5 
                 CCDS 
                 339 
                 C 
                 G 
                 21 
                 Missense 
                 Ala-Gly 
               
               
                 6 
                 CCDS 
                 430 
                 G 
                 T 
                 51 
                 Missense 
                 Glu-Asp 
               
               
                 7 
                 CCDS 
                 462 
                 — 
                 T 
                 62 
                 Frameshift 
                 TA-Thr 
               
               
                 8 
                 CCDS 
                 480 
                 A 
                 T 
                 68 
                 Missense 
                 Glu-Val 
               
               
                 9 
                 CCDS 
                 759 
                 C 
                 T 
                 161 
                 Missense 
                 Ala-Val 
               
               
                 10 
                 CCDS 
                 1128 
                 C 
                 G 
                 284 
                 Missense 
                 Ala-Gly 
               
               
                 11 
                 CCDS 
                 1256 
                 C 
                 A 
                 327 
                 Missense 
                 His-Asn 
               
               
                 12 
                 CCDS 
                 1304 
                 G 
                 A 
                 343 
                 Missense 
                 Ala-Thr 
               
               
                 13 
                 CCDS 
                 1589 
                 C 
                 T 
                 438 
                 Missense 
                 Arg-Trp 
               
               
                 14 
                 CCDS 
                 1597 
                 T 
                 A 
                 440 
                 Synonymous 
                 Leu-Leu 
               
               
                 15 
                 CCDS 
                 1627 
                 A 
                 G 
                 450 
                 Synonymous 
                 Thr-Thr 
               
               
                 16 
                 CCDS 
                 1651 
                 C 
                 T 
                 458 
                 Synonymous 
                 Asn-Asn 
               
               
                 17 
                 CCDS 
                 1652 
                 G 
                 A 
                 459 
                 Missense 
                 Ala-Thr 
               
               
                 18 
                 CCDS 
                 1803 
                 A 
                 G 
                 509 
                 Missense 
                 Asn-Ser 
               
               
                 19 
                 CCDS 
                 1844 
                 T 
                 C 
                 523 
                 Missense 
                 Ser-Pro 
               
               
                 20 
                 CCDS 
                 1849 
                 T 
                 C 
                 524 
                 Synonymous 
                 Asp-Asp 
               
               
                 21 
                 CCDS 
                 1879 
                 A 
                 G 
                 534 
                 Synonymous 
                 Thr-Thr 
               
               
                 22 
                 CCDS 
                 1882 
                 A 
                 G 
                 535 
                 Synonymous 
                 Gln-Gln 
               
               
                 23 
                 CCDS 
                 1911 
                 T 
                 G 
                 545 
                 Missense 
                 Val-Gly 
               
               
                 24 
                 CCDS 
                 1940 
                 C 
                 G 
                 555 
                 Missense 
                 Pro-Ala 
               
               
                 25 
                 CCDS 
                 1949 
                 A 
                 G 
                 558 
                 Missense 
                 Ser-Gly 
               
               
                 26 
                 CCDS 
                 1982 
                 T 
                 C 
                 569 
                 Missense 
                 Tyr-His 
               
               
                 27 
                 CCDS 
                 2000 
                 G 
                 C 
                 575 
                 Missense 
                 Ala-Pro 
               
               
                 28 
                 CCDS 
                 2002 
                 C 
                 T 
                 575 
                 Synonymous 
                 Ala-Ala 
               
               
                 29 
                 CCDS 
                 2008 
                 G 
                 C 
                 577 
                 Synonymous 
                 Pro-Pro 
               
               
                 30 
                 CCDS 
                 2026 
                 C 
                 T 
                 583 
                 Synonymous 
                 Ser-Ser 
               
               
                 31 
                 CCDS 
                 2031 
                 G 
                 T 
                 585 
                 Missense 
                 Arg-Leu 
               
               
                 32 
                 3′UTR 
                 2106 
                 C 
                 T 
               
               
                 33 
                 3′UTR 
                 2137 
                 G 
                 A 
               
               
                 34 
                 3′UTR 
                 2142 
                 A 
                 G 
               
               
                 35 
                 3′UTR 
                 2163 
                 C 
                 T 
               
               
                 36 
                 3′UTR 
                 2294 
                 C 
                 T 
               
               
                 37 
                 3′UTR 
                 2390 
                 A 
                 G 
               
               
                 38 
                 3′UTR 
                 2391 
                 T 
                 A 
               
               
                 39 
                 3′UTR 
                 2627 
                 A 
                 G 
               
               
                 40 
                 3′UTR 
                 2691 
                 G 
                 T 
               
               
                 41 
                 3′UTR 
                 3036 
                 G 
                 T 
               
               
                 42 
                 3′UTR 
                 3102 
                 A 
                 G 
               
               
                 43 
                 3′UTR 
                 3240 
                 C 
                 T 
               
               
                 44 
                 3′UTR 
                 3265 
                 C 
                 G 
               
               
                 45 
                 3′UTR 
                 3266 
                 C 
                 T 
               
               
                 46 
                 3′UTR 
                 3290 
                 A 
                 G 
               
               
                 47 
                 3′UTR 
                 3358 
                 C 
                 T 
               
               
                 48 
                 3′UTR 
                 3366 
                 A 
                 T 
               
               
                 49 
                 3′UTR 
                 3578 
                 C 
                 T 
               
               
                 50 
                 3′UTR 
                 3632 
                 — 
                 C 
               
               
                 51 
                 3′UTR 
                 3646 
                 C 
                 T 
               
               
                 52 
                 3′UTR 
                 3670 
                 A 
                 G 
               
               
                 53 
                 3′UTR 
                 3690 
                 C 
                 T 
               
               
                 54 
                 3′UTR 
                 3708 
                 A 
                 G 
               
               
                 55 
                 3′UTR 
                 3735 
                 A 
                 G 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 SNPs of the GATA6 Ad isoform 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Contig 
                 Poly- 
                 Codon 
                   
                 Protein 
               
               
                 S. No. 
                 Region 
                 Position 
                 reference 
                 morphism 
                 Position 
                 Function 
                 residue 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 5′UTR 
                 138 
                 C 
                 G 
                   
                   
                   
               
               
                 2 
                 5′UTR 
                 228 
                 G 
                 A 
               
               
                 3 
                 5′UTR 
                 255 
                 G 
                 C 
               
               
                 4 
                 5′UTR 
                 262 
                 C 
                 G 
               
               
                 5 
                 5′UTR 
                 274 
                 C 
                 G 
               
               
                 6 
                 5′UTR 
                 365 
                 G 
                 T 
               
               
                 7 
                 5′UTR 
                 397 
                 — 
                 T 
               
               
                 8 
                 5′UTR 
                 415 
                 A 
                 T 
               
               
                 9 
                 CCDS 
                 694 
                 C 
                 T 
                 15 
                 Missense 
                 Ala-Val 
               
               
                 10 
                 CCDS 
                 1063 
                 C 
                 G 
                 138 
                 Missense 
                 Ala- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Gly 
               
               
                 11 
                 CCDS 
                 1191 
                 C 
                 A 
                 181 
                 Missense 
                 His- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Asn 
               
               
                 12 
                 CCDS 
                 1239 
                 G 
                 A 
                 197 
                 Missense 
                 Ala-Thr 
               
               
                 13 
                 CCDS 
                 1524 
                 C 
                 T 
                 292 
                 Missense 
                 Arg- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Trp 
               
               
                 14 
                 CCDS 
                 1532 
                 T 
                 A 
                 294 
                 Synonymous 
                 Leu- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Leu 
               
               
                 15 
                 CCDS 
                 1562 
                 A 
                 G 
                 304 
                 Synonymous 
                 Thr-Thr 
               
               
                 16 
                 CCDS 
                 1586 
                 C 
                 T 
                 312 
                 Synonymous 
                 Asn- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Asn 
               
               
                 17 
                 CCDS 
                 1587 
                 G 
                 A 
                 313 
                 Missense 
                 Ala-Thr 
               
               
                 18 
                 CCDS 
                 1738 
                 A 
                 G 
                 363 
                 Missense 
                 Asn- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Ser 
               
               
                 19 
                 CCDS 
                 1779 
                 T 
                 C 
                 377 
                 Missense 
                 Ser-Pro 
               
               
                 20 
                 CCDS 
                 1784 
                 T 
                 C 
                 378 
                 Synonymous 
                 Asp- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Asp 
               
               
                 21 
                 CCDS 
                 1814 
                 A 
                 G 
                 388 
                 Synonymous 
                 Thr-Thr 
               
               
                 22 
                 CCDS 
                 1817 
                 A 
                 G 
                 389 
                 Synonymous 
                 Gln- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Gln 
               
               
                 23 
                 CCDS 
                 1846 
                 T 
                 G 
                 399 
                 Missense 
                 Val- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Gly 
               
               
                 24 
                 CCDS 
                 1875 
                 C 
                 G 
                 409 
                 Missense 
                 Pro-Ala 
               
               
                 25 
                 CCDS 
                 1884 
                 A 
                 G 
                 412 
                 Missense 
                 Ser-Gly 
               
               
                 26 
                 CCDS 
                 1917 
                 T 
                 C 
                 423 
                 Missense 
                 Tyr-His 
               
               
                 27 
                 CCDS 
                 1935 
                 G 
                 C 
                 429 
                 Missense 
                 Ala-Pro 
               
               
                 28 
                 CCDS 
                 1937 
                 C 
                 T 
                 429 
                 Synonymous 
                 Ala-Ala 
               
               
                 29 
                 CCDS 
                 1943 
                 G 
                 C 
                 431 
                 Synonymous 
                 Pro-Pro 
               
               
                 30 
                 CCDS 
                 1961 
                 C 
                 T 
                 437 
                 Synonymous 
                 Ser-Ser 
               
               
                 31 
                 CCDS 
                 1966 
                 G 
                 T 
                 439 
                 Missense 
                 Arg- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Leu 
               
               
                 32 
                 3′UTR 
                 2041 
                 C 
                 T 
               
               
                 33 
                 3′UTR 
                 2072 
                 G 
                 A 
               
               
                 34 
                 3′UTR 
                 2077 
                 A 
                 G 
               
               
                 35 
                 3′UTR 
                 2098 
                 C 
                 T 
               
               
                 36 
                 3′UTR 
                 2229 
                 C 
                 T 
               
               
                 37 
                 3′UTR 
                 2325 
                 A 
                 G 
               
               
                 38 
                 3′UTR 
                 2326 
                 T 
                 A 
               
               
                 39 
                 3′UTR 
                 2562 
                 A 
                 G 
               
               
                 40 
                 3′UTR 
                 2626 
                 G 
                 T 
               
               
                 41 
                 3′UTR 
                 2971 
                 G 
                 T 
               
               
                 42 
                 3′UTR 
                 3037 
                 A 
                 G 
               
               
                 43 
                 3′UTR 
                 3175 
                 C 
                 T 
               
               
                 44 
                 3′UTR 
                 3200 
                 C 
                 G 
               
               
                 45 
                 3′UTR 
                 3201 
                 C 
                 T 
               
               
                 46 
                 3′UTR 
                 3225 
                 A 
                 G 
               
               
                 47 
                 3′UTR 
                 3293 
                 C 
                 T 
               
               
                 48 
                 3′UTR 
                 3301 
                 A 
                 T 
               
               
                 49 
                 3′UTR 
                 3513 
                 C 
                 T 
               
               
                 50 
                 3′UTR 
                 3567 
                 — 
                 C 
               
               
                 51 
                 3′UTR 
                 3581 
                 C 
                 T 
               
               
                 52 
                 3′UTR 
                 3605 
                 A 
                 G 
               
               
                 53 
                 3′UTR 
                 3625 
                 C 
                 T 
               
               
                 54 
                 3′UTR 
                 3643 
                 A 
                 G 
               
               
                 55 
                 3′UTR 
                 3670 
                 A 
                 G 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 SNPs of the NKX2-1 Em isoform 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Contig 
                 Poly- 
                 Codon 
                   
                 Protein 
               
               
                 S. No. 
                 Region 
                 Position 
                 reference 
                 morphism 
                 Position 
                 Function 
                 residue 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 5′UTR 
                 269 
                 C 
                 T 
                   
                   
                   
               
               
                 2 
                 5′UTR 
                 281 
                 A 
                 G 
               
               
                 3 
                 5′UTR 
                 305 
                 — 
                 A 
               
               
                 4 
                 5′UTR 
                 304 
                 — 
                 AA 
               
               
                 5 
                 CCDS 
                 420 
                 G 
                 A 
                 27 
                 Missense 
                 Val-Met 
               
               
                 6 
                 CCDS 
                 425 
                 C 
                 T 
                 28 
                 Synonymous 
                 Gly-Gly 
               
               
                 7 
                 CCDS 
                 439 
                 G 
                 T 
                 33 
                 Missense 
                 Gly-Val 
               
               
                 8 
                 CCDS 
                 441 
                 C 
                 A 
                 34 
                 Missense 
                 Leu-Ile 
               
               
                 9 
                 CCDS 
                 450 
                 C 
                 T 
                 37 
                 Missense 
                 Pro-Ser 
               
               
                 10 
                 CCDS 
                 486 
                 C 
                 T 
                 49 
                 Missense 
                 Pro-Ser 
               
               
                 11 
                 CCDS 
                 781 
                 G 
                 T 
                 147 
                 Missense 
                 Gly-Val 
               
               
                 12 
                 CCDS 
                 785 
                 C 
                 T 
                 148 
                 Synonymous 
                 Asp-Asp 
               
               
                 13 
                 CCDS 
                 825 
                 A 
                 C 
                 162 
                 Synonymous 
                 Arg-Arg 
               
               
                 14 
                 CCDS 
                 950 
                 G 
                 T 
                 203 
                 Synonymous 
                 Thr-Thr 
               
               
                 15 
                 CCDS 
                 1169 
                 G 
                 A 
                 276 
                 Synonymous 
                 Ala-Ala 
               
               
                 16 
                 CCDS 
                 1305 
                 G 
                 A 
                 322 
                 Missense 
                 Gly-Ser 
               
               
                 17 
                 CCDS 
                 1344 
                 G 
                 T 
                 335 
                 Missense 
                 Ala-Ser 
               
               
                 18 
                 CCDS 
                 1448 
                 G 
                 A 
                 369 
                 Synonymous 
                 Arg-Arg 
               
               
                 19 
                 3′UTR 
                 1458 
                 C 
                 T 
               
               
                 20 
                 3′UTR 
                 1467 
                 C 
                 T 
               
               
                 21 
                 3′UTR 
                 1489 
                 G 
                 T 
               
               
                 22 
                 3′UTR 
                 1552 
                 G 
                 T 
               
               
                 23 
                 3′UTR 
                 1633 
                 A 
                 G 
               
               
                 24 
                 3′UTR 
                 1634 
                 A 
                 G 
               
               
                 25 
                 3′UTR 
                 1640 
                 — 
                 T 
               
               
                 26 
                 3′UTR 
                 1641 
                 — 
                 GT 
               
               
                 27 
                 3′UTR 
                 1643 
                 — 
                 &gt;6 bp 
               
               
                 28 
                 3′UTR 
                 1667 
                 A 
                 T 
               
               
                 29 
                 3′UTR 
                 1673 
                 — 
                 T 
               
               
                 30 
                 3′UTR 
                 1678 
                 — 
                 T 
               
               
                 31 
                 3′UTR 
                 1748 
                 — 
                 C 
               
               
                 32 
                 3′UTR 
                 1750 
                 — 
                 C 
               
               
                 33 
                 3′UTR 
                 1831 
                 A 
                 T 
               
               
                 34 
                 3′UTR 
                 1893 
                 G 
                 T 
               
               
                 35 
                 3′UTR 
                 1916 
                 — 
                 A 
               
               
                 36 
                 3′UTR 
                 1917 
                 — 
                 A 
               
               
                 37 
                 3′UTR 
                 1934 
                 C 
                 G/T 
               
               
                 38 
                 3′UTR 
                 2099 
                 C 
                 G 
               
               
                 39 
                 3′UTR 
                 2319 
                 C 
                 G 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 SNPs of the NKX2-1 Ad isoform 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Contig 
                 Poly- 
                 Codon 
                   
                 Protein 
               
               
                 S. No. 
                 Region 
                 Position 
                 reference 
                 morphism 
                 Position 
                 Function 
                 residue 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 5′UTR 
                 12 
                 G 
                 T 
                   
                   
                   
               
               
                 2 
                 CCDS 
                 125 
                 G 
                 A 
                 10 
                 Missense 
                 Arg-Gln 
               
               
                 3 
                 CCDS 
                 265 
                 G 
                 A 
                 57 
                 Missense 
                 Val-Met 
               
               
                 4 
                 CCDS 
                 270 
                 C 
                 T 
                 58 
                 Synonymous 
                 Gly-Gly 
               
               
                 5 
                 CCDS 
                 284 
                 G 
                 T 
                 63 
                 Missense 
                 Gly-Val 
               
               
                 6 
                 CCDS 
                 286 
                 C 
                 A 
                 64 
                 Missense 
                 Leu-Ile 
               
               
                 7 
                 CCDS 
                 295 
                 C 
                 T 
                 67 
                 Missense 
                 Pro-Ser 
               
               
                 8 
                 CCDS 
                 331 
                 C 
                 T 
                 79 
                 Missense 
                 Pro-Ser 
               
               
                 9 
                 CCDS 
                 626 
                 G 
                 T 
                 177 
                 Missense 
                 Gly-Val 
               
               
                 10 
                 CCDS 
                 630 
                 C 
                 T 
                 178 
                 Synonymous 
                 Asp-Asp 
               
               
                 11 
                 CCDS 
                 670 
                 A 
                 C 
                 192 
                 Synonymous 
                 Arg-Arg 
               
               
                 12 
                 CCDS 
                 795 
                 G 
                 T 
                 233 
                 Synonymous 
                 Thr-Thr 
               
               
                 13 
                 CCDS 
                 1014 
                 G 
                 A 
                 306 
                 Synonymous 
                 Ala-Ala 
               
               
                 14 
                 CCDS 
                 1150 
                 G 
                 A 
                 352 
                 Missense 
                 Gly-Ser 
               
               
                 15 
                 CCDS 
                 1189 
                 G 
                 T 
                 365 
                 Missense 
                 Ala-Ser 
               
               
                 16 
                 CCDS 
                 1293 
                 G 
                 A 
                 399 
                 Synonymous 
                 Arg-Arg 
               
               
                 17 
                 3′UTR 
                 1303 
                 C 
                 T 
               
               
                 18 
                 3′UTR 
                 1312 
                 C 
                 T 
               
               
                 19 
                 3′UTR 
                 1334 
                 G 
                 T 
               
               
                 20 
                 3′UTR 
                 1397 
                 G 
                 T 
               
               
                 21 
                 3′UTR 
                 1478 
                 A 
                 G 
               
               
                 22 
                 3′UTR 
                 1479 
                 A 
                 G 
               
               
                 23 
                 3′UTR 
                 1478 
                 — 
                 &gt;6 bp 
               
               
                 24 
                 3′UTR 
                 1485 
                 — 
                 T 
               
               
                 25 
                 3′UTR 
                 1486 
                 — 
                 GT 
               
               
                 26 
                 3′UTR 
                 1488 
                 — 
                 &gt;6 bp 
               
               
                 27 
                 3′UTR 
                 1512 
                 A 
                 T 
               
               
                 28 
                 3′UTR 
                 1518 
                 — 
                 T 
               
               
                 29 
                 3′UTR 
                 1523 
                 — 
                 T 
               
               
                 30 
                 3′UTR 
                 1593 
                 — 
                 C 
               
               
                 31 
                 3′UTR 
                 1595 
                 — 
                 C 
               
               
                 32 
                 3′UTR 
                 1676 
                 A 
                 T 
               
               
                 33 
                 3′UTR 
                 1738 
                 G 
                 T 
               
               
                 34 
                 3′UTR 
                 1761 
                 — 
                 A 
               
               
                 35 
                 3′UTR 
                 1762 
                 — 
                 A 
               
               
                 36 
                 3′UTR 
                 1779 
                 C 
                 G/T 
               
               
                 37 
                 3′UTR 
                 1944 
                 C 
                 G 
               
               
                 38 
                 3′UTR 
                 2164 
                 C 
                 G 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 SNPs of the FOXA2 Em isoform 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Contig 
                 Poly- 
                 Codon 
                   
                 Protein 
               
               
                 S. No. 
                 Region 
                 Position 
                 reference 
                 morphism 
                 Position 
                 Function 
                 residue 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 5′UTR 
                 168 
                 — 
                 &gt;6 bp 
                   
                   
                   
               
               
                 2 
                 CCDS 
                 208 
                 T 
                 C 
                 8 
                 Missense 
                 Leu-Pro 
               
               
                 3 
                 CCDS 
                 289 
                 G 
                 A 
                 35 
                 Missense 
                 Ser-Asn 
               
               
                 4 
                 CCDS 
                 361 
                 G 
                 A 
                 59 
                 Missense 
                 Ser-Asn 
               
               
                 5 
                 CCDS 
                 368 
                 G 
                 A 
                 61 
                 Synonymous 
                 Ser-Ser 
               
               
                 6 
                 CCDS 
                 374 
                 C 
                 T 
                 63 
                 Synonymous 
                 Asn-Asn 
               
               
                 7 
                 CCDS 
                 379 
                 G 
                 A 
                 65 
                 Missense 
                 Ser-Asn 
               
               
                 8 
                 CCDS 
                 383 
                 G 
                 A 
                 66 
                 Synonymous 
                 Ala-Ala 
               
               
                 9 
                 CCDS 
                 404 
                 G 
                 T 
                 73 
                 Synonymous 
                 Ser-Ser 
               
               
                 10 
                 CCDS 
                 459 
                 G 
                 A 
                 92 
                 Missense 
                 Ala-Thr 
               
               
                 11 
                 CCDS 
                 481 
                 C 
                 T 
                 99 
                 Missense 
                 Ser-Leu 
               
               
                 12 
                 CCDS 
                 483 
                 G 
                 C 
                 100 
                 Missense 
                 Ala-Pro 
               
               
                 13 
                 CCDS 
                 494 
                 C 
                 T 
                 103 
                 Synonymous 
                 Ala-Ala 
               
               
                 14 
                 CCDS 
                 529 
                 G 
                 A 
                 115 
                 Missense 
                 Ser-Asn 
               
               
                 15 
                 CCDS 
                 564 
                 A 
                 G 
                 127 
                 Missense 
                 Met-Val 
               
               
                 16 
                 CCDS 
                 577 
                 C 
                 G 
                 131 
                 Missense 
                 Ala-Gly 
               
               
                 17 
                 CCDS 
                 584 
                 C 
                 T 
                 133 
                 Synonymous 
                 Tyr-Tyr 
               
               
                 18 
                 CCDS 
                 590 
                 C 
                 A 
                 135 
                 Missense 
                 Asn-Lys 
               
               
                 19 
                 CCDS 
                 610 
                 T 
                 C 
                 142 
                 Missense 
                 Met-Thr 
               
               
                 20 
                 CCDS 
                 623 
                 G 
                 C 
                 146 
                 Synonymous 
                 Ala-Ala 
               
               
                 21 
                 CCDS 
                 641 
                 C 
                 T 
                 152 
                 Synonymous 
                 Arg-Arg 
               
               
                 22 
                 CCDS 
                 650 
                 G 
                 A 
                 155 
                 Synonymous 
                 Lys-Lys 
               
               
                 23 
                 CCDS 
                 659 
                 G 
                 T 
                 158 
                 Missense 
                 Arg-Ser 
               
               
                 24 
                 CCDS 
                 674 
                 C 
                 T 
                 163 
                 Synonymous 
                 His-His 
               
               
                 25 
                 CCDS 
                 773 
                 G 
                 T 
                 196 
                 Missense 
                 Met-Ile 
               
               
                 26 
                 CCDS 
                 845 
                 C 
                 T 
                 220 
                 Synonymous 
                 Asn-Asn 
               
               
                 27 
                 CCDS 
                 1040 
                 A 
                 G 
                 285 
                 Synonymous 
                 Gly-Gly 
               
               
                 28 
                 CCDS 
                 1075 
                 C 
                 T 
                 297 
                 Missense 
                 Ala-Val 
               
               
                 29 
                 CCDS 
                 1186 
                 C 
                 T 
                 334 
                 Missense 
                 Ala-Val 
               
               
                 30 
                 CCDS 
                 1188 
                 G 
                 C 
                 335 
                 Missense 
                 Ala-Pro 
               
               
                 31 
                 CCDS 
                 1240 
                 C 
                 T 
                 352 
                 Missense 
                 Ala-Val 
               
               
                 32 
                 CCDS 
                 1242 
                 G 
                 A 
                 353 
                 Missense 
                 Ala-Thr 
               
               
                 33 
                 CCDS 
                 1243 
                 C 
                 G 
                 353 
                 Missense 
                 Ala-Gly 
               
               
                 34 
                 CCDS 
                 1304 
                 A 
                 C 
                 373 
                 Missense 
                 Glu-Asp 
               
               
                 35 
                 CCDS 
                 1374 
                 AG 
                 — 
                 397 
                 Frameshift 
                 Ser-Pro 
               
               
                 36 
                 CCDS 
                 1391 
                 A 
                 G 
                 402 
                 Synonymous 
                 Gln-Gln 
               
               
                 37 
                 CCDS 
                 1408 
                 T 
                 C 
                 408 
                 Missense 
                 Leu-Pro 
               
               
                 38 
                 CCDS 
                 1414 
                 C 
                 T 
                 410 
                 Missense 
                 Ala-Val 
               
               
                 39 
                 CCDS 
                 1432 
                 A 
                 C 
                 416 
                 Missense 
                 His-Pro 
               
               
                 40 
                 CCDS 
                 1458 
                 C 
                 A 
                 425 
                 Missense 
                 Pro-Thr 
               
               
                 41 
                 CCDS 
                 1475 
                 G 
                 A 
                 430 
                 Missense 
                 Met-Ile 
               
               
                 42 
                 CCDS 
                 1487 
                 G 
                 C 
                 434 
                 Synonymous 
                 Thr-Thr 
               
               
                 43 
                 CCDS 
                 1522 
                 C 
                 G 
                 446 
                 Missense 
                 Ala-Gly 
               
               
                 44 
                 CCDS 
                 1539 
                 C 
                 G 
                 452 
                 Missense 
                 Gln-Glu 
               
               
                 45 
                 3′UTR 
                 1582 
                 G 
                 T 
               
               
                 46 
                 3′UTR 
                 1583 
                 A 
                 G 
               
               
                 47 
                 3′UTR 
                 1594 
                 C 
                 T 
               
               
                 48 
                 3′UTR 
                 1627 
                 A 
                 G 
               
               
                 49 
                 3′UTR 
                 1631 
                 A 
                 G 
               
               
                 50 
                 3′UTR 
                 1687 
                 A 
                 G 
               
               
                 51 
                 3′UTR 
                 1723 
                 A 
                 C 
               
               
                 52 
                 3′UTR 
                 1737 
                 — 
                 G 
               
               
                 53 
                 3′UTR 
                 1738 
                 — 
                 G 
               
               
                 54 
                 3′UTR 
                 1754 
                 A 
                 G 
               
               
                 55 
                 3′UTR 
                 1812 
                 A 
                 G 
               
               
                 56 
                 3′UTR 
                 1831 
                 A 
                 T 
               
               
                 57 
                 3′UTR 
                 1838 
                 — 
                 T 
               
               
                 58 
                 3′UTR 
                 1940 
                 A 
                 C 
               
               
                 59 
                 3′UTR 
                 1966 
                 — 
                 G/T 
               
               
                 60 
                 3′UTR 
                 1970 
                 — 
                 A 
               
               
                 61 
                 3′UTR 
                 2070 
                 A 
                 T 
               
               
                 62 
                 3′UTR 
                 2083 
                 A 
                 G 
               
               
                 63 
                 3′UTR 
                 2084 
                 — 
                 T 
               
               
                 64 
                 3′UTR 
                 2093 
                 — 
                 T 
               
               
                 65 
                 3′UTR 
                 2105 
                 A 
                 C 
               
               
                 66 
                 3′UTR 
                 2112 
                 C 
                 T 
               
               
                 67 
                 3′UTR 
                 2200 
                 C 
                 T 
               
               
                 68 
                 3′UTR 
                 2388 
                 A 
                 G 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 SNPs of the FOXA2 Em isoform 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Contig 
                 Poly- 
                 Codon 
                   
                 Protein 
               
               
                 S. No. 
                 Region 
                 Position 
                 reference 
                 morphism 
                 Position 
                 Function 
                 residue 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 5′UTR 
                 5 
                 C 
                 T 
                   
                   
                   
               
               
                 2 
                 5′UTR 
                 37 
                 G 
                 T 
               
               
                 3 
                 5′UTR 
                 65 
                 C 
                 T 
               
               
                 4 
                 5′UTR 
                 68 
                 A 
                 C 
               
               
                 5 
                 5′UTR 
                 70 
                 A 
                 G 
               
               
                 6 
                 5′UTR 
                 88 
                 A 
                 G 
               
               
                 7 
                 5′UTR 
                 128 
                 C 
                 T 
               
               
                 8 
                 CCDS 
                 195 
                 T 
                 C 
                 2 
                 Missense 
                 Leu-Pro 
               
               
                 9 
                 CCDS 
                 276 
                 G 
                 A 
                 29 
                 Missense 
                 Ser-Asn 
               
               
                 10 
                 CCDS 
                 348 
                 G 
                 A 
                 53 
                 Missense 
                 Ser-Asn 
               
               
                 11 
                 CCDS 
                 355 
                 G 
                 A 
                 55 
                 Synonymous 
                 Ser-Ser 
               
               
                 12 
                 CCDS 
                 361 
                 C 
                 T 
                 57 
                 Synonymous 
                 Asn-Asn 
               
               
                 13 
                 CCDS 
                 366 
                 G 
                 A 
                 59 
                 Missense 
                 Ser-Asn 
               
               
                 14 
                 CCDS 
                 370 
                 G 
                 A 
                 60 
                 Synonymous 
                 Ala-Ala 
               
               
                 15 
                 CCDS 
                 391 
                 G 
                 T 
                 67 
                 Synonymous 
                 Ser-Ser 
               
               
                 16 
                 CCDS 
                 446 
                 G 
                 A 
                 86 
                 Missense 
                 Ala-Thr 
               
               
                 17 
                 CCDS 
                 468 
                 C 
                 T 
                 93 
                 Missense 
                 Ser-Leu 
               
               
                 18 
                 CCDS 
                 470 
                 G 
                 C 
                 94 
                 Missense 
                 Ala-Pro 
               
               
                 19 
                 CCDS 
                 481 
                 C 
                 T 
                 97 
                 Synonymous 
                 Ala-Ala 
               
               
                 20 
                 CCDS 
                 516 
                 G 
                 A 
                 109 
                 Missense 
                 Ser-Asn 
               
               
                 21 
                 CCDS 
                 551 
                 A 
                 G 
                 121 
                 Missense 
                 Met-Val 
               
               
                 22 
                 CCDS 
                 564 
                 C 
                 G 
                 125 
                 Missense 
                 Ala-Gly 
               
               
                 23 
                 CCDS 
                 571 
                 C 
                 T 
                 127 
                 Synonymous 
                 Tyr-Tyr 
               
               
                 24 
                 CCDS 
                 577 
                 C 
                 A 
                 129 
                 Missense 
                 Asn-Lys 
               
               
                 25 
                 CCDS 
                 597 
                 T 
                 C 
                 136 
                 Missense 
                 Met-Thr 
               
               
                 26 
                 CCDS 
                 610 
                 G 
                 C 
                 140 
                 Synonymous 
                 Ala-Ala 
               
               
                 27 
                 CCDS 
                 628 
                 C 
                 T 
                 146 
                 Synonymous 
                 Arg-Arg 
               
               
                 28 
                 CCDS 
                 637 
                 G 
                 A 
                 149 
                 Synonymous 
                 Lys-Lys 
               
               
                 29 
                 CCDS 
                 646 
                 G 
                 T 
                 152 
                 Missense 
                 Arg-Ser 
               
               
                 30 
                 CCDS 
                 661 
                 C 
                 T 
                 157 
                 Synonymous 
                 His-His 
               
               
                 31 
                 CCDS 
                 760 
                 G 
                 T 
                 190 
                 Missense 
                 Met-Ile 
               
               
                 32 
                 CCDS 
                 832 
                 C 
                 T 
                 214 
                 Synonymous 
                 Asn-Asn 
               
               
                 33 
                 CCDS 
                 1027 
                 A 
                 G 
                 279 
                 Synonymous 
                 Gly-Gly 
               
               
                 34 
                 CCDS 
                 1062 
                 C 
                 T 
                 291 
                 Missense 
                 Ala-Val 
               
               
                 35 
                 CCDS 
                 1173 
                 C 
                 T 
                 328 
                 Missense 
                 Ala-Val 
               
               
                 36 
                 CCDS 
                 1175 
                 G 
                 C 
                 329 
                 Missense 
                 Ala-Pro 
               
               
                 37 
                 CCDS 
                 1227 
                 C 
                 T 
                 346 
                 Missense 
                 Ala-Val 
               
               
                 38 
                 CCDS 
                 1229 
                 G 
                 A 
                 347 
                 Missense 
                 Ala-Thr 
               
               
                 39 
                 CCDS 
                 1230 
                 C 
                 G 
                 347 
                 Missense 
                 Ala-Gly 
               
               
                 40 
                 CCDS 
                 1291 
                 A 
                 C 
                 367 
                 Missense 
                 Gly-Glu 
               
               
                 41 
                 CCDS 
                 1361 
                 AG 
                 — 
                 391 
                 Frameshift 
                 Ser-Pro 
               
               
                 42 
                 CCDS 
                 1378 
                 A 
                 G 
                 396 
                 Synonymous 
                 Gln-Gln 
               
               
                 43 
                 CCDS 
                 1395 
                 T 
                 C 
                 402 
                 Missense 
                 Leu-Pro 
               
               
                 44 
                 CCDS 
                 1401 
                 C 
                 T 
                 404 
                 Missense 
                 Ala-Val 
               
               
                 45 
                 CCDS 
                 1419 
                 A 
                 C 
                 410 
                 Missense 
                 His-Pro 
               
               
                 46 
                 CCDS 
                 1445 
                 C 
                 A 
                 419 
                 Missense 
                 Pro-Thr 
               
               
                 47 
                 CCDS 
                 1462 
                 G 
                 A 
                 424 
                 Missense 
                 Met-Ile 
               
               
                 48 
                 CCDS 
                 1474 
                 G 
                 C 
                 428 
                 Synonymous 
                 Thr-Thr 
               
               
                 49 
                 CCDS 
                 1509 
                 C 
                 G 
                 440 
                 Missense 
                 Ala-Gly 
               
               
                 50 
                 CCDS 
                 1526 
                 C 
                 G 
                 446 
                 Missense 
                 Gln-Glu 
               
               
                 51 
                 3′UTR 
                 1569 
                 G 
                 T 
               
               
                 52 
                 3′UTR 
                 1570 
                 A 
                 G 
               
               
                 53 
                 3′UTR 
                 1581 
                 C 
                 T 
               
               
                 54 
                 3′UTR 
                 1614 
                 A 
                 G 
               
               
                 55 
                 3′UTR 
                 1618 
                 A 
                 G 
               
               
                 56 
                 3′UTR 
                 1674 
                 A 
                 G 
               
               
                 57 
                 3′UTR 
                 1710 
                 A 
                 C 
               
               
                 58 
                 3′UTR 
                 1724 
                 — 
                 G 
               
               
                 59 
                 3′UTR 
                 1725 
                 — 
                 G 
               
               
                 60 
                 3′UTR 
                 1741 
                 A 
                 G 
               
               
                 61 
                 3′UTR 
                 1799 
                 A 
                 G 
               
               
                 62 
                 3′UTR 
                 1818 
                 A 
                 T 
               
               
                 63 
                 3′UTR 
                 1825 
                 — 
                 T 
               
               
                 64 
                 3′UTR 
                 1927 
                 A 
                 C 
               
               
                 65 
                 3′UTR 
                 1953 
                 — 
                 G/T 
               
               
                 66 
                 3′UTR 
                 1957 
                 — 
                 A 
               
               
                 67 
                 3′UTR 
                 2057 
                 A 
                 T 
               
               
                 68 
                 3′UTR 
                 2070 
                 A 
                 G 
               
               
                 69 
                 3′UTR 
                 2071 
                 — 
                 T 
               
               
                 70 
                 3′UTR 
                 2080 
                 — 
                 T 
               
               
                 71 
                 3′UTR 
                 2092 
                 A 
                 C 
               
               
                 72 
                 3′UTR 
                 2099 
                 C 
                 T 
               
               
                 73 
                 3′UTR 
                 2187 
                 C 
                 T 
               
               
                 74 
                 3′UTR 
                 2375 
                 A 
                 G 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 SNPs of the ID2 Em isoform 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Contig 
                 Poly- 
                 Codon 
                   
                 Protein 
               
               
                 S. No. 
                 Region 
                 Position 
                 reference 
                 morphism 
                 Position 
                 Function 
                 residue 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 5′UTR 
                 6 
                 C 
                 T 
                   
                   
                   
               
               
                 2 
                 5′UTR 
                 43 
                 A 
                 G 
               
               
                 3 
                 5′UTR 
                 53 
                 A 
                 G 
               
               
                 4 
                 5′UTR 
                 55 
                 C 
                 G 
               
               
                 5 
                 5′UTR 
                 154 
                 C 
                 G/T 
               
               
                 6 
                 CCDS 
                 195 
                 C 
                 T 
                 4 
                 Missense 
                 Phe-Phe 
               
               
                 7 
                 CCDS 
                 209 
                 C 
                 T 
                 9 
                 Missense 
                 Ser-Phe 
               
               
                 8 
                 CCDS 
                 224 
                 G 
                 A 
                 14 
                 Missense 
                 Ser-Asn 
               
               
                 9 
                 CCDS 
                 237 
                 C 
                 T 
                 18 
                 Synonymous 
                 His-His 
               
               
                 10 
                 CCDS 
                 263 
                 C 
                 A 
                 27 
                 Missense 
                 Thr-Asn 
               
               
                 11 
                 CCDS 
                 286 
                 C 
                 T 
                 35 
                 Synonymous 
                 Leu-Leu 
               
               
                 12 
                 CCDS 
                 360 
                 G 
                 A 
                 59 
                 Synonymous 
                 Val-Val 
               
               
                 13 
                 CCDS 
                 399 
                 C 
                 T 
                 72 
                 Synonymous 
                 Ile-Ile 
               
               
                 14 
                 CCDS 
                 405 
                 C 
                 T 
                 74 
                 Synonymous 
                 Asp-Asp 
               
               
                 15 
                 CCDS 
                 485 
                 C 
                 T 
                 101 
                 Missense 
                 Thr-Met 
               
               
                 16 
                 CCDS 
                 501 
                 C 
                 G/T 
                 106 
                 Synonymous 
                 Leu-Leu 
               
               
                 17 
                 CCDS 
                 544 
                 C 
                 T 
                 121 
                 Missense 
                 Pro-Ser 
               
               
                 18 
                 CCDS 
                 547 
                 T 
                 A 
                 122 
                 Missense 
                 Ser-Thr 
               
               
                 19 
                 3′UTR 
                 605 
                 A 
                 G 
               
               
                 20 
                 3′UTR 
                 662 
                 C 
                 G 
               
               
                 21 
                 3′UTR 
                 665 
                 G 
                 T 
               
               
                 22 
                 3′UTR 
                 716 
                 A 
                 T 
               
               
                 23 
                 3′UTR 
                 757 
                 C 
                 T 
               
               
                 24 
                 3′UTR 
                 871 
                 A 
                 G 
               
               
                 25 
                 3′UTR 
                 876 
                 A 
                 G 
               
               
                 26 
                 3′UTR 
                 975 
                 — 
                 &gt;6 bp 
               
               
                 27 
                 3′UTR 
                 1085 
                 — 
                 &gt;6 bp 
               
               
                 28 
                 3′UTR 
                 1115 
                 A 
                 G 
               
               
                 29 
                 3′UTR 
                 1119 
                 — 
                 AT 
               
               
                 30 
                 3′UTR 
                 1149 
                 C 
                 T 
               
               
                 31 
                 3′UTR 
                 1151 
                 A 
                 T 
               
               
                 32 
                 3′UTR 
                 1251 
                 — 
                 CA 
               
               
                 33 
                 3′UTR 
                 1333 
                 A 
                 G 
               
               
                 34 
                 3′UTR 
                 1350 
                 C 
                 G 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 SNPs of the ID2 Ad isoform 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Contig 
                 Poly- 
                 Codon 
                   
                 Protein 
               
               
                 S. No. 
                 Region 
                 Position 
                 reference 
                 morphism 
                 Position 
                 Function 
                 residue 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 5 
                 5′UTR 
                 93 
                 C 
                 G/T 
                   
                   
                   
               
               
                 6 
                 CCDS 
                 134 
                 C 
                 T 
                 4 
                 Missense 
                 Phe-Phe 
               
               
                 7 
                 CCDS 
                 148 
                 C 
                 T 
                 9 
                 Missense 
                 Ser-Phe 
               
               
                 8 
                 CCDS 
                 163 
                 G 
                 A 
                 14 
                 Missense 
                 Ser-Asn 
               
               
                 9 
                 CCDS 
                 176 
                 C 
                 T 
                 18 
                 Synonymous 
                 His-His 
               
               
                 10 
                 CCDS 
                 202 
                 C 
                 A 
                 27 
                 Missense 
                 Thr-Asn 
               
               
                 11 
                 CCDS 
                 225 
                 C 
                 T 
                 35 
                 Synonymous 
                 Leu-Leu 
               
               
                 12 
                 CCDS 
                 299 
                 G 
                 A 
                 59 
                 Synonymous 
                 Val-Val 
               
               
                 13 
                 CCDS 
                 338 
                 C 
                 T 
                 72 
                 Synonymous 
                 Ile-Ile 
               
               
                 14 
                 CCDS 
                 344 
                 C 
                 T 
                 74 
                 Synonymous 
                 Asp-Asp 
               
               
                 15 
                 CCDS 
                 424 
                 C 
                 T 
                 101 
                 Missense 
                 Thr-Met 
               
               
                 16 
                 CCDS 
                 440 
                 C 
                 G/T 
                 106 
                 Synonymous 
                 Leu-Leu 
               
               
                 17 
                 CCDS 
                 483 
                 C 
                 T 
                 121 
                 Missense 
                 Pro-Ser 
               
               
                 18 
                 CCDS 
                 486 
                 T 
                 A 
                 122 
                 Missense 
                 Ser-Thr 
               
               
                 19 
                 3′UTR 
                 544 
                 A 
                 G 
               
               
                 20 
                 3′UTR 
                 601 
                 C 
                 G 
               
               
                 21 
                 3′UTR 
                 604 
                 G 
                 T 
               
               
                 22 
                 3′UTR 
                 655 
                 A 
                 T 
               
               
                 23 
                 3′UTR 
                 696 
                 C 
                 T 
               
               
                 24 
                 3′UTR 
                 810 
                 A 
                 G 
               
               
                 25 
                 3′UTR 
                 815 
                 A 
                 G 
               
               
                 26 
                 3′UTR 
                 914 
                 — 
                 &gt;6 bp 
               
               
                 27 
                 3′UTR 
                 1024 
                 — 
                 &gt;6 bp 
               
               
                 28 
                 3′UTR 
                 1054 
                 A 
                 G 
               
               
                 29 
                 3′UTR 
                 1058 
                 — 
                 AT 
               
               
                 30 
                 3′UTR 
                 1088 
                 C 
                 T 
               
               
                 31 
                 3′UTR 
                 1090 
                 A 
                 T 
               
               
                 32 
                 3′UTR 
                 1190 
                 — 
                 CA 
               
               
                 33 
                 3′UTR 
                 1272 
                 A 
                 G 
               
               
                 34 
                 3′UTR 
                 1289 
                 C 
                 G 
               
               
                   
               
            
           
         
       
     
     A control sample according to the present invention is a sample from a healthy control subject. Such a sample can be obtained for example from a subject known to be a healthy subject. It is also possible to generate a control sample according to the present invention as a mixture of samples obtained from several healthy subjects, for example from a group of 10, 20, 30, 50, 100 or even up to 1000 healthy subjects. A control sample according to the present invention can be generated for example from age-matched and or gender-matched healthy control subjects. A control sample according to the present invention can also be generated for example in vitro to mimic a control sample obtained from one or several healthy subjects. 
     Control samples can, inter alia, be healthy tissues (i.e. biopsies) from diseased individuals/subjects. “Healthy tissue from diseased individuals/subjects” can refer to tissue that is pathologically classified as “normal” or “healthy” and/or that is distant or adjacent to a (suspected) tumor. For example, the “healthy tissue from diseased individuals/subjects” can be obtained e.g. by biopsy from adjacent healthy tissue of (suspected) cancer patients. 
     For example, the “healthy tissue” can be obtained from the subject(s) to be assessed in accordance with the present invention for suffering from cancer or being prone to suffering from cancer. In another example, the “healthy tissue” can be obtained from other diseased patients (e.g. patients that have already been diagnosed to suffer from cancer by conventional means and methods or patients that have a history of cancer); in that case, “healthy tissue” is not obtained from subject(s) to be assessed in accordance with the present invention for suffering from cancer or being prone to suffering from cancer. 
     Thus, also “healthy tissue from (a) diseased individual(s)” can be used as a control sample in accordance with the present invention. 
     Control samples can, inter alia, be EBCs from healthy individuals. The term “healthy individuals” as used herein can refer to individuals with no history of cancer, i.e. individuals that did not suffer from cancer or that do currently (i.e. at the time the control sample is obtained) not suffer from cancer. Thus, “healthy tissue/sample” (i.e. tissue (e.g. a biopsy) or another sample (e.g. EBC) obtained from a healthy individual” can be used as a control sample in accordance with the present invention. 
     A subject according to the present invention is preferably a human subject. The subject according to the present invention can be a human subject which has an increased likelihood of suffering from cancer. Such an increased likelihood of suffering from cancer can for example result from certain exposures to cancerogens, for example through the habit of smoking. 
     The “amount of said specific transcription isoform” according to the present invention can be a relative amount or an absolute amount. The relative amount can be determined relative to a control sample. To determine the “amount of said specific transcription isoform”, the absolute or relative amount of a reference gene or reference protein can be determined in the sample from the subject and in the control sample. Non-limiting examples of reference genes/proteins are TUBA1A1 (Uniprot-ID: Q71U36, Gene-ID: 7846), HPRT1 (Uniprot-ID: P00492, Gene-ID: 3251), ACTB (Uniprot-ID: P60709, Gene-ID: 60), HMBS (Uniprot-ID: P08397, Gene-ID: 3145), RPL13A (Uniprot-ID: Q9BSQ6, Gene-ID: 23521) and UBE2A (Uniprot-ID: P49459, Gene-ID: 7319). 
     The herein provided method can be used to stratify/assess subjects according to the tumor/cancer grade. It can be helpful to assess whether a patient is suffering from Grade I, Grade II or Grade III tumor/cancer in order to decide which therapeutic intervention is warranted. 
     The definition of Grade I, Grade II and Grade III tumor is based on TNM classification recommended by the American Joint Committee on Cancer (Goldstraw P. et al. (2007) J Thorac Oncol. 2(8):706-14; Beadsmoore C J and Screaton N J (2003) Eur J Radiol. 45(1):8-17; Mountain C F (1997) Chest. 111(6):1710-7.), which is incorporated herein by reference. 
     Herein, lung cancer is preferred, in particular non-small cell lung cancer or small cell lung cancer. Particularly preferred is non-small cell lung cancer. 
     It is known by the person skilled in the art that genes can contain single nucleotide polymorphisms. The specific transcription factor Em isoform sequences of the present invention encompass all naturally occurring sequences of the respective isoform independent of the number and nature of the SNPs in said sequence. To relate to currently known SNPs, the specific transcription factor Ad isoform sequences of the present invention are defined such that they contain up to 55 (in the case of GATA6) or up to 38 (in the case of NKX2-1), up to 74 (in the case of FOXA2) or up to 30 (in the case of ID2) additions, deletions or substitutions of the nucleic acid sequences defined by SEQ ID NOs: 5, 6, 7 and 8, respectively, to also cover the respective Ad transcripts of carriers of different nucleotides at the respective SNPs. The SNPs of tables 2, 4, 6 and 8 may occur in the Ad isoforms of the present invention in any combination. For example, a (genetic) variant of the GATA6 Ad isoform to be used herein may comprise a nucleic acid sequence of SEQ ID NO:5, whereby the “C” residue at position 694 of SEQ ID NO:5 is substituted by “T”. Further variants of the isoforms to be used herein are apparent from Tables 1 to 8 to the person skilled in the art. 
     The GATA6 Ad isoform according to the invention is the GATA6 Ad isoform comprising the nucleic acid sequence of SEQ ID NO: 5 or the GATA6 Ad isoform comprising a nucleic acid sequence with up to 55; preferably up to 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20; even more preferably up to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7. 6, 5, 4, 3 or 2; or even furthermore preferably only 1 addition(s), deletion(s) or substitution(s) of SEQ ID NO: 5. The GATA6 Ad isoform can also be defined as the GATA6 Ad isoform comprising the nucleic acid sequence of SEQ ID NO: 5 or the GATA6 Ad isoform comprising the nucleic acid sequence of SEQ ID NO: 5 with additions, deletions or substitutions at any of positions 138; 228; 255; 262; 274; 365; 397; 415; 694; 1063; 1191; 1239; 1524; 1532; 1562; 1586; 1587; 1738; 1779; 1784; 1814; 1817; 1846; 1875; 1884; 1917; 1935; 1937; 1943; 1961; 1966; 2041; 2072; 2077; 2098; 2229; 2325; 2326; 2562; 2626; 2971; 3037; 3175; 3200; 3201; 3225; 3293; 3301; 3513; 3567; 3581; 3605; 3625; 3643 or 3670. The GATA6 Ad isoform according to the invention can also be defined as the GATA6 Ad isoform comprising the nucleic acid sequence of SEQ ID No: 5 or the GATA6 Ad isoform comprising a nucleic acid sequence with at least 85% homology to SEQ ID No: 5, preferably up to 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% homology to SEQ ID No: 5; even more preferably up to 99% homology to SEQ ID No: 5. 
     The NKX2-1 Ad isoform according to the invention is the NKX2-1 Ad isoform comprising the nucleic acid sequence of SEQ ID NO: 6 or the NKX2-1 Ad isoform comprising a nucleic acid sequence with up to 38; preferably up to 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10; even more preferably up to 9, 8, 7, 6, 5, 4, 3, or 2; or even furthermore preferably only 1 addition(s), deletion(s) or substitution(s) of SEQ ID NO: 6. The NKX2-1 Ad isoform can also be defined as the NKX2-1 Ad isoform comprising the nucleic acid sequence of SEQ ID NO: 6 or the Nkx2-1 isoform Ad comprising the nucleic acid sequence of SEQ ID NO: 6 with additions, deletions or substitutions at any of positions 12; 125; 265; 270; 284; 286; 295; 331; 626; 630; 670; 795; 1014; 1150; 1189; 1293; 1303; 1312; 1334; 1397; 1478; 1479; 1478; 1485; 1486; 1488; 1512; 1518; 1523; 1593; 1595; 1676; 1738; 1761; 1762; 1779; 1944 or 2164. The NKX2-1 Ad isoform according to the invention can also be defined as the NKX2-1 Ad isoform comprising the nucleic acid sequence of SEQ ID No: 6 or the NKX2-1 Ad isoform comprising a nucleic acid sequence with at least 90% homology to SEQ ID No: 6, preferably up to 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% homology to SEQ ID No: 6; even more preferably up to 99% homology to SEQ ID No: 6. 
     The FOXA2 Ad isoform according to the invention is the FOXA2 Ad isoform comprising the nucleic acid sequence of SEQ ID NO: 7 or the FOXA2 Ad isoform comprising a nucleic acid sequence with up to 74; preferably up to 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53 52, 51, 50, 49, 48 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20; even more preferably up to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7. 6, 5, 4, 3 or 2; or even furthermore preferably only 1 addition(s), deletion(s) or substitution(s) of SEQ ID NO: 7. The FOXA2 Ad isoform can also be defined as the FOXA2 Ad isoform comprising the nucleic acid sequence of SEQ ID NO: 7 or the FOXA2 Ad isoform comprising the nucleic acid sequence of SEQ ID NO: 7 with additions, deletions or substitutions at any of positions 5; 37; 65; 68; 70; 88; 128; 195; 276; 348; 355; 361; 366; 370; 391; 446; 468; 470; 481; 516; 551; 564; 571; 577; 597; 610; 628; 637; 646; 661; 760; 832; 1027; 1062; 1173; 1175; 1227; 1229; 1230; 1291; 1361; 1378; 1395; 1401; 1419; 1445; 1462; 1474; 1509; 1526; 1569; 1570; 1581; 1614; 1618; 1674; 1710; 1724; 1725; 1741; 1799; 1818; 1825; 1927; 1953; 1957; 2057; 2070; 2071; 2080; 2092; 2099; 2187 or 2375. The FOXA2 Ad isoform according to the invention can also be defined as the FOXA2 Ad isoform comprising the nucleic acid sequence of SEQ ID No: 7 or the FOXA2 Ad isoform comprising a nucleic acid sequence with at least 93% homology to SEQ ID No: 7, preferably up to 92%, 93%, 94%, 95%, 96%, 97% or 98% homology to SEQ ID No: 7; even more preferably up to 99% homology to SEQ ID No: 7. 
     The ID2 Ad isoform according to the invention is the ID2 Ad isoform consisting the nucleic acid sequence of SEQ ID NO: 8 or the ID2 Ad isoform consisting of a nucleic acid sequence with up to 30; preferably up to 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10; even more preferably up to 9, 8, 7, 6, 5, 4, 3, or 2; or even furthermore preferably only 1 addition(s), deletion(s) or substitution(s) of SEQ ID NO: 8. The ID2 Ad isoform can also be defined as the ID2 Ad isoform consisting the nucleic acid sequence of SEQ ID NO: 8 or the ID2 Ad isoform consisting the nucleic acid sequence of SEQ ID NO: 8 with additions, deletions or substitutions at any of positions 93; 134; 148; 163; 176; 202; 225; 299; 338; 344; 424; 440; 483; 486; 544; 601; 604; 655; 696; 810; 815; 914; 1024; 1054; 1058; 1088; 1090; 1190; 1272 or 1289. The ID2 Ad isoform according to the invention can also be defined as the ID2 Ad isoform comprising the nucleic acid sequence of SEQ ID No: 8 or the ID2 Ad isoform comprising a nucleic acid sequence with at least 51% homology to SEQ ID No: 8, preferably up to 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% homology to SEQ ID No: 8; even more preferably up to 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID No: 8. 
     The term “cancer patient” as used herein refers to a patient that is suspected to suffer from cancer or being prone to suffer from cancer. The cancer to be treated in accordance with the present invention can be a solid cancer or a liquid cancer. Non-limiting examples of cancers which can be treated according to the present invention are lung cancer, ovarian cancer, colorectal cancer, kidney cancer, bone cancer, bone marrow cancer, bladder cancer, prostate cancer, esophagus cancer, salivary gland cancer, pancreas cancer, liver cancer, head and neck cancer, CNS (especially brain) cancer, cervix cancer, cartilage cancer, colon cancer, genitourinary cancer, gastrointestinal tract cancer, pancreas cancer, synovium cancer, testis cancer, thymus cancer, thyroid cancer and uterine cancer. 
     Preferably, the cancer patient according to the present invention is a patient suffering from lung cancer, such as non-small cell lung cancer (NSCLC) or small cell lung cancer (SLC). Particularly preferably, the patient suffers non-small cell lung cancer (NSCLC). Even more preferably, the cancer patient is a patient suffering from adenocarcinoma. The patient may also suffer from a squamous cell carcinoma or a large cell carcinoma. The adenocarcinoma can be a bronchoalveolar carcinoma. 
     The amount of the specific transcription factor isoform according to the invention can be measured for example by a polymerase chain reaction-based method, an in situ hybridization-based method, or a microarray. If the amount of the specific transcription factor isoform according to the invention is measured via a polymerase chain reaction-based method, it is preferably measured via a quantitative reverse transcriptase polymerase chain reaction. 
     The method of assessing whether a subject suffers from cancer or is prone to suffering from cancer according to the invention may comprise the contacting of a sample with primers, wherein said primers can be used for amplifying the respective specific transcription factor isoforms. 
     Primers for the polymerase chain reaction-based measurement of the amount of the specific transcription factor isoforms according to the invention may encompass the use of primers being selected from the Table 9. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Examples of primer pairs for the amplification, detection and/or 
               
               
                 quantification of the amount of specific transcription factor isoforms 
               
            
           
           
               
               
               
            
               
                   
                 Primers 
                 Primers for Human (5′→3′) (For 
               
               
                 Gene 
                 for Human (5′→3′) 
                 RNA from tissue sections) 
               
               
                   
               
               
                 Gata6-Em Fwd 
                 SEQ ID NO 9: 
                 SEQ ID NO 10: 
               
               
                   
                 CTCGGCTTCTCTCCGCGCCTG 
                 TTGACTGACGGCGGCTGGTG 
               
               
                   
               
               
                 Gata6-Em Rev 
                 SEQ ID NO 11: 
                 SEQ ID NO 12: 
               
               
                   
                 AGCTGAGGCGTCCCGCAGTTG 
                 CTCCCGCGCTGGAAAGGCTC 
               
               
                   
               
               
                 Gata6-Ad Fwd 
                 SEQ ID NO 13: 
                 SEQ ID NO 14: 
               
               
                   
                 GCGGTTTCGTTTTCGGGGAC 
                 AGGACCCAGACTGCTGCCCC 
               
               
                   
               
               
                 Gata6-Ad Rev 
                 SEQ ID NO 15: 
                 SEQ ID NO 16: 
               
               
                   
                 AAGGGATGCGAAGCGTAGGA 
                 CTGACCAGCCCGAACGCGAG 
               
               
                   
               
               
                 Nkx2-1-Em Fwd 
                 SEQ ID NO 17: 
                 SEQ ID NO 18: 
               
               
                   
                 AAACCTGGCGCCGGGCTAAA 
                 CAGCGAGGCTTCGCCTTCCC 
               
               
                   
               
               
                 Nkx2-1-Em Rev 
                 SEQ ID NO 19: 
                 SEQ ID NO 20: 
               
               
                   
                 GGAGAGGGGGAAGGCGAAGCC 
                 TCGACATGATTCGGCGGCGG 
               
               
                   
               
               
                 Nkx2-1-Ad Fwd 
                 SEQ ID NO 21: 
                 SEQ ID NO 22: 
               
               
                   
                 AGCGAAGCCCGATGTGGTCC 
                 TCCGGAGGCAGTGGGAAGGC 
               
               
                   
               
               
                 Nk2-1-Ad Rev 
                 SEQ ID NO 23: 
                 SEQ ID NO 24: 
               
               
                   
                 CCGCCCTCCATGCCCACTTTC 
                 GACATGATTCGGCGGCGGCT 
               
               
                   
               
               
                 Foxa2-Var1 Fwd 
                 SEQ ID NO 25: 
                 SEQ ID NO 26: 
               
               
                   
                 TGCCATGCACTCGGCTTCCAG 
                 CAGGGAGAGGGAGGGCGAGA 
               
               
                   
               
               
                 Foxa2-Var1 Rev 
                 SEQ ID NO 27: 
                 SEQ ID NO 28: 
               
               
                   
                 TCATGTTGCCCGAGCCGCTG 
                 CCCCCACCCCCACCCTCTTT 
               
               
                   
               
               
                 Foxa2-Var2 Fwd 
                 SEQ ID NO 29: 
                 SEQ ID NO 30: 
               
               
                   
                 CTGCTAGAGGGGCTGCTTGCG 
                 CGCTTCTCCCGAGGCCGTTC 
               
               
                   
               
               
                 Foxa2-Var2 Rev 
                 SEQ ID NO 31: 
                 SEQ ID NO 32: 
               
               
                   
                 ACGGCTCGTGCCCTTCCATC 
                 TAACTCGCCCGCTGCTGCTC 
               
               
                   
               
               
                 Id2-Var1 Fwd 
                 SEQ ID NO 33: 
                 SEQ ID NO 34: 
               
               
                   
                 AACCCCTGTGGACGACCCGA 
                 TGCGGATAAAAGCCGCCCCG 
               
               
                   
               
               
                 Id2-Var1 Rev 
                 SEQ ID NO 35 
                 SEQ ID NO 36: 
               
               
                   
                 GCCCGGGTCTCTGGTGATGC 
                 AGCTAGCTGCGCTTGGCACC 
               
               
                   
               
               
                 Id2-Var2 Fwd 
                 SEQ ID NO 37: 
                 SEQ ID NO 38: 
               
               
                   
                 CTGCGGTGCTGAACTCGCCC 
                 CCCCCTGCGGTGCTGAACTC 
               
               
                   
               
               
                 Id2-Var2 Rev 
                 SEQ ID NO 39: 
                 SEQ ID NO 40: 
               
               
                   
                 GACGAGCGGGCGCTTCCATT 
                 TAACTCGCCCGCTGCTGCTC 
               
               
                   
               
            
           
         
       
     
     The diagnostic methods can be used, for example, in combination with (i.e. subsequently prior to or simultaneously with) other diagnostic techniques, like CT (short for computer tomography) and CXR (short for chest radiograph, colloquially called chest X-ray (CXR)). 
     The herein provided methods for the diagnosis of a patient group and the therapy of this selected patient group is particularly useful for high risk subjects/patients or patient groups, such as those that have a hereditary history and/or are exposed to tobacco smoke, environmental smoke, cooking fumes, indoor smoky coal emissions, asbestos, some metals (e.g. nickel, arsenic and cadmium), radon (particularly amongst miners) and ionizing radiation. These subjects/patients may particularly profit from an early diagnosis and, hence, treatment of the cancer in accordance with the present invention. 
     A method of treating a patient according to the present invention may comprise
     a) obtaining a sample from a patient;   b) selecting a cancer patient according to any of the above mentioned statistical methods of assessing whether a subject suffers from cancer or is prone to suffering from cancer;   c) administering to said cancer patient an effective amount of an anti-cancer agent.   

     The present invention also provides a method of treating a patient, said method comprising
     a) selecting a cancer patient according to any of the above mentioned statistical methods of assessing whether a subject suffers from cancer or is prone to suffering from cancer   b) administering to said cancer patient an effective amount of an anti-cancer agent, wherein the cancer agent is for example selected from the group of agents comprising Oxalaplatin, Gemcitabine (Gemzar), Paclitaxel (Taxol), Vincristine (Oncovin) and a composition for use in medicine comprising an inhibitor of
       i) the GATA6 Em isoform comprising the nucleic acid sequence of SEQ ID No: 1 or the GATA6 Em isoform comprising the nucleic acid sequence with up to 55 additions, deletions or substitutions of SEQ ID NO: 1;   ii) the NKX2-1 Em isoform comprising the nucleic acid sequence of SEQ ID No: 2 or the NKX2-1 Em isoform comprising the nucleic acid sequence with up to 39 additions, deletions or substitutions of SEQ ID NO: 2.   iii) the FOXA2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 3 or the FOXA2 Em isoform comprising nucleic acid sequence with up to 68 additions, deletions or substitutions of SEQ ID NO: 3; and/or   iv) the ID2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 4 or the ID2 Em isoform comprising nucleic acid sequence with up to 34 additions, deletions or substitutions of SEQ ID NO: 4.   
       

     The present invention relates to a pharmaceutical composition comprising an agent for the treatment or the prevention of cancer, wherein for the patient suffering from cancer has been determined by a statistical method of the present invention and wherein the method of treatment comprises the step of determining whether or not the patient suffers from cancer. Preferably, the pharmaceutical composition according to the present invention comprises an agent for the treatment or the prevention of lung cancer, wherein for the patient lung cancer has been determined by a method of the present invention and wherein the method of treatment comprises the step of determining whether or not the patient suffers from lung cancer 
     For example, the pharmaceutical composition to be used herein in the treatment of patients selected according to the statistical methods provide herein can an inhibitor of
     i) the GATA6 Em isoform comprising the nucleic acid sequence of SEQ ID No: 1 or the GATA6 Em isoform comprising the nucleic acid sequence with up to 55 additions, deletions or substitutions of SEQ ID NO: 1;   ii) the NKX2-1 Em isoform comprising the nucleic acid sequence of SEQ ID No: 2 or the NKX2-1 Em isoform comprising the nucleic acid sequence with up to 39 additions, deletions or substitutions of SEQ ID NO: 2;   iii) the FOXA2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 3 or the FOXA2 Em isoform comprising nucleic acid sequence with up to 68 additions, deletions or substitutions of SEQ ID NO: 3; and/or   iv) the ID2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 4 or the ID2 Em isoform comprising nucleic acid sequence with up to 34 additions, deletions or substitutions of SEQ ID NO: 4.   

     It is surprisingly found that the Em isoforms of the transcription factors of the present invention have an oncogenic potential (see Examples 4, 6 and 7). Further, it is shown that their reduction leads to the prevention of the development of tumors and allows treating cancer (see example 7). Thus, the present invention relates to inhibitors of the Em isoforms of the transcription factors GATA6, NKX2-1, FOXA2 and ID2. In particular, the present invention relates to agents that allow reducing the amount of the Em isoform of the transcription factors GATA6, NKX2-1, FOXA2 and ID2. The present invention also relates to activators of the Ad isoform of the transcription factors GATA6, NKX2-1, FOXA2 and ID2. Examples of such activators are agents, which activate the promoter of the Ad isoform of the respective transcription factors. 
     The inhibitors of
     i) the GATA6 Em isoform comprising the nucleic acid sequence of SEQ ID No: 1 or the GATA6 Em isoform comprising the nucleic acid sequence with up to 55 additions, deletions or substitutions of SEQ ID NO: 1;   ii) the NKX2-1 Em isoform comprising the nucleic acid sequence of SEQ ID No: 2 or the NKX2-1 Em isoform comprising the nucleic acid sequence with up to 39 additions, deletions or substitutions of SEQ ID NO: 2,   iii) the FOXA2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 3 or the FOXA2 Em isoform comprising nucleic acid sequence with up to 68 additions, deletions or substitutions of SEQ ID NO: 3; or   iv) the ID2 Em isoform comprising the nucleic acid sequence of SEQ ID No: 4 or the ID2 Em isoform comprising nucleic acid sequence with up to 34 additions, deletions or substitutions of SEQ ID NO: 4
 
according to the present invention can for example comprise siRNAs (small interfering RNAs) or shRNAs (small hairpin RNAs) targeting said specific transcription factor Em isoforms.
   

     The person skilled in the art knows how to design siRNAs and shRNAs, which specifically target the specific transcription factor Em isoforms of the present invention. Examples of such specific siRNAs and shRNAs targeting the specific transcription factor Em isoforms of the present invention are depicted in Tables 10 and 11. 
     
       
         
           
               
             
               
                 TABLE 10 
               
               
                   
               
               
                 Examples of siRNA sequences for the knockdown of Gata6 Em 
               
               
                   
               
             
            
               
                 Gata6 
               
            
           
           
               
               
               
            
               
                 Target Sequence 
                 Sense strand siRNA 
                 Antisense strand siRNA 
               
               
                   
               
               
                 AATCAGGAGCGCAGGCTGCAG 
                 SEQ ID NO: 41 
                 SEQ ID NO: 43 
               
               
                 (SEQ ID NO. 58) 
                 UCAGGAGCGCAGGCUGCAGtt 
                 CUGCAGCCUGCGCUCCUGA 
               
               
                   
                   
                 tt 
               
               
                   
               
               
                 AAGAGGCGCCTCCTCTCTCCT 
                 SEQ ID NO: 42 
                 SEQ ID NO: 44 
               
               
                 (SEQ ID NO. 59) 
                 GAGGCGCCUCCUCUCUCCUtt 
                 AGGAGAGAGGAGGCGCCU 
               
               
                   
                   
                 Ctt 
               
               
                   
               
            
           
           
               
            
               
                 Foxa2 
               
            
           
           
               
               
               
            
               
                 Target Sequence 
                 Sense strand siRNA 
                 Antisense strand siRNA 
               
               
                   
               
               
                 AAACCGCCATGCACTCGGCTT 
                 SEQ ID NO: 45 
                 SEQ ID NO: 46 
               
               
                 (SEQ ID NO. 60) 
                 ACCGCCAUGCACUCGGCUUtt 
                 AAGCCGAGUGCAUGGCGG 
               
               
                   
                   
                 Utt 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
               
                   
               
               
                 Examples of shRNA sequences for the knockdown of 
               
               
                 Nkx2-1 
               
               
                   
               
             
            
               
                 Nkx2-1 
               
               
                 shHairpin sequence (5′-3′) 
               
               
                   
               
               
                 SEQ ID NO: 47 
               
               
                 CCGGCCCATGAAGAAGAAAGCAATTCTCGAGAATTGCTTTCTTCTTCAT 
               
               
                 GGGTTTTTG 
               
               
                   
               
               
                 SEQ ID NO: 48 
               
               
                 GTACCGGGGGATCATCCTTGTAGATAAACTCGAGTTTATCTACAAGGAT 
               
               
                 GATCCCTTTTTTG 
               
               
                   
               
               
                 SEQ ID NO: 49 
               
               
                 CCGGATTCGGAATCAGCTAGCAATTCTCGAGAATTGCTAGCTGATTCCG 
               
               
                 AATTTTTTG 
               
               
                   
               
            
           
         
       
     
     The amount of the specific transcription factor isoform according to the present invention can be determined on the polypeptide level. 
     The amount of the specific transcription factor isoforms according to the invention can be assessed on the polypeptide level using known quantitative methods for the assessment of polypeptide levels. For example, ELISA (Enzyme-linked Immunosorbent Assay)-based, gel-based, blot-based, mass spectrometry-based, or flow cytometry-based methods can be used for measuring the amount of the specific transcription factor isoforms on the polypeptide level according to the invention. 
     It is apparent to the person skilled in the art that the specific transcription factor isoforms of the present invention can show certain sequence varieties between different subjects of the same ancestry and in particular between subjects of different ancestry. Non-limiting examples of the polymorphisms of the cancer specific isoforms of the present invention are given in Tables 12 and 13. 
     
       
         
           
               
             
               
                 TABLE 12 
               
               
                   
               
               
                 Examples of polymorphisms in the sequences of GATA6, Em and Ad isoforms in 
               
               
                 dependence of the ancestry of a subject (CEU: Utah residents with Northern and Western 
               
               
                 European ancestry from the CEPH collection; CHB: Han Chinese in Beijing, China; JPT: 
               
               
                 Japanese in Tokyo, Japan; YRI: Yoruban in Ibadan, Nigeria) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 S. No 
                 Region 
                 Position in Gata6 Em 
                 Position in Gata6 Ad 
                 Polymorphism 
                 Population 
                 Frequency of T 
                 Frequency of C 
               
               
                   
               
               
                 1 
                 CCDS 
                 1982 
                 1917 
                 T/C 
                 CEU 
                 100% 
                 0% 
               
               
                   
                   
                   
                   
                   
                 JPT 
                 100% 
                 0% 
               
               
                   
                   
                   
                   
                   
                 YRI 
                 100% 
                 0% 
               
               
                   
               
               
                 S. No 
                 Region 
                 Position in Gata6 Em 
                 Position in Gata6 Ad 
                 Polymorphism 
                 Population 
                 Frequency of G 
                 Frequency of A 
               
               
                   
               
               
                 2 
                 3′UTR 
                 2137 
                 2072 
                 G/A 
                 CEU 
                 56% 
                 44% 
               
               
                   
                   
                   
                   
                   
                 CHB 
                 57% 
                 43% 
               
               
                   
                   
                   
                   
                   
                 JPT 
                 65% 
                 35% 
               
               
                   
                   
                   
                   
                   
                 YRI 
                 45% 
                 55% 
               
               
                   
               
               
                 S. No 
                 Region 
                 Position in Gata6 Em 
                 Position in Gata6 Ad 
                 Polymorphism 
                 Population 
                 Frequency of A 
                 Frequency of G 
               
               
                   
               
               
                 3 
                 3′UTR 
                 2142 
                 2077 
                 A/G 
                 CEU 
                  97% 
                 3% 
               
               
                   
                   
                   
                   
                   
                 CHB 
                  90% 
                 10%  
               
               
                   
                   
                   
                   
                   
                 JPT 
                 100% 
                 0% 
               
               
                   
                   
                   
                   
                   
                 YRI 
                 100% 
                 0% 
               
               
                   
               
               
                 S. No 
                 Region 
                 Position in Gata6 Em 
                 Position in Gata6 Ad 
                 Polymorphism 
                 Population 
                 Frequency of T 
                 Frequency of A 
               
               
                   
               
               
                 4 
                 3′UTR 
                 2391 
                 2326 
                 T/A 
                 CEU 
                 100% 
                 0% 
               
               
                   
                   
                   
                   
                   
                 CHB 
                 100% 
                 0% 
               
               
                   
                   
                   
                   
                   
                 JPT 
                 100% 
                 0% 
               
               
                   
                   
                   
                   
                   
                 YRI 
                 100% 
                 0% 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
               
                   
               
               
                 Examples of polymorphisms in the sequences of FOXA2 variant 1 and 2 in 
               
               
                 dependence of the ancestry of a subject (ASW: African ancestry in Southwest USA; CEU: 
               
               
                 Utah residents with Northern and Western European ancestry from the CEPH collection; CHB: 
               
               
                 Han Chinese in Beijing, China; CHD: Chinese in Metropolitan Denver, Colorado; GIH: 
               
               
                 Gujarati Indians in Houston, Texas; JPT: Japanese in Tokyo, Japan; LWK: Luhya in Webuye, 
               
               
                 Kenya; MEX: Mexican ancestry in Los Angeles, California; MKK: Maasai in Kinyawa, 
               
               
                 Kenya; TSI: Tuscan in Italy; YRI: Yoruban in Ibadan, Nigeria) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 S. No 
                 Region 
                 Position in Foxa2 Em 
                 Position in Foxa2 Ad 
                 Polymorphism 
                 Population 
                 Frequency of T 
                 Frequency of C 
               
               
                   
               
               
                 1 
                 CCDS 
                 1408 
                 1395 
                 T/C 
                 CEU 
                 100% 
                 0% 
               
               
                   
                   
                   
                   
                   
                 CHB 
                 100% 
                 0% 
               
               
                   
                   
                   
                   
                   
                 JPT 
                 100% 
                 0% 
               
               
                   
                   
                   
                   
                   
                 YRI 
                 100% 
                 0% 
               
               
                   
               
               
                 S. No 
                 Region 
                 Position in Foxa2 Em 
                 Position in Foxa2 Ad 
                 Polymorphism 
                 Population 
                 Frequency of A 
                 Frequency of G 
               
               
                   
               
               
                 1 
                 3′UTR 
                 1627 
                 1614 
                 A/G 
                 ASW 
                 38% 
                 62% 
               
               
                   
                   
                   
                   
                   
                 CEU 
                 96% 
                  4% 
               
               
                   
                   
                   
                   
                   
                 CHB 
                 84% 
                 16% 
               
               
                   
                   
                   
                   
                   
                 CHD 
                 84% 
                 16% 
               
               
                   
                   
                   
                   
                   
                 JPT 
                 77% 
                 23% 
               
               
                   
                   
                   
                   
                   
                 GIH 
                 89% 
                 11% 
               
               
                   
                   
                   
                   
                   
                 LWK 
                 27% 
                 73% 
               
               
                   
                   
                   
                   
                   
                 MEX 
                 92% 
                  8% 
               
               
                   
                   
                   
                   
                   
                 MKK 
                 40% 
                 60% 
               
               
                   
                   
                   
                   
                   
                 TSI 
                 91% 
                  9% 
               
               
                   
                   
                   
                   
                   
                 YRI 
                 20% 
                 80% 
               
               
                   
               
            
           
         
       
     
     In certain aspects, the present invention provides a kit for use in carrying out the statistical method of the present invention. The kit of the present invention may comprise primers and further reagents necessary for a qPCR analysis. The respective primers may be selected from the list in Table 9. 
     While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. It will be understood that changes and modifications may be made by those of ordinary skill within the scope and spirit of the following claims. In particular, the present invention covers further embodiments with any combination of features from different embodiments described above and below. 
     The invention also covers all further features shown in the figures individually although they may not have been described in the afore or following description. Also, single alternatives of the embodiments described in the figures and the description and single alternatives of features thereof can be disclaimed from the subject matter of the other aspect of the invention. 
     Furthermore, in the claims the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. A single unit may fulfill the functions of several features recited in the claims. The terms “essentially”, “about”, “approximately” and the like in connection with an attribute or a value particularly also define exactly the attribute or exactly the value, respectively. Any reference signs in the claims should not be construed as limiting the scope. 
    
    
     
       The present invention is further described by reference to the following non-limiting figures and examples. Unless otherwise indicated, established methods of recombinant gene technology were used as described, for example, in Sambrook, Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (2001)) which is incorporated herein by reference in its entirety. 
       The Figures show: 
         FIG. 1 : Embryonic isoforms of GATA6 and NKX2-1 are highly expressed in human lung cancer cell lines and in a mouse model of experimental metastasis. (A) Schematic representation of the gene structure of human GATA6 and NKX2-1. In silico analysis of the indicated genes (top) shows an identical arrangement with two promoters (grey boxes) driving the expression of two distinct transcripts (middle and bottom; exons as black and coding region as white boxes). GATA6, GATA Binding Factor 6; NKX2-1, also known as Ttf1, Thyroid transcription factor 1; Em, Embryonic; Ad, Adult. (B) The two transcript isoforms are differentially regulated during lung cancer and show complementary expression. Isoform specific gene expression analysis was performed for both genes by q-RT PCR in control donor lung tissue (Ctrl) and lung cancer cell lines, A549, A427 (adenocarcinoma) and H322 (bronchoalveolar carcinoma). Rel nor exp, relative expression normalized to TUBA1A. Error bars, standard error of the mean (s.e.m.), n=5. (C) High expression of Em-isoform of Gata6 and Nkx2-1 in a mouse model for tumor metastasis. Isoform specific expression analysis was performed in lungs from control mice (n=3) injected with PBS (Ctrl) and lung tumors (Tum) that developed in mice (n=5) after tail vein injection of 1 million LLC1 cells. Representative are shown the results from one control and two experimental (Tuml, 2) mice. Data are represented as in B. 
         FIG. 2 : Expression ratios of Em- by Ad-isoforms of GATA6 and NKX2-1 as a biomarker for lung cancer diagnosis. (A and B) Isoform specific expression of GATA6 (A) and NKX2-1 (B) was monitored by qRT-PCR after total RNA isolation from formalin fixed paraffin embedded (FFPE) lung tissue sections from control donors (Ctrl, n=34) or lung cancer (LC, n=63) patients. The Em/Ad ratio for both genes is plotted. Samples are normalized to TUB1A1 Each point represents one sample, black points represent adenocarcinoma, blue points represent squamous cell carcinoma, orange point represents adenosquamous carcinoma, red point represents large cell carcinoma, horizontal line in the middle represents the mean and the error bars represent the standard error mean (s.e.m). P values after one-way ANOVA. (C and D) High Em/Ad ratio is conserved among ethnic groups (C) and gender (D). CHB, Han Chinese in Beijing, Ctrl n=7 and LC n=32; CEU, Utah residents with ancestry from northern and western Europe, Ctrl n=19 and LC n=18; MXL, Mexican ancestry in Los Angeles, Ctrl n=8 and LC n=13; Male Ctrl n=8 and LC n=20; Female Ctrl n=4 and LC n=21. Data are represented as in A. (E) Expression of Em-isoform correlates with LC grade. Ratio of Em/Ad was monitored in lung tissue samples of control donor (Ctrl, n=7) cancer patients of Grade I (n=12), II (n=14) and III (n=5). Samples were staged according to the TNM Classification recommended by the International Union Against Cancer (UICC, 7th edition). Data are represented as in A. 
         FIG. 3 : Detection of Em- and Ad-isoforms of GATA6 and NKX2-1 in exhaled breath condensate as non-invasive method for lung cancer diagnosis. (A) Isoform specific expression of GATA6 (left) and NKX2-1 (right) was monitored by qRT-PCR after total RNA isolation from EBCs from control donors (Ctrl, n=22) or lung cancer (LC, n=48) patients. The Em/Ad ratio for both genes is plotted. Samples are normalized to TUB1A1. Each point represents one sample, pink points represent samples of first diagnosis, horizontal line in the middle represents the mean and the error bars represent the standard error mean (s.e.m). P values after one-way ANOVA. (B) Correlation between the values obtained from lung tissue sample and EBC for each patient. The GATA6 (left) and NKX2-1 (right) Em/Ad ratio for both lung tissue (y-axis) and EBC (x-axis) samples were log 2 transformed and plotted. The linear regression was also plotted for both. Red dots, patients where the values from both sample types were significantly different. 
         FIG. 4 : Reliable diagnosis of lung cancer patients using a combination of GATA6 and NKX2-1. (A). The (log) Em/Ad ratio of GATA6 (x-axis) and NKX2-1 (y-axis) of control donors (filled and open circles) and lung cancer patients (triangles) are used to construct a linear SVM classifier, whose decision boundary is the solid line. The LC score is the distance to this boundary (dotted lines: points having LC score±1). A positive LC score indicates lung cancer (light grey shading), a negative LC score indicates a normal lung (dark grey shading). The only misclassified sample is a control sample indicated as an open circle. (B) LC score provides a clear separation of the Ctrl and LC samples. The log transformed LC score was plotted for each sample. Each point represents one sample, the horizontal line in the middle represents the mean and the error bars, standard error mean (s.e.m). The dotted line at 0 represents the decision boundary. (C) Discriminatory power of the Em/Ad ratios alone (dotted line: GATA6, dashed line: NKX2-1) and the LC score (solid line) assessed by an ROC curve. The diamond on the LC score ROC curve represents the “point of operation” (performance) of the SVM classifier 38 . 
         FIG. 5 : Optimization of EBC based expression analysis for lung cancer diagnosis. (A) EBC as a promising source of biomarkers for lung diseases. Water vapor is rapidly diffused from the airway lining fluid (both bronchial and alveolar) into the expiratory flow. Droplet formation (nonvolatile biomarkers) takes place in the airway lining fluid, while respiratory gases (volatile biomarkers) are from both the airspaces and the airways. Modified from 20 . (B) RTube is more suitable for RNA isolation as compared to TurboDECCS. Two main EBC collection devices were compared for the total RNA yield (y-axis, ng) obtained using the QIAGEN RNeasy Micro kit using 500 μl EBC as starting material. Data are represented as mean±s.e.m, n=6. P values after one-way ANOVA. (C) 500 μl of EBC is optimal for RNA isolation. 
       Total RNA isolation with the RNeasy Micro kit was compared using 200, 350, 500 and 1000 μl starting EBC volume. Data are represented as in B, n=4. (D) At least 75 ng of starting RNA is required for reliable diagnosis using EBC for isoform specific expression analysis. Different amounts of RNA (x-axis, ng) were used for cDNA synthesis by RT reaction and subsequently isoform specific expression analysis. The GATA6 (left) and NKX2-1 (right) Em/Ad ratio is plotted for both control (square) and lung cancer samples (triangle). 
         FIG. 6 : Specific PCR amplification of both isoforms of GATA6. (A) 
       Amplification efficiency for each primer pair was calculated using serial dilutions of the cDNA template. Primer efficiency was assessed by plotting the cycle threshold values (Ct, y-axis) against the logarithm (base 10) of the fold dilution (log (Quantity), x-axis). Primer efficiency was calculated using the slope of the linear function. Data points represent mean Ct values of triplicates. (B) Dissociation curve analysis of the PCR products was performed by constantly monitoring the fluorescence with increasing temperatures from 60° C. to 95° C. Melt curves were generated by plotting the negative first derivative of the fluorescence (−d/dT (Fluorescence) 520 nm) versus temperature (degree Celsius, ° C.). (C) Specific PCR amplification was also demonstrated by agarose gel electrophoresis. PCR products after quantitative RT-PCR were analyzed by agarose gel electrophoresis. +, specific PCR reaction using EBC template; −, no RT control; M, 100 bp DNA ladder. (D) Sequencing of the PCR products of GATA6 Em and Ad demonstrates specific PCR amplification of both isoforms using EBC as template. Five clones for each primer pair (GATA6 Em and Ad) were sequenced and aligned to the reference sequence (top row, yellow highlighted). Sequence similarities are represented as dots. 
         FIG. 7 : Specific PCR amplification of both isoforms of NKX2-1. (A) 
       Amplification efficiency for each primer pair was calculated using serial dilutions of the cDNA template. Primer efficiency was assessed by plotting the cycle threshold values (Ct, y-axis) against the logarithm (base 10) of the fold dilution (log (Quantity), x-axis). Primer efficiency was calculated using the slope of the linear function. Data points represent mean Ct values of triplicates. (B) Dissociation curve analysis of the PCR products was performed by constantly monitoring the fluorescence with increasing temperatures from 60° C. to 95° C. Melt curves were generated by plotting the negative first derivative of the fluorescence (−d/dT (Fluorescence) 520 nm) versus temperature (degree Celsius, ° C.). (C) Specific PCR amplification was also demonstrated by agarose gel electrophoresis. PCR products after quantitative RT-PCR were analyzed by agarose gel electrophoresis. +, specific PCR reaction using EBC template; −, no RT control; M, 100 bp DNA ladder. (D) Sequencing of the PCR products of NKX2-1 Em and Ad demonstrates specific PCR amplification of both isoforms using EBC as template. Five clones for each primer pair (NKX2-1 Em and Ad) were sequenced and aligned to the reference sequence (top row, yellow highlighted). Sequence similarities are represented as dots. 
         FIG. 8 : EBC based lung cancer diagnosis correlates with classical methods. Representative pictures of (A) chest X-ray and (B) low-dose helical computed tomography (CT) scans from patients with lung cancer. (C) Immunohistochemistry analysis of adjacent normal (upper panel) and tumor tissue (lower panel) from a representative LC patient with the indicated antibodies. PAN-KRT, Pan Cytokeratin; NKX2-1, also known as TTF1, Thyroid transcription factor 1; DAPI, nucleus. Scale bar, 10 μm. (D) Expression analysis of known tumor suppressor and oncogenes in EBCs of healthy donors and LC patients. CDKNA2, also known as P16, cyclin-dependent kinase inhibitor 2A; TP53, tumor protein p53; MYC, v-myc avian myelocytomatosis viral oncogene homolog. Data are represented as in  FIG. 2A . 
     
    
    
     THE EXAMPLES ILLUSTRATE THE INVENTION 
     Example 1: Detection of Embryonic Isoforms of GATA6 and NKX2-1 in Exhaled Breath Condensate as Non-Invasive Method for Lung Cancer Diagnosis 
     Summary 
     BACKGROUND: Identification of reliable biomarkers and development of non-invasive detection methods for lung cancer are critical to improve prognosis of the disease. 
     METHODS: RNA isolation was performed from human lung tissue and exhaled breath condensates from control donors and lung cancer patients. The Em/Ad expression ratio of GATA6 and NKX2-1 was determined by qRT-PCR. Statistical analysis using R was performed to determine the separating line for the two groups of samples and to evaluate the efficiency of our diagnostic method. 
     RESULTS: We show that two different mRNAs are expressed from both GATA6 and NKX2-1. The expression of both transcripts from the same gene is complementary and differentially regulated during both embryonic lung development and lung cancer. One transcript is expressed during early embryonic lung development (Em-isoform), while the second transcript is expressed in later stages and in the adult lung (Ad-isoform). We detected an enrichment of the Em-isoform in lung cancer tissues, suggesting that the detection of these transcripts could be a powerful tool for early lung cancer diagnosis. The Em- to Ad-expression ratio of both GATA6 and NKX2-1 in RNA from exhaled breath condensates can be used as a non-invasive, specific and sensitive diagnostic tool. A SVM classifier was used to combine the Em/Ad ratios of GATA6 and NKX2-1 of each EBC sample to create a more powerful tool for the diagnosis of lung cancer. 
     CONCLUSIONS: The SVM calculates a simple linear score, LC score, that could be used as a clinical score for lung cancer detection. 
     Glossary 
     Exhaled breath condensate: Exhaled breath condensate (EBC) is a non-invasive method of sampling the airways, allowing biomarkers of airway inflammation and oxidative stress to be measured. It is collected by cooling the exhaled breath to −20° C., resulting in condensation of the aerosol particles. 
     Gene expression analysis: Determination of the level of messenger RNA (mRNA) transcribed from specific genes. Different techniques can be used for this type of analysis, such as quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), Northern Blot, arraybased expression analysis and, more recently, RNA sequencing. In the present manuscript we focus on qRT-PCR based expression analysis that consists of total RNA isolation, RT reaction for the synthesis of cDNA and qPCR amplification using gene specific primers. 
     Isoform: Different versions of mRNA from the same gene that arise by either alternative splicing or differential promoter usage. 
     Polymerase chain reaction: A laboratory technique used to amplify DNA sequences. Short, synthetic complementary DNA sequences called primers are used to selectively amplify the specific portion of the genome. The temperature of the sample is repeatedly raised and lowered to facilitate the copying of the target DNA sequence by a DNA-replication enzyme. Theoretically, the technique doubles the amount of target DNA molecule per cycle. 
     TNM staging criteria: The TNM system is one of the most widely used cancer staging systems. 
     It is based on the size and/or extent (reach) of the primary tumor (T), the amount of spread to nearby lymph nodes (N), and the presence of metastasis (M) or secondary tumors formed by the spread of cancer cells to other parts of the body. A number is added to each letter to indicate the size and/or extent of the primary tumor and the degree of cancer spread. 
     10-fold cross validation: A validation method in which the model is fitted on 90 percent of the samples and then the classification of the remaining 10 percent of the samples is predicted. The procedure is repeated 10 times such that each sample acts as a test sample once. The average error rate of all 10 parts is an estimate of the method&#39;s classification error. 
     Introduction 
     We postulated that many of the mechanisms involved in embryonic development are recapitulated during LC initiation. To this end, two transcription factors that are key regulators of embryonic lung development, GATA6 (GATA Binding Factor 6) and NKX2-1 (NK2 homeobox 1, also known as Ttf-1, Thyroid transcription factor-1) 7-10 , and have been implicated in LC formation and metastasis 11-16  were analyzed. Here we show that two different mRNAs are expressed from each the GATA6 and the NKX2-1 gene. Furthermore, the expression of both transcripts from the same gene is complementary and differentially regulated during embryonic lung development as well as in LC. One transcript is expressed in early stages of embryonic lung development (Em-isoform), whereas the second transcript is expressed in later developmental stages and in the adult lung (Ad-isoform). We detected an enrichment of the Em-isoform in LC, even at early stages, making the detection of these embryonic specific transcripts a powerful tool for cancer diagnosis. Moreover, we demonstrate that isoform specific expression analysis of GATA6 and NKX2-1 in exhaled breath condensates (EBCs) can be used as a non-invasive, specific and sensitive method for both early LC diagnosis. 
     Methods 
     Study Population 
     The patients were studied according to protocols approved by the institutional review board and ethical committee of Regional Hospital of High Specialties of Oaxaca (HRAEO) which belongs to the Ministry of Health in Mexico (HRAEO—CIC-CEI 006/13), Union Hospital Hong Kong (EC003) and Medicine Faculty of the Justus Liebig University in Giessen, Germany (AZ.111/08-eurIPFreg). All cases were reviewed by an expert panel of pulmonologists and oncologists in the different cohorts according to the current diagnostic criteria for morphological features and immunophenotypes recommended by the International Union Against Cancer (UICC, 7 th  edition). 
     LC tissue was obtained from 63 patients who had primary lung tumors in the last five years (Table 1). Control lung tissue was taken from macroscopically healthy adjacent regions of the lung of 15 patients. Control donor lung tissue was also obtained from 19 age-matched individuals, who have had no diagnosis or family history of LC. 
     EBCs were also collected from 48 LC patients that were currently undergoing diagnostic evaluation for LC (Table 1). EBC collection was performed prior to transbronchial biopsy. Further, control EBC was also collected from 22 age matched control individuals with no prior history of LC or any other lung diseases. All participants provided written informed consent. 
     Cell Culture and Mouse Experiments 
     In this study we used human lung adenocarcinoma cell lines (A549; CCL-185 and A427; HTB-53) and a human bronchoalveolar carcinoma cell line (H322; CRL-5806). In addition,  Mus musculus  Lewis Lung cancer cell line (LLC1; CRL-1642) were used in a mouse model of experimental metastasis 17 , wherein 1 million LLC1 cells were injected into the tail vein of experimental mice (n=5) in 100 μl sterile phosphate buffer saline (PBS). Control mice (n=3) were injected with 100 μl sterile PBS. 
     Gene Expression Analysis by qRT-PCR 
     Total RNA was isolated from cell lines using the RNeasy Mini kit (Qiagen). Human lung tissue samples were obtained as formalin fixed paraffin embedded (FFPE) tissues, from which total RNA was isolated using the RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Ambion). 
     Total RNA isolation from EBC was performed using 500 μl of sample with the RNeasy Micro Kit (Qiagen). Complementary DNA (cDNA) was synthetized using the High Capacity cDNA Reverse Transcription kit (Applied Biosystem) and quantitative real time PCR reactions were performed using SYBR® Green on the Step One plus Real-time PCR system (Applied Biosystems) using the primers specified in the Supplementary Table 2. 
     Classifier Construction and LC Score 
     Log-transformed Em/Ad ratios of GATA6 and NKX2-1 were used as independent variables to predict LC. A linear kernel support vector machine (SVM) 39  was used to construct a linear classifier. SVM learning was done with the default parameters, without any adjustments. We preferred SVM to linear discriminant analysis (LDA), which might be the more obvious choice for low dimensional classification tasks, because the control and the LC samples did not show a Gaussian-like distribution, which is an underlying assumption of LDA. The SVM finds a robust separating line and the distance to this line is our decision score, which we call LC score. The LC score can be conveniently calculated as 
     
       
         
           
             
               LC 
                
               
                   
               
                
               Score 
             
             = 
             
               
                 
                   - 
                   0.607 
                 
                 * 
                 
                   
                     log 
                     2 
                   
                    
                   
                     ( 
                     
                       
                         Em 
                          
                         
                             
                         
                          
                         GAT 
                          
                         
                             
                         
                          
                         A 
                          
                         
                             
                         
                          
                         6 
                       
                       
                         Ad 
                          
                         
                             
                         
                          
                         GAT 
                          
                         
                             
                         
                          
                         A 
                          
                         
                             
                         
                          
                         6 
                       
                     
                     ) 
                   
                 
               
               - 
               
                 1.431 
                  
                 
                     
                 
                  
                 
                   
                     log 
                     2 
                   
                    
                   
                     ( 
                     
                       
                         
                           Em 
                            
                           
                               
                           
                            
                           NKX 
                            
                           
                               
                           
                            
                           2 
                         
                         - 
                         1 
                       
                       
                         
                           Ad 
                            
                           
                               
                           
                            
                           NKX 
                            
                           
                               
                           
                            
                           2 
                         
                         - 
                         1 
                       
                     
                     ) 
                   
                 
               
               - 
               1.916 
             
           
         
       
     
     or comprising a prefactor of (−1) for illustrative purposes of 
     
       
         
           
             
               LC 
                
               
                   
               
                
               Score 
             
             = 
             
               
                 ( 
                 
                   - 
                   1 
                 
                 ) 
               
               * 
               
                 
                   ( 
                   
                     
                       
                         - 
                         0.607 
                       
                       * 
                       
                         
                           log 
                           2 
                         
                          
                         
                           ( 
                           
                             
                               Em 
                                
                               
                                   
                               
                                
                               GAT 
                                
                               
                                   
                               
                                
                               A 
                                
                               
                                   
                               
                                
                               6 
                             
                             
                               Ad 
                                
                               
                                   
                               
                                
                               GAT 
                                
                               
                                   
                               
                                
                               A 
                                
                               
                                   
                               
                                
                               6 
                             
                           
                           ) 
                         
                       
                     
                     - 
                     
                       1.431 
                        
                       
                           
                       
                        
                       
                         
                           log 
                           2 
                         
                          
                         
                           ( 
                           
                             
                               
                                 Em 
                                  
                                 
                                     
                                 
                                  
                                 NKX 
                                  
                                 
                                     
                                 
                                  
                                 2 
                               
                               - 
                               1 
                             
                             
                               
                                 Ad 
                                  
                                 
                                     
                                 
                                  
                                 NKX 
                                  
                                 
                                     
                                 
                                  
                                 2 
                               
                               - 
                               1 
                             
                           
                           ) 
                         
                       
                     
                     - 
                     1.916 
                   
                   ) 
                 
                 . 
               
             
           
         
       
     
     Results 
     Embryonic Isoforms of GATA6 and NKX2-1 are Highly Expressed in Human Lung Cancer Cell Lines and in a Mouse Model of Experimental Metastasis. 
     In silico analysis of GATA6 and NKX2-1 revealed a common gene structure ( FIG. 1A , top). Two promoters were predicted in each of the genes, one 5′ of the first exon and the other one in the first intron. Further analysis showed that each of the predicted promoters was surrounded by CpG islands (greater than 200 bp, with more than 50% CG), suggesting that these might be epigenetically regulated, functional promoters. Indeed, expression analysis showed that each gene gave rise to two distinct transcripts ( FIG. 1A , bottom) driven by different promoters. In silico analysis of the murine ortholog genes demonstrated a similar structure as in humans, which highlights that the identified gene structure was maintained during evolution and is conserved among species, reflecting its relevance. Expression analysis by qRT-PCR during mouse lung development revealed that the expression of both isoforms of the same gene was complementary and differentially regulated, with the Em-isoform being mainly expressed during early developmental stages, and the Ad-isoform being expressed at later stages and in the adult lung (data not shown). Interestingly, isoform specific expression analysis ( FIG. 1B ) in control donor lung tissue (Ctrl), human lung adenocarcinoma (A549, A427) and human bronchoalveolar carcinoma (H322) cell lines showed that in these cancer cell lines the expression of the Em isoforms of GATA6 and NKX2-1 was always higher than the expression of the Ad-isoforms. In control human lung tissue, we observed the opposite results, in which the Ad-isoforms were expressed at higher levels than the Em-isoforms. Moreover, in a mouse model of experimental metastasis ( FIG. 1C ) 17 , in which LLC1 cells were injected into the tail vein to induce tumor formation in the mouse lung 21 days later, we detected elevated expression of the Em-isoforms of Gata6 and Nkx2-1 in the tumors when compared to healthy lung tissue (Ctrl). Summarizing, our results suggest that the Em-isoforms of GATA6 and NKX2-1 are relevant during LC formation. 
     Expression Ratios of Em- by Ad-Isoforms of GATA6 and NKX2-1 as a Biomarker for Lung Cancer Diagnosis. 
     To confirm that a similar increase in the expression levels of the Em-isoforms of GATA6 and NKX2-1 occurs in LC patients, we analyzed human lung tissues from control donors and LC patients ( FIG. 2A-B ). The pathological diagnosis of the 63 lung tissue samples was considered as the standard against which the gene expression based molecular diagnosis was compared (Table 1). Isoform specific expression analysis based on qRT-PCR showed that the Em-isoforms of GATA6 and NKX2-1 were enriched in LC tissues as compared to control donor tissue, consistent with our previous results ( FIGS. 1B-C ). In order to facilitate comparability, we decided to use the expression of the Ad-isoform as an internal control and calculated the Em to Ad expression ratio (Em/Ad) for each sample to minimize the effect of individual variations among the different LC specimens. In control lung tissue, Em/Ad was 0.624±0.065 (n=34) for GATA6 and 0.475±0.044 (n=34) for NKX2-1. Interestingly, Em/Ad increased in the LC tissue to 2.63±0.194 (n=63, P&lt;0.001) for GATA6 and to 2.075±0.22 (n=63; P&lt;0.001) for NKX2-1, supporting that an increased Em/Ad expression ratio of GATA6 and NKX2-1 could be used as marker for LC diagnosis. The diagnostic accuracy of the Em/Ad expression ratios of GATA6 and NKX2-1 was maintained after sample grouping by ethnicity ( FIG. 2C ) or by gender ( FIG. 2D ). Furthermore, sample grouping based on TNM classification recommended by the International Union Against Cancer (UICC, 7th edition) ( FIG. 2E ) revealed that the Em/Ad expression ratios of GATA6 and NKX2-1 increased progressively with advancing stages of LC from Grade I (2.395±0.257; P&lt;0.001 for GATA6 and 1.878±0.129; P&lt;0.001 for NKX2-1) through Grade II (3.436±0.243; P&lt;0.001 for GATA6 and 2.589±0.257; P=0.002 for NKX2-1) till Grade III (1.838±0.598; P=0.003 for GATA6 and 3.787±0.392; P&lt;0.001 for NKX2-1). 
     Detection of Em- and Ad-Isoforms of GATA6 and NKX2-1 in Exhaled Breath Condensate as Non-Invasive Method for Lung Cancer Diagnosis. 
     EBC is a promising source of biomarkers for lung diseases since the condensed droplets contain a mixture of nonvolatile biomarkers such as adenosine, prostaglandins, leukotriene, cytokines, etc. and water soluble volatile biomarkers such as nitrogen oxides 18-27.  We optimized different steps and parameters to establish a reliable protocol for qRT-PCR based expression analysis in EBCs ( FIG. 5A-D ). We also demonstrated the specificity of the different qRTPCR products detected in the EBCs ( FIGS. 6A-D  and  7 A-D). Using the optimized conditions, we performed an isoform specific expression analysis of GATA6 and NKX2-1 in EBCs from control donors and LC patients ( FIG. 3A ). In control donor EBCs, the Em/Ad ratio was 0.255±0.02 (n=22) for GATA6 and 0.336±0.02 (n=22) for NKX2-1. In accordance with our previous results using lung tissues, the Em/Ad ratio increased in the EBCs of LC patients to 1.59±0.15 (n=48, P&lt;0.0001) for GATA6 and to 1.625±0.15 (n=48; P&lt;0.0001) for NKX2-1. Remarkably, we were able to anticipate the diagnosis of six LC patients (first diagnosis represented as pink points in the plots) measured in a blinded manner. Hence, our results support the concept that an increased Em/Ad expression ratio of GATA6 and NKX2-1 in the EBCs could be used as non-invasive technique for LC diagnosis. 
     To further validate our findings, EBC based expression analysis was directly compared with LC tissues from the same patient ( FIG. 3B ). The GATA6 (left) and NKX2-1 (right) Em/Ad ratios obtained from both types of samples of the same individuals were comparable and demonstrated a strong positive correlation. Moreover, we compared the classical methods for LC diagnosis directly with EBC based expression analysis ( FIG. 8 ). The pathological and molecular diagnosis correlated with the increased Em/Ad of GATA6 and NKX2-1 in all cases that we tested. 
     Reliable Diagnosis of Lung Cancer Patients Using a Combination of GATA6 and NKX2-1. 
     While the single GATA6 or NKX2-1 isoform ratios predicted LC fairly well ( FIG. 3E ), we combined the two ratios of each EBC sample to create a substantially improved and more powerful tool for the diagnosis of LC. A support vector machine (SVM) classifier achieved 93% accuracy in a 10-fold cross-validation, at 100% sensitivity ( FIG. 4A ). Further, the SVM calculates a simple linear score, which we call LC score, that can be used as a clinical score for LC detection. A sample with an LC score greater than zero is classified as a LC patient while samples with LC score less than zero are classified as control ( FIG. 4B ). The precision of our classification increases with the absolute value of the LC score, in the sense that no misclassifications have been made (yet) for LC scores with an absolute value larger than 1. The individual GATA6 and NKX2-1 isoform ratios, the LC score, and the SVM classification is given in Supplementary Table 3. Furthermore, receiver operating characteristic (ROC) curve analysis confirmed the superiority of the SVM classifier over the single isoforms ratios ( FIG. 4C ). 
     Discussion 
     Early lung cancer diagnosis is crucial to improve patient prognosis and reduce the extremely high case-fatality-rate (95%) 28 . Our work demonstrated that RNA isolated from EBC can be used for qRT-PCR based isoform specific expression analysis of GATA6 and NKX2-1 to determine the Em- by Ad-expression ratio as a non-invasive, specific and sensitive method for early LC diagnosis. We have analyzed 97 human lung tissue samples and 70 EBCs from three cohorts located in different continents and detected increased Em/Ad of GATA6 and NKX2-1 in NSCLC samples independent of the ethnic group, gender and NSCLC subtype. When compared to standard expression analysis, the use of isoform ratios incorporate an additional normalization step to our diagnosis method that makes it robust and reproducible by reducing variability coming from both biological and/or technical parameters. 
     Although the single Em/Ad ratios of GATA6 or NKX2-1 were sufficient to detect LC ( FIG. 3E ), the LC score, which combines the two Em/Ad ratios of each EBC, constitutes a substantially improved tool for the diagnosis of LC, as shown by the ROC analysis ( FIG. 4C ). Our calculation method based on a SVM classifier achieved 93% accuracy in a 10-fold crossvalidation, at 100% sensitivity ( FIG. 4A ). Thus, the method proposed by us may find application in the screening of high risk groups, which includes current and former smokers, individuals exposed to environmental smoke, cooking fumes, indoor smoky coal emissions, asbestos, some metals (e.g. nickel, arsenic and cadmium), radon and ionizing radiation 29-31 . 
     Currently, CT and CXR are used to screen such high risk groups. CT imaging has been shown to be considerably superior to CXR in the identification of small pulmonary nodules 32 . However, despite the success of CT imaging for early LC diagnosis, it suffers from serious limitations, including a high detection rate of benign non calcified nodules (&gt;90% of participants) resulting in follow-up CT scans, biopsies and frequently unnecessary resection of the benign non calcified nodules 33 . Routine implementation of EBC based molecular diagnosis may improve and complement the success of CT and CXR for early LC diagnosis, and especially help to distinguish between false and true positives. 
     Microarray based analysis of LC samples not only led to identification of gene expression profiles that are associated with NSCLC subtypes 34,35 , but also accurately predicted the clinical outcome 36,37 . Although the method proposed here did not discriminate between different NSCLC subtypes, it may be superior to previous approaches of molecular and clinical LC diagnosis due to its higher sensitivity and accuracy, straightforward and fast protocol, noninvasiveness and relative low price. However, a combination of the method proposed here with the existing clinical and molecular methods of LC diagnosis will help to safely settle a LC diagnosis at an earlier, hence curable, stage of the disease. The method of LC diagnosis proposed here could be further refined to discriminate between different NSCLC subtypes by incorporating EBC based expression analysis of known markers of the different subtypes. Furthermore, it might be combined with other markers for the detection of hyper-proliferative non-cancer related diseases as idiopathic pulmonary fibrosis (IPF) or chronic obstructive pulmonary disease (COPD). Interestingly, the current method could be extended to cancer detection in other organs utilizing the expression ratio of developmentally regulated transcript isoforms of the corresponding members of the GATA and/or NKX families of transcription factors in the respective tissue. Lastly, it could be used to monitor the response of a patient to specific treatments in order to fine-tune the therapy to improve the prognosis. 

 
     
       
         
           
               
             
               
                 Supplement TABLE 2 
               
               
                   
               
               
                 Primer sequences used for the analysis of GATA6 and NKX2-1. 
               
               
                   
               
             
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
       
      
      
     
     The following alternative Supplement Table 3 shows also values for the individual ratios of GATA6, NKX2-1 and the LC score, wherein the LC score has been calculated using a a prefactor of (−1) for illustrative purposes. 

 
     Supplementary Results 
       FIG. 5 : Optimization of EBC Based Expression Analysis for Lung Cancer Diagnosis. 
     EBC consists of three main components ( FIG. 5A ): distilled water condensed from the gas phase (&gt;99%), droplets aerosolized from the airway lining fluid and water soluble respiratory gases (the last two make the remaining 1%) 18,19  EBC is a promising source of biomarkers for lung diseases since the condensed droplets contain a mixture of both nonvolatile biomarkers such as adenosine, prostaglandins, leukotriene, cytokines, etc. and water soluble volatile biomarkers such as nitrogen oxides that diffuse from both airspace and airway lining fluid 20-27 . EBCs are typically collected through cooling devices. Here, we tested two of the most commonly used devices for EBC collection for their suitability for subsequent RNA extraction ( FIG. 5B ). Using the same conditions for EBC collection and RNA extraction, the RTube showed a yield of 573±48 ng RNA per 500 μl EBC (n=6), whereas the TurboDECCS showed a lower yield of 292±42 ng RNA per 500 μl EBC (n=6; P=0.001). Thus, we continued collecting the samples with the RTube and tested different EBC volumes to determine the best for RNA extraction ( FIG. 5C ). The RNA yield increased with the EBC volume following a sigmoid curve that reached a plateau at 573±48 ng RNA using 500 μl EBC. RNA yield did not improve further when more than 500 μl of EBC volume was used as starting material. In addition, conditions for cDNA synthesis by reverse transcription and qPCR amplification were optimized using 500 μl EBC collected with the RTube (data not shown). Further, serial dilution of the RNA template was used to determine the minimal material required for reliable diagnosis of cancer based on the Em/Ad ratio of GATA6 and NKX2-1 ( FIG. 5D ). The expression ratio remained stable for both control donor as well as LC EBC samples until 75 ng of RNA starting material. Decreasing the starting material below 75 ng resulted in suboptimal detection of the Em-isoform in the control and the Ad-isoform in the LC group which led to distorted ratios. Using the optimized conditions, we performed isoform specific expression analysis of GATA6 and NKX2-1 in EBCs. 
     FIG.  6 : Specific PCR Amplification of Both Isoforms of GATA6. 
     FIG.  7 : Specific PCR Amplification of Both Isoforms of NKX2-1. 
     The specificity of the different qRT-PCR products detected in the EBCs ( FIGS. 7A-D  and  8 A-D) was demonstrated by dissociation curve analysis, electrophoretic gel analysis and sequencing of the different qRT-PCR products. 
       FIG. 8 : EBC Based Lung Cancer Diagnosis Correlates with Classical Methods. 
     The classical methods for lung cancer diagnosis were directly compared with EBC based expression analysis. Pulmonary nodules were clearly identified by CXR (Supplementary  FIG. 8A  left) and low-dose helical CT (right) in the patients with elevated Em/Ad of GATA6 and NKX2-1. Furthermore, immunostaining on sections of biopsies from the same patients ( FIG. 8B ) using antibodies specific for the epithelial maker KRT (pan-cytokeratin) and NKX2-1 demonstrated that the nodules were primary adenocarcinomas of the lung. Lastly, to determine that markers that are used for the molecular diagnosis of cancer can be detected in EBC, we analyzed the expression of the tumor suppressor genes CDKN2A (also known as P16 or INK4A) and TP53 and the oncogene MYC in EBCs from control donors and lung cancer patients ( FIG. 8C ). In control donors, expression level of CDKNA2 was 0.6±0.36 (n=5) and it decreased to 0.068±0.09 (n=10; P=0.01) in lung cancer patients. Similarly, TP53 expression in control donors was 0.908±0.52 (n=5) and it decreased to 0.021±0.03 (n=10; P&lt;0.01) in lung cancer patients. Consistently, the expression of MYC increased in lung cancer patients from 0.004±0.002 (n=5) to 0.046±0.034 (n=10; P=0.02). The pathological and molecular diagnosis correlated with the increased Em/Ad of GATA6 and NKX2-1 in all of the 10 cases from which we obtained the EBCs. 
     Supplementary Methods 
     Study Population: 
     Samples were collected in three different cohorts located in different continents (America, Asia and Europe), allowing us to investigate ethnic differences. Inclusion criteria for the present study were primary lung tumor samples including lung adenocarcinoma (Grades 1, 2, 3), lung squamous cell carcinoma (Grades 1, 2, 3), large cell carcinoma and adenosquamous carcinoma (Table 1). All tumors were graded according to the Bloom-Richardson and the TNM grading system recommended by the International Union Against Cancer (UICC, 7th edition). Secondary lung tumors and lung cancer samples older than 5 years were excluded. 
     In accordance with the general prevalence, the majority of the samples here represented adenocarcinoma (73.0% and 54.1% for lung cancer tissue and EBC, respectively), followed by squamous cell carcinoma (14.2% and 20.8% for lung cancer tissue and EBC, respectively) (Table 1). Correlating with the disease incidence, the majority of the patients were in the age group of 50-70 years and both male and female patients were equally represented (Supplementary Table 1). Further, the majority of the patients were in the early stage of the disease (Stage I-II) and only a very small minority (6% and 8% for tissues and EBC respectively) had a recurrent disease (Supplementary Table 1). 
     Exhaled Breath Condensate Collection 
     EBC collection was performed using the RTube (Respiratory Research) as described online (http://www.respiratoryresearch.com/products-rtube-how.htm) with some modifications. As a precaution to avoid contaminants from the mouth, donors were asked to refrain from eating, drinking (except water) and smoking up to 3 hours before EBC collection and were asked to rinse their mouth with fresh water just prior to collection. All donors used a nose clamp to avoid nasal contaminants and breathing was only through the mouthpiece. EBCs were collected for 10 min for each donor and immediately stored at −80° C. in 500 μl aliquots. All steps during the collection and processing of EBCs were performed under RNase-free conditions, which is critical to ensure the integrity and high quality of the samples. 
     Cell Culture and Mouse Experiments 
     Cell lines were cultured in medium and conditions recommended by the American Type Culture Collection (ATCC). Cells were used for the preparation of RNA (QIAGEN RNeasy plus mini kit) and protein extracts. 
     Five to 6 weeks old C57BL6 mice were used throughout this study. Animals were housed under controlled temperature and lighting [12/12-hour light/dark cycle], fed with commercial animal feed and water ad libitum. For the mouse model of experimental metastasis, LLC1 cell suspension of 1 million cells/100 μl was prepared in sterile phosphate buffer saline (PBS). Control mice (n=3) were injected with 100 μl PBS whereas experimental mice (n=5) with 100 μl of cell suspension into the tail vein of each mouse. The development of tumors was monitored 21 days post injection. Lung tissue was harvested from each mouse separately for RNA isolation and isoform specific expression analysis. 
     Mouse work was performed in compliance with the German Law for Welfare of Laboratory Animals. The permission to perform the experiments presented in this study was obtained from the Regional Council (Regierungspräsidium in Darmstadt, Germany). The numbers of the permissions are V54-19c20/15-B2/345; IVMr46-53r30.03.MPP04.12.02 and IVMr46-53r30.03.MPP06.12.01. Animals were killed for scientific purposes according to the law mentioned above which comply with national and international regulations. 
     Statistical Analysis 
     Cell line and mouse experiments were performed three times. Statistical analyses were performed using Excel Solver. Samples were analyzed at least in triplicates. The data are represented as mean±Standard Error (mean±s.e.m). For human samples, each point on the graph represents an individual sample while the horizontal line represents the median±Standard Error (median±s.e.m.). One-way analysis of variance (ANOVA) was used to determine the levels of difference between the groups and P values for significance. 
     Gene Expression Analysis by qRT-PCR 
     Total RNA was isolated from cell lines using the RNeasy Mini kit (Qiagen. Human lung tissue samples were obtained as formalin fixed paraffin embedded (FFPE) tissues and 8 sections of 10 μm thickness were used for total RNA isolation using the RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Ambion). Total RNA isolation from EBC was performed using 500 μl of sample and the RNeasy Micro Kit (Qiagen). Complementary DNA (cDNA) was synthetized using the High Capacity cDNA Reverse Transcription kit (Applied Biosystem) and 0.5-0.7m (EBC) or 1 μg (cell lines, mice and human lung cancer tissue) total RNA. Quantitative real time PCR reactions were performed using SYBR® Green on the Step One plus Real-time PCR system (Applied Biosystems) using the primers specified in the Supplementary Table 2. Briefly, 1× concentration of the SYBR green master mix, 250 nM each forward and reverse primer and 3.5 μl (EBC) or 1 μl (cell lines, mice and human lung cancer tissue) from a 6 fold diluted RT reaction were used for the gene specific qPCR reaction. The PCR results were normalized with respect to the housekeeping gene alpha 1a Tubulin (TUBA1A). 
     Example 2: Further Validation of the Detection of Embryonic Isoforms of GATA6 and NKX2-1 in Exhaled Breath Condensate as Non-Invasive Method for Lung Cancer Diagnosis 
     Further validation of the LC score classifier was performed on an independent set of samples (EBCs) consisting of 22 previously unseen samples (10 controls and 12 LC patient EBCs,  FIG. 23 ). These EBCs were collected mimicking conditions of clinical use, e.g. they were collected in different centers by different operators according to optimized SOP. The protocol and algorithm were followed exactly as described in Example 1 to compute the LC Score. Performance assessment of the LC score classifier by applying it to this independently collected set of EBCs confirmed its high performance by achieving an accuracy of 91%, sensitivity of 77%, and a specificity of 95%. Receiver operating characteristic (ROC) curve analysis based on all EBCs together (training and validation  FIG. 24 ) showed an area under the curve (AUC) of 0.8153409 for NKX2-1, 0.9204545 for GATA6 and 0.9397727 for the LC score. 
     FIG.  23 : 
     The log 2-transformed Em/Ad ratio of GATA6 (x-axis) and NKX2-1 (y-axis) of controls (light grey circles) and LC patients (black circles) for the new validation set were plotted. The solid line represents the decision boundary determined by a linear support vector machine (SVM) classifier combining the Em/Ad ratios of GATA6 and NKX2-1 of each sample. Filled circle, sample classified correctly; empty circle, sample classified wrong. LC score is the distance to the boundary. 
     FIG.  24 : 
     Discriminatory power of the Em/Ad ratios of GATA6 (grey line), NKX2-1 (grey dashed line) and the improved LC score (black line) assessed by receiver operating characteristic (ROC) curve analysis based on both sets of EBCs together (training and validation). The orange diamond represents the “point of operation” (performance) of the SVM classifier. 
     The present invention refers to the following nucleotide and amino acid sequences: 
     The sequences provided herein are available in the NCBI database and can be retrieved from www.ncbi.nlm.nih.gov/sites/entrez?db=gene; Theses sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods wherein homologous sequences, and variants of the concise sequences provided herein are used. Preferably, such “variants” are genetic variants. 
     The following exemplary sequences relate to additional marker(s) that can be used in accordance with the present invention for classifying cancer, for example, for classifying lung cancer into subtypes of lung cancer. 
     The following markers are upregulated in adenocarcinoma: 
     
       
         
           
               
             
               
                   
               
             
            
               
                 SEQ ID No. 65: 
               
               
                 Nucleotide sequence encoding  Homo sapiens  Surfactant protein A: 
               
               
                 PMID 11707590 
               
            
           
           
               
               
               
            
               
                 gene symbol 
                 Alias and additional info 
                   
               
               
                 SFTPA1 
                 Surfactant protein A 
                   
               
               
                 Accession number 
                 Transcript variant 
                   
               
               
                 NM_001093770.2 
                 surfactant protein A1 (SFTPA1),  
                   
               
               
                   
                 transcript variant 2 
                   
               
            
           
           
               
            
               
                 SEQ ID No. 66: 
               
               
                 Amino acid sequence of  Homo sapiens  Surfactant protein A: 
               
            
           
           
               
               
               
            
               
                 NP_001087239.2 
                 surfactant protein A1 (SFTPA1),  
                   
               
               
                   
                 transcript variant 2 
                   
               
            
           
           
               
            
               
                 SEQ ID No. 67: 
               
               
                 Nucleotide sequence encoding  Homo sapiens  Surfactant protein A: 
               
            
           
           
               
               
            
               
                 Accession number 
                 Transcript variant 
               
            
           
           
               
               
               
            
               
                 NM_001164644.1 
                 surfactant protein A1 (SFTPA1),  
                   
               
               
                   
                 transcript variant 3 
                   
               
            
           
           
               
            
               
                 SEQ ID No. 68: 
               
               
                 Amino acid sequence of  Homo sapiens  Surfactant protein A: 
               
            
           
           
               
               
               
            
               
                 NP_001158116.1 
                 surfactant protein A1 (SFTPA1), 
                   
               
               
                   
                 transcript variant 3 
                   
               
            
           
           
               
            
               
                 SEQ ID No. 69: 
               
               
                 Nucleotide sequence encoding  Homo sapiens  Surfactant protein A: 
               
            
           
           
               
               
               
            
               
                 Accession number 
                 Transcript variant 
                   
               
               
                 NM_01164645.1 
                 surfactant protein A1 (SFTPA1),  
                   
               
               
                   
                 transcript variant 5 
                   
               
            
           
           
               
            
               
                 SEQ ID No. 70: 
               
               
                 Amino acid sequence of  Homo sapiens  Surfactant protein A: 
               
            
           
           
               
               
               
            
               
                 NP_001158117.1 
                 surfactant protein A1 (SFTPA1),  
                   
               
               
                   
                 transcript variant 5 
                   
               
            
           
           
               
            
               
                 SEQ ID No. 71: 
               
               
                 Nucleotide sequence encoding  Homo sapiens  Surfactant protein A: 
               
            
           
           
               
               
            
               
                 Accession number 
                 Transcript variant 
               
               
                 NM_001164646.1 
                 surfactant protein A1 (SFTPA1),  
               
               
                   
                 transcript variant 6 
               
            
           
           
               
            
               
                 SEQ ID No. 72: 
               
               
                 Amino acid sequence of  Homo sapiens  Surfactant protein A: 
               
            
           
           
               
               
               
            
               
                 NP_001158118.1 
                 surfactant protein A1 (SFTPA1),  
                   
               
               
                   
                 transcript variant 6 
                   
               
            
           
           
               
            
               
                 SEQ ID No. 73: 
               
               
                 Nucleotide sequence encoding  Homo sapiens  Surfactant protein A: 
               
            
           
           
               
               
               
            
               
                 Accession number 
                 Transcript variant 
                   
               
               
                 NM_001164647.1 
                 surfactant protein A1 (SFTPA1),  
                   
               
               
                   
                 transcript variant 4 
                   
               
            
           
           
               
            
               
                 SEQ ID No. 74: 
               
               
                 Amino acid sequence of  Homo   sapiens  Surfactant protein A: 
               
            
           
           
               
               
               
            
               
                 NP_001158119.1 
                 surfactant protein A1 (SFTPA1),  
                   
               
               
                   
                 transcript variant 4 
                   
               
            
           
           
               
            
               
                 SEQ ID No. 75: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  Surfactant protein A: 
               
            
           
           
               
               
               
            
               
                 Accession number 
                 Transcript variant 
                   
               
               
                 NM_005411.4 
                 surfactant protein A1 (SFTPA1),  
                   
               
               
                   
                 transcript variant 1 
                   
               
            
           
           
               
            
               
                 SEQ ID No. 76: 
               
               
                 Amino acid sequence of  Homo   sapiens  Surfactant protein A: 
               
            
           
           
               
               
               
            
               
                 gene symbol 
                 Alias and additional info 
                   
               
               
                 NP_005402.3 
                 surfactant protein A1 (SFTPA1), 
                   
               
               
                   
                 transcript variant 1 
                   
               
            
           
           
               
            
               
                 SEQ ID No. 77: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  Surfactant protein B: 
               
            
           
           
               
               
               
            
               
                 gene symbol 
                 Alias and additional info 
                   
               
               
                 SFTPB 
                 Surfactant protein B 
                   
               
               
                 Accession number 
                 Transcript variant 
                   
               
               
                 NM_000542.3 
                 pulmonary surfactant-associated  
                   
               
               
                   
                 protein B precursor 
                   
               
            
           
           
               
            
               
                 This variant (1) is the longer transcript. Both variants 1 and 2 encode the same protein. 
               
               
                 SEQ ID No. 78: 
               
               
                 Amino acid sequence of  Homo   sapiens  Surfactant protein B: 
               
            
           
           
               
               
               
            
               
                 NP_000533.3 
                 pulmonary surfactant-associated  
                   
               
               
                   
                 protein B precursor 
                   
               
            
           
           
               
            
               
                 SEQ ID No. 79: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  Surfactant protein B: 
               
            
           
           
               
               
               
            
               
                 NM_198843.2 
                 pulmonary surfactant-associated 
                   
               
               
                   
                 protein B precursor 
                   
               
               
                   
                 Alias and additional info 
                   
               
            
           
           
               
            
               
                 This variant (2) lacks an internal segment in the 3&#39; UTR, as compared to variant 1.  
               
               
                 Both variants 1 and 2 encode the same protein 
               
               
                 SEQ ID No. 80: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  napsin A aspartic peptidase: 
               
            
           
           
               
               
               
            
               
                 NAPSA  
                 napsin A  
                 NM_004851.1 
               
               
                   
                 aspartic peptidase 
                   
               
            
           
           
               
            
               
                 SEQ ID No. 81: 
               
               
                 Amino acid sequence of  Homo   sapiens  napsin A aspartic peptidase: 
               
            
           
           
               
               
               
            
               
                 napsin A aspartic peptidase 
                 NP_004842.1 
                   
               
            
           
           
               
            
               
                 The following markers are upregulated in Squamous cell carcinoma. 
               
               
                 SEQ ID No. 82: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  tumor protein p63: 
               
            
           
           
               
               
               
            
               
                 PMID 21623384 
                   
                   
               
               
                 gene symbol 
                 Alias and additional info 
                   
               
               
                 TP63 
                 tumor protein p63 
                   
               
               
                 Accession number 
                 Transcript variant 
                   
               
               
                 NM_001114978.1 
                 tumor protein p63 (TP63),  
                   
               
               
                   
                 transcript variant 2 
                   
               
            
           
           
               
               
            
               
                 SEQ ID No. 83: 
                   
               
            
           
           
               
            
               
                 Amino acid sequence of  Homo   sapiens  tumor protein p63: 
               
            
           
           
               
               
            
               
                 NP_001108450.1 
                   Homo   sapiens  tumor protein p63 (TP63), transcript variant 2 
               
            
           
           
               
            
               
                 SEQ ID No. 84: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  tumor protein p63: 
               
               
                 tumor protein p63 (TP63), transcript variant 3 
               
               
                 NM_001114979.1 
               
               
                 SEQ ID No. 85: 
               
               
                 Amino acid sequence of  Homo   sapiens  tumor protein p63: 
               
            
           
           
               
               
            
               
                 NP_001108451.1 
                   Homo   sapiens  tumor protein p63 (TP63), transcript variant 3 
               
            
           
           
               
            
               
                 SEQ ID No. 86: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  tumor protein p63: 
               
            
           
           
               
               
            
               
                 NM_001114980.1 
                 tumor protein p63 (TP63), transcript variant 4 
               
            
           
           
               
            
               
                 SEQ ID No. 87: 
               
               
                 Amino acid sequence of  Homo   sapiens  tumor protein p63: 
               
            
           
           
               
               
            
               
                 NP_001108452.1 
                   Homo   sapiens  tumor protein p63 (TP63), transcript variant 4 
               
            
           
           
               
            
               
                 SEQ ID No. 88: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  tumor protein p63: 
               
            
           
           
               
               
            
               
                 NM_001114981.1 
                 tumor protein p63 (TP63), transcript variant 5 
               
            
           
           
               
            
               
                 SEQ ID No. 89: 
               
               
                 Amino acid sequence of  Homo   sapiens  tumor protein p63: 
               
            
           
           
               
               
            
               
                 NP_001108453.1 
                   Homo   sapiens  tumor protein p63 (TP63), transcript variant 5 
               
            
           
           
               
            
               
                 SEQ ID No. 90: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  tumor protein p63: 
               
            
           
           
               
               
            
               
                 NM_001114982.1 
                 tumor protein p63 (TP63), transcript variant 6 
               
            
           
           
               
            
               
                 SEQ ID No. 91: 
               
               
                 Amino acid sequence of  Homo   sapiens  tumor protein p63: 
               
            
           
           
               
               
            
               
                 NP_001108454.1 
                   Homo   sapiens  tumor protein p63 (TP63), transcript variant 6 
               
            
           
           
               
            
               
                 SEQ ID No. 92: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  tumor protein p63: 
               
            
           
           
               
               
            
               
                 NM_003722.4 
                 tumor protein p63 (TP63), transcript variant 1 
               
            
           
           
               
            
               
                 SEQ ID No. 93: 
               
               
                 Amino acid sequence of  Homo   sapiens  tumor protein p63: 
               
            
           
           
               
               
            
               
                 NP_003713.3 
                   Homo   sapiens  tumor protein p63 (TP63), transcript variant 1 
               
            
           
           
               
            
               
                 SEQ ID No. 94: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  keratin 5: 
               
            
           
           
               
               
               
            
               
                 KRT5  
                 keratin 5 
                 NM_000424.3 
               
            
           
           
               
            
               
                 SEQ ID No. 95: 
               
               
                 Amino acid sequence of  Homo   sapiens  keratin 5: 
               
               
                 keratin 5 NP_000415.2 
               
               
                 SEQ ID No. 96: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  keratin 6: 
               
            
           
           
               
               
               
            
               
                 KRT6A 
                 keratin6 
                 NM_005554.3 
               
            
           
           
               
            
               
                 SEQ ID No. 97: 
               
               
                 Amino acid sequence of  Homo   sapiens  keratin 6: 
               
            
           
           
               
               
               
            
               
                 KRT6A 
                 keratin6 
                 NP_005545.1 
               
            
           
           
               
            
               
                 SEQ ID No. 98: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  keratin 7: 
               
            
           
           
               
               
               
            
               
                 KRT7 
                 keratin 7 
                 NM_005556.3 
               
            
           
           
               
            
               
                 SEQ ID No. 99: 
               
               
                 Amino acid sequence of  Homo   sapiens  keratin 7: 
               
            
           
           
               
               
               
            
               
                 KRT7 
                 keratin 7 
                 NP_005547.3 
               
            
           
           
               
            
               
                 Nucleotide sequence of  Homo   sapiens  hsa-miR9 and related isoforms: 
               
               
                 SEQ ID No. 100: 
               
               
                 PMID 23999427 
               
            
           
           
               
               
            
               
                 hsa-miR9 
                 micro RNA miR9 
               
               
                 NR_029691.1 
                   Homo   sapiens  microRNA 
               
               
                 SEQ ID No. 101: 
                 9-1 (MIR9-1) 
               
               
                 NR_030741.1 
                   Homo   sapiens  microRNA  
               
               
                   
                 9-2 (MIR9-2) 
               
            
           
           
               
            
               
                 SEQ ID No. 102: 
               
            
           
           
               
               
            
               
                 NR_029692.1 
                   Homo   sapiens  microRNA  
               
               
                   
                 9-3 (MIR9-3) 
               
            
           
           
               
            
               
                 The following marker is downregulated in adenocarcinoma: 
               
               
                 SEQ ID No. 103: 
               
               
                 Nucleotide sequence of  Homo   sapiens  hsa-let7-d: 
               
            
           
           
               
               
            
               
                 ″17437991, 24305048 
                   
               
            
           
           
               
               
               
            
               
                 ″  hsa-1et7-d  
                  microRNA let-7d (MIRLET7D) 
                 NR_029481.1 
               
               
                   
               
            
           
         
       
     
     The following markers are upregulated in metastatic adenocarcinoma: 
     
       
         
           
               
             
               
                   
               
             
            
               
                 SEQ ID No. 104: 
               
               
                 Nucleotide sequence encoding  Homo sapiens  VEGFA: 
               
               
                 VEGFA 
               
               
                 NM_001025366.2-vascular endothelial growth factor A isoform a 
               
               
                 SEQ ID No. 105: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001020537.2 
               
               
                 SEQ ID No. 106: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001025367.2  vascular endothelial growth factor A isoform c 
               
               
                 SEQ ID No. 107: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001020538.2 
               
               
                 SEQ ID No. 108: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001025368.2  vascular endothelial growth factor A isoform d 
               
               
                 SEQ ID No. 109: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001020539.2 
               
               
                 SEQ ID No. 110: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001025369.2  vascular endothelial growth factor A isoform e 
               
               
                 SEQ ID No. 111: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001020540.2 
               
               
                 SEQ ID No. 112: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001025370.2  vascular endothelial growth factor A isoform f 
               
               
                 SEQ ID No. 113: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001020541.2 
               
               
                 SEQ ID No. 114: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001033756.2  vascular endothelial growth factor A isoform g 
               
               
                 SEQ ID No. 115: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001028928.1 
               
               
                 SEQ ID No. 116: 
               
               
                 Nucleotide sequence encoding Homo sapiens VEGFA: 
               
               
                 NM_001171622.1  vascular endothelial growth factor A isoform h 
               
               
                 SEQ ID No. 117: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001165093.1 
               
               
                 SEQ ID No. 118: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001171623.1  vascular endothelial growth factor A isoform i precursor 
               
               
                 SEQ ID No. 119: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001165094.1 
               
               
                 SEQ ID No. 120: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001171624.1  vascular endothelial growth factor A isoform j precursor 
               
               
                 SEQ ID No. 121: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001165095.1 
               
               
                 SEQ ID No. 122: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001171625.1  vascular endothelial growth factor A isoform k precursor 
               
               
                 SEQ ID No. 123: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001165096.1 
               
               
                 SEQ ID No. 124: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001171626.1  vascular endothelial growth factor A isoform l precursor 
               
               
                 SEQ ID No. 125: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001165097.1 
               
               
                 SEQ ID No. 126: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001171627.1  vascular endothelial growth factor A isoform m precursor 
               
               
                 SEQ ID No. 127: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001165098.1 
               
               
                 SEQ ID No. 128: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001171628.1  vascular endothelial growth factor A isoform n precursor 
               
               
                 SEQ ID No. 129: 
               
               
                 Amino acid sequence of Homo sapiens VEGFA: 
               
               
                 Amino acid-NP_001165099.1 
               
               
                 SEQ ID No. 130: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001171629.1  vascular endothelial growth factor A isoform o precursor 
               
               
                 SEQ ID No. 131: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001165100.1 
               
               
                 SEQ ID No. 132: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001171630.1  vascular endothelial growth factor A isoform p precursor 
               
               
                 SEQ ID No. 133: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001165101.1 
               
               
                 SEQ ID No. 134: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001204384.1  vascular endothelial growth factor A isoform q precursor 
               
               
                 SEQ ID No. 135: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001191313.1 
               
               
                 SEQ ID No. 136: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001204385.1  vascular endothelial growth factor A isoform r 
               
               
                 SEQ ID No. 137: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001191314.1 
               
               
                 SEQ ID No. 138: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_001287044.1  vascular endothelial growth factor A isoform s 
               
               
                 SEQ ID No. 139: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid-NP_001273973.1 
               
               
                 SEQ ID No. 140: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFA: 
               
               
                 NM_003376.5 vascular endothelial growth factor A isoform b 
               
               
                 SEQ ID No. 141: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFA: 
               
               
                 Amino acid- NP_003367.4 
               
               
                 SEQ ID No. 142: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFB: 
               
               
                 VEGFB 
               
               
                 NM_001243733.1  vascular endothelial growth factor B isoform VEGFB-167 precursor 
               
               
                 SEQ ID No. 143: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFB: 
               
               
                 Amino acid-NP_001230662.1 
               
               
                 SEQ ID No. 144: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFB: 
               
               
                 NM_003377.4  vascular endothelial growth factor B isoform VEGFB-186 precursor 
               
               
                 SEQ ID No. 145: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFB: 
               
               
                 Amino acid-NP_003368.1 
               
               
                 SEQ ID No. 146: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFD: 
               
               
                 VEGFD (FIGF, c-fos induced growth factor) 
               
               
                 NM_004469.4vascular endothelial growth factor D preproprotein 
               
               
                 SEQ ID No. 147: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFD: 
               
               
                 Amino acid-NP_004460.1 
               
               
                 SEQ ID No. 148: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  VEGFC: 
               
            
           
           
               
               
               
            
               
                 11707590 VEGFC  
                 Vascular endothelial growth factor C  
                 NM_005429.4 
               
            
           
           
               
            
               
                 SEQ ID No. 149: 
               
               
                 Amino acid sequence of  Homo   sapiens  VEGFC: 
               
            
           
           
               
               
               
            
               
                 VEGFC  
                 Vascular endothelial growth factor C  
                 NP_005420.1 
               
            
           
           
               
            
               
                 SEQ ID No. 150: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  PLAUR 
               
            
           
           
               
               
            
               
                 11707590 PLAUR plasminogen activator urokinase receptor  
                 NM_001005376.2 
               
               
                   plasminogen activator, urokinase receptor (PLAUR), transcript variant 2 
                   
               
               
                 SEQ ID No. 151: 
                   
               
               
                 Amino acid sequence of  Homo   sapiens  PLAUR 
                   
               
            
           
           
               
            
               
                 PLAUR  plasminogen activator urokinase receptor  NP_001005376.1   Homo   sapiens   
               
               
                 plasminogen activator, urokinase receptor (PLAUR), transcript variant 2 
               
               
                 SEQ ID No. 152: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  PLAUR 
               
               
                 11707590 PLAUR plasminogen activator urokinase receptor NM_001005377.2plasminogen 
               
               
                 activator, urokinase receptor (PLAUR), transcript variant 3 
               
               
                 SEQ ID No. 153: 
               
               
                 Amino acid of  Homo   sapiens  PLAUR 
               
               
                 PLAUR  plasminogen activator urokinase receptor  Homo   sapiens  plasminogen activator, 
               
               
                 urokinase receptor (PLAUR), transcript variant 3 
               
               
                 SEQ ID No. 154: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  PLAUR 
               
               
                 11707590 PLAUR plasminogen activator urokinase receptor plasminogen activator,  
               
               
                 urokinase receptor (PLAUR), transcript variant 4 
               
               
                 SEQ ID No. 155: 
               
               
                 Amino acid sequence of  Homo   sapiens  PLAUR 
               
               
                 PLAUR  plasminogen activator urokinase receptor NP_001287966.1   Homo   sapiens   
               
               
                 plasminogen activator, urokinase receptor (PLAUR), transcript variant 4 
               
               
                 SEQ ID No. 156: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  PLAUR 
               
               
                 11707590 PLAUR plasminogen activator urokinase receptor plasminogen activator,  
               
               
                 urokinase receptor (PLAUR), transcript variant 1 
               
               
                 SEQ ID No. 157: 
               
               
                 Amino acid sequence of  Homo   sapiens  PLAUR 
               
               
                 PLAUR   plasminogen activator urokinase receptor 
               
               
                        Homo sapiens  plasminogen activator, urokinase receptor (PLAUR), 
               
               
                 NP_002650.1 transcript variant 1 
               
               
                   
               
            
           
         
       
     
     The following marker is upregulated in Large cell lung cancer 
     
       
         
           
               
             
               
                   
               
             
            
               
                 SEQ ID No. 158: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  HMGA1 
               
               
                 19903768 HMGA1  NM_002131.3   Homo   sapiens  high mobility group 
               
               
                 AT-hook 1 (HMGA1), transcript variant 2 
               
               
                 SEQ ID No. 159: 
               
               
                 Amino acid sequence of  Homo   sapiens  HMGA1 
               
               
                 HMGA1   NP_002122.1  Homo   sapiens  high mobility group AT-hook 1 (HMGA1), 
               
               
                 transcript variant 2 
               
               
                 SEQ ID No. 160: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  HMGA1 
               
               
                   Homo   sapiens  high mobility group AT-hook 1 (HMGA1), transcript variant 1 
               
               
                 19903768  HMGA1 NM_145899.2   Homo   sapiens  high mobility group AT-hook 1 
               
               
                 (HMGA1), transcript variant 1 
               
               
                 SEQ ID No. 161: 
               
               
                 Amino acid sequence of  Homo   sapiens  HMGA1 
               
               
                 HMGA1 NP_665906.1   Homo   sapiens  high mobility group AT-hook 1 (HMGA1), 
               
               
                 transcript variant 1 
               
               
                 SEQ ID No. 162: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  HMGA1 
               
               
                 19903768  HMGA1 NM_145901.2   Homo   sapiens  high mobility group AT-hook 1 
               
               
                 (HMGA1), transcript variant 3 
               
               
                 SEQ ID No. 163: 
               
               
                 Amino acid sequence of  Homo   sapiens  HMGA1 
               
               
                 HMGA1 NP_665908.1   Homo   sapiens  high mobility group AT-hook 1 (HMGA1), 
               
               
                 transcript variant 3 
               
               
                 SEQ ID No. 164: 
               
               
                 Nucleotide sequence encoding  Homo   sapiens  HMGA1 
               
               
                 19903768 HMGA1 
               
               
                 NM_145902.2  Homo   sapiens  high mobility group AT-hook 1 (HMGA1), transcript 
               
               
                 variant 4 
               
               
                 SEQ ID No. 165: 
               
               
                 Amino acid sequence of  Homo   sapiens  HMGA1 
               
               
                 HMGA1  Homo   sapiens  high mobility group AT-hook 1 (HMGA1), transcript variant 4 
               
               
                 SEQ ID No. 166: 
               
               
                 19903768  HMGA1 NM_145903.2   Homo   sapiens  high mobility group AT-hook 1 
               
               
                 (HMGA1), transcript variant 5 
               
               
                 SEQ ID No. 167: 
               
               
                 Amino acid sequence of  Homo   sapiens  HMGA1 
               
               
                 NP_665910.1  Homo   sapiens  high mobility group AT-hook 1 (HMGA1), transcript variant 5 
               
               
                 SEQ ID No. 168: 
               
               
                 19903768  HMGA1  NM_145905.2   Homo   sapiens  high mobility group  
               
               
                 AT-hook 1 (HMGA1), transcript variant 7 
               
               
                 SEQ ID No. 169: 
               
               
                 Amino acid sequence of  Homo   sapiens  HMGA1 
               
               
                 HMGA1 NP_665912.1   Homo   sapiens  high mobility group AT-hook 1 (HMGA1), 
               
               
                 transcript variant 7 
               
               
                   
               
            
           
         
       
     
     Genomic Alterations 
       
     
       
         
           
               
             
               
                   
               
               
                 Genomic alterations 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 PMID 
                   
                   
               
               
                   
                 18794081 KRAS G12D 
                 G --&gt; CIT transversion  
               
               
                   
                   
                 at codon for Exon 12  
               
               
                   
                   
                 Adenocarcinoma 
               
               
                   
                 21471965 KRAS G12D// 
                 R172H Substitution in p53  
               
               
                   
                 p53 mutations 
                 (Li-Fraumeni syndrome, PMID 
               
               
                   
                   
                 15607981) 
               
               
                   
                   
                 Metastatic Adenocarcinoma 
               
               
                   
                 18794081 KRAS G12D 
                 G --&gt; A transition 
               
               
                   
                   
                 Adenocarcinoma in never smokers 
               
               
                   
                 1324794 p53 mutations,  
                 Adenocarcinoma or Squamous  
               
               
                   
                 translocations 
                 cell carcinoma 
               
               
                   
                 15737014 EGFR T790M 
                 mutation in exon 20, codon 790 
               
               
                   
                   
                 Drug resistant Adenocarcinoma, 
               
               
                   
                   
                 patients relapse after tyrosine  
               
               
                   
                   
                 kinase inhibitors 
               
               
                   
                 21665149 p53 mutations//Rb-/- 
                 Small cell carcinoma 
               
               
                   
               
            
           
         
       
     
     The following table provides more detailed information in relation to genomic alterations: 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Amino acid  
                 Genomic  
                 Cancer 
                   
               
               
                 change/Gene 
                 Alteration 
                 classification 
                 Reference 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 KRAS G12D 
                 G → C/T transversion 
                 Adenocarcinoma 
                 (Riely, Kris et al. 2008) 
               
               
                   
                 G → A transition 
                 Adenocarcinoma in 
                 (Winslow, Dayton et al. 
               
               
                   
                   
                 never smokers 
                 2011) 
               
               
                 p53 
                 Mutations and 
                 Adenocarcinoma or 
                 (Kishimoto,  
               
               
                   
                 translocations 
                 Squamous cell 
                 Murakami et al. 1992) 
               
               
                   
                   
                 carcinoma 
                   
               
               
                 P53 R172H 
                   
                 Li-Fraumeni 
                 (Lang, Iwakuma  
               
               
                 Substitution in p53 
                   
                 syndrome 
                 et al. 2004) 
               
               
                 KRAS G12D//p53 
                   
                 Metastatic 
                   
               
               
                 mutations 
                   
                 Adenocarcinoma 
                   
               
               
                 EGFR T790M 
                 Mutations in exon 20, 
                 Drug resistant 
                 (Pao, Miller et al. 2005) 
               
               
                   
                 codon 790 
                 Adenocarcinoma, 
                   
               
               
                   
                   
                 patients relapse 
                   
               
               
                   
                   
                 after tyrosine kinase 
                   
               
               
                   
                   
                 inhibitors 
                   
               
               
                 p53 mutations//Rb-/- 
                   
                 Small cell  
                 (Sutherland, Proost et 
               
               
                   
                   
                 carcinoma 
                 al. 2011) 
               
               
                   
               
            
           
         
       
     
     REFERENCES 
     
         
         1. Herbst R S, Heymach J V, Lippman S M. Lung cancer. The New England journal of medicine 2008; 359:1367-80. 
         2. Hoffman P C, Mauer A M, Vokes E E. Lung cancer. Lancet 2000; 355:479-85. 
         3. Hyde L, Hyde C I. Clinical manifestations of lung cancer. Chest 1974; 65:299-306. 
         4. Strauss G M, Dominioni L. Chest X-ray screening for lung cancer: overdiagnosis, endpoints, and randomized population trials. Journal of surgical oncology 2013; 108:294-300. 
         5. D&#39;Urso V, Doneddu V, Marchesi I, et al. Sputum analysis: non-invasive early lung cancer detection. Journal of cellular physiology 2013; 228:945-51. 
         6. Travis W D, Brambilla E, Noguchi M, et al. Diagnosis of lung cancer in small biopsies and cytology: implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Archives of pathology &amp; laboratory medicine 2013; 137:668-84. 
         7. Keijzer R, van Tuyl M, Meijers C, et al. The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development. Development 2001; 128:503-11. 
         8. Tian Y, Zhang Y, Hurd L, et al. Regulation of lung endoderm progenitor cell behavior by miR302/367. Development 2011; 138:1235-45. 
         9. Zhang Y, Rath N, Hannenhalli S, et al. GATA and Nkx factors synergistically regulate tissue-specific gene expression and development in vivo. Development 2007; 134:189-98. 
         10. Kolla V, Gonzales L W, Gonzales J, et al. Thyroid transcription factor in differentiating type II cells: regulation, isoforms, and target genes. American journal of respiratory cell and molecular biology 2007; 36:213-25. 
         11. Guo M, Akiyama Y, House M G, et al. Hypermethylation of the GATA genes in lung cancer. Clinical cancer research: an official journal of the American Association for Cancer Research 2004; 10:7917-24. 
         12. Gorshkova E V, Kaledin V I, Kobzev V F, Merkulova T I. Codon 12 region of mouse K-ras gene is the site for in vitro binding of transcription factors GATA-6 and NF-Y. Biochemistry Biokhimiia 2005; 70:1180-4. 
         13. Lindholm P M, Soini Y, Myllarniemi M, et al. Expression of GATA-6 transcription factor in pleural malignant mesothelioma and metastatic pulmonary adenocarcinoma. Journal of clinical pathology 2009; 62:339-44. 
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     All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by a person skilled in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.