Patent Publication Number: US-2011053804-A1

Title: Gene Signatures for the Prognosis of Cancer

Description:
This application claims the benefit of the filing date of U.S. Provisional Application No. 61/072,851, filed on Apr. 3, 2008, the disclosure of which is incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention provides a gene expression signature useful to predict the risk of lung metastases in a breast cancer patient. Moreover, the signature of the present invention is useful to predict the time duration of lung metastasis free survival in a cancer patient. Further, this invention can predict the likelihood of responsiveness to anti-transforming growth factor beta (TGFβ) pathway therapy of a specific cancer tumor. 
     BACKGROUND OF THE INVENTION 
     Metastasis refers to the spread of cancerous cells from the site of the primary tumor to non-contiguous organs. The presence or absence of metastasis often determines treatment as well as survival. The prediction of metastatic potential is thus an important component of cancer management. The overexpression or underexpression of certain genes has been shown to be related to the propensity of tumors to metastasize to the lungs (Minn et al., 2005) or bones (Kang et al., 2003b; Lynch et al., 2005; Yin et al., 1999). In some cases, the overexpression of certain genes has been linked to the production of, or responsiveness to, certain mediators that can actually influence the tumor cells and confer on them the ability to seed other organs and survive there. The microenvironment of the tumor, including the presence of cytokines, growth factors and proteases, could influence the ability of tumor cells to metastasize (Sleeman et al., 2007, McSherry et al., 2007). The cytokine TGFβ that has been implicated in the modulation of tumor progression in various experimental systems (Akhurst and Derynck, 2001; Bierie and Moses, 2006; Dumont and Arteaga, 2003; Siegel and Massagué, 2003; Wakefield and Roberts, 2002). Low expression levels of TGFβ receptors in ER− tumors is associated with better overall outcome (Buck et al., 2004), whereas overexpression of TGFβ is associated with a high incidence of distant metastasis (Dalal et al., 1993). 
     A previously described gene signature has been shown to be predictive of lung metastasis of cancer. The LMS is a set of 17 genes (Table 1) whose expression in ER− tumors indicates a high risk of pulmonary relapse in patients (Minn et al., 2007). Several of these genes have been validated as mediators of lung metastasis (Gupta et al., 2007a; Gupta et al., 2007b; Gupta, 2007; Minn et al., 2005). 
     SUMMARY OF THE INVENTION 
     The cytokine TGFβ, in the tumor microenvironment, primes cancer cells for metastasis to the lungs. A TGFβ gene response signature reveals a clinical association between TGFβ activity in primary estrogen receptor negative (ER−) tumors and risk of lung metastasis. Further, combining the gene signature of the present invention with the known lung metastasis signature (LMS) increases the predictive value of the LMS considerably. 
     TGFβ response status (TBRS) can be determined by comparing expression levels of a panel of genes from cancer cells to the expression levels of the same genes in epithelial cell lines before and after induction with TGFβ. While a total of 153 genes were found to be involved in the response, smaller subsets of these genes may be used to determine the signature. 
     The TBRS provides a method of diagnosing metastatic potential of cancer comprising obtaining a diagnostic signature from cancer cells indicative of the metastatic potential of the cancer cells, wherein this diagnostic signature is obtained by measuring levels in cancer cells from the patient of five or more markers selected from the group of genes typifying the TGFβ response in human epithelial cells. This diagnostic signature is compared to a control signature; and based on the comparison, a prognosis of a high risk for metastasis is given if the diagnostic signature is different from the control signature by at least a threshold amount. 
     This method may be used in melanoma, breast cancer, colon carcinoma, and other types of cancer. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIGS. 1A-C . (A) and (B) Kaplan-Meier curves representing the probability of cumulative lung (left panel) or bone (right panel) metastasis-free survival for this cohort. Tumors are categorized according to their TBRS and ER status. (C) Lung metastasis-free survival restricted to patients with ER− negative tumors. Patients were categorized according to their TBRS and LMS status. 
         FIGS. 2A-E . (A) Schematic of lung metastasis assay from an orthotopic breast cancer inoculation. (B) Immunoblots using indicated antibodies were performed on whole-cell extracts from control, Smad4 knockdown, and Smad4-Rescue LM2 cells. (C) Mice injected with 5×10 5  cells into the fourth mammary fat pads were measured for tumor size at day 28. (D) Blood from tumor-bearing mice was isolated and red blood cells lysed. RNA from the remaining cells was extracted for qRT-PCR. The presence of circulating tumor cells was assessed as a function of human-specific GAPDH expression relative to murine β2-microglobulin, in 3 mL of mouse blood perfusate. (E) Bioluminescent quantification of lung metastases elicited from orthotopically implanted breast tumors. Orthotopic tumors were grown to approximately 300 mm 3 , mastectomies were performed and lung metastases were quantified using bioluminescence imaging seven days later. 
         FIGS. 3A-D . (A) LM2 cells and a clinically-derived pleural effusion sample (CN34.2A) were pretreated with TGFβ for 6 h. LM2 (2×10 5 ) and CN34.2A (4×10 5 ) cells were injected into the lateral tail vein and lung colonization was analyzed by in vivo bioluminescence imaging. (B) Bar graph represents 24 h time point measurements of the normalized photon flux from animals injected with either LM2 or CN34.2A cells. (C) Bone metastasis assays were performed by intracardiac injection of LM2 or BoM-1833 cells (3×10 4 ). Samples were pretreated with 100 μM of TGFβ for 6 h and compared to an untreated control. (D) Bar graphs represent seven-day time point analysis of the normalized photon flux from the mouse hind limbs. 
         FIGS. 4A-C . (A) Microarray and qRT-PCR analysis for the four epithelial cell lines treated with TGFβ. Fold change values for the TGFβ induction of ANGPTL4 are indicated. (B) Box-and-whisker plot comparing ANGPTL4 and NEDD9 TBRS-negative and -positive ER-negative tumors from the MSK/EMC cohorts. (C) TGFβ-induced changes in the mRNA expression of LMS genes in a panel of clinically derived pleural effusion samples and LM2 cells. Cells were treated with 100 μM of TGFβ for 3 h and analyzed by qRT-PCR using primers for the indicated genes. ER status for each is shown. 
         FIGS. 5A-J . Box-and-whisker plots comparing the RNA expression of TGFβ1, TGFβ2, TGFβ3, and LTBP1 TGFβR1, TGFβR2, TGFβR3, SMAD2, SMAD3 and SMAD4 in TBRS positive or negative primary breast tumors. 
         FIGS. 6A-B  Kaplan-Meier curves of brain and liver metastasis-free survival from ER negative breast cancer patients either TBRS positive or negative. 
         FIGS. 7A-D  Kaplan-Meier curves of ER negative breast cancer patients comparing TBRS status and other markers of poor clinical outcome such as large size, poor-prognosis signature positive, wound signature positive, and basal molecular subtype tumor designation. 
         FIGS. 8A-D  Kaplan-Meier curves of breast cancer patients comparing LMS status and other markers of poor clinical outcome such as large size, poor-prognosis signature positive, wound signature positive, and basal molecular subtype designation. 
         FIGS. 9A-C  Analysis of  FIG. 1  data using ER status determined by microarray probe levels rather than using clinical pathology designations. 
         FIG. 10  Kaplan-Meier curves of melanoma patients comparing 153 gene TBRS signatures. 
         FIGS. 11A-B  Kaplan-Meier curves of melanoma patients comparing 20 gene TBRS signatures. 
     
    
    
     DESCRIPTION OF THE INVENTION 
     The present invention is based on the identification of a set of genes that can predict the risk of lung metastases developing in a cancer patient. 
     Method for Diagnosing Metastatic Potential of Cancer Cells 
     One embodiment of the present invention is a method of determining the TGFβresponse status (TBRS) in cancer cells from a cancer patient. In another embodiment of the present invention, expression levels of all the 153 genes listed in Table 2 (TBRS153) are evaluated in a sample of cancer cells from a cancer patient using Affymetrix chips and these expression levels are, then compared, using statistical analysis, to the expression levels of the same genes in four epithelial cell lines before and after induction with TGFβ. According to the results of this comparison, the tumor of the patient is classified as either TBRS+ or TBRS− based on whether markers are at a higher or lower level than a threshold level. If the tumor is classified as TBRS+ and the tumor is further determined to be estrogen receptor negative, the cancer patient is determined to have a higher risk of lung metastasis than if the tumor was classified as TBRS−. This TBRS or signature can be determined with a combination of the 153 genes, in addition to looking at additional genes. 
     It will be appreciated that the determination of specific numerical values for the threshold is dependent on the particular tests that are included in the formation of the signatures, and the level of risk that is to be assigned as high risk. By way of example, however, when the markers are tested using the procedures described below, and the sum of the expression levels is the genetic signature, the threshold level is suitably the sum of the expression levels of the control signature plus one standard deviation for the control signature. 
     A cancer cell with a TBRS+ signature that is also estrogen receptor negative is more likely to metastasize. Therefore, it would be useful to test a cancer cell that is TBRS+ for the presence of estrogen receptor. 
     Another embodiment of the invention is a subset of the 153 genes listed in Table 2 comprising 50 genes (Table 3), the expression levels of those 50 genes, when used in combination, have been found to allow correlation with the risk of lung metastasis in ER− cancer patients that are positive for LMS (Table 1), or that have tumors larger than 2 cm or that are positive for the wound signature or that have a basal subtype of ER− tumors. 
     Another embodiment of the invention is a subset of the 50 genes listed in Table 3 comprising 20 genes (Table 4), the expression levels of those 20 genes, when used in combination, have been found to allow correlation with the risk of lung metastasis in ER− cancer patients that are positive for LMS, or that have tumors larger than 2 cm or that are positive for the wound signature or that have a basal subtype of ER− tumors. 
     Another embodiment of the invention is a subset of at least 5 genes out of the 50 genes listed in Table 3 the expression levels of which, when used together, have been found to correlate with the risk of lung metastasis in ER− cancer patients that are positive for LMS, or that have tumors larger than 2 cm or that are positive for the wound signature or that have a basal subtype of ER− tumors. Many different gene sets of less than the 50 genes of Table 2 can be generated from the group of 50 genes in the TBRS-50, as many of these 50 genes individually show a p value of &lt;0.05 in the correlation of their expression with TGFβ responsiveness. Tables 4 and 5 list different TBRS signatures with associated p values. It should be noted that certain genes are more commonly used in these signatures with high correlation levels. For example, BHLHB2, COL4A1 are used in five out of the six panels, CCDC93, JAG1, JUN, NR2F2, RAI2, RBMS1, ZFP36L1 are all used in four out of the six, and AGXT2L1, ALOX5AP, C6orf145, FAT4, FHL3, GADD45B, HMOX1, SERPINE1, SMTN, SMURF1, SPSB1, TNFRSF12A are used in three out of the six. 
     The expression levels of the genes used in the signatures of the present invention can be determined with commercially available test materials, as described below. However, it will be appreciated that the specific method of determination is not critical and that any method may be used. When a new method or platform for determining gene expression levels is used, a new standard curve for establishing TBRS positively and negativity of a sample has to be established using a large number of tumor samples, the number of tumor samples needed depending on the level of statistical confidence required. 
     Another embodiment of the present invention is a method to determine if a cancer patient would benefit from anti TGFβ therapy, the method comprising determining whether the cancer cells from that patient are TBRS positive or negative using any of the gene signatures of the present invention. A TBRS positive result makes the patient likely to benefit from anti TGFβ therapy. 
     The ER− tumors that are independently assessed to be LMS+ and TBRS+ are determined to have higher risk (50%) of lung metastasis. Therefore, it would be useful to test a TBRS+ cancer cell for LMS as well. In addition, it would be useful to test a TBRS+ cancer cell for LMS and estrogen receptor. 
     If a cancer is identified as one that has a high risk for metastasis to the lungs, management of this patient can take a more defined approach and involve: 
     (1) more aggressive treatment because the tumor has a higher risk of metastasis; 
     (2) more frequent follow-ups, with a focus on diagnostic imaging procedures in the lung 
     Since there appears to be a link between responsiveness to TGFβ and metastatic potential, the genes comprising the TGFβ response signature or their encoded proteins, are suitable targets for therapy. Therapy can be achieved by reducing the expression of an overexpressed gene, or by directly inhibiting the expressed protein. Targeting of one or more of these genes, for example using antisense or RNAi techniques could reduce lung metastasis activity. 
     Several studies have probed the link between TGFβ and lung metastasis. The study that is the basis of this invention sought to clarify the link between TGFβ responsiveness and metastatic potential. Using gene expression analysis, it was found that there is a definite connection between response to TGFβ and probability of metastasis to the lungs but not to other organs such as bone or liver. The basis for this connection seems to be TGFβ induction of angiopoietin-like 4 (ANGPTL4) in cancer cells that are about to enter the circulation which enhances their subsequent retention in the lungs 
     Moreover, the TBRS status of a tumor can be used to determine which cancer patient should be treated with therapies aimed at reducing or eliminating TGFβ pathway signaling. If a cancer patient is determined to be TBRS+ using any of the gene signatures of the present invention then that patient can be said to be eligible for, and have a high chance of benefiting from, such anti TGFβ therapy or therapies. 
     Kit for Diagnosing Metastatic Potential for Cancer Cells 
     A kit may be used to determine metastatic potential. The kit contains reagents for determining expression levels of at least five markers selected from the group of genes typifying the TGFβ response in human epithelial cells. This kit may contain a gene chip with a set five or more markers selected from the group of genes typifying the TGFβ response in human epithelial cells. The gene chip could be an Affymatrix chip that detects the expression level of the desired markers. 
     The kit may also contain a plate with wells for determining the expression level of five or more markers. This plate would have separate wells with reagents for each marker. 
     Development of a TGFβ Response Bioinformatics Classifier 
     In order to investigate the role of TGFβ in cancer progression, we set out to develop a bioinformatics classifier that would identify human tumors containing a high level of TGFβ activity. A gene expression signature typifying the TGFβ response in human epithelial cells was obtained from transcriptomic analysis of four human cell lines. These cell lines include HaCaT keratinocytes, HPL1 immortalized lung epithelial cells, MCF10A breast epithelial cells, and MDA-MB-231 breast carcinoma cells. The cells were treated with TGFβ1 for 3 h in order to capture direct TGFβ gene responses (Kang et al., 2003a). The resulting 153-gene TGFβ response signature (TBRS) (174 probe sets; Table 2) was used to generate a classifier by means of “meta-gene” analysis with the cell lines as references (Bild et al., 2006). The meta-gene analysis resulted in a continuous variable ranging from 0 to 1 that designates the relative level of TGFβ pathway activity in tissue samples. Using 0.5 as a threshold, most tumors could be unambiguously assigned to a TBRS− class or a TBRS+ class. When applied to metastatic lesions extracted from bones, lungs and other sites representing the natural metastatic spectrum of human breast cancer, the TBRS classifier identified TGFβ activity in a 38/67 of these samples (Table 6), which is in agreement with previous observations of activated Smad in a majority of human bone metastasis samples (Kang et al., 2005). 
     Development of a TGFβ Response Bioinformatics Classifier with Smaller Gene Sets 
     Among the 153 genes, 50 are univariately correlated with lung metastasis in ER− tumors. This is determined by the following procedures:
     1) Find the express values of each of the 153 genes in each of the tumor microarray;   2) Find the lung metastasis incidences and the length of lung metastasis-free-survival for each corresponding patient (from whom the tumor was resected);   3) Fit this information in a Cox proportional hazards regression model (to examine whether there is significant correlation between the expression of these genes with lung metastasis incidences or length of lung metastasis-free survival);   4) The 50 individual genes that yielded significant correlations (p&lt;0.05) were collected to form the TBRS50.   

     Combinations of any 5 or more genes from TBRS50 can also predict the risk of lung metastasis in ER− cancer patients. To try a particular combination (hereafter denoted as TBRS-x) from TBRS50 or from the original TBRS153, follow the procedures below:
     1) Find the expression values of the genes in TBRS-x in each of the ER− tumors;   2) Hierarchically cluster the tumors based on the gene expression values of TBRS-x. We used R statistical software, “gplots” package and “heatmap.2” command, with the default parameter setting.   3) A cluster of tumors that overexpress all the upregulated genes in TBRS-x and underexpress all the downregulated genes in TBRS-x can be readily distinguished from the rest of tumors. And the tumors in this cluster are called TBRS+ and the rest called TBRS−;
       TBRS+ and TBRS− tumors were compared in terms of number of lung metastasis incidences and the length of lung metastasis-free survival in the corresponding ER− patients. A p value is calculated based on log-likelihood to denote the significance of the difference. We used R statistical software, “survival” package and “survdiff” command to implement this comparison.   
       

     By using this protocol any expert in the art can find gene signatures that can determine statistically significant TBRS that can be used in the present invention. As an example, but without being limited, Table 5 lists 5 different TBRS signatures (and its associated p values) that can be used in the present invention. Ideally one would choose a signature with the highest p value but in choosing a specific signature other considerations might be more important, for example a signature that uses few genes to be able to create simpler or cheaper signatures that are more easily incorporated into a commercial product. 
     Hierarchical clustering was performed on the MSK/EMC cohort with the indicated pathological and genomic markers including the TBRS, the lung metastasis signature (LMS), the wound response signature (Wound), the 70-gene prognosis signature (70-gene), size (Size&gt;2 cm), the basal molecular subtype (Basal), and the ER status. 
     TGFβ Activity in Primary Breast Tumors is Selectively Linked to Lung Metastasis 
     We applied the TBRS classifier to a series of primary breast carcinomas that were analyzed on the same microarray platform (Minn et al., 2007; Minn et al., 2005; Wang et al., 2005). This series includes 82 tumors collected at Memorial Sloan-Kettering Cancer Center (MSK cohort) and 286 tumors from the Erasmus Medical Center (EMC cohort). Both cohorts comprised a mix of breast cancer subtypes, with tumors in the MSK cohort being more locally advanced than those in the EMC cohort (Minn et al., 2007). Out of a combined total of 368 patients, 39 patients developed lung metastases and 83 developed bone metastasis after a median follow-up of 10 years, with some patients developing metastasis in both sites. TBRS+ tumors were similarly distributed between estrogen receptor-positive (ER+) and ER− tumors. Microarray analysis revealed that the TBRS+ tumors expressed significantly higher mRNA levels for TGFα1, TGFβ2, and the latent TGFβ activating factor, LTBP1. TBRS− tumors had lower mRNA levels for type II TGFβ receptor, Smad3 and Smad4. The expression level of other TGFβ pathway components was independent of TBRS status. 
     TBRS status in ER+ tumors did not correlate with distant metastasis. However, in ER− tumors there was a striking association between TBRS+ status and relapse to the lungs ( FIG. 1A ). This association was observed regardless of whether the tumor ER status was assigned using the clinical pathology reports, which are based on immunohistochemical analysis ( FIG. 1A ), or using a microarray probe level designation ( FIG. 9A ). No link was observed between TBRS status and bone metastasis ( FIGS. 1A ,  9 A) or liver metastasis, and a brain metastasis association did not attain statistical significance ( FIG. 6 ). In univariate as well as multivariate analyses, the expression level of TGFβ pathway components was much inferior to the TBRS at linking these tumors with metastasis outcome (Table 7). These results indicate that TGFβ activity in ER− breast tumors is selectively associated with lung metastasis. 
     Cooperation Between TGFβ and the Lung Metastasis Signature 
     The association of TBRS with lung relapse prompted us to search for links between the TBRS and a previously described lung metastasis signature (LMS) (Minn et al., 2005). The LMS is a set of 17 genes whose expression in ER− tumors indicates a high risk of pulmonary relapse in patients (Minn et al., 2007). Several of these genes have been validated as mediators of lung metastasis (Gupta et al., 2007a; Gupta et al., 2007b; Gupta, 2007; Minn et al., 2005). The TBRS+ subset of ER− tumors partially overlapped the LMS+ subset. Remarkably, tumors that were positive for both the TBRS and LMS were associated with a high risk of pulmonary relapse, whereas single-positive tumors were not ( FIG. 1B ). Within poor-prognosis tumor subsets defined by other features, such as size&gt;2 cm, basal subtype gene-expression signature (Sorlie et al., 2003), 70-gene poor prognosis signature (van de Vijver et al., 2002), or wound signature (Chang et al., 2005), TBRS status was associated with risk of lung metastasis in nearly every case. The TBRS performed independently of these other prognostic features ( FIG. 7 ), as did the LMS ( FIG. 8  (Minn et al., 2007). 
     TGFβ Signaling in Mammary Tumors Enhances Lung Metastatic Dissemination 
     To functionally test whether TGFβ signaling in primary tumors contributes to lung metastases, we used a xenograft model of ER− breast cancer metastasis (Minn et al., 2005). The MDA-MB-231 cell line was established from the pleural fluid of a patient with ER− metastatic breast cancer (Cailleau et al., 1978). MDA-MB-231 cells have a functional Smad pathway and evade TGFβ growth inhibitory responses though alterations downstream of Smads (Gomis et al., 2006). The lung metastatic subpopulation LM2-4175 (henceforth LM2) was isolated by in vivo selection of MDA-MB-231 cells (Minn et al., 2005). We perturbed the TGFβ pathway in LM2 cells by overexpressing a kinase-defective, dominant-negative mutant form of the TGFβ type I receptor (Weis-Garcia and Massagué, 1996), or by reducing the expression of Smad4, which is an essential partner of Smad2/3 in the formation of transcriptional complexes (Massaguéet al., 2005). Using a validated SMAD4 short-hairpin RNA (shRNA) (Kang et al., 2005) we reduced Smad4 levels by 80-90% in LM2 cells ( FIG. 2B ). As a control, we generated SMAD4 rescue cells by expressing a shRNA-resistant SMAD4 cDNA in SMAD4 knockdown cells ( FIG. 2B ). 
     Neither the dominant negative TGFβ receptor nor the Smad4 knockdown decreased mammary tumor growth as determined by tumor volume measurements, or the extent of tumor cell passage into the circulation, as determined by qRT-PCR analysis of human GAPDH mRNA in blood cellular fractions ( FIGS. 2C ,  2 D). Tumors inoculated into the mammary glands of immunocompromised mice and allowed to grow to 300 mm 3 , were surgically removed and the emergence of distant metastases after the mastectomy was determined ( FIG. 2A ). Inactivation of TGFβsignaling markedly inhibited the lung metastatic activity of the tumors as determined by quantitative luciferase bio-luminescence imaging ( FIG. 2E ) (Ponomarev et al., 2004) and histological examination. These results suggest that the canonical TGFβ pathway enhances mammary tumor dissemination to the lungs. 
     TGFβ Primes Tumor Cells to Seed of Lung Metastases 
     We wondered whether TGFβ within the tumor microenvironment could endow tumor cells with the ability to seed the lungs as these cells enter the circulation. To test this possibility, we mimicked the exposure of tumor cells to TGFβ by incubating LM2 cells with TGFβ for 6 h prior to inoculation of these cells into the tail vein of mice. Interestingly, this pre-treatment with TGFβ significantly increased the lung colonizing activity of LM2 cells, as determined by a higher retention of these cells in the lungs 24 h after inoculation ( FIG. 3A ). In this time frame LM2 cells extravasate into the lung parenchyma (Gupta et al., 2007a). A similar effect was observed when we carried out this experiment with malignant cells (CN34.2A) obtained from the pleural fluid of a breast cancer patient treated at MSKCC. The pre-treatment with TGFβ increased the lung seeding activity of LM2 and CN34.2A cells three- and five-fold, respectively ( FIG. 3B ). The initial advantage provided by a transient exposure to TGFβ was sustained but not expanded during the ensuing outgrowth of metastatic colonies ( FIG. 3A ). 
     To investigate the selectivity of this lung metastasis-priming effect, we tested the effect of TGFβ pre-incubation on the establishment of bone metastases. LM2 cells have limited bone metastatic activity in addition to their high lung metastatic activity (Minn et al., 2005). The pre-treatment of LM2 cells with TGFβ prior to their inoculation into the arterial circulation did not increase the ability of these cells to form bone metastases ( FIG. 3C ). We also tested the effect of TGFβ on the metastatic activity of an MDA-MB-231 sub-population (BoM-1833) that is highly metastatic to bone (Kang et al., 2003b) and responsive to TGFβ (Kang et al., 2005). Pre-incubation of BoM-1833 cells with TGFβ did not increase their ability to form bone metastases ( FIG. 3C ), and had no discernible effect on the early seeding of the bones ( FIG. 3D ). Thus, TGFβ stimulation primes tumor cells for an early step in lung metastasis but not bone metastasis, which is concordant with the selective association of TBRS+ status in primary tumors with risk of lung metastasis in clinical cohorts (refer to  FIG. 1A ). 
     Assessment of TBRS Status 
     The assessment of TBRS status can be carried out in one of two ways. The first method involves the performance of a “meta-gene” analysis based on the TBRS50 gene set and using the cell lines as references (Bild et al., 2006). For each tumor, a number between 0 and 1 is derived, indicating the likelihood that the TGFβ signaling is active in that tumor. The tumor being tested is also assigned a score based on the gene expression of TBRS50 and thus TBRS status is determined. The second method involves clustering tumors with known TBRS50 expression levels based on these levels and identifying where a tumor from a patient with unknown TBRS status fits into this cluster map. 
     Protocol for Assessing TBRS Status in a Patient with a Tumor Using Affymetrix Chips 
     Example 1 
     Protocol I, Metagene Analysis 
     The tumor sample is profiled with Affymetrix U133Plus2 or U133a chips, using 5 ug RNA and the standard protocol recommended by the manufacturer. The data are pre-processed using RMA algorithm (available in affy package). The median of expression values of all genes are set to 0. The expression values of TBRS50 are found. The cell line data are merged with the patient data to get a matrix of 13×50 (designated as tbrs hereafter), where there are 13 columns (12 cell lines plus one patient) and 50 rows (gene expression values of the TBRS50 genes). Principle component analysis (PCA) is performed on the matrix (using command prcomp(t(tbrs)), default setting). The first principle component of the 12 cell lines derived from above analysis is used to train a Bayesian probit model using the package MCMCpack, command MCMCprobit, with parameters: thin=100, burnin=100000, mcmc=100000, seed=6032005. The first principle component of the patient (derived from step 8) is fit to the probit model in the above step, using the script in appendix.in R. A score between 0 and 1 will be generated. A score above 0.5 can be considered as positive for the TBRS. 
     Protocol II, Unsupervised Clustering: 
     The tumor sample is profiled with Affymetrix U133Plus2 or U133a chips, using 5 ug RNA and the standard protocol recommended by the manufacturer. The data are pre-processed using RMA algorithm (available in affy package). The median of expression values of all genes are set to 0. The expression values of TBRS50 are found. The MSK and Erasmus datasets are obtained from the GEO database (accession numbers are GSE2603 and GSE2034, respectively). The two datasets are combined. The tumor data are normalized the same way as above. The subset patients with negative ER status are located (annotations available with the datasets at the GEO website). The TBRS50 in ER− patients are obtained. The datasets from the 368 tumors are combined with the data from the patient with the unknown TBRS status to get a matrix of N+1 columns and 50 rows (N=number of ER− patients). The matrix is clustered using command heatmap.2 in package gplots. For better visualization, the following parameters can be used: scale=“row”, col=greenred(100), trace=“none”. Two major clusters will be revealed. One will display significantly higher expression of the vast majority of the TBRS and will be designated as TBRS+. The other cluster will be denoted as “TBRS-”. The patient with unknown TBRS status will be located on this map and it will be possible to determine the cluster at which it is located. The TBRS status of this patient will be determined according to the cluster it resides. 
     Note: All algorithms were implemented in R statistical software. 
     Script for Fitting the MCMCprobit Model: 
       
                                            predict.mean &lt;- function(A, B)           {             A &lt;- as.matrix(A)             I &lt;- dim(A)[1]             y &lt;- rep(NA, I)             for(i in 1:I)             {               x &lt;- c(1, A[i,])               eta &lt;- as.vector(x)%*%t(as.matrix(B))               p &lt;- pnorm(eta)               y[i] &lt;- mean(p)             }             y           }                       A: principal components of the patient;           B: the MCMCprobit model.            
Protocol for Assessing TBRS Status in a Patient with a Tumor Using Hybridization Technique Other than Affymetrix Chips
 
     Each time a new method of determining the RNA levels for TBRS50 is used, a new standard curve has to be set up first to estimate the distribution of each gene among patients. A gene could be easily detected using hybridization based technology or with polymerase chain reaction technology, but the absolute intensity of signal in a single patient can be influenced by many factors, including the efficiency of hybridization, the amount and quality of total RNA loaded, etc. All these need to be normalized using a statistically significant number of patients, preferentially with known clinical outcomes (just like points on a standard curve). Then additional patients could be interrogated and compared with the standard curve. Then the prognosis can be made. 
     Combining TBRS and LMS Signatures for Better Prediction of Lung Metastasis Prognosis 
     When the same set of tumors was tested for TBRS positively and LMS positively, there was a subset of ER− tumors that tested positive for both. Remarkably, tumors that were positive for both the TBRS and LMS were associated with a high risk of pulmonary relapse, whereas single-positive tumors were not. So, for a more refined approach towards predicting lung metastasis potential of cancers, the patient sample can be tested for TBRS as well as LMS status. The tumors that test positive for both signatures can be treated with a view to minimizing future lung metastasis. 
     Example 1 
     TGFβ Response Gene-Expression Signature and TBRS Classifier 
     Cell lines with and without TGFβ1 treatment (3 h, 100 μM) were subject to expression profiling using Affymetrix U133A or U133 plus2 microchips. Microarray results were pre-processed using RMA algorithm (carried with affy package of R statistical program). The first comparison was conducted between all TGFβtreated samples versus all untreated samples. Three hundred and fifty genes that yielded a p value of 0.05 or less (after Benjamini and Hochberg correction for multiple tests) were kept. Among these genes, we chose to focus on the genes that are significantly changed in at least two different cell lines when the cell lines are considered separately. This step resulted in 174 probe sets corresponding to 153 distinct human genes, which were collectively designated as the TGFβ gene response signatures. 
     To generate a TBRS classifier, we carried out a “meta-gene” analysis based on this gene set and using the cell lines as references (Bild et al., 2006) and references therein. In short, expression values of the 153 TGFβ responsive genes in cell lines were linearly transformed and encapsulated into one or two “Meta genes”. A Bayesian Probit model was then trained based the cell line data and applied to the Meta genes of the tumor samples. For each tumor, a number between 0 and 1 was derived, indicating the likelihood that the TGFβ signaling is active in that tumor. 
     Example 2 
     Cell Culture and Reagents 
     MDA-MB-231 and its metastatic derivatives LM2-4175 and BoM-1833 have been described previously (Kang et al., 2003b; Minn et al., 2005). Breast carcinoma cells were isolated from the pleural effusion of patients with metastatic breast cancer treated at our institution upon written consent obtained following IRB regulations as previously described (Gomis et al., 2006). BCN samples were obtained and treated as per Hospital clinic de Barcelona guidelines (CEIC-approved). 
     TGFβ and TGFβ-receptor inhibition used 100 pM TGFβ1 (R&amp;D Systems) for 3 or 6 h as indicated and 10 μM SB431542 (Tocris) with 24 h pretreatment. Epithelial cell lines were treated for 3 h with BMP2 (25 ng/mL, R&amp;D), Wnt3a (50 ng/mL, R&amp;D), FGF (5 ng/mL, Sigma), EGF (100 ng/mL, Invitrogen), IL6 (20 ng/mL, R&amp;D), VEGF-165 (100 ng/mL, R&amp;D), and IL1β (100 ng/mL, R&amp;D). Conditioned media experiments were performed by growing cells in serum-deprived media for 48 hours. Recombinant human Angpt14 (Biovendor) was used at 2.5 μg/mL for 24 h. 
     Example 3 
     RNA Isolation, Labeling, and Microarray Hybridization 
     Methods for RNA extraction, labeling and hybridization for DNA microarray analysis of the cell lines have been described previously (Kang et al., 2003b; Minn et al., 2005). The EMC and MSK tumor cohorts and their gene expression data have been previously described (Minn et al., 2007; Minn et al., 2005; Wang et al., 2005). Bone or lung recurrence at any time is indicated. 
     Example 4 
     Generation of Retrovirus and Knockdown Cells 
     Knockdown of SMAD4 and ANGPTL4 was achieved using pRetroSuper technology (Brummelkamp et al., 2002) targeting the following 19-nucleotide sequences: 5′-GGTGTGCAGTTGGAATGTA-3′ (SEQ. ID No. 1) (SMAD4) and 5′-GAGGCAGAGTGGACTATTT-3′ (SEQ. ID No. 2) (ANGPTL4). To produce retrovirus for knockdown, the hairpin vector was transfected into the GPG29 amphotropic packaging cell line (Ory et al., 1996). 
     Example 5 
     Immunofluorescence 
     HUVECs were grown to confluence on fibronectin coated chamber slides (BD Biosciences). The cells were fixed for 10 min in 4% paraformaldehyde in PBS, and incubated for 5 min on ice in 0.5% Triton X-100 in PBS. After blocking with 2% BSA, the monolayers were processed for staining with anti-ZO1 (Zymed), anti-beta-catenin (Santa Cruz), rhodamine phalloidin (Molecular Probes) for F-actin staining and DAPI (Vector Labs) for nuclear staining. Fluorescence images were obtained using an Axioplan2 microscopy system (Zeiss). 
     Example 6 
     Animal Studies 
     All animal work was done in accordance with a protocol approved by the MSKCC Institutional Animal Care and Use Committee. NOD/SCID female mice (NCI) age-matched between 5-7 weeks were used for xenografting studies. For experimental metastasis assays from orthotopic inoculations, the tumors were extracted from both mammary glands when they each reached 300 mm 3 , approximately 30 days. Seven days after mastectomies, lung metastases were monitored and quantified using non-invasive bioluminescence as previously described (Minn et al., 2005). 
     Example 7 
     In Vivo Lung Permeability Assays 
     To observe in vivo permeability of lung blood vessels, tumor cells were labeled by incubating with 5 μM cell tracker green (Invitrogen) for 30 min and inoculated into the lateral tail vein. One day post inoculation, mice were injected intravenously with rhodamine-conjugated dextran (70 kDa, Invitrogen) at 2 mg per 20 g body weight. After 3 h, mice were sacrificed; lungs were extracted and fixed by intra-tracheal injection of 5 mL of 4% PFA. Lungs were fixed-frozen and 10 μm sections were taken to be examined by fluorescence microscopy for vascular leakage. Images were acquired on an Axioplan2 microscopy system (Zeiss). To analyze, a uniform ROI of approximately 3 nuclei in diameter was drawn around the tumor cells and applied to each image. A second larger ROI was also applied with similar results. Signal from the ROI was quantified using Volocity (Improvision). 
     Example 8 
     Statistical Analysis 
     Results are reported as mean±standard error of the mean unless otherwise noted. Comparisons between continuous variables were performed using an unpaired one-sided t-test. Statistics for the orthotopic lung metastasis assays were performed using log-transformation of raw photon flux. 
     Example 9 
     Cell Culture and Reagents 
     HaCaT were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin, and fungizone. MCF-10A cells were maintained in a 1:1 mixture of DMEM and Ham&#39;s F12 supplemented with 5% horse serum, 10 μg/ml insulin (Sigma), 0.5 μg/ml hydrocortisone (Sigma), 0.02 μg/ml epidermal growth factor (Sigma), and antibiotics. HPL1 cells were maintained in Ham&#39;s F12 supplemented with 1% FBS, 5 μg/ml insulin, 0.5 μg/ml hydrocortisone, 5 μg/ml transferrin (Sigma), 2×10 10 M triiode thyronine, and antibiotics. All tumor cell lines were cultured in DMEM supplemented with 10% FBS, glutamine, penicillin, streptomycin and fungizone. The pleural effusion samples were centrifuged at 1,000 r.p.m. for 10 min, cell pellets were re-suspended in PBS and treated with ACK lysis buffer to lyse blood cells. A fraction of these cells underwent negative selection to remove leukocytes (CD45 +  and CD15 +  cells), and EpCam-positive cells were sorted from the population upon recovery in tissue culture for 24 h. Human vascular endothelial cells (HUVECs) (ScienCell) were cultured in complete ECM media (ScienCell) and used between passages 3-6. The retroviral packaging cell line GPG29 was maintained in DMEM containing 10% FBS supplemented with puromycin, G418, doxycycline, penicillin, streptomycin and fungizone. Transfections were done using standard protocols with Lipofectamine 2000 (Invitrogen). After transfection, GPG29 cells were cultured in DMEM containing 10% FBS. 
     Example 10 
     RNA Isolation, Labeling, and Microarray Hybridization 
     The tissues for microarray analysis were obtained from therapeutic procedures performed as part of routine clinical management. Samples were snap-frozen in liquid nitrogen and stored at −80 C. Each sample was examined histologically in cryostat sections stained with hematoxylin and eosin. Regions were dissected manually from the frozen block to provide a consistent tumor cell content of greater than 70% in tissues used for analysis. RNA was extracted from frozen tissues by homogenization in TRIzol reagent (Gibco/BRL) and evaluated for integrity. Complementary DNA was synthesized from total RNA by using a dT primer tagged with a T7 promoter. The RNA target was synthesized by transcription in vitro and labeled with biotinylated nucleotides (Enzo Biochem). The labeled target was assessed by hybridization to Test3 arrays (Affymetrix). All gene expression analysis was performed with an HG-U133A GeneChip (Affymetrix). Gene expression was quantified with MAS 5.0 or GCOS (Affymetrix). All studies involving patient materials or data were conducted under protocols approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center, and that of the Hospital Clinic de Barcelona. 
     For the cell culture experiments, total RNA was prepared from 5×10 6  cultured cells that were untreated or treated with TGFβ. Twenty-five micrograms of total RNA was used to prepare cRNA probe using a Custom Superscript Kit (Invitrogen) and the BioArray HighYield RNA Transcript Labeling Kit (Enzo). Each sample was hybridized with an Affymetrix Human Genome U133A microarray for 16 hr at 45° C. 
     Example 11 
     Generation of Retrovirus and Knockdown Cells 
     Viruses were collected 48 and 72 h after transfection, filtered, and concentrated by ultracentrifugation. Concentrated retrovirus was used to infect cells in the presence of 8 μg ml −1  polybrene, typically resulting in a transduction rate of over 80%. Infected cells were selected with puromycin or hygromycin. To generate knockdown-rescue cell lines, we used a similar method to produce virus encoding complementary DNAs for overexpression of the RNAi-targeted genes, along with a hygromycin or puromycin selectable marker. The overexpressing retrovirus vector, pBabe, was used to super-infect previously generated knockdown cells that were subsequently selected with either hygromycin or puromycin. 
     Example 12 
     Analysis of mRNA and Protein Expression 
     Total RNA from subconfluent MDA-MB-231 cells was collected and purified using the RNeasy kit (Qiagen). Four-hundred nanograms of total purified RNA was subjected to a reverse transcriptase reaction according to the Hi-Capacity Archive kit (Applied Biosystems). cDNA corresponding to approximately 4 ng of starting RNA was used in three replicates for quantitative PCR. Indicated Taqman gene expression assays (Applied Biosystems) and the Taqman universal PCR master mix (Applied Biosystems) were used to quantify expression. Quantitative expression data were acquired and analyzed using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). For immunoblotting, we used previously described methods (Calonge and Massagué, 1999). Briefly, proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad) that were immunoblotted with mouse monoclonal antibodies that recognize Smad4 (Cell Signaling) and α-tubulin (Sigma). For analysis of secreted protein expression, cells were plated in triplicate at 90% confluency in 12-well plates, incubated in DMEM 0.2% FBS, and conditioned media was collected 72 h later. Media was cleared of cells by centrifuging at 2,000 r.p.m. for 5 min. Angpt14 concentrations were analyzed in conditioned media using an ELISA kits (BioVendor). 
     Example 13 
     Immunostaining 
     For visualizing lung metastases, mice were killed and perfused with PBS and 4% paraformaldehyde through the left ventricle. Lungs were fixed and paraffin-embedded. Immunohistochemical staining for vimentin (Novocastra) was performed on paraffin-embedded lung sections by the MSKCC Molecular Cytology Core Facility. Brightfield microscopic images were collected using an Axioplan2 microscopy system (Zeiss). 
     Example 14 
     Animal Studies 
     For primary tumor analysis, 5×10 5  viable single cells were re-suspended in a 1:1 mixture of PBS and growth-factor-reduced Matrigel (BD Biosciences) and injected orthotopically into both mammary gland number four in a total volume of 100 μL as previously described (Minn et al., 2005). Primary tumor growth rates were analyzed by measuring tumour length (L) and width (W), and calculating tumor volume based on the formula  II LW 2 /6. For lung colonization assays, 2×10 5  cells were re-suspended in 0.1 ml PBS and injected into the lateral tail vein. Lung metastatic progression was again monitored and quantified using bioluminescence. For bone metastasis, 30,000 cells in PBS were injected into the left ventricle of anaesthetized mice (100 mg kg −1  ketamine, 10 mg kg −1  xylazine). For priming assays, cells were switched to low media (0.2% FBS) for 12 hours and then treated with 100 μM of TGFβ for 6 hours. Mice were inoculated with 200,000 and 30,000 cells for lung and bone assays, respectively. Mice were imaged for luciferase activity immediately after injection to exclude any that were not successfully xenografted. Lymph node analysis was performed by ex vivo bioluminescent imaging of the peri-aortic lymph nodes. 
     TBRS in Metastatic Melanomas 
     Tissue samples were taken from patients with metastatic melanomas. The tissues were taken from skin or lymph node metastases of melanomas. The data analyzed are publicly available at Gene Expression Omnibus (GEO) database, accession number is GSE8401. The clinical annotations of these patients were published in Xu et al., 2008, Mol. Cancer Res., 6(5): 760-769. 
       FIG. 10  shows Kaplan-Meier curves obtained using the 153 gene signature. TBRS was applied to 52 metastatic malignant melanomas using the same approach described in Padua et al., 2008 and the previous section of this application. Patients were classified into two groups. The group with a positive pattern of TBRS expression (TBRS+) exhibits worse overall survival compared to TBRS− group, as shown by the Kaplan-Meier curves (median survival 5 months vs. 40 months). 
       FIGS. 11A and 11B  show Kaplan-Meier curves obtained using 20 gene signatures.  FIG. 11A  used the genes listed in Run 1 of Table 5 and  FIG. 11B  used the genes listed in Run 2 of Table 5. Again, the group with a positive pattern of TBRS expression (TBRS+) exhibits worse overall survival compared to TBRS− group. 
     TBRS in Colon Cancers 
     TBRS was applied to patients in accession number GSE5206. 35 patients are classified as TBRS+, among which 11 developed recurrences (31.4%). 65 patients are classified as TBRS−, among which only 1 patient developed recurrence (1.54%). The difference is highly significant (p=2.7e-5, Fisher&#39;s Exact Test). The description of GSE5206 can be found together with the microarray data at GEO: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE5206. This shows that the TBRS+ signature is implicated in recurrence on colon cancer. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 17-gene classifier (LM17) to predict the expression of the lung metastasis 
               
               
                 signature. 
               
               
                 Shown are the genes used in class prediction. In leave-one-out cross 
               
               
                 validation, support vector machine yielded a 97% correct classification rate, with a 
               
               
                 permutation p-value of less than 0.00005. Shown for each gene is the p-value to test the 
               
               
                 null hypothesis that the gene is differentially expressed between the classes used during 
               
               
                 training. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Parametric 
                   
                   
                 Gene 
               
               
                   
                 p-value 
                 Description 
                 GB acc 
                 symbol 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 &lt;0.000001 
                 singed ( Drosophila )-like (sea urchin fascin 
                 NM_003088 
                 FSCN1 
               
               
                   
                   
                 homolog like) 
               
               
                 2 
                 &lt;0.000001 
                 matrix metalloproteinase 1 (interstitial collagenase) 
                 NM_002421 
                 MMP1 
               
               
                 3 
                 &lt;0.000001 
                 PPAR (gamma) angiopoietin related protein 
                 NM_016109 
                 ANGPTL4 
               
               
                 4 
                 0.000006 
                 adipose specific 2 
                 NM_006829 
                 APM2 
               
               
                 5 
                 0.00002 
                 GRO1 oncogene (melanoma growth stimulating 
                 NM_001511 
                 CXCL1 
               
               
                   
                   
                 activity, alpha) 
               
               
                 6 
                 0.000355 
                 prostaglandin-endoperoxide synthase 2 
                 NM_000963 
                 PTGS2 
               
               
                   
                   
                 (prostaglandin G/H synthase and cyclooxygenase) 
               
               
                 7 
                 0.000444 
                 keratin, hair, basic, 1 
                 NM_002281 
                 KRTHB1 
               
               
                 8 
                 0.000506 
                 vascular cell adhesion molecule 1 
                 NM_001078 
                 VCAM1 
               
               
                 9 
                 0.000627 
                 retinoic acid receptor responder (tazarotene 
                 NM_004585 
                 RARRES3 
               
               
                   
                   
                 induced) 3 
               
               
                 10 
                 0.001263 
                 latent transforming growth factor beta binding 
                 NM_000627 
                 LTBP1 
               
               
                   
                   
                 protein 1 
               
               
                 11 
                 0.004365 
                 kynureninase (L-kynurenine hydrolase) 
                 NM_003937 
                 KYNU 
               
               
                 12 
                 0.005179 
                 chemokine (C—X—C motif), receptor 4 (fusin) 
                 NM_003467 
                 CXCR4 
               
               
                 13 
                 0.006426 
                 lymphocyte antigen 6 complex, locus E 
                 NM_002346 
                 LY6E 
               
               
                 14 
                 0.007153 
                 inhibitor of DNA binding 1, dominant negative 
                 NM_002165 
                 ID1 
               
               
                   
                   
                 helix-loop-helix protein 
               
               
                 15 
                 0.010871 
                 mannosidase, alpha, class 1A, member 1 
                 NM_005907 
                 MAN1A1 
               
               
                 16 
                 0.032361 
                 enhancer of filamentation 1 (cas-like docking; Crk- 
                 NM_006403 
                 NEDD9 
               
               
                   
                   
                 associated substrate related) 
               
               
                 17 
                 0.03713 
                 epiregulin 
                 NM_001432 
                 EREG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 The 153 genes in the TBRS. Gene symbols, gene description, and TGFβ 
               
               
                 induction levels are displayed. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 MDA-MB- 
                   
                   
                   
                   
               
               
                 Probes 
                 231 
                 HaCaT 
                 HPL1 
                 MCF10A 
                 Symbol 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 213497_at 
                 1.88 
                 2.14 
                 2.24 
                 1.53 
                 ABTB2 
               
               
                 206466_at 
                 1.09 
                 2.90 
                 17.93 
                 1.18 
                 ACSBG1 
               
               
                 222090_at 
                 0.97 
                 0.55 
                 0.44 
                 0.46 
                 ADCK2 
               
               
                 206170_at 
                 0.37 
                 0.40 
                 0.73 
                 0.52 
                 ADRB2 
               
               
                 207294_at 
                 0.98 
                 0.69 
                 0.35 
                 0.06 
                 AGTR2 
               
               
                 221008_s_at 
                 1.01 
                 0.76 
                 0.15 
                 0.34 
                 AGXT2L1 
               
               
                 220842_at 
                 1.01 
                 1.03 
                 0.13 
                 0.04 
                 AHI1 
               
               
                 204174_at 
                 1.28 
                 1.36 
                 2.19 
                 2.02 
                 ALOX5AP 
               
               
                 222108_at 
                 4.26 
                 1.81 
                 5.54 
                 1.51 
                 AMIGO2 
               
               
                 217630_at 
                 1.02 
                 0.53 
                 0.16 
                 0.47 
                 ANGEL2 
               
               
                 221009_s_at 
                 7.58 
                 2.18 
                 40.35 
                 6.26 
                 ANGPTL4 
               
               
                 207087_x_at 
                 1.02 
                 1.23 
                 7.13 
                 4.36 
                 ANK1 
               
               
                 217888_s_at 
                 2.23 
                 1.02 
                 1.45 
                 2.01 
                 ARFGAP1 
               
               
                 207606_s_at 
                 0.40 
                 1.10 
                 0.50 
                 0.54 
                 ARHGAP12 
               
               
                 212614_at 
                 0.40 
                 0.41 
                 0.31 
                 0.42 
                 ARID5B 
               
               
                 217579_x_at 
                 1.04 
                 1.69 
                 9.19 
                 2.77 
                 ARL6IP2 
               
               
                 216197_at 
                 1.07 
                 0.64 
                 0.40 
                 0.05 
                 ATF7IP 
               
               
                 218631_at 
                 0.49 
                 0.76 
                 0.42 
                 0.37 
                 AVPI1 
               
               
                 220470_at 
                 1.13 
                 1.05 
                 5.04 
                 4.41 
                 BET1L 
               
               
                 201169_s_at 
                 3.67 
                 2.21 
                 9.39 
                 2.27 
                 BHLHB2 
               
               
                 201170_s_at 
                 2.99 
                 3.37 
                 7.51 
                 2.45 
                 BHLHB2 
               
               
                 206119_at 
                 1.15 
                 1.66 
                 2.13 
                 4.44 
                 BHMT 
               
               
                 209920_at 
                 1.72 
                 2.35 
                 2.18 
                 1.29 
                 BMPR2 
               
               
                 210214_s_at 
                 2.22 
                 1.45 
                 2.14 
                 2.61 
                 BMPR2 
               
               
                 206787_at 
                 0.98 
                 0.72 
                 0.12 
                 0.06 
                 BRDT 
               
               
                 218723_s_at 
                 0.50 
                 0.56 
                 0.05 
                 0.24 
                 C13orf15 
               
               
                 217508_s_at 
                 1.87 
                 1.73 
                 2.62 
                 1.47 
                 C18orf25 
               
               
                 221533_at 
                 1.18 
                 1.18 
                 2.04 
                 3.22 
                 C3orf28 
               
               
                 219474_at 
                 2.00 
                 1.80 
                 2.46 
                 2.14 
                 C3orf52 
               
               
                 212923_s_at 
                 2.12 
                 2.21 
                 1.61 
                 1.52 
                 C6orf145 
               
               
                 220513_at 
                 1.09 
                 1.95 
                 7.90 
                 2.09 
                 C6orf148 
               
               
                 209688_s_at 
                 1.63 
                 1.36 
                 2.07 
                 2.30 
                 CCDC93 
               
               
                 219774_at 
                 2.14 
                 1.26 
                 1.04 
                 2.52 
                 CCDC93 
               
               
                 203645_s_at 
                 0.96 
                 0.76 
                 0.36 
                 0.15 
                 CD163 
               
               
                 208592_s_at 
                 1.23 
                 1.37 
                 4.40 
                 4.29 
                 CD1E 
               
               
                 211861_x_at 
                 1.09 
                 0.68 
                 0.48 
                 0.08 
                 CD28 
               
               
                 202284_s_at 
                 3.18 
                 2.70 
                 2.44 
                 1.11 
                 CDKN1A 
               
               
                 218929_at 
                 0.59 
                 0.96 
                 0.37 
                 0.47 
                 CDKN2AIP 
               
               
                 203973_s_at 
                 0.21 
                 0.57 
                 0.44 
                 0.53 
                 CEBPD 
               
               
                 207331_at 
                 1.08 
                 2.05 
                 10.46 
                 1.02 
                 CENPF 
               
               
                 207980_s_at 
                 0.12 
                 0.34 
                 0.17 
                 0.23 
                 CITED2 
               
               
                 209357_at 
                 0.13 
                 0.19 
                 0.22 
                 0.19 
                 CITED2 
               
               
                 205295_at 
                 1.11 
                 0.65 
                 0.47 
                 0.23 
                 CKMT2 
               
               
                 202310_s_at 
                 4.75 
                 1.33 
                 1.90 
                 5.05 
                 COL1A1 
               
               
                 211980_at 
                 2.07 
                 1.47 
                 2.64 
                 4.02 
                 COL4A1 
               
               
                 211981_at 
                 1.84 
                 1.40 
                 2.99 
                 2.27 
                 COL4A1 
               
               
                 211964_at 
                 1.27 
                 1.19 
                 2.08 
                 2.08 
                 COL4A2 
               
               
                 221900_at 
                 1.21 
                 1.10 
                 2.01 
                 3.43 
                 COL8A2 
               
               
                 209101_at 
                 4.01 
                 5.88 
                 2.12 
                 5.20 
                 CTGF 
               
               
                 206775_at 
                 1.22 
                 1.07 
                 3.13 
                 2.26 
                 CUBN 
               
               
                 203923_s_at 
                 1.13 
                 0.76 
                 0.07 
                 0.19 
                 CYBB 
               
               
                 202887_s_at 
                 0.50 
                 0.93 
                 0.79 
                 0.47 
                 DDIT4 
               
               
                 215252_at 
                 1.12 
                 1.56 
                 2.08 
                 3.67 
                 DNAJC7 
               
               
                 40612_at 
                 0.94 
                 0.55 
                 0.15 
                 0.30 
                 DOPEY1 
               
               
                 218995_s_at 
                 3.76 
                 2.13 
                 1.50 
                 1.03 
                 EDN1 
               
               
                 206127_at 
                 2.01 
                 1.56 
                 2.11 
                 1.35 
                 ELK3 
               
               
                 221773_at 
                 2.09 
                 1.90 
                 3.03 
                 1.62 
                 ELK3 
               
               
                 201328_at 
                 1.72 
                 3.35 
                 2.86 
                 6.83 
                 ETS2 
               
               
                 201329_s_at 
                 1.75 
                 2.65 
                 1.72 
                 3.95 
                 ETS2 
               
               
                 219427_at 
                 0.61 
                 0.70 
                 0.50 
                 0.39 
                 FAT4 
               
               
                 204988_at 
                 1.01 
                 0.55 
                 0.18 
                 0.36 
                 FGB 
               
               
                 218818_at 
                 2.21 
                 1.19 
                 1.60 
                 2.51 
                 FHL3 
               
               
                 204135_at 
                 2.28 
                 1.38 
                 1.23 
                 2.44 
                 FILIP1L 
               
               
                 220326_s_at 
                 1.30 
                 1.58 
                 2.06 
                 2.10 
                 FLJ10357 
               
               
                 58780_s_at 
                 1.50 
                 1.81 
                 2.22 
                 2.19 
                 FLJ10357 
               
               
                 210316_at 
                 0.96 
                 0.48 
                 1.15 
                 0.14 
                 FLT4 
               
               
                 219316_s_at 
                 0.85 
                 0.48 
                 0.87 
                 0.17 
                 FLVCR2 
               
               
                 218618_s_at 
                 2.01 
                 1.25 
                 1.26 
                 2.09 
                 FNDC3B 
               
               
                 203592_s_at 
                 1.90 
                 2.20 
                 3.24 
                 1.71 
                 FSTL3 
               
               
                 209414_at 
                 1.45 
                 1.17 
                 2.08 
                 2.73 
                 FZR1 
               
               
                 209416_s_at 
                 1.47 
                 1.31 
                 2.00 
                 2.02 
                 FZR1 
               
               
                 207574_s_at 
                 3.88 
                 3.79 
                 2.80 
                 1.45 
                 GADD45B 
               
               
                 209304_x_at 
                 3.37 
                 3.21 
                 2.45 
                 1.30 
                 GADD45B 
               
               
                 209305_s_at 
                 3.38 
                 3.06 
                 2.58 
                 1.11 
                 GADD45B 
               
               
                 215248_at 
                 1.16 
                 1.21 
                 2.99 
                 7.32 
                 GRB10 
               
               
                 214438_at 
                 1.17 
                 2.07 
                 1.55 
                 4.48 
                 HLX 
               
               
                 203665_at 
                 3.05 
                 1.29 
                 6.19 
                 1.70 
                 HMOX1 
               
               
                 204111_at 
                 1.02 
                 0.65 
                 0.30 
                 0.19 
                 HNMT 
               
               
                 205580_s_at 
                 2.08 
                 1.14 
                 2.06 
                 1.47 
                 HRH1 
               
               
                 208937_s_at 
                 1.04 
                 0.54 
                 0.38 
                 0.34 
                 ID1 
               
               
                 206924_at 
                 9.37 
                 2.46 
                 4.91 
                 2.70 
                 IL11 
               
               
                 207952_at 
                 1.04 
                 0.65 
                 0.66 
                 0.12 
                 IL5 
               
               
                 204686_at 
                 0.41 
                 0.80 
                 0.67 
                 0.50 
                 IRS1 
               
               
                 209098_s_at 
                 2.26 
                 2.06 
                 1.76 
                 2.45 
                 JAG1 
               
               
                 41387_r_at 
                 1.22 
                 1.64 
                 2.02 
                 2.06 
                 JMJD3 
               
               
                 201464_x_at 
                 2.19 
                 2.15 
                 2.62 
                 1.82 
                 JUN 
               
               
                 201466_s_at 
                 2.81 
                 2.58 
                 5.10 
                 2.65 
                 JUN 
               
               
                 201473_at 
                 7.33 
                 2.65 
                 8.17 
                 1.87 
                 JUNB 
               
               
                 218651_s_at 
                 2.19 
                 3.08 
                 3.96 
                 2.86 
                 LARP6 
               
               
                 221011_s_at 
                 2.32 
                 1.87 
                 5.40 
                 6.95 
                 LBH 
               
               
                 218604_at 
                 1.62 
                 2.08 
                 2.67 
                 1.23 
                 LEMD3 
               
               
                 218574_s_at 
                 3.41 
                 1.13 
                 5.25 
                 1.67 
                 LMCD1 
               
               
                 204089_x_at 
                 1.40 
                 1.56 
                 2.18 
                 2.04 
                 MAP3K4 
               
               
                 208210_at 
                 1.00 
                 0.69 
                 0.21 
                 0.41 
                 MAS1 
               
               
                 214696_at 
                 2.02 
                 1.46 
                 2.26 
                 1.51 
                 MGC14376 
               
               
                 202519_at 
                 2.05 
                 1.67 
                 1.35 
                 2.54 
                 MLXIP 
               
               
                 216303_s_at 
                 1.02 
                 0.58 
                 0.52 
                 0.34 
                 MTMR1 
               
               
                 213906_at 
                 0.41 
                 0.85 
                 0.83 
                 0.42 
                 MYBL1 
               
               
                 202431_s_at 
                 1.02 
                 0.40 
                 0.39 
                 0.37 
                 MYC 
               
               
                 201496_x_at 
                 1.06 
                 0.66 
                 0.11 
                 0.31 
                 MYH11 
               
               
                 218149_s_at 
                 0.49 
                 0.84 
                 0.72 
                 0.30 
                 NA 
               
               
                 207760_s_at 
                 2.50 
                 1.11 
                 2.43 
                 1.91 
                 NCOR2 
               
               
                 202607_at 
                 1.35 
                 1.11 
                 4.21 
                 2.02 
                 NDST1 
               
               
                 202150_s_at 
                 2.74 
                 3.08 
                 3.05 
                 3.56 
                 NEDD9 
               
               
                 201695_s_at 
                 2.12 
                 1.26 
                 1.14 
                 2.08 
                 NP 
               
               
                 206699_x_at 
                 1.00 
                 1.43 
                 2.09 
                 22.54 
                 NPAS1 
               
               
                 209120_at 
                 0.19 
                 0.64 
                 0.49 
                 0.34 
                 NR2F2 
               
               
                 215073_s_at 
                 0.32 
                 0.60 
                 0.32 
                 0.40 
                 NR2F2 
               
               
                 213824_at 
                 0.91 
                 0.71 
                 0.50 
                 0.23 
                 OLIG2 
               
               
                 221868_at 
                 1.08 
                 0.61 
                 0.32 
                 0.18 
                 PAIP2B 
               
               
                 206594_at 
                 1.05 
                 1.80 
                 3.47 
                 2.08 
                 PASK 
               
               
                 221918_at 
                 2.17 
                 2.27 
                 1.34 
                 1.61 
                 PCTK2 
               
               
                 205463_s_at 
                 2.30 
                 1.53 
                 7.00 
                 1.73 
                 PDGFA 
               
               
                 218691_s_at 
                 2.43 
                 1.50 
                 18.06 
                 1.15 
                 PDLIM4 
               
               
                 202464_s_at 
                 2.02 
                 2.15 
                 1.83 
                 1.65 
                 PFKFB3 
               
               
                 212134_at 
                 2.06 
                 1.36 
                 2.85 
                 1.96 
                 PHLDB1 
               
               
                 204612_at 
                 1.31 
                 2.09 
                 1.15 
                 8.96 
                 PKIA 
               
               
                 204958_at 
                 1.20 
                 1.48 
                 6.24 
                 2.08 
                 PLK3 
               
               
                 209740_s_at 
                 0.87 
                 0.83 
                 0.49 
                 0.24 
                 PNPLA4 
               
               
                 218849_s_at 
                 2.22 
                 1.41 
                 2.83 
                 1.57 
                 PPP1R13L 
               
               
                 202879_s_at 
                 1.29 
                 2.10 
                 1.42 
                 2.16 
                 PSCD1 
               
               
                 202880_s_at 
                 1.22 
                 2.09 
                 2.08 
                 2.09 
                 PSCD1 
               
               
                 206977_at 
                 0.96 
                 1.23 
                 15.95 
                 4.12 
                 PTH 
               
               
                 219812_at 
                 1.18 
                 1.19 
                 2.43 
                 2.52 
                 PVRIG 
               
               
                 216719_s_at 
                 1.06 
                 0.50 
                 0.16 
                 0.93 
                 RAB11FIP4 
               
               
                 219440_at 
                 1.10 
                 0.79 
                 0.21 
                 0.16 
                 RAI2 
               
               
                 203749_s_at 
                 3.39 
                 1.93 
                 4.10 
                 1.46 
                 RARA 
               
               
                 206850_at 
                 1.23 
                 1.34 
                 4.49 
                 2.30 
                 RASL10A 
               
               
                 203748_x_at 
                 1.42 
                 1.33 
                 2.02 
                 2.21 
                 RBMS1 
               
               
                 207266_x_at 
                 1.42 
                 1.36 
                 2.18 
                 2.09 
                 RBMS1 
               
               
                 209868_s_at 
                 1.42 
                 1.37 
                 2.07 
                 2.06 
                 RBMS1 
               
               
                 212099_at 
                 3.92 
                 2.05 
                 4.73 
                 3.16 
                 RHOB 
               
               
                 205158_at 
                 0.71 
                 0.78 
                 0.48 
                 0.26 
                 RNASE4 
               
               
                 202627_s_at 
                 3.67 
                 11.31 
                 7.36 
                 7.64 
                 SERPINE1 
               
               
                 202656_s_at 
                 0.20 
                 0.77 
                 0.56 
                 0.45 
                 SERTAD2 
               
               
                 202657_s_at 
                 0.23 
                 0.59 
                 0.38 
                 0.53 
                 SERTAD2 
               
               
                 201739_at 
                 3.23 
                 2.29 
                 4.88 
                 1.11 
                 SGK 
               
               
                 206675_s_at 
                 1.26 
                 4.80 
                 8.33 
                 16.58 
                 SKIL 
               
               
                 217591_at 
                 2.02 
                 3.25 
                 3.13 
                 2.52 
                 SKIL 
               
               
                 217685_at 
                 0.96 
                 0.76 
                 0.44 
                 0.11 
                 SLC16A3 
               
               
                 207298_at 
                 1.23 
                 1.19 
                 3.02 
                 2.01 
                 SLC17A3 
               
               
                 204790_at 
                 4.17 
                 5.95 
                 10.80 
                 6.39 
                 SMAD7 
               
               
                 210357_s_at 
                 1.60 
                 2.29 
                 2.40 
                 3.03 
                 SMOX 
               
               
                 207390_s_at 
                 1.40 
                 1.36 
                 2.01 
                 3.00 
                 SMTN 
               
               
                 212666_at 
                 1.62 
                 1.66 
                 2.30 
                 2.02 
                 SMURF1 
               
               
                 212668_at 
                 1.32 
                 2.10 
                 1.24 
                 4.46 
                 SMURF1 
               
               
                 215458_s_at 
                 1.49 
                 1.31 
                 2.00 
                 2.36 
                 SMURF1 
               
               
                 219480_at 
                 2.01 
                 1.38 
                 6.11 
                 0.90 
                 SNAI1 
               
               
                 219257_s_at 
                 2.36 
                 1.66 
                 2.11 
                 2.09 
                 SPHK1 
               
               
                 209875_s_at 
                 1.00 
                 0.90 
                 0.15 
                 0.23 
                 SPP1 
               
               
                 219677_at 
                 2.15 
                 1.26 
                 1.27 
                 2.11 
                 SPSB1 
               
               
                 217991_x_at 
                 1.43 
                 1.10 
                 2.43 
                 2.29 
                 SSBP3 
               
               
                 216917_s_at 
                 1.05 
                 0.50 
                 0.72 
                 0.20 
                 SYCP1 
               
               
                 212796_s_at 
                 1.73 
                 1.29 
                 1.81 
                 2.26 
                 TBC1D2B 
               
               
                 211462_s_at 
                 1.04 
                 1.24 
                 8.78 
                 2.08 
                 TBL1Y 
               
               
                 208398_s_at 
                 2.09 
                 1.40 
                 1.33 
                 2.17 
                 TBPL1 
               
               
                 221866_at 
                 1.47 
                 1.35 
                 5.80 
                 2.91 
                 TFEB 
               
               
                 50221_at 
                 1.82 
                 2.25 
                 4.16 
                 2.41 
                 TFEB 
               
               
                 211154_at 
                 0.99 
                 0.49 
                 0.80 
                 0.08 
                 THPO 
               
               
                 217875_s_at 
                 5.44 
                 2.96 
                 31.67 
                 1.62 
                 TMEPAI 
               
               
                 208296_x_at 
                 2.11 
                 1.33 
                 1.24 
                 2.01 
                 TNFAIP8 
               
               
                 218368_s_at 
                 1.36 
                 2.00 
                 2.11 
                 1.39 
                 TNFRSF12A 
               
               
                 210987_x_at 
                 2.00 
                 1.27 
                 2.11 
                 1.43 
                 TPM1 
               
               
                 212664_at 
                 1.08 
                 1.66 
                 2.37 
                 3.15 
                 TUBB4 
               
               
                 205807_s_at 
                 2.92 
                 1.85 
                 2.23 
                 2.75 
                 TUFT1 
               
               
                 221098_x_at 
                 1.03 
                 0.50 
                 0.73 
                 0.06 
                 UTP14A 
               
               
                 211527_x_at 
                 2.56 
                 1.45 
                 8.61 
                 21.20 
                 VEGFA 
               
               
                 221423_s_at 
                 2.00 
                 1.11 
                 1.19 
                 2.16 
                 YIPF5 
               
               
                 208078_s_at 
                 3.17 
                 2.49 
                 2.47 
                 2.05 
                 ZEB1 
               
               
                 211962_s_at 
                 2.07 
                 2.07 
                 1.44 
                 1.14 
                 ZFP36L1 
               
               
                 211965_at 
                 1.56 
                 2.28 
                 1.43 
                 3.34 
                 ZFP36L1 
               
               
                 203520_s_at 
                 0.81 
                 0.82 
                 0.50 
                 0.16 
                 ZNF318 
               
               
                 221123_x_at 
                 0.42 
                 0.96 
                 0.93 
                 0.50 
                 ZNF395 
               
               
                 206810_at 
                 1.02 
                 0.56 
                 0.23 
                 0.25 
                 ZNF44 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 a 55 probe, unique 50 gene signature of all the genes that have univariate 
               
               
                 associations with lung metastases. In ER− tumors: p = 0.0003 (originally 0.0003); In 
               
               
                 LMS+ tumors: p = 0.000012 (originally &lt; 0.0001) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Probes 
                 Direction 
                 Corr.w.tgfb.act 
                 Univariate.c 
                 Univariate.p 
                 Symbol 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 203665_at 
                 1 
                 0.329058167 
                 0.6606244 
                 2.16544E−07 
                 HMOX1 
               
               
                 211527_x_at 
                 1 
                 0.675990296 
                 0.391253582 
                 4.48942E−05 
                 VEGFA 
               
               
                 202627_s_at 
                 1 
                 0.618184128 
                 0.583283049 
                 5.72343E−05 
                 SERPINE1 
               
               
                 218818_at 
                 1 
                 0.198070604 
                 1.861044172 
                 7.42988E−05 
                 FHL3 
               
               
                 218618_s_at 
                 1 
                 0.588177262 
                 0.941372511 
                 8.04662E−05 
                 FNDC3B 
               
               
                 211981_at 
                 1 
                 0.65332303 
                 0.76720787 
                 9.15415E−05 
                 COL4A1 
               
               
                 221423_s_at 
                 1 
                 0.779285281 
                 1.003581089 
                 0.000104407 
                 YIPF5 
               
               
                 221009_s_at 
                 1 
                 0.167244747 
                 0.699276019 
                 0.000125438 
                 ANGPTL4 
               
               
                 218368_s_at 
                 1 
                 0.745860924 
                 0.73385478 
                 0.00014557 
                 TNFRSF12A 
               
               
                 209101_at 
                 1 
                 0.465010719 
                 0.687912875 
                 0.000317061 
                 CTGF 
               
               
                 201169_s_at 
                 1 
                 0.781081029 
                 0.507509886 
                 0.000545689 
                 BHLHB2 
               
               
                 201464_x_at 
                 1 
                 0.117512488 
                 0.8944621 
                 0.000792739 
                 JUN 
               
               
                 201329_s_at 
                 1 
                 0.395700392 
                 0.909128827 
                 0.000854726 
                 ETS2 
               
               
                 219774_at 
                 1 
                 0.097559726 
                 1.506265364 
                 0.000898283 
                 CCDC93 
               
               
                 211964_at 
                 1 
                 0.280608916 
                 0.941178307 
                 0.001090799 
                 COL4A2 
               
               
                 215458_s_at 
                 1 
                 0.390897808 
                 1.093314224 
                 0.001102154 
                 SMURF1 
               
               
                 219677_at 
                 1 
                 0.243015361 
                 1.129040601 
                 0.001353386 
                 SPSB1 
               
               
                 209868_s_at 
                 1 
                 0.358172959 
                 0.697085183 
                 0.001534684 
                 RBMS1 
               
               
                 207390_s_at 
                 1 
                 0.621201404 
                 0.805769931 
                 0.00163468 
                 SMTN 
               
               
                 209098_s_at 
                 1 
                 0.629640726 
                 0.647901004 
                 0.001856884 
                 JAG1 
               
               
                 202519_at 
                 1 
                 0.090323369 
                 1.026190707 
                 0.003185206 
                 MLXIP 
               
               
                 202150_s_at 
                 1 
                 0.627467963 
                 0.900781359 
                 0.003297804 
                 NEDD9 
               
               
                 221868_at 
                 −1 
                 −0.287063403 
                 −0.561604122 
                 0.003443685 
                 PAIP2B 
               
               
                 219440_at 
                 −1 
                 −0.420481258 
                 −0.45681289 
                 0.003543637 
                 RAI2 
               
               
                 211980_at 
                 1 
                 0.115960349 
                 0.942133326 
                 0.004472386 
                 COL4A1 
               
               
                 209305_s_at 
                 1 
                 0.610149662 
                 0.621448467 
                 0.00745052 
                 GADD45B 
               
               
                 201695_s_at 
                 1 
                 0.168137981 
                 0.755554958 
                 0.016916518 
                 NP 
               
               
                 203748_x_at 
                 1 
                 0.004854785 
                 0.620751896 
                 0.019243033 
                 RBMS1 
               
               
                 221008_s_at 
                 −1 
                 −0.248892145 
                 −0.447909816 
                 0.020871059 
                 AGXT2L1 
               
               
                 204174_at 
                 1 
                 0.182789341 
                 0.542718952 
                 0.022888449 
                 ALOX5AP 
               
               
                 207266_x_at 
                 1 
                 0.034497397 
                 0.597727868 
                 0.023298492 
                 RBMS1 
               
               
                 212923_s_at 
                 1 
                 0.054488441 
                 0.824774431 
                 0.023791129 
                 C6orf145 
               
               
                 220842_at 
                 −1 
                 −0.016216259 
                 −0.331869172 
                 0.026528843 
                 AHI1 
               
               
                 210987_x_at 
                 1 
                 0.203854585 
                 0.726674532 
                 0.028998356 
                 TPM1 
               
               
                 217591_at 
                 1 
                 0.541379915 
                 0.603351772 
                 0.029257568 
                 SKIL 
               
               
                 201170_s_at 
                 1 
                 0.09163886 
                 0.644830027 
                 0.029455813 
                 BHLHB2 
               
               
                 210357_s_at 
                 1 
                 0.058575277 
                 0.504354773 
                 0.032482652 
                 SMOX 
               
               
                 201473_at 
                 1 
                 0.519203348 
                 0.601295839 
                 0.051762653 
                 JUNB 
               
               
                 217991_x_at 
                 1 
                 0.32260368 
                 0.505538738 
                 0.062427707 
                 SSBP3 
               
               
                 211962_s_at 
                 1 
                 0.303134657 
                 0.725288824 
                 0.06986755 
                 ZFP36L1 
               
               
                 206127_at 
                 1 
                 0.463276453 
                 0.532524896 
                 0.076954196 
                 ELK3 
               
               
                 219480_at 
                 1 
                 0.302415429 
                 0.371724664 
                 0.080873546 
                 SNAI1 
               
               
                 215252_at 
                 1 
                 0.404833421 
                 0.392246676 
                 0.08527437 
                 DNAJC7 
               
               
                 218149_s_at 
                 −1 
                 −0.443065001 
                 −0.340459435 
                 0.14103894 
                 NA 
               
               
                 206170_at 
                 −1 
                 −0.500144981 
                 −0.340920044 
                 0.154153385 
                 ADRB2 
               
               
                 209740_s_at 
                 −1 
                 −0.348644355 
                 −0.185608897 
                 0.159872822 
                 PNPLA4 
               
               
                 209416_s_at 
                 1 
                 0.415951285 
                 0.546068301 
                 0.225087286 
                 FZR1 
               
               
                 211965_at 
                 1 
                 0.473642608 
                 0.217399546 
                 0.240922963 
                 ZFP36L1 
               
               
                 217630_at 
                 −1 
                 −0.316244317 
                 −0.140738019 
                 0.480270579 
                 ANGEL2 
               
               
                 209920_at 
                 1 
                 0.458165297 
                 0.116299309 
                 0.503542922 
                 BMPR2 
               
               
                 209120_at 
                 −1 
                 −0.313588976 
                 −0.148157538 
                 0.528961317 
                 NR2F2 
               
               
                 202879_s_at 
                 1 
                 0.375295213 
                 0.070697027 
                 0.561610299 
                 PSCD1 
               
               
                 204686_at 
                 −1 
                 −0.30793376 
                 −0.084058526 
                 0.62988936 
                 IRS1 
               
               
                 203973_s_at 
                 −1 
                 −0.31970473 
                 −0.034431845 
                 0.861159443 
                 CEBPD 
               
               
                 219427_at 
                 −1 
                 −0.300823848 
                 −0.037838178 
                 0.866880412 
                 FAT4 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 20 gene signature. p = 0.0004, when applied to ER− tumors. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Probes 
                 Direction 
                 Corr.w.tgfb.act 
                 Univariate.c 
                 Univariate.p 
                 Symbol 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 203665_at 
                 1 
                 0.329058167 
                 0.6606244 
                 2.16544E−07 
                 HMOX1 
               
               
                 211527_x_at 
                 1 
                 0.675990296 
                 0.391253582 
                 4.48942E−05 
                 VEGFA 
               
               
                 202627_s_at 
                 1 
                 0.618184128 
                 0.583283049 
                 5.72343E−05 
                 SERPINE1 
               
               
                 218818_at 
                 1 
                 0.198070604 
                 1.861044172 
                 7.42988E−05 
                 FHL3 
               
               
                 218618_s_at 
                 1 
                 0.588177262 
                 0.941372511 
                 8.04662E−05 
                 FNDC3B 
               
               
                 211981_at 
                 1 
                 0.65332303 
                 0.76720787 
                 9.15415E−05 
                 COL4A1 
               
               
                 221423_s_at 
                 1 
                 0.779285281 
                 1.003581089 
                 0.000104407 
                 YIPF5 
               
               
                 221009_s_at 
                 1 
                 0.167244747 
                 0.699276019 
                 0.000125438 
                 ANGPTL4 
               
               
                 218368_s_at 
                 1 
                 0.745860924 
                 0.73385478 
                 0.00014557 
                 TNFRSF12A 
               
               
                 209101_at 
                 1 
                 0.465010719 
                 0.687912875 
                 0.000317061 
                 CTGF 
               
               
                 201169_s_at 
                 1 
                 0.781081029 
                 0.507509886 
                 0.000545689 
                 BHLHB2 
               
               
                 201464_x_at 
                 1 
                 0.117512488 
                 0.8944621 
                 0.000792739 
                 JUN 
               
               
                 201329_s_at 
                 1 
                 0.395700392 
                 0.909128827 
                 0.000854726 
                 ETS2 
               
               
                 219774_at 
                 1 
                 0.097559726 
                 1.506265364 
                 0.000898283 
                 CCDC93 
               
               
                 211964_at 
                 1 
                 0.280608916 
                 0.941178307 
                 0.001090799 
                 COL4A2 
               
               
                 215458_s_at 
                 1 
                 0.390897808 
                 1.093314224 
                 0.001102154 
                 SMURF1 
               
               
                 219677_at 
                 1 
                 0.243015361 
                 1.129040601 
                 0.001353386 
                 SPSB1 
               
               
                 209868_s_at 
                 1 
                 0.358172959 
                 0.697085183 
                 0.001534684 
                 RBMS1 
               
               
                 207390_s_at 
                 1 
                 0.621201404 
                 0.805769931 
                 0.00163468 
                 SMTN 
               
               
                 209098_s_at 
                 1 
                 0.629640726 
                 0.647901004 
                 0.001856884 
                 JAG1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Different 20 gene signatures that are able to predict TGFβ responsiveness with 
               
               
                 the associated p-values. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Gene 
                 Run 1 
                 Run 2 
                 Run 3 
                 Run 4 
                 Run 5 
               
               
                   
               
               
                 Symbols 
                 “FHL3 
                 “AHI1” 
                 “IRS1” 
                 “C6orf145” 
                 “CTGF” 
               
               
                   
                 “GADD45B 
                 “C6orf145” 
                 “PSCD1” 
                 “AGXT2L1” 
                 “GADD45B” 
               
               
                   
                 “TNFRSF12A 
                 “IRS1” 
                 “TPM1” 
                 “ADRB2” 
                 “BHLHB2” 
               
               
                   
                 “ETS2 
                 “COL4A1” 
                 “C6orf145” 
                 “PNPLA4” 
                 “SERPINE1” 
               
               
                   
                 “JAG1 
                 “JAG1” 
                 “NEDD9” 
                 “SERPINE1 
                 “NP” 
               
               
                   
                 “SMURF1 
                 “SSBP3” 
                 “HMOX1” 
                 “ZFP36L1” 
                 “ZNF395” 
               
               
                   
                 “FAT4 
                 “ZFP36L1” 
                 “RAI2” 
                 “SKIL” 
                 “MLXIP” 
               
               
                   
                 “NR2F2 
                 “BHLHB2” 
                 “NR2F2” 
                 “JAG1” 
                 “DNAJC7” 
               
               
                   
                 “TPM1 
                 “ADRB2” 
                 “FAT4” 
                 “CCDC93” 
                 “AGXT2L1” 
               
               
                   
                 “JUN 
                 “RAI2” 
                 “SMTN” 
                 “BMPR2” 
                 “RBMS1” 
               
               
                   
                 “CCDC93 
                 “AGXT2L1” 
                 “ZFP36L1” 
                 “ZFP36L1” 
                 “TNFRSF12A 
               
               
                   
                 “HMOX1 
                 “SPSB1 
                 “ALOX5AP 
                 “FAT4” 
                 “CCDC93” 
               
               
                   
                 “RBMS1 
                 “ALOX5AP 
                 “RBMS1” 
                 “JUN” 
                 “VEGFA” 
               
               
                   
                 “BHLHB2 
                 “COL4A1 
                 “SMOX” 
                 “ALOX5AP 
                 “NR2F2” 
               
               
                   
                 “ZFP36L1 
                 “GADD45B 
                 “ANGPTL4” 
                 “FZR1” 
                 “COL4A1” 
               
               
                   
                 “SSBP3 
                 “SMURF1” 
                 “PAIP2B” 
                 “NR2F2” 
                 “FNDC3B” 
               
               
                   
                 “ZNF395 
                 “BHLHB2” 
                 “BHLHB2” 
                 “FHL3” 
                 “SPSB1” 
               
               
                   
                 “SKIL” 
                 “JUNB” 
                 “RBMS1” 
                 “COL4A1” 
                 “PNPLA4” 
               
               
                   
                 “FZR1” 
                 “SMTN” 
                 “JUN” 
                 “CEBPD” 
                 “PAIP2B” 
               
               
                   
                 “RAI2” 
                 “NEDD9” 
                 “COL4A1” 
                 “RAI2” 
                 “BMPR2” 
               
               
                 p values 
                 0.006 
                 4.96E+00 
                 9.61E−05 
                 0.002 
                 3.32E−05 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 TBRS status was assessed in each of the 
               
               
                 breast cancer metastasis samples submitted for microarray analysis. 
               
               
                 The percentage of TBRS positive samples in each 
               
               
                 metastasis site is shown. 
               
            
           
           
               
               
               
            
               
                   
                 Site 
                 TBRS+ (%) 
               
               
                   
                   
               
               
                   
                 Lung 
                 14/18 (78) 
               
               
                   
                 Bone 
                  8/16 (50) 
               
               
                   
                 Brain 
                  6/19 (32) 
               
               
                   
                 Liver 
                   5/5 (100) 
               
               
                   
                 Other sites* 
                  5/9 (56) 
               
               
                   
                 All sites 
                 38/67 (58) 
               
               
                   
                   
               
               
                   
                 *Includes ovary, duodenum and chest wall 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Univariate and multivariate analyses correlating TGFβ pathway 
               
               
                 components and incidence of lung metastasis in the breast cancer 
               
               
                 patient cohorts. P-values for these associations are listed. 
               
            
           
           
               
               
               
            
               
                   
                 All tumors 
                 ER− tumors 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 TGFBR1 
                 0.7740 
                 0.2560 
               
               
                   
                 TGFBR2 
                 0.1700 
                 0.0110 
               
               
                   
                 TGFB1 
                 0.5460 
                 0.1800 
               
               
                   
                 TGFB2 
                 0.8750 
                 0.5120 
               
               
                   
                 TGFB3 
                 0.001 a   
                 0.0271 a   
               
               
                   
                 Smad2 
                 0.1990 
                 0.0425 
               
               
                   
                 Smad3 
                 0.3090 
                 0.0111 
               
               
                   
                 Smad4 
                 0.0937 
                 0.1780 
               
               
                   
                 TGFBR2 + Smad3 b   
                 0.3380 
                 0.0106 
               
               
                   
                 All (except TGFB3) b   
                 0.6260 
                 0.0533 
               
               
                   
                 TBRS 
                 0.0133 
                 &lt;0.0001 
               
               
                   
                   
               
               
                   
                   a Negative correlation 
               
               
                   
                   b Multivariate analyses using Cox proportional hazards regression model 
               
            
           
         
       
     
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