# Patent

## Title
Molecular markers for lung and colorectal carcinomas

## Abstract

Molecular markers for lung and colorectal carcinomas and methods of using them in blood sample assays are disclosed. The method comprises measuring the expression of the markers in a blood sample from a subject for detecting the presence and/or severity of lung and/or colorectal cancer, and for monitoring and/or assessing the prognosis of the subject's response to a cancer therapy. Also disclosed are kits for detecting, diagnosing, and/or monitoring lung or colorectal carcinomas.

## Background
REFERENCE TO RELATED APPLICATION
The present application claims the priority to U.S. Provisional Application Ser. No. 61/099,008, filed Sep. 22, 2008, which is herein incorporated by reference in its entirety.


FIELD OF THE INVENTION
The present invention relates generally to tumor-associated molecular markers, and more specifically to tumor-associated molecular markers in peripheral blood.
BACKGROUND OF THE INVENTION
There are two main types of lung cancer, non-small cell lung cancer (NSCLC) and small cell lung cancer. NSCLC accounts for about 80% of lung cancers. Three most common types of NSCLC in the United States are adenocarcinoma/bronchoalveolar (35-40%), squamous cell carcinoma (25-30%) and large-cell carcinoma (10-15%). Lung cancer is most easily and successfully treated if it is caught early. An early-stage cancer is less likely to have grown to a large size or to have spread to other parts of the body (metastasized). Large or metastasized cancers are much more difficult to be treated.
Colorectal cancer, also called colon cancer or large bowel cancer, includes cancerous growths in the colon, rectum and appendix. It is the third most common form of cancer and the second leading cause of cancer-related death in the Western world. Many colorectal cancers are thought to arise from adenomatous polyps in the colon. These mushroom-shaped growths are usually benign, but some may develop into cancer over time. The majority of the time the diagnosis of localized colon cancer is through colonoscopy.
Diagnostic markers for early stage lung and colorectal cancers will have a significant impact on the morbidity and mortality of these diseases. Detection of cancer cell-specific biomarkers provides an effective screening strategy. It can also screen for and detect post-operative residual tumor cells, and for occult metastases, an early indicator of tumor recurrence. Early detection can thus improve survival in patients before symptoms are detectable clinically while undergoing treatment and while in remission. Certain markers were known in the art to make predictions of a patient's cancer risk using tumor tissues, however, it was not predicted nor predictable that the same markers could be detected in blood (see “DETAILED DESCRIPTION OF THE INVENTION; Detections of cancer gene markers in tissue versus blood samples”).
Therefore, a heretofore unaddressed need exists in the art to address the aforementioned deficiencies and inadequacies, especially in connection with the method of detecting lung and/or colorectal cancers from peripheral blood samples.
SUMMARY OF THE INVENTION
One aspect of the invention relates to a method for detecting the presence and/or severity of lung and/or colorectal cancer. The method comprises:

    
    
        (a) obtaining a test sample of bodily fluid comprising a nucleic acid from a subject;
        (b) measuring the expression level of at least one cancer gene marker selected from:
        
            (i) the group consisting of: DUSP6, EIF2S3, GRB2, RNF4, MMD, MCM4, NF1 and MDM2; or
            (ii) the group consisting of: DUSP6, EIF2S3, MDM2, NF1, MMD, RNF4, GRB2, and EXT2;
        
        
        (c) normalizing the expression level of the at least one cancer gene marker to a housekeeping gene;
        (d) applying the normalized expression level of the at least one cancer gene marker to a logistic regression prediction model which calculates the probability of cancer and/or cancer recurrence risk; and
        (e) determining the presence and/or severity of lung and/or colorectal cancer based oil the calculated probability.
    
    


In one embodiment of the invention, the test sample is a blood sample.
In another embodiment of the invention, the expression level of the at least one cancer gene marker is measured by performing a real-time polymerase chain reaction (real-time PCR).
In another embodiment of the invention, measuring step (b) quantifies the mRNA expression level by the cycle number of the test sample [Ct (test)], and wherein normalizing step (c) is performed by subtracting Ct (test) from the mRNA expression level of a housekeeping gene [Ct(HK)], to give a normalized mRNA expression level of the test sample [ΔCt(test)].
In another embodiment of the invention, the housekeeping gene is selected from the group consisting of hypoxanthine phosphoribosyltransferase 1 (HPRT1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
In another embodiment of the invention, step (b) measures the expression level of DUSP6.
In another embodiment of the invention, step (b) measures the expression levels of the following six cancer gene markers: DUSP6, MDM2, NF1, EIF2S3, MMD, and RNF4.
In another embodiment of the invention, the at least one cancer gene marker is selected from the group consisting of DUSP6, EIF2S3, MDM2, NF1, MMD, RNF4, GRB2, and EXT2, and wherein step (e) determines the presence and/or severity of colorectal cancer.
In another embodiment of the invention, step (b) measures the expression level of at least (i) eight cancer gene markers; (ii) seven cancer gene markers; (iii) six cancer gene markers; (iv) five cancer gene markers; (v) four cancer gene markers; (vi) three cancer gene markers; or (vii) two cancer gene markers.
In another embodiment of the invention, step (b) measures the expression level of:

    
    
        (i) the one gene marker: DUSP6;
        (ii) the two gene markers: DUSP6 and EIF2S3;
        (iii) the three gene markers: DUSP6, EIF2S3, and GRB2;
        (iv) the four gene makers: DUSP6, EIF2S3, GRB2, and RNF4;
        (v) the five gene makers: DUSP6, EIF2S3, GRB2, RNF4, and MMD;
        (vi) the six gene makers: DUSP6, EIF2S3, GRB2, RNF4, MMD, and MCM4 or NF1;
        (vii) the seven gene makers: DUSP6, EIF2S3, GRB2, RNF4, MMD, MCM4, and MDM2 or NF1;
        (viii) the three gene markers: DUSP6, EIF2S3, and MDM2;
        (ix) the four gene markers: DUSP6, EIF2S3, MDM2, and NF1;
        (x) the five gene makers: DUSP6, EIF2S3, MDM2, NF1, and MMD;
        (xi) the six gene makers: DUSP6, EIF2S3, MDM2, NF1, MMD, and RNF4;
        (xii) the seven gene makers: DUSP6, EIF2S3, MDM2, NF1, MMD, RNF4, and GRB2; or
        (xiii) the eight gene makers: DUSP6, EIF2S3, MDM2, NF1, MMD, RNF4, GRB2, and EXT2.
    
    


In another embodiment of the invention, the expression level of each cancer gene marker is measured by real-time polymerase chain reaction (real-time PCR) with a pair of primers selected from the group consisting of primer pairs 1 to 9 as follows:

    
    
        (i) DUSP6 (SEQ ID NO: 9)-specific primer pair 1: SEQ ID NOs: 137 and 138, or SEQ ID NOs. 139 and 140;
        (ii) EIF2S3 (SEQ ID NO: 1)-specific primer pair 2: SEQ ID NOs. 17 and 18, SEQ ID NOs. 19 and 20, SEQ ID NOs. 21 and 22, SEQ ID NOs. 23 and 24, SEQ ID NOs. 25 and 26, SEQ ID NOs. 27 and 28, SEQ ID NOs. 29 and 30, or SEQ ID NOs: 31 and 32;
        (iii) MDM2 (SEQ ID NO: 4)-specific primer pair 3: SEQ ID NOs: 75 and 76, SEQ ID NOs: 77 and 78, SEQ ID NOs: 79 and 80, or SEQ ID NOs: 81 and 82;
        (iv) NF1 (SEQ ID NO: 6)-specific primer pair 4: SEQ ID NOs: 97 and 98, SEQ ID NOs: 99 and 100, SEQ ID NOs: 101 and 102, SEQ ID NOs: 103 and 104, SEQ ID NOs: 105 and 106, SEQ ID NOs: 107 and 108, SEQ ID NOs: 109 and 110, SEQ ID NOs: 111 and 112, or SEQ ID NOs: 113 and 114;
        (v) MMD (SEQ ID NO: 7)-specific primer pair 5: SEQ ID NOs: 115 and 116, SEQ ID NOs: 117 and 118, or SEQ ID NOs: 119 and 120;
        (vi) RNF4 (SEQ ID NO: 8)-specific primer pair 6: SEQ ID NOs: 121 and 122, SEQ ID NOs: 123 and 124, SEQ ID NOs: 125 and 126, SEQ ID NOs: 127 and 128, SEQ ID NOs: 129 and 130, SEQ ID NOs: 131 and 132, SEQ ID NOs: 133 and 134, or SEQ ID NOs: 135 and 136;
        (vii) GRB2 (SEQ ID NO: 5)-specific primer pair 7: SEQ ID NOs: 83 and 84, SEQ ID NOs: 85 and 86, SEQ ID NOs: 87 and 88; SEQ ID NOs: 89 and 90, SEQ ID NOs: 91 and 92, SEQ ID NOs: 93 and 94, or SEQ ID NOs: 95 and 96;
        (viii) EXT2 (SEQ ID NO: 2)-specific primer pair 8: SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 50, or SEQ ID NOs: 51 and 52; and
        (ix) MCM4 (SEQ ID NO: 3)-specific primer pair 9: SEQ ID NOs: 53 and 54, SEQ ID NOs: 55 and 56, SEQ ID NOs: 57 and 58, SEQ ID NOs: 59 and 60, SEQ ID NOs: 61 and 62, SEQ ID NOs: 63 and 64, SEQ ID NOs: 65 and 66, SEQ ID NOs: 67 and 68, SEQ ID NOs: 69 and 70, SEQ ID NOs: 71 and 72, or SEQ ID NOs: 73 and 74.
    
    


In another embodiment of the invention, the EIF2S3 (SEQ ID NO: 1)-specific primer pair 2: SEQ ID NOs: 27, 28, and SEQ ID NOs: 31 and 32, the NF1 (SEQ ID NO: 6)-specific primer pair 4: SEQ ID NOs: 103, 104, and/or the GRB2 (SEQ ID NO: 5)-specific primer pair 7: SEQ ID NOs: 91, 92 are selected if step (e) determines the presence and/or severity of lung cancer, and are not selected if step (e) determines the presence and/or severity of colorectal cancer.
In another embodiment of the invention, the EIF2S3 (SEQ ID NO: 1)-specific primer pair 2: SEQ ID NOs: 19, 20, the NF1 (SEQ ID NO: 6)-specific primer pair 4: SEQ ID NOs: 113, 114, the EXT2 (SEQ ID NO: 2)-specific primer pair 8: SEQ ID NOs: 47, 48, and/or the MCM4 (SEQ ID NO: 3)-specific primer pair 9: SEQ ID NOs: 67 and 68 are selected if step (e) determines the presence and/or severity of colorectal cancer, and are not selected if step (e) determines the presence and/or severity of lung cancer.
Another aspect of the invention relates to a method for monitoring and/or assessing the prognosis of a patient's response to a cancer therapy. The method comprises the steps of:

    
    
        (a) obtaining samples of bodily fluid comprising a nucleic acid from the patient before and after receiving a cancer therapy for a lung and/or colorectal cancer;
        (b) measuring the expression level of at least one cancer gene marker selected from the group consisting of DUSP6, EIF2S3, GRB2, RNF4, MMD, MCM4, NF1, and MDM2;
        (c) normalizing the expression level of at least one cancer gene marker to a housekeeping gene;
        (d) applying the normalized expression level of at least one cancer gene marker to a logistic regression prediction model which calculates the probability of cancer and/or cancer recurrence risk; and
        (e) evaluating the response by comparing the calculated probabilities from the samples, and thereby monitoring and/or assessing the prognosis of a patient's response to a cancer therapy;
    
    


wherein a decrease in the probability after receiving the cancer therapy is indicative of a positive response to the therapy.
Another aspect of the invention relates to a method for monitoring and/or assessing the prognosis of a patient's response to a cancer therapy. The method comprises the steps of:

    
    
        (a) obtaining a first sample of bodily fluid comprising a nucleic acid from the patient before receiving a cancer therapy for a lung and/or colorectal cancer;
        (b) obtaining a second sample of bodily fluid comprising a nucleic acid from the patient after receiving the therapy;
        (c) measuring the expression level of at least one cancer gene marker selected from the group consisting of DUSP6, EIF2S3, GRB2, RNF4, MMD, MCM4, NF1, MDM2 in the first and second samples; and
        (d) comparing the measured expression levels in the first and second samples and thereby monitoring and/or assessing the prognosis of the patient's response to the cancer therapy; wherein:
        
            (i) an increase in the expression level(s) of DUSP6, GRB2, MCM4 and/or NF1 in the second sample as compared to the level(s) of the corresponding gene marker(s) in the first sample is an indication that the subject is at risk of developing lung cancer and/or lung cancer recurrence;
            (ii) an increase in the expression level of MDM2 in the second sample as compared to the level of the corresponding marker in the first sample is an indication that the subject is at risk of lung cancer recurrence; and
            (iii) an increase in the expression level of EIF2S3, MMD, and/or RNF4 in the second sample as compared to the level of the corresponding marker(s) in the first sample is an indication that the subject is not at risk of developing lung cancer.
        
        
    
    


Further another aspect of the invention relates to a method for detecting the presence and/or severity of lung and/or colorectal cancer. The method comprises:

    
    
        (a) obtaining a test sample of bodily fluid comprising a nucleic acid from a subject;
        (b) measuring the expression level of at least one cancer gene marker selected from:
        
            (i) the group consisting of: DUSP6, EIF2S3, GRB2, RNF4, MMD, MCM4, NF1, and MDM2; or
            (ii) the group consisting of: DUSP6, EIF2S3, MDM2, NF1, MMD, RNF4, GRB2, and EXT2; and
        
        
        (c) comparing the expression level of at least one cancer gene marker to an expression level of a corresponding cancer gene marker in a sample of bodily fluids from a non-cancerous control, and thereby detecting the presence and/or severity of lung and/or colorectal cancer; wherein:
        
            (i) an increase in the expression level of DUSP6, GRB2, MCM4 and/or NF1 in the test sample as compared to the level of the corresponding marker in the control is an indication that the subject is at risk of developing lung cancer;
            (ii) an increase in the expression level of MDM2 in the test sample as compared to the level of the corresponding marker in the control is an indication that the subject is at risk of lung cancer recurrence;
            (iii) a decrease in the expression level of EIF2S3, MMD, and/or RNF4 in the test sample as compared to the level of the corresponding marker in the control is an indication that the subject is at risk of developing lung cancer;
            (iv) an increase in the expression level of DUSP6, GRB2, MDM2 and/or NF1 in the test sample as compared to the level of the corresponding marker in the control is an indication that the subject is at risk of developing colorectal cancer; and
            (v) a decrease in the expression level of EIF2S3, MMD, EXT2, and/or RNF4 in the test sample as compared to the level of the corresponding marker in the control is an indication that the subject is at risk of developing colorectal cancer.
        
        
    
    


The method may further comprises (a) normalizing the expression level of at least one cancer gene marker to a housekeeping gene; (b) applying the normalized expression level of at least one cancer gene marker to a logistic regression prediction model which calculates the probability of cancer and/or cancer recurrence risk; and (c) determining the presence and/or severity of lung and/or colorectal cancer based on the calculated probability.
Further another aspect of the invention relates to a kit for use in the aforementioned method for detecting the presence and/or severity of lung and/or colorectal cancer. The kit comprises one or more than one primer pair selected from the group consisting of cancer gene marker-specific primer pairs as follows:

    
    
        (i) a DUSP6 (SEQ ID NO: 9)-specific primer pair;
        (ii) an EIF2S3 (SEQ ID NO: 1)-specific primer pair;
        (iii) an MDM2 (SEQ ID NO: 4)-specific primer pair;
        (iv) a NF1 (SEQ ID NO: 6)-specific primer pair;
        (v) an MMD (SEQ ID NO: 7)-specific primer pair;
        (vi) an RNF4 (SEQ ID NO: 8)-specific primer pair;
        (vii) a GRB2 (SEQ ID NO: 5)-specific primer pair;
        (viii) an EXT2 (SEQ ID NO: 2)-specific primer pair; and
        (ix) an MCM4 (SEQ ID NO: 3)-specific primer pair
    
    


Yet another aspect of the invention relates to a kit for use in the aforementioned method for detecting the presence and/or severity of lung and/or colorectal cancer. The kit comprises one or more than one primer pair selected from the group consisting of cancer gene marker-specific primer pairs 1-9 as follows:

    
    
        (i) DUSP6 (SEQ ID NO: 9)-specific primer pair 1: SEQ ID NOs. 137 and 138, or SEQ ID NOs. 139 and 140;
        (ii) EIF2S3 (SEQ ID NO: 1)-specific primer pair 2: SEQ ID NOs. 17 and 18, SEQ ID NOs. 19 and 20, SEQ ID NOs. 21 and 22, SEQ ID NOs. 23 and 24, SEQ ID NOs. 25 and 26, SEQ ID NOs. 27 and 28, SEQ ID NOs. 29 and 30, or SEQ ID NOs: 31 and 32;
        (iii) MDM2 (SEQ ID NO: 4)-specific primer pair 3: SEQ ID NOs: 75 and 76, SEQ ID NOs: 77 and 78, SEQ ID NOs: 79 and 80, or SEQ ID NOs: 81 and 82;
        (iv) NF1 (SEQ ID NO: 6)-specific primer pair 4: SEQ ID NOs: 97 and 98, SEQ ID NOs: 99 and 100, SEQ ID NOs: 101 and 102, SEQ ID NOs: 103 and 104, SEQ ID NOs: 105 and 106, SEQ ID NOs: 107 and 108, SEQ ID NOs: 109 and 110, SEQ ID NOs: 111 and 112, or SEQ ID NOs: 113 and 114;
        (v) MMD (SEQ ID NO: 7)-specific primer pair 5: SEQ ID NOs: 115 and 116, SEQ ID NOs: 117 and 118, or SEQ ID NOs: 119 and 120;
        (vi) RNF4 (SEQ ID NO: 8)-specific primer pair 6: SEQ ID NOs: 121 and 122, SEQ ID NOs: 123 and 124, SEQ ID NOs: 125 and 126, SEQ ID NOs: 127 and 128, SEQ ID NOs: 129 and 130, SEQ ID NOs: 131 and 132, SEQ ID NOs: 133 and 134, or SEQ ID NOs: 135 and 136;
        (vii) GRB2 (SEQ ID NO: 5)-specific primer pair 7: SEQ ID NOs: 83 and 84, SEQ ID NOs: 85 and 86, SEQ ID NOs: 87 and 88; SEQ ID NOs: 89 and 90, SEQ ID NOs: 91 and 92, SEQ ID NOs: 93 and 94, or SEQ ID NOs: 95 and 96;
        (viii) EXT2 (SEQ ID NO: 2)-specific primer pair 8: SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 50, or SEQ ID NOs: 51 and 52; and
        (ix) MCM4 (SEQ ID NO: 3)-specific primer pair 9: SEQ ID NOs: 53 and 54, SEQ ID NOs: 55 and 56, SEQ ID NOs: 57 and 58, SEQ ID NOs: 59 and 60, SEQ ID NOs: 61 and 62, SEQ ID NOs: 63 and 64, SEQ ID NOs: 65 and 66, SEQ ID NOs: 67 and 68, SEQ ID NOs: 69 and 70, SEQ ID NOs: 71 and 72, or SEQ ID NOs: 73 and 74.
    
    


The kit may further comprise a primer pair 10 that is specific to a housekeeping gene.
In one embodiment of the invention, the kit further comprises an HPRT1-specific primer pair 10: SEQ ID NOs: 153 and 154.
These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.
The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph of a receiver operating characteristic (ROC) curve for the prediction model PM-7 (N=300) with Area under curve (AUC) of 0.93136.
FIG. 2 is a graph of an ROC curve for prediction model PM-14 for N=272 with AUC=0.93448.
FIG. 3 is a graph of an ROC curve for prediction models using different molecular marker or combination of multiple markers listed in Table 15.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.
As used herein, “around”, “about” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term “around”, “about” or “approximately” can be inferred if not expressly stated.
The term “gene” as used herein refers to a locatable region of genomic sequence. In cells, a gene is a portion of DNA that contains both “coding” sequences that determine what the gene does, and “non-coding” sequences that determine when the gene is active (expressed). A gene is a union of genomic sequences encoding a coherent set of potentially overlapping functional products. The molecules resulting from gene expression, whether RNA or protein, are known as gene products.
The term “genetic marker” as used herein refers to alteration in DNA that may indicate an increased risk of developing a specific disease or disorder.
The term “gene expression” means the production of a protein or a functional RNA from its gene.
The term “gene signature” or “Genetic signatures” are characteristic patterns of gene activity in cells.
As used herein, a “housekeeping gene” is a typically a constitutive gene that is transcribed at a relatively constant level across many or all known conditions. The housekeeping gene's products are typically needed for maintenance of the cell. It is generally assumed that their expression is unaffected by experimental conditions. Housekeeping genes that have been tested by applicants of this invention using clinical tumor/normal tissues, spike-in cultured cancer cells in the blood of healthy person, or clinical blood samples (lung cancer and controls) are GAPDH (glyceraldehyde 3-phosphate dehydrogenase; NM—002046; SEQ ID NO: 155), YWHAH (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide; NM—003405; SEQ ID NO: 156), SFRS8 (Homo sapiens splicing factor, arginine/serine-rich 8 (suppressor-of-white-apricot homolog, Drosophila; NM—004592; SEQ ID NO: 157), UBA3 (Ubiquitin-activating enzyme 3; NM—003968; SEQ ID NO: 158), RPS24 (ribosomal protein S24; NM—033022; SEQ ID NO: 159), RPL13 (ribosomal protein L13; NM—000977; SEQ ID NO: 160) PGK1 (phosphoglycerate kinase 1; NM—000291; SEQ ID NO: 161) (Also see Dheda et al. (2004) Biotechniques 37(1): 112-4, 116, 118-9; Human Reference Gene Panel (Roche); Vandesompele et al. (2002) Genome Biology 3(7):research0034.1-0034.11).
The term “prognosis” means a forecasting of the probable course and outcome of a disease, esp. of the chances of recovery.
The term “primer” refers to a strand of nucleic acid that serves as a starting point for DNA replication.
The term “overexpressed” refers to a state wherein there exists any measurable increase over normal or baseline levels. For example, a molecule that is overexpressed in a disease is one that is manifested in a measurably higher level in the presence of the disease than in the absence of the disease.
The term “underexpressed” refers to a state wherein there exists any measurable decrease over normal or baseline levels. For example, a molecule that is underexpressed in a disease is one that is manifested in a measurably lower level in the presence of the disease than in the absence of the disease.
The terms “detecting” and “diagnosing” are used interchangeably.
The term “normal tissue samples” or “control” refers to lung or colorectal tissue and/or body fluid from a subject determined to be negative for lung or colorectal cancer.
The terms “individual,” “host,” “patient,” and “subject,” used interchangeably herein, refer to a mammal, including, but not limited to, murines, simians, humans, non-human primates, felines, canines, equines, bovines, porcines, and ovines.
Detections of Cancer Gene Markers in Tissue Versus Blood Samples
The invention relates to detection of cancer-associated nucleic acid in blood samples using real time-PCR assays. The discoveries of a panel of gene markers that may be measured in blood samples for detecting lung and/or colorectal cancer risks are unexpected. It has been known that certain cancer gene markers in tumor tissues may be used for predicting a patient's cancer risk. It was, however, not predicted nor predictable that the same markers could be detected in blood samples (See U.S. application Ser. No. 11/437,607. which is incorporated by reference in its entirety) discloses a list of 12 genes (with Hazard Ratio greater than 1) that were thought to be “RISK” factors and thus tested in blood samples. The four risk genes for lung cancer prognosis, HGF, HMMR, ErbB3 and DLG2, resulted in much lower expression levels of mRNA because their Ct values were each greater than 30. In addition, these four genes sometimes were not measurable in blood samples. Furthermore, it was unexpected that the experimental results showed that two genes, MMD, RNF4, seemed to play a protective role in the logistic regression model of the current application (See U.S. application Ser. No. 11/437,607).
Further proofs that cancer markers in the tissue samples are not predicted nor predictable in the blood samples are the following: EIF2S3 was considered to be a protective gene in the present prediction models in blood assays, while it was as a risk gene for metastasis of lung cancer when the test was performed on tissue samples. MCM4 was identified as a tumor-associated marker in lung cancer tissue. It was deemed to be a risk factor in blood studies for lung cancer, but seemed to act protectively in the full model of colorectal cancer (Table 14). The expression level GRB2was positively correlated to the metastasis of lung cancer. The relative transcript of GRB2 gene represented a risk for lung cancer and colorectal cancer based on the odds ratio in Tables 4 and 9, but acted as a protective factor in the full model of colorectal cancer (Table 14). CPEB4 acted as a risk factor in the tissue sample studies, but a protective factor in the blood sample assays (Table 14). POLDIP2 was selected as a protective gene in the tissue sample studies, but a risk factor in the blood sample assays (Table 14).
Thus, the gene markers disclosed herein as cancer markers in blood samples for diagnosis and predictions of cancer risks are unexpected results.
Lung and Colorectal Cancer-Associated Genetic Markers
The invention relates to identification and applications of genetic signatures for detecting, diagnosing lung and/or colorectal cancers, monitoring therapeutic response, and prognosis prediction, such as recurrence possibility. Expression levels of lung and colorectal cancer gene markers were detected and measured in blood samples from patients and controls.
In one embodiment, expression levels of 8 genetic markers were examined in the blood samples using real-time PCR. Three genes, DUSP6, MCM4 and NF1, showed higher expression in the blood samples collected from the lung and/or colorectal cancer patients, which indicated that these three genes were associated with lung and/or colorectal cancers. Therefore, they can serve as genetic markers for predicting the risk of lung and/or colorectal cancer. Using statistical approach based on the mRNA expression levels of the 8 genes, several prediction models were built to predict the risk of one getting lung and/or colorectal cancer. Table 1 lists lung and colorectal cancer gene markers.








TABLE 1







Lung and colorectal cancer-associated gene marker
SEQ









Full name
Symbol
ID NO












Eukaryotic translation initiation factor 2,
EIF2S3
1


subunit 3 gamma




Exostoses (multiple) 2
EXT2
2


Minichromosome maintenance complex
MCM4
3


component 4




Mdm2, transformed 3T3 cell double minute 2,
MDM2
4


p53 binding protein (mouse)




Growth factor receptor-bound protein 2
GRB2
5


Neurofibromin 1 (neurofibromatosis, von
NF1
6


Recklinghausen disease, Watson disease)




Monocyte to macrophage differentiation-associated
MMD
7


Ring finger protein 4
RNF4
8


Dual specificity phosphatase 6
DUSP6
9


Cytoplasmic polyadenylation element binding
CPEB4
10


protein 4




Wee+ (S. pombe) homolog
WEE1
11


Interferon regulatory factor 4
IRF4
12


Signal transducer and activator of transcription 2,
STAT2
13


113 kD




zinc finger protein 264
ZNF264
14


DNA polymerase delta interacting protein 2
POLDIP2
15


Hypoxanthine phosphoribosyltransferase 1*
HPRT1
16









Expression of cancer gene markers can be detected by a variety of means including reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, TAQMAN assay, Northern blotting, in situ hybridization, and microarray technology.
PCR primers may be designed to be specific for the polynucleotide of genetic markers disclosed. Alternatively, primers may be designed to cross react with related polynucleotides, e.g., to allow hybridization to variants of cancer genetic markers. PCR-based assays of transcript expression profiles of genes may detect a single polynucleotide or multiple polynucleotides simultaneously (“multiplex” PCR). The amplified products may be detected by electrophoresis. Individual PCR products corresponding to cancer genetic markers may be identified by electrophoretic mobility. Alternatively, a PCR primer may be labeled for detecting PCR products. A primer comprising a fluorescent label may be used and the PCR product detected by detecting fluorescence. Where multiple polynucleotides are detected simultaneously, a mixture of primers each with a distinct label may be used in a PCR reaction. The products produced are detected based on the label. For example, fluorescent labels each with unique emission spectra may be used for labeling primers.
Polynucleotides expressed by the cancer marker genes may be detected by SAGE and by Massively Parallel Signature Sequencing (MPSS). See e.g., Brenner et al., “Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays,” Nature Biotechnology 18: 630-34 (2000).
Prediction Models
Statistical analysis of expression profiling of investigated genes may be used to predict the risk of an individual getting lung and/or colorectal cancers. An odds ratio (OR) is used as an estimate of the relative risk for lung and/or colorectal cancer. The odds ratio is a measure of effect size, describing the strength of association or non-independence between two binary data values. It is used as a descriptive statistic and plays an important role in logistic regression.
The odds ratio is defined as the ratio of the odds of an event occurring in one group to the odds of it occurring in another group. In statistics, the odds is the probability of an event occurring divided by the probability of an event not occurring. The odds ratio is a way of comparing whether the probability of a certain event is the same for two groups. An odds ratio of 1 implies that the event is equally likely to occur in both groups. An odds ratio greater than one implies that the event is more likely to occur in the first group. An odds ratio less than one implies that the event is less likely to occur in the first group.
If the probabilities of the event in each of the groups are p1 (first group) and p2 (second group), then the odds ratio is:




  
    
      p
      1
    
    /
    
      (
      
        1
        -
        
          p
          1
        
      
      )
    
  
  
    
      p
      2
    
    /
    
      (
      
        1
        -
        
          p
          2
        
      
      )
    
  




Logistic regression (sometimes called the logistic model or logit model) is used for prediction of the probability of occurrence of an event by fitting data to a logistic curve. The logistic regression model gives the probability that the response occurs as an exponential function of independent variables. The model is written in terms of a probability (P), which is in the range from 0 to 1.
The logistic regression begins with the logistic function:




  
    f
    ⁡
    
      (
      Y
      )
    
  
  =
  
    
      1
      
        1
        +
        
          exp
          
            -
            Y
          
        
      
    
    =
    P
  




The “input” is Y and the “output” is f(Y), i.e., P. The output is confined to values between 0 and 1. The variable Y represents the exposure to some set of risk factors, while f(Y) represents the probability (P) of a particular outcome, given that set of risk factors. The variable Y is a measure of the total contribution of all the risk factors used in the model and is known as the logit. The variable Y is usually defined as Y=β0+β1 x1+β2 x2+ . . . +βk xk, where β0 is called the “intercept” and β1, β2, β3, and so on, are called the “regression coefficients” of x1, x2, x3, respectively. The intercept is the value of Y when the value of all risk factors is zero (i.e., the value of z in someone with no risk factors). Each of the regression coefficients describes the size of the contribution of that risk factor. A positive regression coefficient means that that risk factor increases the probability of the outcome, while a negative regression coefficient means that risk factor decreases the probability of that outcome; a large regression coefficient means that the risk factor strongly influences the probability of that outcome; while a near-zero regression coefficient means that that risk factor has little influence on the probability of that outcome. Logistic regression is a useful way of describing the relationship between one or more risk factors and an outcome.
In the present invention, a person will be suspected of having “lung or colorectal cancer”, if the calculated probability (P) is greater than 0.5. However, the setting of the cutoff value for probability for having a higher sensitivity or specificity of prediction model can be achieved.
Further, the clinical performance of a laboratory test can be described in terms of diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups (Zweig and Campbell, 1993, Receiver-Operating Characteristic (ROC) plots. Clinical Chemistry 39:561-577). Terms commonly used for evaluation of clinical performances of a diagnostic test include sensitivity, specificity, efficiency, accuracy, utility, usefulness, and efficacy.
ROC plots may be used. ROC plots provide a pure index of accuracy by demonstrating the limits of a test's ability to discriminate between alternative states of health over the complete spectrum of operating conditions. A ROC curve is a graphical plot of the sensitivity vs. (1-specificity) for a binary classifier system as its discrimination threshold is varied (FIG. 1). The ROC curve can also be presented equivalently by plotting the fraction of true positives (TPR=true positive rate) vs. the fraction of false-positives (FPR=false positive rate). The closer a ROC curve is to the upper left-hand corner of the graph, the more accurate it is, because the true-positive rate is 1 and the false-positive rate is 0. ROC curves are useful for evaluating the clinical utility of a diagnostic test based on a molecular marker.
The value of the area under the ROC curve (AUC) indicates the tests' ability (prediction model) to discriminate the disease (lung or colorectal cancer) group from normal subjects. The greater is the AUC, the better is the diagnostic test. Generally, the AUC of 0.7 to 0.8 is a marginally useful test, 0.8 to 0.9 is a good test, and those tests with an area greater than 0.9 are excellent (Nakamur et al. (2004), “Cancer Diagnostics, Current and Future Trends” Humana Press, Totowa, N.J., USA; p 403).
These tumor markers can further be used in combination, e.g., in a panel or a prediction model that comprises two or more markers. A panel of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 markers may be used. It is likely that many lung cancers will overexpress at least one of the gene markers DUSP6, MDM2, MCM4, NF1 and GRB2, and/or underexpress at least one of the markers EIF2S3, MMD and RNF4, and many colorectal cancers will overexpress at least one of MDM2, GRB2, NF1 and DUSP6. and/or underexpress at least one of EIF2S3, EXT2, MMD and RNF4. Thus, combining these markers into a prediction model will provide a comprehensive screen for certain cancers.
A kit may contain, in separate containers, one or more primer pairs comprising polynucleotides sequences that are complementary to the mRNAs of genes comprising nucleotide sequences of SEQ ID NO: 1-9 or at least 90% identical to the nucleotide sequences of SEQ ID NO: 1-9.
EXAMPLES
Without intent to limit the scope of the invention, exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action.
Example 1
Procedures for Blood Test for Lung and Colorectal Cancer Molecular Markers
Preparation of mononuclear cells. Peripheral blood (5-8 ml) collected from lung and colorectal cancer patients, and healthy subjects was used to isolate mononuclear cell (MNC) fraction using BD VACUTAINER® CPTTM tube (BD, USA) according to the manufacturer instructions. The MNC fraction was supplemented with PBS buffer with three volumes of the original whole blood sample and followed by centrifugation at 2000 rpm for 10 min. The MNC fraction was further washed with 1 ml of PBS followed by centrifugation at 2000 rpm for 5 min. The final pellet was supplemented with 2 ml of Super RNAPURETM reagent (contained in SUPERRNAPURETM kit, Genesis, Taiwan).
Real-time PCR analysis of cancer marker gene expression. Total RNA was extracted from the MNC fraction using Super RNAPURETM kit according to the manufacturer's instructions. The RNA pellet was dissolved in DEPC-treated water and stored at −80° C. until use. The RNA quality was ascertained with gel electrophoresis using 1% agarose and also with OD260/OD280 ratio greater than 1.7. Around 1 μg of total RNA was used for cDNA synthesis with random hexamer primers (Amersham Bioscience, UK) and SUPERSCRIPTTMII reverse transcriptase (Invitrogen, USA). The reaction mixture and conditions for reverse transcription reaction were according to the manufacturer's instructions.
Table 1 lists 16 genes that were chosen as lung and colorectal cancer molecular markers for real-time PCR analysis of mRNA expression level. They are: (1) eukaryotic translation initiation factor 2, subunit 3 gamma, 52 kDa (EIF2S3), (2) exostoses (multiple) 2 (EXT2), (3) minichromosome maintenance complex component 4 (MCM4), (4) Mdm2, transformed 3T3 cell double minute 2, p 53 binding protein (mouse) (MDM2), (5) growth factor receptor-bound protein 2 (GRB2), (6) neurofibromin 1 (neurofibromatosis, von Recklinghausen disease, Watson disease) (NF1), (7) monocyte to macrophage differentiation-associated (MMD), (8) ring finger protein 4 (RNF4), (9) dual specificity Is phosphatase 6 (DUSP6), (10) cytoplasmic polyadenylation element binding protein 4 (CPEB4), (11) wee+ (S. pombe) homolog (WEE1), (12) interferon regulatory factor 4 (IRF4), (13) signal transducer and activator of transcription 2, 113 kD (STAT2), (14) zinc finger protein 264 (ZNF264), (15) DNA polymerase delta interacting protein 2 (POLDIP2), and (16) hypoxanthine phosphoribosyltransferase 1 (HPRT1).
Real-time PCR analysis of mRNA expression level of each gene was performed using Roche LIGHTCYCLER® 1.5 according to the manufacturer instructions. A total volume of 20 μl reaction mixture contained 20-100 ng of cDNA, primer, Probe (Universal ProbeLibrary probe), and Master Mix LIGHTCYCLER® TaqMan® Master Mix (Roche, Germany). Amplification was performed after 10 min at 95° C., which was followed by 40 cycles of 5 sec at 95° C., 20 sec at 60° C., and with a final extension at 72° C. for 1 sec. The control was performed for each batch by performing RT-PCR on a reaction mixture without cDNA to confirm no contamination in the assay.
Table 2 lists the primer pairs and probes used for synthesizing each of the amplicons in RT-PCR. The combination of a gene-specific primer pair and a nucleotide probe selected from UNIVERSAL PROBELIBRARYTM (F. Hoffmann-La Roche Ltd, Basel, Switzerland) could generate mores gene-specific amplicon during the Real-time PCR assay.











TABLE 2





Gene
Positions of
Forward Primer/
Reverse Primer/
Probe


(SEQ ID NO.)
amplicon
SEQ ID NO.
SEQ ID NO.
No.*





















EIF2S3
1143-1388
EIF2S3-F17
17
EIF2S3-R18
18
47


(1)
477-554
EIF2S3-F19
19
EIF2S3-R20
20
30



 4-111
EIF2S3-F21
21
EIF2S3-R22
22
78



310-423
EIF2S3-F23
23
EIF2S3-R24
24
32



 989-1056
EIF2S3-F25
25
EIF2S3-R26
26
1



778-853
EIF2S3-F27
27
EIF2S3-R28
28
76



2071-2194
EIF2S3-F29
29
EIF2S3-R30
30
72



1784-1872
EIF2S3-F31
31
EIF2S3-R32
32
2, 30,








51,








75


EXT2
1255-1502
EXT2-F33
33
EXT2-R34
34
85


(2)
1639-1747
EXT2-F35
35
EXT2-R36
36
37



1208-1275
EXT2-F37
37
EXT2-R38
38
69



2471-2543
EXT2-F39
39
EXT2-R40
40
22



2359-2444
EXT2-F41
41
EXT2-R42
42
49



2403-2488
EXT2-F43
43
EXT2-R44
44
76



2484-2593
EXT2-F45
45
EXT2-R46
46
77



2483-2591
EXT2-F47
47
EXT2-R48
48
31



1471-1585
EXT2-F49
49
EXT2-R50
50
76



1756-2037
EXT2-F51
51
EXT2-R52
52
43


MCM4
2071-2164
MCM4-F53
53
MCM4-R54
54
42


(3)
1326-1410
MCM4-F55
55
MCM4-R56
56
64



1022-1097
MCM4-F57
57
MCM4-R58
58
9



2263-2375
MCM4-F59
59
MCM4-R60
60
20



429-524
MCM4-F61
61
MCM4-R62
62
33



1572-1667
MCM4-F63
63
MCM4-R64
64
41



1609-1728
MCM4-F65
65
MCM4-R66
66
47



588-648
MCM4-F67
67
MCM4-R68
68
84



1988-2115
MCM4-F69
69
MCM4-R70
70
74



563-743
MCM4-F71
71
MCM4-R72
72
84



 892-1102
MCM4-F73
73
MCM4-R74
74
9


MDM2
218-425
MDM2-F75
75
MDM2-R76
76
68


(4)
598-667
MDM2-F77
77
MDM2-R78
78
18



256-318
MDM2-F79
79
MDM2-R80
80
68



852-988
MDM2-F81
81
MDM2-R82
82
21


GRB2
 313-535#
GRB2-F83
83
GRB2-R84
84
66


(5)
 599-674##
GRB2-F85
85
GRB2-R86
86
7



 503-591##
GRB2-F87
87
GRB2-R88
88
21



  799-868###
GRB2-F89
89
GRB2-R90
90
66



  420-502###
GRB2-F91
91
GRB2-R92
92
13



 504-612##
GRB2-F93
93
GRB2-R94
94
29



 635-745##
GRB2-F95
95
GRB2-R96
96
6


NF1
5509-5581
NF1-F97
97
NF1-R98
98
56


(6)
1560-1619
NF1-F99
99
NF1-R100
100
9



6233-6302
NF1-F101
101
NF1-R102
102
60



583-655
NF1-F103
103
NF1-R104
104
61



3475-3547
NF1-F105
105
NF1-R106
106
84



7293-7367
NF1-F107
107
NF1-R108
108
6



2314-2391
NF1-F109
109
NF1-R110
110
18



10791-10883
NF1-F111
111
NF1-R112
112
81



8538-8665
NF1-F113
113
NF1-R114
114
21


MMD
279-365
MMD-F115
115
MMD-R116
116
19


(7)
749-822
MMD-F117
117
MMD-R118
118
19



671-761
MMD-F119
119
MMD-R120
120
65


RNF4
514-623
RNF4-F121
121
RNF4-R122
122
43


(8)
322-397
RNF4-F123
123
RNF4-R124
124
22



416-515
RNF4-F125
125
RNF4-R126
126
67



377-478
RNF4-F127
127
RNF4-R128
128
38



 96-205
RNF4-F129
129
RNF4-R130
130
1



524-607
RNF4-F131
131
RNF4-R132
132
75



 755-1010
RNF4-F133
133
RNF4-R134
134
16



548-756
RNF4-F135
135
RNF4-R136
136
43


DUSP6
789-912
DUSP6-F137
137
DUSP6-R138
138
66


(9)
1117-1331
DUSP6-F139
139
DUSP6-R140
140
22


CPEB4
3169-3308
CPEB4-F141
141
CPEB4-R142
142
33


(10)








WEE1
2745-2994
WEE1-F143
143
WEE1-R144
144
56


(11)








IRF4
1247-1345
IRF4-F145
145
IRF4-R146
146
55


(12)








STAT2
1868-1960
STAT2-F147
147
STAT2-R148
148
68


(13)








ZNF264
478-594
ZNF264-F149
149
ZNF264-
150
79


(14)



R150




POLDIP2
788-941
POLDIP2-
151
POLDIP2-
152
20


(15)

F151

R152




HPRT1**
218-319
HPRT1-F153
153
HPRT1-R154
154
73


(16)





*Probe No. indicates Universal ProbLibrary probe TM number.


**HPRT1 serves as a reference gene.


#Sequence of primer 184-1R (SEQ ID NO. 149) is perfect match to variant 2 (NM_203506), with one mismatch at 5′ end to variant 1 (SEQ ID NO. 5, NM_002086). Both variant mRNAs could be amplified using 184-1F/184-1R primer pair.


##Only variant 1 (SEQ ID NO. 5, NM_002086) could be amplified using the primer pair.


###Both variant mRNAs (SEQ ID No. 5, NM_002086 and NM_203506) could get the same amplicon.






Example 2
Blood Test for Lung Cancer-Associated Molecular Markers
Materials and Methods
Blood sample collections from lung cancer patients. One hundred fifty patients with histologically confirmed lung cancer were enrolled at 3 hospitals in this study. Forty lung cancer patients among them were enrolled at the National Taiwan University Hospital (Taipei, Taiwan; Area A), 30 patients at the Tri-Service General Hospital (Taipei, Taiwan; Area A), and 80 patients at the Taichung Veterans General Hospital (Taichung, Taiwan; Area B) between April 2006 and March 2007. The last group included 28 patients with recurrence of lung cancer. Tables 3 lists the detailed clinicopathological features of all (n=150) and new incidents of lung cancer patients (n=122). All patients were enrolled in a prospective investigational protocol approved by the Institutional Review Board (IRB) of each hospital, respectively.








TABLE 3








Characteristic










First cohort
Second cohort



All lung cancer patients
New lung cancer patients











Area
A
B
A
B





Patient No. (%)
70
80
70
52


Age (Mean ± SD)
66.3 ± 12.6
63.4 ± 12.8
66.3 ± 12.6
63.5 ± 12.6
















Gender
Male
39
(26%)
54
(36%)
39
(32%)
37
(31%)



Female
31
(21%)
26
(17%)
31
(25%)
15
(12%)


Stage
I
13
(9%)
15
(10%)
13
(11%)
7
(6%)



II
2
(1%)
12
(8%)
2
(1%)
9
(7%)



III
22
(15%)
23
(15%)
22
(18%)
12
(10%)



IV
33
(22%)
30
(20%)
33
(27%)
24
(20%)


Cell
Adenocarcinomas
51
(34.0%)
56
(37.3%)
51
(72.9%)
38
(73.1%)


Type
Squamous cell
9
(6.0%)
16
(10.7%)
9
(12.8%)
7
(13.5%)



carcinomas











Other NSCLC*
5
(3.3%)
1
(0.7%)
5
(7.1%)
1
(1.9%)



SCLC**
1
(0.7%)
0

1
(1.5%)
0




Others
4
(2.6%)
7
(4.7%)
4
(5.7%)
6
(11.5%)





*NSCLC stands for non-small cell lung cancer.


**SCLC stands for small cell lung cancer.






Control samples. Seventy eight peripheral blood samples were collected from normal volunteers consisting of 28 males and 50 females with an average age of 60.9±11.0 in Area A (Taipei, Taiwan). In Area B (Taichung, Taiwan), 72 peripheral blood samples were collected from normal volunteers (without clinical cancer disease) consisting of 24 males and 48 females with an average age of 55.3±9.3. Informed consent of each sample donor was obtained.
Eight milliliters of peripheral blood per patient was collected using BD VACUTAINER® CPTTM tube (BD, USA). Preparation of MNC from blood samples, total RNA extraction and RT-PCR analysis of cancer marker gene expression were performed according to the methods disclosed in Example 1.
Data normalization and statistical analysis. Real-time or quantitative PCR (qPCR) techniques rely on the ability to detect the PCR product at each cycle during the exponential phase. Real-time instrumentation, which couples fluorescence detection and thermal cycling, measures the change of signal (in relative fluorescence units, RFU) at every cycle. Results obtained during the exponential phase give the best estimate of the amount of starting material. An amplification threshold is set within the early exponential phase. The cycle number at which the amplification curve crosses this threshold is the cycle threshold (Ct) of the sample. The Ct value decreases linearly with an increasing quantity of the input DNA template and can be used as a quantitative measure of mRNA expression of a gene analyzed.
Relative quantities of mRNA expression for each gene were used for statistical analysis using Statistical program SAS version 9.1.3 Service Pack 3. Data were normalized as follows: ΔCt(test)=Ct(HK)−Ct(test), where Ct (test) stands for the cycle number of a gene analyzed, Ct(HK) stands for the cycle number of the endogenous housekeeping gene HPRT1 (HK).
Results
A chi-square test and analysis of variance (ANOVA) were used to analyze the data. ANOVA gives a statistical test of whether the means of several groups are all equal and determines whether any significant differences exist among two or more groups of subjects on one or more factors. Messenger RNA expression levels of all investigated genes in the control group were shown to be significantly correlated with geographical parameters, while only the mRNA expression levels of EIF2S3, MDM2, and DUSP6 in lung cancer patients were significantly correlated. In this study, “geographical” effect was controlled because the control group's blood samples were collected in the same two “geographical” areas as those of the lung cancer patient group.
Multiple logistic regression was applied to evaluate the correlation between gene expression level and lung cancer since more than one independent variable was included in the prediction equation. The data from the first and second cohort studies were analyzed separately for statistical significance.
The first cohort had a sample number (N) of 300, including 150 lung cancer patients (new and recurrent cases) and 150 controls. The statistic results from the first study cohort indicated that the mRNA expression levels of the following seven genes significantly correlated (p<0.05) with lung cancer: EIF2S3, MCM4, MDM2, GRB2, MMD, RNF4 and DUSP6 (Table 4).
In Table 4, an OR greater than 1 indicates that a patient with a relatively high mRNA expression level of investigated gene, such as MCM4, MDM2, GRB2 or DUSP6, is more likely classified under the lung cancer group. The OR of 11.873 for mRNA expression level of GRB2 gene means a person in the lung cancer group is over 10 times more likely to develop lung cancer than a person in the control group, where ΔCt(test) for GRB2 mRNA is increased by one unit. An OR less than 1 indicates a person with a relatively high mRNA expression level of EIF2S3, MMD or RNF4 genes is less likely to develop lung cancer.
The second cohort had a sample number (N) of 272, including 122 new incidents of lung cancer and the same control group (150) as the first cohort's. The second cohort included only new cases without the 28 recurrent cases. The OR from the second study cohort indicated that the following seven genes significantly correlated with lung cancer: EIF2S3, MCM4, GRB2, NF1, MMD RNF4 and DUSP6. Of these genes, 6 genes except the NF1 gene showed the correlation in both study cohorts (Table 4). The MCM4, GRB2, NF1 and DUSP6 genes with relatively higher mRNA expression levels were considered as risk factors for lung cancer, while EIF2S3, MMD, and RNF4 genes were considered as protective genes (Table 4).
The mRNA expression level of MDM2 was significantly associated with the 28 recurrent cases of lung cancer, which indicated that the MDM2 gene expression might be correlated with the lung cancer recurrence. A comparison of statistical analysis of the first and second study cohort data indicated that the NF1 gene expression might more likely correlate with the occurrence of lung cancer.












TABLE 4









First cohort

Second cohort




SEQ
N = 300

N = 272













Gene
ID NO.
OR*
P value
OR
P value















EIF2S3
1
0.053
<0.0001
0.004
<0.0001


MCM4
3
2.999
0.0024
2.252
0.0293


MDM2
4
2.528
0.0272
—
—


GRB2
5
11.873
<0.0001
14.724
<0.0001


NF1
6
—
—
4.628
0.007


MMD
7
0.443
0.0004
0.326
<.0001


RNF4
8
0.147
<0.0001
0.160
<0.0001


DUSP6
9
5.301
<0.0001
8.722
<0.0001





*OR stands for odds ratio.






Example 3
Gene Signature and Prediction of Clinical Outcome in Lung Cancer
Prediction models were generated based on the mRNA expression levels of investigated genes for predicting the risk of getting lung cancer, evaluating therapeutic response to a particular drug or treatment. Equations for prediction models were derived by using a step-wise variable selection method of multiple regression approach and with the criteria of p-value less than 0.1.
The DUSP6 gene was chosen first by the statistical analysis program using logistic regression to form a prediction model for N=300 and N=272. The gene EIF2S3 was then added to the prediction model for the same analysis. Other significant genes were serially added and processed as mentioned above until the optimal validity indexes were fulfilled for the study cohorts of N=300 and N=272, such as sensitivity>80%, specificity>85%, accuracy>85% and AUC (area under the ROC)>0.9.
Table 5 shows calculations of validity indices, such as sensitivity, specificity and accuracy. When a person is tested for cancer, the test outcome can be either positive (sick) or negative (healthy). Sensitivity=(No. of True Positives)/(No. of True Positives+False Negatives); Specificity=(No. of True Negatives)/(No. of True Negatives+False Positives); Accuracy=(No. of True Positives+No. of True Negatives)/(No. of True Positives+False Negatives+False Positives+True Negatives).







TABLE 5







Condition (e.g., disease) as


determined by “Gold” standard










True
False














Test
Positive
True Positive
False Positive
Positive


outcome

(TP)
(FP)
Predictive Value






(PPV)



Negative
False
True Negative
Negative




Negative
(TN)
Predictive Value




(FN)

(NPV)




Sensitivity
Specificity
Accuracy





One-Gene Signature: DUSP6






An increase in the relative mRNA expression level of the DUSP6 gene was detected in the peripheral blood samples of lung cancer patients, but not in the samples from normal controls. An elevated mRNA expression level of DUSP6 was highly associated with lung cancer for both study cohorts. In addition, the odds ratio of 5.3 and 8.7 for the mRNA expression level of the DUSP6 gene (Table 4) means that a person with a relatively higher mRNA expression level of the DUSP6 gene is about 5- and 9-times more at risk of developing lung cancer than a person with lower gene expression in study cohort N=300 and N=272, respectively.
The measurement of the DUSP6 gene mRNA expression is sufficient as a single variable in the prediction model PM-1 and PM-8 for the study cohorts N=300 and N=272, respectively. Both models delivered good validity indexes, such as sensitivity=69-72%, specificity=80-83%, accuracy=72-77%, and AUC=79-81% (Tables 6 and 7). These results indicated that the relative mRNA expression level of the DUSP6 gene can be potentially used as a molecular index for detection of lung cancer because an AUC of 0.8 to 0.9 is generally considered as a good test.
The DUSP6 gene can be further applied as a prognostic marker (index) for monitoring the therapeutic response, recurrence, and survival because the higher mRNA quantity of DUSP6 was consistently found in peripheral blood samples obtained from lung cancer patients, but not in the samples obtained from normal controls. For example, a reduction in the mRNA expression of the DUSP6 gene can be a direct or an indirect result from a positive therapeutic response or as an indication for a lower possibility of recurrence, better prognosis, and a longer survival period.







TABLE 6







Prediction models (PM) for study cohort n = 300


based on multiple regression analysis with step-wise approach














Gene Set for







Model
Prediction
SEQ ID






ID
Model (PM)
NO:
Sensitivity %
Specificity %
Accuracy %
AUC
















PM-1
DUSP6
9
72.0
80.0
72.0
0.79013


PM-2
DUSP6, EIF2S3
9, 1
77.3
80.7
79.0
0.86998


PM-3
DUSP6, EIF2S3,
9, 1, 5
78.0
86.0
82.0
0.89249



GRB2







PM-4
DUSP6, EIF2S3,
9, 1, 5, 8
82.0
88.7
85.3
0.90824



GRB2, RNF4







PM-5
DUSP6, EIF2S3,
9, 1, 5,
81.3
88.0
84.7
0.91849



GRB2, RNF4,
8, 7







MMD,







PM-6
DUSP6, EIF2S3,
9, 1, 5,
82.7
88.7
85.7
0.92676



GRB2, RNF4,
8, 7, 3







MMD, MCM4







PM-7
DUSP6, EIF2S3,
9, 1, 5,
82
88.0
85.0
0.93136



GRB2, RNF4,
8, 7, 3, 4







MMD, MCM4,








MDM2





The prediction model equations were as follow:


(1) PM-1: Y = −2.9954 + 1.5474 × DUSP6;


(2) PM-2: Y = 2.2095 + 2.0365 × DUSP6 − 1.7257 × EIF2S3;


(3) PM-3: Y = 1.4289 + 1.4017 × DUSP6 − 2.5814 × EIF2S3 + 1.9511 × GRB2;


(4) PM-4: Y = 3.1608 + 1.5836 × DUSP6 − 2.7234 × EIF2S3 + 2.5838 × GRB2 − 1.3237 × RNF4;


(5) PM-5: Y = 3.5445 + 1.7293 × DUSP6 − 2.3917 × EIF2S3 + 2.7266 × GRB2 − 1.6062 × RNF4 − 0.7211 × MMD;


(6) PM-6: Y = 6.0403 + 1.7820 × DUSP6 − 2.4374 × EIF2S3 + 2.5568 × GRB2 − 1.8151 × RNF4 − 0.7617 × MMD + 1.0296 × MCM4;


(7) PM-7: Y = 9.0793 + 1.6680 × DUSP6 − 2.9325 × EIF2S3 + 2.4742 × GRB2 − 1.9206 × RNF4 − 0.8141 × MMD + 1.0983 × MCM4 + 0.9274 × MDM2.













TABLE 7







Prediction models (PM) for study cohort n = 272


based on multiple regression analysis with step-wise approach














Gene Set for







Model
Prediction
SEQ ID






ID
Model (PM)
NO:
Sensitivity %
Specificity %
Accuracy %
AUC





PM-8
DUSP6
9
69.7
83.3
77.2
0.81328


PM-9
DUSP6, EIF2S3
9, 1
76.2
86.7
82.0
0.87221


PM-10
DUSP6, EIF2S3,
9, 1, 5
73.0
87.3
80.9
0.89423



GRB2







PM-11
DUSP6, EIF2S3,
9, 1, 5, 8
79.5
90.7
85.7
0.90623



GRB2, RNF4







PM-12
DUSP6, EIF2S3,
9, 1, 5,
78.7
88.7
84.2
0.92383



GRB2, RNF4.
8, 7







MMD







PM-13
DUSP6, EIF2S3,
9, 1, 5,
78.7
88.0
83.8
0.92948



GRB2, RNF4,
8, 7, 6







MMD, NFI







PM-14
DUSP6, EIF2S3,
9, 1, 5,
80.3
90.0
85.7
0.93448



GRB2, RNF4,
8, 7, 6, 3







MMD, NFI,








MCM4





The prediction model equations were as follow:


(1) PM-8: Y = −3.8260 + 1.8525 × DUSP6;


(2) PM-9: Y = 1.4569 − 1.7287 × EIF2S3 + 2.3448 × DUSP6;


(3) PM-10: Y = 0.6016 − 2.4501 × EIF2S3 + 1.7576 × GRB2 + 1.7607 × DUSP6;


(4) PM-11: Y = 2.1254 − 2.5506 × EIF2S3 + 2.3450 × GRB2 − 1.2167 × RNF4 + 1.8972 × DUSP6;


(5) PM-12: Y = 2.7212 − 2.2022 × EIF2S3 + 2.5379 × GRB2 − 0.8757 × MMD − 15887 × RNF4 + 2.1095 × DUSP6;


(6) PM-13: Y = 4.7530 − 3.1491 × EIF2S3 + 1.5388 × NFI + 2.8198 × GRB2 − 1.1177 × MMD − 1.7172 × RNF4 + 2.1433 × DUSP6;


(7) PM-14: Y = 6.7266 − 3.2199 × EIF2S3 + 0.8119 × MCM4 + 1.5322 × NFI + 2.6894 × GRB2 − 1.1209 × MMD − 1.8324 × RNF4 + 2.1659 × DUSP6.






Two-Gene Signature: DUSP6, EIF2S3

The EIF2S3 gene was chosen to improve the prediction performances for both study cohorts. The relative mRNA expression level of EIF2S3 gene seemed to be negatively correlated with the incidence of lung cancer. An extremely low odds ratio (0.004 and 0.053, Table 4) for the EIF2S3 gene indicated that an increase in one unit of ΔCt(test) can result in a reduction of 99.6% (=(1−0.004)×100%) and 94.7% (=(1-0.053)×100%) probability for having lung cancer in the study cohort of N=272 and N=300, respectively.
The prediction models PM-2 and PM-9 each contained two molecular markers, EIF2S3 and DUSP6 genes, to discriminate lung cancer patients and non-cancer control samples with a high specificity. 87% for PM-9 and 81% for PM-2. The three validity indexes sensitivity, accuracy, and AUC of both models using a two-gene signature were increased about 6-9% as compared to the models using one-gene signature (Tables 6 and 7).
Using a two-gene signature prediction model gave a better specificity than a one-gene signature prediction model for the study cohort of N=272, while the specificity remained the same for the study cohorts of N=300.
The value of the area under the ROC curve (AUC=0.86998 for N=300; AUC=0.87221 for N=272) for the prediction model using a two-gene signature presented an assay with a good diagnostic accuracy. Potential clinical utilities should include applications for detecting lung cancer, monitoring the therapeutic response and prognosis, such as the recurrence possibility within a certain follow-up period and survival.
Three-Gene Signature: DUSP6, EIF2S3, GRB2
The prediction model using a 3-gene signature (PM-3) gave an increased validity indexes in the study cohort of N=300 as compared to that using a two-gene signature (PM-2, Table 6). In the study cohort of N=272, however, the prediction model using a three-gene signature (PM-10) only slightly improved the specificity as compared to that using a two-gene signature (PM-9) (Table 7).
A higher AUC, LIP to 0.89, of both models based on a three-gene signature indicated an increased potential for clinical uses in detecting lung cancer, monitoring the therapeutic response and prognosis, such as the recurrence possibility within a certain follow-up period and survival.
Four-Gene Signature: DUSP6, EIF2S3, GRB2, RNF4
The mRNA expression level of the RNF4 gene was negatively correlated with lung cancer and was the fourth significant factor added to the modeling (PM-4 and PM-11; Tables 6 and 7). The odds ratios of 0.147 and 0.16 for the RNF4 gene in N=300 and N=272 cohorts indicated that the probability of having lung cancer can reduce 85.3%, i.e., (1−0.147)×100%, and 84%, i.e., (1−0.16)×100%, respectively, with an increase by one unit of ΔCt(test).
A lung cancer patient can be accurately discriminated from a non-cancerous individual by models based on a 4-gene signature since the AUC in both models were greater than 0.9, which is generally considered as an excellent test. Other validity indexes of both models also met the performance of a good diagnostic test, such as 80-82% sensitivity, 89-91% specificity, and 86% accuracy (Tables 6 and 7).
The potential clinical uses of a four-gene signature include development of tests for detecting lung cancer, monitoring the therapeutic response and prognosis, such as the recurrence possibility within a certain follow-up period and survival.
Five-Gene Signature: DUSP6, EIF2S3, GRB2, RNF4, MMD
The gene MMD was added as the fifth cancer-associated molecular marker to form the prediction model PM-5 and PM-12 (Tables 6 and 7). The relative mRNA expression levels of MMD, EIF2S3 and RNF4 genes were negatively correlated with lung cancer, since the OR was 0.443, 0.053 and 0.147 for N=300 cohort and 0.326, 0.004 and 0.160 for N=272 cohort, respectively (Table 4). These genes should therefore be considered as protective genes for lung cancer in their predictive models.
The diagnostic performance of a five-gene signature in both prediction models (PM-5 and PM-12) was slightly improved as compared to the models using a four-gene signature (PM-4 and PM-11). Only the AUC value increased from 0.908 to 0.918 for N=300 and from 0.906 to 0.923 for N=272 cohorts, whereas other validity indexes, sensitivity, specificity and accuracy in both models were almost the same. The five-gene signature can also be applied to develop tests for detecting lung cancer, monitoring the therapeutic response and prognosis, such as the recurrence possibility within a certain follow-up period and survival.
Six-Gene Signature: DUSP6, EIF2S3, GRB2, RNF4, MMD, MCM4/or NF1
The gene MCM4 was selected as the sixth cancer-associated market in the prediction model PM-6 for the study cohort of N=300, while the gene NF1 was added in the prediction model PM-13 for the study cohort of N=272. Based on the OR of 2.999 for N=300 and 2.252 for N=272 cohorts, a person with an increase by one unit of ΔCt(test) for the relative mRNA expression of MCM4 gene was 2-3 times more at risk of developing lung cancer. The higher mRNA expression of NF1 gene was also represented as a risk factor for lung cancer disease.
The diagnostic performance of the prediction model PM-6 was slightly better than PM-5 for the study cohort of N=300 as to an increased sensitivity, specificity, accuracy and AUC (Table 6). The prediction model PM-13 for the study cohort of N=272 showed almost the same performance as the prediction model PM-12 (Table 7). Both six-gene signature prediction models can potentially be used for the development of clinical tests for detecting lung cancer, monitoring the therapeutic response and prognosis, such as the recurrence possibility within a certain follow-up period and survival.
Seven-Gene Signature: DUSP6, EIF2S3, GRB2, RNF4, MMD, MCM4 and MDM2 or NF1
The gene MDM2 was the seventh molecular marker added to the aforementioned 6 genes, DUSP6, EIF2S3, GRB2, RNF4, MMD and MCM4, in the prediction model PM-7 for the study cohort of N=300 using logistic multiple regression (Table 6). The higher mRNA expression of the MDM2 gene was significantly correlated to lung cancer only when the study cohort included recurrent and new incident cases. It, however, disappeared from the list of significant molecular factors after removal of recurrent cases. The result indicated that the mRNA expression level of the MDM2 gene might play a special role in the progression of lung cancer recurrence.
Based on the study cohort of N=300, the prediction model PM-7 using a seven-gene signature was optimized for clearly discriminating those lung cancer patients from normal controls with an excellent diagnostic performance, such as 82% sensitivity, 88% specificity, 85% accuracy and an estimated AUC of 0.93136. FIG. 1 shows the ROC curve for the prediction model PM-7 and the value of the estimated area (C).
The prediction model PM-14 for the study cohort of N=272 contained seven lung cancer-associated molecular markers: DUSP6, EIF2S3, GRB2, RNF4, MMD, NF1 and MCM4 genes (Table 7). A higher mRNA expression level of the NF1 gene appeared to be a significant risk factor for lung cancer based on the study cohort of N=272, which included only new cases of lung cancer.
The prediction model PM-14 showed an excellent diagnostic performance with 80% sensitivity, 90% specificity, 86% accuracy and an estimated AUC of 0.93448, similar to that of the prediction model PM-7. The validity indexes such as specificity and AUC of the prediction model PM-14 were slightly higher than those of the prediction model PM-7 (Table 7). FIG. 2 shows the ROC curve for the prediction model PM-7 and the value of the estimated area under the ROC curve (AUC).
Example 4
Procedures for Blood Test for Colorectal Cancer-Associated Molecular Markers
Fifty patients with histologically confirmed colorectal cancer were enrolled in a prospective investigational protocol, which was approved by the Institutional Review Board at the Cheng Hsin Rehabilitation Medical Center (Taipei, Taiwan). Table 8 lists the detailed clinicopathological features of the patients.
Eight milliliters of peripheral blood of each patient was collected using BD VACUTAINER® CPTTM tubes (BD, USA). For control samples, statistical analysis was performed on assay data of 78 peripheral blood samples of normal volunteers (without clinical cancer) collected in Taipei (Taiwan). The control group contained 28 males and 50 females with an average age of 60.9±11.0.
Total RNA was extracted from blood samples and cDNA was then synthesized as described in Example 1. The mRNA expression of sixteen genes EIF2S3, EXT2, MCM4, MDM2, GRB2, NF1, MMD, RNF4, DUSP6, CPEB4, WEE1, IRF4, STAT2, ZNF264, POLDIP2, and HPRT1 (as reference gene) in each sample was quantified using real-time PCR assay as described in Example 1. Data normalization and statistical analysis of experimental data were performed as described in Example 2.







TABLE 8







Clinicopathologic Characteristic of all colorectal cancer patients


(N = 50)










Characteristic
No. of Patients (%)






Area A
 50 (100%)



Age(Mean ± SD)
66.3 ± 12.6



Gender




Male
29 (58%)



Female
21 (42%)



Stage




I
10 (20%)



II
10 (20%)



III
15 (30%)



IV
11 (22%)



Others
4 (8%)









Results

Chi-square test and analysis of variance (ANOVA) of the mRNA expression levels of investigated genes obtained from the colorectal cancer patient group and from the control group were performed to examine the correlation and independency of variants.
Age and gender-associated gene expression was identified neither in colorectal cancer group nor in control group. The effects derived from the geographical factor were excluded because both control and colorectal cancer samples were collected in the same geographical area.
The mRNA expression levels of EIF2S3, MDM2, GRB2, NF1, MMD, RNF4, and DUSP6 significantly correlated with colorectal cancer with p-value<0.05 (Table 9). Although the p-value for the mRNA expression level of the EXT2 gene was 0.06, close to 0.05, it was used as a colorectal cancer-associated molecular marker for modeling.
An odds ratio greater than 1 indicates that an individual with a higher mRNA expression level of an investigated gene such as MDM2, GRB2, NF1 or DUSP6 is more likely to develop colorectal cancer than an individual with a lower expression level of the same gene. An odds ratio of less than 1 indicates that an individual with a lower mRNA expression level of an investigated gene such as EIF2S3, EXT2, MMD, or RNF4 is more likely to develop colorectal cancer. In other words, the higher mRNA expression levels of MDM2, GRB2, NF1 and DUSP6 genes in peripheral blood samples were considered as risk factors for colorectal cancer, while the higher mRNA expression of EIF2S3, EXT2, MMD and RNF4 genes seemed to play a protective role.







TABLE 9







Colorectal cancer-associated genetic markers












Gene
SEQ ID NO:
OR
P value















EIF2S3
1
0.006
<0.0001***



EXT2
2
0.240
0.0623



MDM2
4
17.745
0.0009**



GRB2
5
6.993
0.0153*



NF1
6
42.825
0.0032**



MMD
7
0.223
0.0003**



RNF4
8
0.195
0.0117*



DUSP6
9
5.309
0.0019**


*p < 0.05;


**p < 0.01;


***p < 0.001






Example 5
Gene Signatures for Colorectal Cancer 
The statistical approach used for predicting colorectal cancer, evaluating the therapeutic response and prognosis was as described in Example 2. Briefly, the prediction models were derived based on the mRNA expression of investigated genes. Equations for prediction models were derived by using a step-wise variable selection method of multiple regression approach and with the criteria of p-value<0.1 (Table 9). The DUSP6 gene was chosen as the first gene by the statistical analysis program to form a prediction model. Then the EIF2S3 gene was added to the prediction model to perform the same analysis procedures. Other significant genes were serially added and processed as mentioned above until optimal validity indexes were fulfilled, such as >80% sensitivity, >85% specificity, >85% accuracy and an AUC (area under the ROC ) of >0.9 (Table 10).







TABLE 10







Prediction models (PM) for study cohort n = 128


based on multiple regression analysis with step-wise approach













Model
Gene Set for
SEQ ID






ID
Prediction Model
NO:
Sensitivity %
Specificity %
Accuracy %
AUC





PM-15
DUSP6
9
58.0
82.1
72.7
0.77077


PM-16
DUSP6, EIF2S3
9, 1
64.0
84.6
76.6
0.82615


PM-17
DUSP6, EIF2S3,
9, 1, 4
74.0
87.2
82.0
0.88679



MDM2







PM-18
DUSP6, EIF2S3,
9, 1, 4, 6
70.0
87.2
80.5
0.90641



MDM2, NF1







PM-19
DUSP6, EIF2S,
9, 1, 4, 6, 7
74.0
89.7
83.6
0.91962



MDM2, NF1, MMD







PM-20
DUSP6, EIF2S3,
9, 1, 4, 6,
80.0
89.7
85.9
0.93795



MDM2, NF1, MMD,
7, 8







RNF4







PM-21
DUSP6, EIF2S3,
9, 1, 4, 6,
74.0
91.0
84.4
0.94410



MDM2, NF1, MMD,
7, 8, 5







RNF4, GRB2







PM-22
DUSP6, EIF2S3,
9, 1, 4, 6,
76.0
88.5
83.6
0.94551



MDM2, NF1, MMD,
7, 8, 5, 2







RNF4, GRB2, EXT2





The prediction model equations were as follows.


(1) PM-15: Y = −3.5600 + 1.5766 × DUSP6;


(2) PM-16: Y = 1.8263 − 1.5721 × EIF2S3 + 1.6581 × DUSP6;


(3) PM-17: Y = 8.5768 − 2.9997 × EIF2S3 + 2.5961 × MDM2 + 1.2722 × DUSP6;


(4) PM-18: Y = 9.1597 − 3.9042 × EIF2S3 + 1.9563 × MDM2 + 2.4926 × NF1 + 1.3385 × DUSP6;


(5) PM-19: Y = 9.9823 − 3.9749 × EIF2S3 + 1.8787 × MDM2 + 2.8088 × NF1 − 0.7192 × MMD + 1.6132 × DUSP6;


(6) PM-20: Y = 14.9816 − 4.0103 × EIF2S3 + 2.5131 × MDM2 + 2.6702 × NF1 − 1.1964 × MMD − 1.7016 × RNF4 + 2.0085 × DUSP6;


(7) PM-21: Y = 15.0677 − 4.6737 × EIF2S3 + 2.4380 × MDM2 + 1.3340 × GRB2 + 3.2085 × NF1 − 1.3968 × MMD − 1.9878 × RNF4 + 1.7477 × DUSP6:


(8) PM-22: Y = 13.9614 − 5.1493 × EIF2S3 − 1.428 × EXT2 + 2.8761 × MDM2 + 1.9449 × GRB2 + 3.7571 × NF1 − 1.4987 × MMD − 1.6345 × RNF4 + 1.6694 × DUSP6.






One-Gene Signature: DUSP6

A consistent increase in the mRNA expression of the DUSP6 gene was found in the peripheral blood samples obtained from the colorectal cancer patients but not from the normal controls. The mRNA expression level of the DUSP6 gene was significantly correlated with colorectal cancer (p-value=0.0019). An odds ratio of 5.3 for the DUSP6 gene meant that a person with a relatively high mRNA expression level of DUSP6 is about 5-times more at risk of developing colorectal cancer than a person with a lower expression level (decrease of ΔCt(test) by one unit).
The measurement of the DUSP6 gene mRNA expression level was sufficient in the prediction model PM-15 and delivered a high specificity (0.82) and other good validity indexes as shown in Table 10. The results indicated that the mRNA expression level of the DUSP6 gene can be potentially used as a molecular marker for detecting colorectal cancer disease since an AUC of 0.7 to 0.8 is generally considered as a marginally useful test.
The mRNA expression level of the DUSP6 gene can be further applied as an index for monitoring the therapeutic response and as a prognostic marker for evaluation of the recurrence possibility within a certain follow-up period and survival. For example, a reduction in the mRNA expression level of the DUSP6 gene can be a direct or an indirect result from a positive therapeutic response or as an indication for a lower possibility of recurrence, better prognosis and a longer survival period.
Two-Gene Signature: DUSP6, EIF2S3
The mRNA expression level of the EIF2S3 gene was selected as the second colorectal cancer-associated molecular marker (p-value<0.0001) to improve the prediction performance during modeling (Table 10). An extremely low odds ratio of 0.006 for the EIF2S3 gene indicated that an increase in one unit of ΔCt(test) could result in a reduction of 99.4% ((1−0.006)*100) probability for having colorectal cancer.
The prediction model PM-16, which was based on the mRNA expression levels of the EIF2S3 and DUSP6 genes, discriminated the colorectal cancer patient from the control with a high specificity (nearly 85%). Other diagnostic performance characters of the PM-16 model, such as the sensitivity, accuracy and AUC, increased about 5-10% as compared to the PM-15 model, which contained only one molecular marker, DUSP6.
The value of AUC for the prediction model PM-16 was 0.82615 (Table 10), which met the criteria for a good diagnostic test. Potential clinical utilities of a two-gene signature test include applications for detecting colorectal cancer, monitoring the therapeutic response and prognosis prediction, i.e., the recurrence possibility within a certain follow-up period and survival.
Three-Gene Signature: DUSP6, EIF2S3, MDM2
The MDM2 gene (p-value=0.0009), which had a high odds ratio of 17.745, was the third cancer-associated molecular marker added for modeling of PM-16. A person with a relatively high mRNA expression level of the MDM2 gene is about 17-times more at risk of developing colorectal cancer than a person with a lower mRNA expression level (decrease of ΔCt(test) by one unit).
A 10 percent increase in the sensitivity was achieved using the PM-17 model as compared to the PM-16 model. Other three validity indexes, specificity, accuracy and AUC, of this model met the criteria for a diagnostic test with a good performance. An AUC of 0.88679 in the PM-17 model indicated potential clinical uses in developing detection assays for colorectal cancer, monitoring the therapeutic response and prognosis prediction, i.e., the recurrence possibility within a certain follow-up period and survival.
Four-Gene Signature: DUSP6, EIF2S3, MDM2, NF1
The NF1 gene was the fourth colorectal cancer-associated molecular marker (p-value=0.0032) added for modeling. The prediction model PM-18 represented a four-gene signature (Table 10). A very high odds ratio of 42.825 for NF1 provided information that an individual with a relatively high mRNA expression level is approximately 43-times more at risk of developing colorectal cancer than those with a lower expression level (decrease of ΔCt(test) by one unit).
An AUC of 0.90641 was obtained for PM-18, which is generally considered as an excellent diagnostic test. Furthermore, the high specificity (87%) and accuracy (81%) of this model also met the perfomiance requirement of a good diagnostic test. The potential clinical uses of a four-gene signature test include development of a diagnostic test for colorectal cancer, monitoring the therapeutic response and prognosis prediction, i.e., the recurrence possibility within a certain follow-up period and survival.
Five-Gene Signature: DUSP6, EIF2S3, MDM2, NF1, MMD
The MMD gene was added as the fifth colorectal cancer-associated molecular marker (p-value=0.0003) for the model PM-19. A higher mRNA expression level of the MMD gene was considered to have a protective effect against colorectal cancer based on the odds ratio of 0.223 (less than 1).
The diagnostic performance of PM-19 based on a five-gene signature was improved as compared to that using a four-gene signature (PM-18) since all the four validity indexes of test quality, i.e., the sensitivity, specificity, accuracy and AUC, were increased (Table 10). The five-gene signature can be applied to develop detection tests for colorectal cancer, monitoring the therapeutic response, and prognosis prediction, i.e., the recurrence possibility within a certain follow-up period and survival.
Six-Gene Signature: DUSP6, EIF2S3, MDM2, NF1, MMD, RNF4
The RNF4 gene was the sixth significant factor (p-value=0.0117) for optimization of the prediction model. Based on the odds ratio (0.195), there was an 80.5%, i.e., (1−0.195)×100) probability for decreasing the colorectal cancer risk with an increase in one unit of ΔCt(test), which was the normalized mRNA expression level for the RNF4 gene.
The validity indexes of the model PM-20 fulfilled the criteria of an excellent performance for diagnostic tests with 80% sensitivity, 90% specificity, 86% accuracy, and an AUC (i.e., area under the ROC) of 0.94. The six-gene signature test can potentially be used for development of clinical tests for detecting colorectal cancer, monitoring the therapeutic response and prognosis, i.e., the recurrence possibility within a certain follow-up period and survival.
Seven-Gene Signature: DUSP6, EIF2S3, MDM2, NF1, MMD, RNF4, GRB2
The GRB2 gene was chosen as the seventh molecular marker (p-value=0.0153) for further optimization of the prediction efficacy of the model. A high odds ratio (6.993) for the GRB2 gene meant an individual with a relatively high mRNA expression level of the GRB2 gene will be approximately 7-times more at risk of developing colorectal cancer than an individual with a lower mRNA expression level (decrease of ΔCt(test) by one unit).
The high specificity (91%), accuracy (84%) and AUC (0.94410) indicated that a seven-gene signature test can be an excellent test for diagnostic use rather than for screening purpose since the sensitivity was 74%. The adjustment of cutoff value can further increase the test's sensitivity. The seven-gene signature test can potentially be used for detecting colorectal cancer, monitoring the therapeutic response and prognosis, such as the recurrence within a certain period and survival.
Eight-Gene Signature: DUSP6, EIF2S3, MDM2, NF1, MMD, RNF4, GRB2, EXT2
The EXT2 gene has been assumed as a colorectal cancer-associated molecular marker with a p-value=0.0623, very close to the value of significance. The mRNA expression of the EXT2 gene appeared to play a protective role for colorectal cancer because of its odds ratio at 0.24.
The diagnostic accuracy of the eight-gene signature test of PM-22 was excellent with respect to its high AUC at 0.94551. Taken together with other validity indexes of the PM-22 model, the eight-gene signature test can potentially be used for development of tests for detecting colorectal cancer, monitoring the therapeutic response and prognosis of colorectal cancer.
Example 6 
Primers and Probes for Quantitative Measurement of Cancer-Associated Gene Expression
Table 2 lists the information on primers and probes used for quantitative measurement of mRNA levels of cancer-associated genes. To test the specificity of the primers, cDNA templates were prepared from ten different lung and colon carcinoma cell lines purchased from the Food industry Research and Development Institute (FIRDI, Hsinshu, Taiwan) and American Type Culture Collection (ATCC; Manassas, USA). The cultures were maintained under the conditions described in the instruction manual. The list of mammalian cell lines used for the preparation of cDNA templates were as follows: the human colon adenocarcinoma cell lines CC-M1 (FIRDI/BCRC 60448), DLD-1 (FIRDI/BCRC 60132), LS174T (FIRDI/BCRC 60053), the human lung carcinoma cell line A549 (FIRDI/BCRC 60074), the human lung adenocarcinoma cell line NCI-H23 (ATCC/CRL 5800), the human lung squamous cell carcinoma cell line NCI-H520 (FIRDI/BCRC 60124), the human prostate carcinoma cell line PC3 (FIRDI/BCRC 60122), the human breast carcinoma cell line MCF-7 (FIRDI/BCRC 60436), the human normal prostate cell line PZ-HPV-7 (FIRDI/BCRC 60136), and the human normal lung cell line W138 (FIRDI/BCRC 60047).
Extraction of the total mRNA from cell lines and synthesis of cDNA using reverse transcription were performed as described in Example 1. Three different pools of cDNA templates were prepared for PCR amplification tests. The first pool was the lung cDNA pool, which contained equal amount of cDNA prepared from three lung cancer cell lines, A549, NCI-H23 and NCI-H520. The second pool was the colon cDNA pool, which contained equal amounts of cDNA prepared from three colon cancer cell lines, CC-M1, DLD-1, and LS174T. The third pool was a cDNA mix, which consisted of equally mixed cDNA from seven different sources of cancer and normal cell lines, including A549, LS174T, MCF-7, CC-M1, PZ-HPV-7, PC3 and W138.
Three programs used for designing primers and probes included the web-based PROBEFINDERTM software, Primer Select (Lasergene, DNASTAR, USA) and PRIMER EXPRESS® Software, LIGHTCYCLER® Probe Design Software 2.0. The PROBEFINDERTM software from the UNIVERSAL PROBELIBRARYTM Assay Design Center helped select an optimal combination of a UNIVERSAL PROBELIBRARYTM probe (F. Hoffmann-La Roche Ltd, Basel, Switzerland) and a gene-specific primer set for gene expression analysis using a real-time PCR assay. PROBELIBRARYTM probes contain only 8-9 nucleotides in length and often target sequences near exon-exon junctions. Therefore, the total RNA sample contaminated with genomic DNA during preparation would not interfere with the measurement of transcript level of an investigated gene. Table 2 lists information on specific primer pairs, probe sets and theoretical sequences of respective corresponding amplified products.
For every amplification PCR test, the reaction mixture contained 50 ng of cDNA template, 0.2 μM each of forward and reverse primers and TAQTM DNA Polymerase Kit (TAKARATM; Japan). Amplification reaction was performed using MJ Research PTC-100TM (Global Medical Instrumentation, Inc, MN, USA). The PCR conditions were 50° C. for 2 min, 95° C. for 10 min, 44 cycles of 95° C. for 15 sec and 60° C. for 1 min. The amplified products were analyzed using 3% agarose gel electrophoresis for confirmation of specificity.
Results
Most of the tested primer pairs were highly specific or specific for amplification of expected products. No amplicon was observed for the negative control (i.e., without the cDNA template) (Table 11). Some primer pairs, such as EIF2S3-F19/R20 (NOs: 19 and 20), EXT2-F47/R48 (SEQ ID NOs: 47 and 48), MCM4-F67/R68 (NOs. 67 and 68) and NF1-F113/R114 (SEQ ID NOs. 113 and 114), might not be suitable for assaying samples containing lung cancer cells, they were however suitable for assaying colorectal cancer. Table 2 lists the SEQ ID NOs. of primers.
Some primer pairs, such as EIF2S3-F27/R28 (SEQ ID NOs. 27 and 28), EIF2S3-F31/R32 (SEQ ID NOs. 31 and 32), GRB2-F91/R92 (SEQ ID NOs. 91 and 92) and NF1-F103/R104 (SEQ ID NOs. 103 and 104), did not show to be useful for assaying samples containing colorectal cancer cells but were suitable for assaying lung cancer.







TABLE 11*







Specificity test results of designed primer pairs















Size








of expected
Lung
Colon




Primer Pair

amplicon
cDNA
cDNA
cDNA



Forward/Reverse
SEQ ID NO.
(bp)
pool
pool
mix
NTC










EIF2S3 (SEQ ID NO: 1)













EIF2S3-F19/R20
19/20
78
NN
H
H
NN


EIF2S3-F21/R22
21/22
108
O
H
H
NN


EIF2S3-F23/R24
23/24
114
O
H
O
NN


EIF2S3-F25/R26
25/26
68
H
H
O
NN


EIF2S3-F27/R28
27/28
76
H
NN
NN
NN


EIF2S3-F29/R30
29/30
124
H
H
O
NN


EIF2S3-F31/R32
31/32
89
O
NN
H
NN







EXT2 (SEQ ID NO: 2)













EXT2-F35/R36
35/36
109
O
H
O
NN


EXT2-F37/R38
37/38
68
H
H
H
NN


EXT2-F39/R40
39/40
73
H
H
H
NN


EXT2-F41/R42
41/42
86
H
H
O
NN


EXT2-F43/R44
43/44
86
O
H
H
NN


EXT2-F45/R46
45/46
110
O
H
H
NN


EXT2-F47/R48
47/48
109
NN
O
O
NN


EXT2-F49/R50
49/50
115
O
H
O
NN


EXT2-F51/R52
51/52
282
H
H
H
NN







MCM4 (SEQ ID NO: 3)













MCM4-F55/R56
55/56
85
H
O
H
NN


MCM4-F57/R58
57/58
76
H
H
H
NN


MCM4-F59/R60
59/60
113
H
H
O
NN


MCM4-F61/R62
61/62
96
H
O
H
NN


MCM4-F63/R64
63/64
96
O
O
H
NN


MCM4-F65/R66
65/66
120
H
H
H
NN


MCM4-F67/R68
67/68
61
NN
H
O
NN


MCM4-F69/R70
69/70
128
O
O
O
NN


MCM4-F71/R72
71/72
181
H
H
H
NN


MCM4-F73/R74
73/74
211
H
H
H
NN







MDM2 (SEQ ID NO: 4)













MDM2-F77/R78
77/78
70
O
H
H
NN


MDM2-F79/R80
79/80
63
H
H
O
NN


MDM2-F81/R82
81/82
137
H
H
H
NN







GRB2 (SEQ ID NO: 5)













GRB2-F85/R86
85/86
76
H
H
H
NN


GRB2-F87/R88
87/88
89
O
H
NN
NN


GRB2-F89/R90
89/90
70
H
H
O
NN


GRB2-F91/R92
91/92
83
H
NN
NN
NN


GRB2-F93/R94
93/94
109
O
H
H
NN


GRB2-F95/R96
95/96
111
H
H
H
NN







NF1 (SEQ ID NO: 6)













NF1-F99/R100
 99/100
60
H
H
H
NN


NF1-F101/R102
101/102
70
H
H
H
NN


NF1-F103/R104
103/104
73
H
NN
H
NN


NF1-F105/R106
105/106
73
H
H
H
NN


NF1-F107/R108
107/108
75
H
H
H
NN


NF1-F109/R110
109/110
78
H
H
H
NN


NF1-F111/R112
111/112
93
O
O
O
NN


NF1-F113/R114
113/114
128
NN
H
H
NN







MMD (SEQ ID NO: 7)













MMD-F117/R118
117/118
74
H
H
H
NN


MMD-F119/R120
119/120
91
O
H
O
NN







RNF4 (SEQ ID NO: 8)













RNF4-F123/R124
123/124
76
O
H
O
NN


RNF4-F125/R126
125/126
100
H
H
H
NN


RNF4-F127/R128
127/128
102
H
H
H
NN


RNF4-F129/R130
129/130
110
O
H
H
NN


RNF4-F131/R132
131/132
84
H
H
H
NN


RNF4-F133/R134
133/134
256
O
H
H
NN


RNF4-F135/R136
135/136
209
H
H
H
NN







DUSP6 (SEQ ID NO: 9)













DUSP6-F139/R140
139/140
215
H
H
O
NN





*The full names for abbreviations used in Table 11 are as follows:


“NTC” for negative control assay containing the same reaction mixture but no template;


“H” for highly specific, only one single product obtained;


“O” for specific, amplified product was not shown in the NTC assay;


“NTC” for no expected amplicon obtained.






Example 7
Colorectal Cancer Marker Gene Expression Analysis Based on Case-Control Studies
Fifteen molecular markers were further investigated for detecting colorectal cancer in subjects using case-control studies, in which the cases were colorectal cancer patients and the controls were normal subjects without clinical cancer.
Methods
Sixty-five patients with histologically confirmed colorectal cancer were enrolled in a prospective investigational protocol approved by the Institutional Review Board at the Cheng Hsin Rehabilitation Medical Center (Taipei, Taiwan). In this example, 15 colorectal cancer patients were included in addition to the patient population disclosed in Example 4 (Table 8). The peripheral blood of each patient was collected as described in Examples 2. Table 12 lists the detailed clinicopathological features of the patient population.







TABLE 12







Clinicopathologic characteristics of colorectal cancer patients


(N = 65)










Characteristics
No. of Patients (%)






Area A
65 (100%) 



Age(Mean ± SE)
62.63 (1.47)    



Gender




Male
35 (53.8%)



Female
30 (46.2%)



Stage




0
3 (4.6%)



I
14 (21.5%)



II
13 (20%)  



III
17 (26.2%)



IV
13 (20%)  



Others
5 (7.7%)









Sixty-five normal volunteers (without cancer) as controls were chosen from the control population in Area A (Taipei, Taiwan) described in Example 2, including 35 males and 30 females with an average age (SE) of 59.55 ( 1.63). Sample preparations, total RNA extraction, reverse transcription reaction, quantification of mRNA expression levels using real-time PCR assay and raw data normalization were as described in Example 1.
A matched case-control design with one control for each patient was applied in the study (N=130). The matching criteria included the age and gender. Sixty five consecutive patients were identified with colorectal cancer who met the inclusion criteria, while 65 controls with matched age (±3) and gender were included. Chi-square test and t-test were employed to confirm gender and age distributions between cases and controls. The mRNA expression levels of 15 investigated genes were tested statistically between cases and controls using t-test. The logistic regression models and odds ratios were used to develop a model using a combination of investigated genes to predict whether subjects had colorectal cancer. The ROC curve and AUC indicated the probability of each investigated gene and/or a combination of multiple genes in predicting whether subjects (patients) had colorectal cancer. The statistical α level was 0.05.
Table 13 lists the mRNA expression levels of nine genes, RNF4, GRB2, MDM2, DUSP6, NF1, IRF4, EIF2S3, EXT2, and POLDIP2, in cases and controls (with p-value<0.05) and indications of significant difference between case and control subjects using t-test (Table 13).







TABLE 13







Difference in gene mRNA expression between colorectal


cancer patients and controls using t-test










Average of Normalized



Variable
Cycle Number (SD)












and
SEQ ID
Colorectal Cancer
Controls



Gene
NO.
Patients (N = 65)
(N = 65)
P value
















Male

35
(53.8%)
35
(53.8%)
1.000


Age (SD)

62.83
(1.47)
59.55
(1.63)
0.138


MCM4
 3
−1.2174
(0.07213)
−1.3571
(0.07946)
0.106


ZNF264
14
−1.9862
(0.05972)
−1.9766
(0.09086)
0.732


RNF4
 8***
2.7128
(0.08647)
2.2608
(0.08484)
<0.001


GRB2
 5*
2.4745
(0.07682)
2.3145
(0.08847)
0.050


MDM2
 4**
−0.4900
(0.06590)
−0.2260
(0.07570)
0.001


STAT2
13
2.1217
(0.07906)
2.2074
(0.09667)
0.694


WEE1
11
−0.1528
(0.09475)
−0.2843
(0.08316)
0.059


DUSP6
 9***
1.6312
(0.07803)
2.1391
(0.09396)
<0.001


CPEB4
10
1.5274
(0.07096)
1.6942
(0.09829)
0.274


MMD
 7
1.8295
(0.13526)
1.7954
(0.14593)
0.784


NF1
 6*
0.7423
(0.07101)
0.9678
(0.05011)
0.030


IRF4
12*
0.7186
(0.07178)
0.5135
(0.11368)
0.032


EIF2S3
 1***
3.6342
(0.08035)
3.3588
(0.06314)
<0.001


EXT2
 2**
−0.2835
(0.07266)
−0.5194
(0.06489)
0.001


POLDIP2
15*
2.7789
(0.07937)
2.5734
(0.06925)
0.026





*p < 0.05;


**p < 0.01;


***p < 0.001






Logistic Regression Model

The mRNA expression levels of the six genes RNF4, MDM2, DUSP6, MMD, NF1 and EIF2S3 were statistically significant to discriminate whether patients had colorectal cancer in Logistic Regression analysis (Table 14). The three genes GRB2, EXT2 and POLDIP2 were no longer significant after controlling other investigated genes in the Logistic Regression analysis and might have a weaker correlation with colorectal cancer than the above-mentioned six genes. When the age and gender were controlled in both case and control populations, the expression level of the GRB2 gene was not significantly associated with colorectal cancer (p-value=0.125; Table 14). The expression of the GRB2 gene might have an interaction with the age or gender. When the age and gender were not controlled in Example 4, up-regulation of the GRB32 gene was associated with colorectal cancer (Table 9). However, the colorectal cancer and normal subjects in Example 4 had a similar range of age (66.3±12.6 vs. 60.9±11.0).
The mRNA expression levels of three genes MDM2, DUSP6, and NF1 were found to increase, while the other three genes RNF4, MMD and EIF2S3 were found to decrease, in the peripheral blood of colorectal cancer patients. The MDM2, DUSP6, and NF1 three genes could thus be considered as risk genes for colorectal cancer.
The odds ratios (95% confidence intervals) of significantly up-regulated genes MDM2, DUSP6 and NF1 were 9.19 (1.93 ̃43.67), 6.017 (1.864 ̃19.415) and 84.164 (6.596 ̃1073.92), respectively, while those of significantly down-regulated genes RNF4, MMD and EIF2S3 were 0.072 (0.016 ̃0.32), 0.385 (0.168 ̃0.877) and 0.039 (0.007 ̃0.209), respectively. The prediction model based on all 15 genes is as follows: Y=9.999−0.928×MCM4+0.763×ZNF264−2.636×RNF4−1.437×GRB2+2.218×MDM2+1.216×STAT2+0.066×WEE1+1.795×DUSP6−0.153×CPEB4−0.955×MMD+4.433×NF1+0.081×IRF4−3.248×EIF2S3−0.973×EXT2+1.539×POLDIP2. It has 82.3% accuracy rate, 81.5% sensitivity and 83.1% specificity.







TABLE 14







Prediction model based on mRNA expression of each gene


using logistic regression model (82.3% accuracy)










SEQ ID
95% C.I. of OR













Gene
NO.
B
P value
OR
Upper
Lower
















MCM4
 3
−0.928
0.165
0.395
0.107
1.466


ZNF264
14
0.763
0.233
2.146
0.612
7.526


RNF4
 8**
−2.636
0.001
0.072
0.016
0.321


GRB2
 5
−1.437
0.125
0.238
0.038
1.493


MDM2
 4**
2.218
0.005
9.190
1.934
43.669


STAT2
13
1.216
0.074
3.373
0.891
12.772


WEE1
11
0.066
0.924
1.069
0.272
4.193


DUSP6
 9**
1.795
0.003
6.017
1.864
19.415


CPEB4
10
−0.153
0.761
0.858
0.321
2.297


MMD
 7**
−0.955
0.023
0.385
0.168
0.879


NF1
 6***
4.433
0.001
84.164
6.596
1073.920


IRF4
12
0.081
0.880
1.085
0.377
3.122


EIF2S3
 1***
−3.248
0.000
0.039
0.007
0.209


EXT2
 2
−0.973
0.279
0.378
0.065
2.200


POLDIP2
15
1.539
0.076
4.662
0.852
25.522


Constant

9.999
0.014
22012.838





B: regression coefficient;


OR: odds ratio;


C.I.: confidence interval;


*p < 0.05;


**p < 0.01;


***p < 0.001.






Step-Wise Logistic Regression Model

The step-wise logistic regression analysis showed that the mRNA expression levels of the six genes RNF4, MDM2, DUSP6, MMD, NF1 and EIF2S3 were significant to identify whether subjects had colorectal cancer. The three genes MDM2, DUSP6, and NF1 were significantly up-regulated, and the other three genes, RNF4, MMD, and EIF2S3 were down-regulated in colorectal cancer cases. The odds ratios (95% confidence intervals) of significantly up-regulated genes MDM2, DUSP6, and NF1 were 5.694 (1.717 ̃18.885), 6.127 (2.429 ̃15.46) and 34.182 (4.964 ̃235.386), respectively, and those of significantly down-regulated genes RNF4, MMD and EIF2S3 were 0.132 (0.048 ̃0.369), 0.432 (0.241 ̃0.773) and 0.05 (0.012 ̃0.211), respectively. Hosmer-Lemeshow test was insignificant (p=0.281), and accuracy rate was 84.6%.







TABLE 15







Step-wise Logistic Regression Model based on mRNA expression


of colorectal cancer-associated molecular markers










SEQ
95% C.I. of OR













Variable
ID NO.
B
P value
OR
Upper
Lower
















RNF4
8***
−2.022
<0.001
0.132
0.048
0.369


MDM2
4**
1.739
0.004
5.694
1.717
18.885


DUSP6
9***
1.813
<0.001
6.127
2.429
15.460


MMD
7**
−0.840
0.005
0.432
0.241
0.773


NF1
6***
3.532
<0.001
34.182
4.964
235.386


EIF2S3
1***
−3.003
<0.001
0.050
0.012
0.211


Constant

11.231
<0.001
75429.989





*p < 0.05;


**p < 0.01;


***p < 0.001;


B: Regression coefficient;


OR: odds ratio;


C.I.: confidence interval.






Table 16 lists all possible prediction models using multiple regression analysis with the step-wise approach for the case-control study cohort of N=130. The following prediction models have a good diagnostic performance and can be potentially used for development of clinical tests for detecting colorectal cancer, monitoring the therapeutic response, and prognosis, i.e., the recurrence possibility within a certain follow-up (or progress-free) period and survival.
One-Gene Signature: DUSP6, RNF4, MDM2, EIF2S3, NF1 or MMD
Each of the six colorectal cancer-correlated molecular markers DUSP6, RNF4, MDM2, EIF2S3, NF1 and MMD genes is the single variable for the prediction models PM-23, PM-24, PM-25, PM-26, PM-27 and PM-28, respectively (Table 16). The sensitivity, specificity and AUC of the one-gene signature-based prediction model ranged from about 44.6 to about 67.7%, from about 50.8 to about 72.3% and from about 0.486 to about 0.727, respectively. The PM-23 model containing the DUSP6 gene showed the best diagnostic performance, especially the AUC, among other models using the one-gene signature. The mRNA expression level of the DUSP6 gene is sufficient as a single variable in the prediction model PM-23 based on the case-control designed study cohort of N=130. The same result has been shown in the PM-15 model in Example 5, although there was a difference in the population of both study cohorts. The PM-23 model delivered a good validation index AUC of 0.727.
Two-Gene Signature: DUSP6 and RNF4
The mRNA expression level of the RNF4 gene was selected as the second colorectal cancer-associated molecular marker for the prediction model PM-29. The sensitivity of PM-29 (67.7%) was slightly higher than that of PM-16 (64%), while the specificity, accuracy, and AUC of the PM16 model showed a better performance. The PM-29 model can generally be considered as a useful test since its AUC was 0.786.
Three-Gene Signature: DUSP6, RNF4 and MDM2
The MDM2 gene was selected as the third molecular marker, which was the same as the PM-17 model (Example 5), in the prediction model PM-30 based on a 3-gene signature. The diagnostic performance of PM-30 showed an improvement in the sensitivity, accuracy and AUC. The AUC of the PM-30 model was 0.818, which met the criteria for a good diagnostic test.
Four-Gene Signature: DUSP6, RNF4, MDM2, EIF2S3
The mRNA expression level of the EIF2S3 gene, which was down-regulated in most of the colorectal cancer cases, was chosen as the fourth factor in the prediction model PM-31 in addition to DUSP6, RNF4, and MDM2.
The AUC of PM-31 was 0.86, which met the criteria for a good diagnostic test. The other validation indexes such as the sensitivity, specificity and accuracy were higher than 76%.
Five-Gene Signature: DUSP6, RNF4, MDM2, EIF2S3, NF1
The NF1 gene was added as the fifth molecular marker in the prediction model PM-32. The OR (34.2) of the NF1 gene was the highest among the colorectal-associated molecular markers based on the case-control study design (N=130). The same result was observed in the analysis disclosed in Example 5, even though not the same population of the study cohort was used for the statistical analysis.
In addition, the RNF4 gene in the PM-32 model was the only one different colorectal cancer-associated molecular marker chosen in a five-gene signature as compared to the PM-19 model in Example 4. The PM-32 model showed a better sensitivity than did the PM-19 model.
The overall diagnostic performance of the PM-32 model (a five-gene signature) was improved over the PM-31 (four-gene signature). The sensitivity, specificity and accuracy were greater than 81%. Especially, the AUC (0.893) of the PM-32 model almost met the criteria for an excellent diagnostic test.
Six-Gene Signature: DUSP6, RNF4, MDM2, EIF2S3, NF1 and MMD
The MMD gene was selected as the sixth molecular marker for the construction of the prediction model PM-33. The same six colorectal cancer-associated molecular markers were selected both in PM-33 and PM-20 (Example 5) but with different regression coefficients. The statistical analysis based on a careful case-control study cohort might be the reason for the difference.
The validity indices of the PM-33 model fulfilled the criteria of an excellent performance for diagnostic tests with 87.7% sensitivity, 81.5% specificity, 84.6% accuracy and an AUC of 0.912.
Conclusion
The risk of colorectal cancer was related to the up-regulation of the MDM2, DUSP6 and NF1 genes and the down-regulation of the RNF4, MMD and EIF2S3 genes on the basis of a case-control study design (N=130). Patients with the up-regulated expression of the MDM2, DUSP6 and/or NF1 genes were at an increased risk for colorectal cancer. The expression of the RNF4, MMD and EIF2S3 genes appeared to be independent suppressors for colorectal cancer. The same were concluded in Example 4.
The AUC of a one-gene signature-based model for predicting whether the patients had colorectal cancer was ranging from about 49% to about 73%. Enhancements of the AUCs of the prediction models by step-wised additions of next identified colorectal cancer-associated molecular markers were shown in FIG. 3 and Table 16. The accuracy rate (the sensitivity, specificity and accuracy) of tie prediction models could clearly be improved. The optimized prediction model PM-33, which used multiple molecular markers, achieved die best diagnostic performance with the validity indexes: 88% sensitivity, 82% specificity, 85% accuracy, and an AUC of 0.912.







TABLE 16







Diagnostic performance of prediction models for study cohort N = 130 based on multiple


regression analysis with step-wise approach
















P



Model
Gene

SE
Value
95% C.I.
















ID
No.
Sensitivity %
Specificity %
Accuracy %
AUC
(a)
(b)
Lower
Upper



















PM-23
1
67.7
70.8
69.3
0.727
0.045
<.001
0.638
0.816


PM-24
1
63.1
67.7
65.4
0.685
0.047
<.001
0.592
0.777


PM-25
1
63.1
60.0
61.5
0.675
0.047
0.001
0.582
0.768


PM-26
1
56.9
72.3
64.6
0.693
0.046
<.001
0.602
0.784


PM-27
1
58.5
50.8
54.6
0.610
0.049
0.030
0.514
0.707


PM-28
1
44.6
50.8
47.7
0.486
0.051
0.784
0.386
0.586


PM-29
2
67.7
72.3
70.0
0.786
0.040
<.001
0.708
0.864


PM-30
3
78.5
72.3
75.4
0.818
0.037
<.001
0.747
0.890


PM-31
4
76.9
78.5
77.7
0.860
0.032
<.001
0.798
0.923


PM-32
5
83.1
81.5
82.3
0.893
0.028
<.001
0.839
0.947


PM-33
6
87.7
81.5
84.6
0.912
0.026
<.001
0.862
0.963





AUC: area under ROC curve;


C.I.: Confidence Interval;


(a): nonparametric estimation;


(b): null hypothesis is AUC = 0.5.






The prediction model equations were as follows: PM-23=Y=−2.141+1.127×DUSP6; PM-24: Y=2.47×0.988×RNF4; PM-25: Y=0.335+0.982×MDM2; PM-26: Y=3.216−0.916×EIF2S3; PM-27: Y=−0.928+1.07×NF1; PM-28: Y=0.049×0.027×MMD; PM-29: Y=0.512+1.342×DUSP6−1.221×RNF4; PM-30: Y=2.19+1.235×DUSP6−1.602×RNF4+1.527×MDM2; PM-31: Y=8.174+1.185×DUSP6−1.602×RNF4+2.094×MDM2−1.607×EIF2S3; PM-32: Y=9.64+1.287×DUSP6−1.488×RNF4+1.648×MDM2−2.952×EIF2S3+2.93×NF1; PM-33: Y=11.231+1.813×DUSP6−2.022×RNF4+1.739×MDM2−3.003×EIF2S3+3.532×NF1−0.84×MMD.
Initial detection of lung or colorectal cancer in patients with or without symptoms of cancer can be done by a physician. When a physician suspects that a patient may have a lung or colorectal cancer, or is at risk of getting a lung or colorectal cancer, a physician can take a sample of blood or tissue for cancer screening according to one embodiment of the invention. The invention can also be used as a general patient screening tool. The expression levels of cancer-associated genes can be compared before and during the treatment to predict the therapeutic response to a particular cancer treatment. Similarly, the prognosis of lung or colorectal cancer can also be determined by comparing the expression levels of cancer-associated genes.
Table 17 illustrates measurements of mRNA levels of cancer marker genes in two blood samples using real-time PCR. The unit of mRNA quantity is in cycle number (Ct).

















TABLE 17





Sample
HPRT1
EIF2S3
EXT2
MDM2
GRB2
RNF4
MCM4
NF1
MMD
DUSP6







LTS077
26.09
23.52
27.46
25.11
23.80
24.67
27.91
25.23
22.60
23.74


NCI301
25.19
20.83
26.38
26.02
22.82
23.53
27.14
24.16
23.34
24.23









Normalization of measurements for each gene is made by subtracting respective cycle numbers of each gene from the reference gene HPRT1. Using DUSP6 as an example, its normalized mRNA expression level is Ct(HPRT1)−Ct(DUSP6). Table 18 shows the normalized mRNA level for each gene.
















TABLE 18





Sample
EIF2S3
EXT2
MDM2
GRB2
RNF4
MCM4
NF1
MMD
DUSP6
























LTS077
2.57
−1.37
0.98
2.29
1.42
−1.82
0.86
3.49
2.35


NCI301
4.36
−1.19
−0.83
2.37
1.66
−1.95
1.03
1.85
0.96









Applying the normalized data to the prediction models such as PM-1, PM-6 or PM-7 to obtain Y and probability (P) using the logistic regression model.








TABLE 19








PM-7












PM-1
PM-6

Proba-













Sample
Y
Probability
Y
Probability
Y
bility
















LTS077
0.641
0.6550
2.7093
0.9376
4.47
0.9887


NCI301
−1.51
0.181
−3.246
0.0375
−3.847
0.0209









Base on the cut-off value of 0.5 for the probability, sample LTS077 is predicted as “Positive” for lung Cancer, while sample NCI301 is predicted as “Negative” for lung cancer, i.e., normal.
All of the references cited herein are incorporated by reference in their entirety.
The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.
The embodiments and examples were chosen and described in order to explain the principles of the invention and their practical application so as to enable others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which die present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.
Some references, which may include patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is “prior art” to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.

## Claims

1. A method for detecting the presence and/or severity of lung and/or colorectal cancer, comprising the steps of:
(a) obtaining a test sample of bodily fluid comprising a nucleic acid from a subject;
(b) isolating RNA molecules from the sample;
(c) reverse-transcribing the RNA molecules to synthesize cDNA fragments;
(d) amplifying the cDNA fragments of at least six cancer gene markers with primers that comprise a fluorescent label, wherein the at least six cancer gene makers are selected from:
(i) the group consisting of: DUSP6, EIF2S3, MDM2, GRB2, RNF4, NF1, MMD, and MCM4; or
(ii) the group consisting of: DUSP6, EIF2S3, MDM2, GRB2, RNF4, NF1, MMD, and EXT2;

(e) measuring the quantity of the amplified cDNA fragments of the at least six cancer gene markers;
(f) normalizing the measured quantity of the amplified cDNA fragments of each of the at least six cancer gene markers to a housekeeping gene to obtain a normalized quantity of the amplified cDNA fragments of each of the at least six cancer gene markers;
(g) providing a logistic regression prediction model containing a positive coefficient for MDM2, GRB2, MCM4, NF1and DUSP6 each and a negative coefficient for EIF2S3, RNF4, and MMD each;
(h) applying the normalized quantity of the amplified cDNA fragments of each of the at least six cancer gene markers to the logistic regression prediction model to calculate the probability of cancer and/or cancer recurrence risk; and
(i) determining the presence and/or severity of lung and/or colorectal cancer based on the calculated probability.

2. The method of claim 1, wherein the test sample is a blood sample.

3. The method of claim 1, wherein step (d) amplifies the cDNA fragments of the following six cancer gene markers: DUSP6, EIF2S3, MDM2, RNF4, NF1 and MMD.

4. The method of claim 1, wherein step (d) amplifies the cDNA fragments of the following six cancer gene markers: DUSP6, EIF2S3, GRB2, RNF4, MMD and MCM4.

5. The method of claim 1, wherein step (d) amplifies the cDNA fragments of the following eight gene markers: DUSP6, EIF2S3, MDM2, GRB2, RNF4, NF1, MMD, and MCM4.

6. The method of claim 1, wherein step (d) amplifies the cDNA fragments of the following seven cancer gene markers: DUSP6, EIF2S3, MDM2, GRB2, RNF4, NF1and MMD.

7. The method of claim 1, wherein step (d) amplifies the cDNA fragments of the following six cancer gene markers: DUSP6, EIF2S3, GRB2, RNF4, NF1 and MMD.

8. The method of claim 1, wherein step (d) amplifies the cDNA fragments of at least:
(i) eight cancer gene markers;
(ii) seven cancer gene markers; or
(iii) six cancer gene markers.

9. The method of claim 1, wherein step (d) amplifies the cDNA fragments of:
(i) the six gene markers: DUSP6, EIF2S3, GRB2, RNF4, MMD, and MCM4/ or NF1;
(ii) the seven gene markers: DUSP6, EIF2S3, GRB2, RNF4, MMD, MCM4, and MDM2/ or NF1;
(iii) the six gene markers: DUSP6, EIF2S3, MDM2, NF1, MMD, and RNF4;
(iv) the seven gene markers: DUSP6, EIF2S3, MDM2, NF1, MMD, RNF4, and GRB2; or
(v) the eight gene markers: DUSP6, EIF2S3, MDM2, NF1, MMD, RNF4, GRB2, and EXT2.

10. The method of claim 9, wherein the amplifying step is performed by real-time polymerase chain reaction with at least one pair of primers selected from the group consisting of cancer gene marker-specific primer pairs 1 to 9 as follows:
(i) DUSP6(SEQ ID NO: 9)-specific primer pair 1: SEQ ID NOs: 137 and 138, or SEQ ID NOs. 139 and 140;
(ii) EIF2S3(SEQ ID NO: 1)-specific primer pair 2: SEQ ID NOs. 17 and 18, SEQ ID NOs. 19 and 20, SEQ ID NOs. 21 and 22, SEQ ID NOs. 23 and 24, SEQ ID NOs. 25 and 26, SEQ ID NOs. 27 and 28, SEQ ID NOs. 29 and 30, or SEQ ID NOs: 31 and 32;
(iii) MDM2(SEQ ID NO: 4)-specific primer pair 3: SEQ ID NOs: 75 and 76, SEQ ID NOs: 77 and 78, SEQ ID NOs: 79 and 80, or SEQ ID NOs: 81 and 82;
(iv) NF1(SEQ ID NO: 6)-specific primer pair 4: SEQ ID NOs: 97 and 98, SEQ ID NOs: 99 and 100, SEQ ID NOs: 101 and 102, SEQ ID NOs: 103 and 104, SEQ ID NOs: 105 and 106, SEQ ID NOs: 107 and 108, SEQ ID NOs: 109 and 110, SEQ ID NOs: 111 and 112, or SEQ ID NOs: 113 and 114;
(v) MMD (SEQ ID NO: 7)-specific primer pair 5: SEQ ID NOs: 115 and 116, SEQ ID NOs: 117 and 118, or SEQ ID NOs: 119 and 120;
(vi) RNF4 (SEQ ID NO: 8)-specific primer pair 6: SEQ ID NOs: 121 and 122, SEQ ID NOs: 123 and 124, SEQ ID NOs: 125 and 126, SEQ ID NOs: 127 and 128, SEQ ID NOs: 129 and 130, SEQ ID NOs: 131 and 132, SEQ ID NOs: 133 and 134, or SEQ ID NOs: 135 and 136;
(vii) GRB2 (SEQ ID NO: 5)-specific primer pair 7: SEQ ID NOs: 83 and 84, SEQ ID NOs: 85 and 86, SEQ ID NOs: 87 and 88; SEQ ID NOs: 89 and 90, SEQ ID NOs: 91 and 92, SEQ ID NOs: 93 and 94, or SEQ ID NOs: 95 and 96;
(viii) EXT2 (SEQ ID NO: 2)-specific primer pair 8: SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 50, or SEQ ID NOs: 51 and 52; and
(ix) MCM4 (SEQ ID NO: 3)-specific primer pair 9: SEQ ID NOs: 53 and 54, SEQ ID NOs: 55 and 56, SEQ ID NOs: 57 and 58, SEQ ID NOs: 59 and 60, SEQ ID NOs: 61 and 62, SEQ ID NOs: 63 and 64, SEQ ID NOs: 65 and 66, SEQ ID NOs: 67 and 68, SEQ ID NOs: 69 and 70, SEQ ID NOs: 71 and 72, or SEQ ID NOs: 73 and 74.

11. The method of claim 10, wherein the EIF2S3 (SEQ ID NO: 1)-specific primer pair 2: SEQ ID NOs: 27 and 28, or SEQ ID NOs: 31 and 32, the NF1 (SEQ ID NO: 6)-specific primer pair 4: SEQ ID NOs: 103 and 104, and/or the GRB2 (SEQ ID NO: 5)-specific primer pair 7: SEQ ID NOs: 91 and 92 are selected if step (e) determines the presence and/or severity of lung cancer, and are not selected if step (e) determines the presence and/or severity of colorectal cancer.

12. The method of claim 10, wherein the EIF2S3 (SEQ ID NO: 1)-specific primer pair 2: SEQ ID NOs: 19 and 20, the NF1 (SEQ ID NO: 6)-specific primer pair 4: SEQ ID NOs: 113 and 114, the EXT2 (SEQ ID NO: 2)-specific primer pair 8: SEQ ID NOs: 47 and 48, and/or the MCM4 (SEQ ID NO: 3)-specific primer pair 9: SEQ ID NOs: 67 and 68 are selected if step (e) determines the presence and/or severity of colorectal cancer, and are not selected if step (e) determines the presence and/or severity of lung cancer.

13. A method for monitoring and/or assessing the prognosis of a patient's response to a cancer therapy, comprising the steps of:
(a) obtaining samples of bodily fluid comprising a nucleic acid from the patient before and after receiving a cancer therapy for a lung and/or colorectal cancer;
(b) isolating RNA molecules from the sample;
(c) reverse-transcribing the RNA molecules to synthesize cDNA fragments;
(d) amplifying the cDNA fragments of at least six cancer gene markers with primers that comprise a fluorescent label, wherein the at least six cancer gene markers are selected from the group consisting of DUSP6, EIF2S3, MDM2, GRB2, RNF4, NF1, MMD, MCM4;
(e) measuring the quantity of the amplified cDNA fragments of the at least six cancer gene markers;
(f) normalizing the measured quantity of the amplified cDNA fragments of each of the at least six cancer gene markers to a housekeeping gene to obtain a normalized quantity of the amplified cDNA fragments of each of the at least six cancer gene markers;
(g) providing a logistic regression prediction model containing a positive coefficient for MDM2, GRB2, MCM4, NF1 and DUSP6 each and a negative coefficient for EIF2S3, RNF4, and MMD each;
(h) applying the normalized quantity of the amplified cDNA fragments of each of the at least six cancer gene markers to a logistic regression prediction model to calculate the probability of cancer and/or cancer recurrence risk; and
(i) evaluating the response by comparing the calculated probabilities from the samples, and thereby monitoring and/or assessing the prognosis of a patient's response to a cancer therapy;
wherein a decrease in the probability after receiving the cancer therapy is indicative of a positive response to the therapy.

14. The method of claim 13, wherein step (d) amplifies the cDNA fragments of at least:
(i) eight cancer gene markers;
(ii) seven cancer gene markers; or
(iii) six cancer gene markers.

15. The method of claim 13, wherein the test samples are blood samples.

16. A method for detecting the presence and/or severity of lung and/or colorectal cancer, comprising the steps of:
(a) obtaining a test sample of bodily fluid comprising a nucleic acid from a subject;
(b) isolating RNA molecules from the sample;
(c) reverse-transcribing the RNA molecules to synthesize cDNA fragments;
(d) amplifying the cDNA fragments of at least six cancer gene markers with primers that comprise a fluorescent label, wherein the at least six cancer gene markers are selected from:
(i) the group consisting of: DUSP6, EIF2S3, MDM2, GRB2, RNF4, NF1, MMD, MCM4; or
(ii) the group consisting of: DUSP6, EIF2S3, MDM2, GRB2, RNF4, NF1, MMD, and EXT2; and

(e) measuring the quantity of the amplified cDNA fragments of the at least six cancer gene markers;
(f) normalizing the measured quantity of the amplified cDNA fragments of each of the at least six cancer gene markers to a housekeeping gene to obtain a normalized quantity of the amplified cDNA fragments of each of the at least six cancer gene markers; and
(g) comparing the normalized quantity of the amplified cDNA fragments of each of the at least six cancer gene markers to a normalized quantity of the amplified cDNA fragments of each corresponding cancer gene marker in a sample of bodily fluids from a noncancerous control; and
(hi) determining that the subject is at risk of developing lung cancer when there is:
(i) an increase in the normalized quantity of the amplified cDNA fragments of DUSP6, GRB2, MCM4 and NF1 in the test sample as compared to the normalized quantity of the amplified cDNA fragments of the corresponding marker in the control; and
(ii) a decrease in the normalized quantity of the amplified cDNA fragments of EIF2S3, MMD, and RNF4 in the test sample as compared to the normalized quantity of the amplified cDNA fragments of the corresponding marker in the control; or

(hii) determining that the subject is at risk of lung cancer recurrence when there is an increase in the normalized quantity of the amplified cDNA fragments of MDM2 in the test sample as compared to the normalized quantity of the amplified cDNA fragments of the corresponding marker in the control; or
(hiii) determining that the subject is at risk of developing colorectal cancer when there is an increase in the normalized quantity of the amplified cDNA fragments of DUSP6, GRB2, MDM2 and NF1 in the test sample as compared to the normalized quantity of the amplified cDNA fragments of the corresponding marker in the control; or
(hiv) determining that the subject is at risk of developing colorectal cancer when there is a decrease in the normalized quantity of the amplified cDNA fragments of EIF2S3, MMD, EXT2, and RNF4in the test sample as compared to the normalized quantity of the amplified cDNA fragments of the corresponding marker in the control.

17. The method of claim 16, further comprising the steps of:
(a) normalizing the quantity of the amplified cDNA fragments of each of the at least six cancer gene markers to a housekeeping gene to obtain a normalized quantity of the amplified cDNA fragments of each of the at least six cancer gene markers;
(b) applying the normalized quantity of the amplified cDNA fragments of each of the at least six cancer gene markers to a logistic regression prediction model to calculate the probability of cancer and/or cancer recurrence risk; and
(c) determining the presence and/or severity of lung and/or colorectal cancer based on the calculated probability.

18. The method of claim 16, wherein the test sample is a blood sample.

19. A method for detecting the presence and/or severity of lung and/or colorectal cancer, comprising the steps of:
(a) obtaining a test sample of bodily fluid comprising a nucleic acid from a subject;
(b) isolating RNA molecules from the sample;
(c) reverse-transcribing the RNA molecules to synthesize cDNA fragments;
(d) amplifying the cDNA fragments of a set of cancer gene markers with primers that comprise a fluorescent label, wherein the set of cancer gene markers consists of 4, 5, 6, 7, or 8 genes selected from the group consisting of: DUSP6, EIF2S3, MDM2, GRB2, RNF4, NF1, MMD, and MCM4;
(e) measuring the quantity of the amplified cDNA fragments of the 4, 5, 6, 7, or 8 cancer gene markers;
(f) normalizing the measured quantity of the amplified cDNA fragments of each of the 4, 5, 6, 7, or 8 cancer gene markers to a housekeeping gene to obtain a normalized quantity of the amplified cDNA fragments of each of the at least four cancer gene markers;
(g) providing a logistic regression prediction model containing a positive coefficient for MDM2, GRB2, MCM4, NF1and DUSP6 each and a negative coefficient for EIF2S3, RNF4, and MMD each;
(h) applying the normalized quantity of the amplified cDNA fragments of each of the 4, 5, 6, 7, or 8 cancer gene markers to the logistic regression prediction model to calculate the probability of cancer and/or cancer recurrence risk; and
(i) determining the presence and/or severity of lung and/or colorectal cancer based on the calculated probability.

20. The method of claim 19, wherein step (d) amplifies the cDNA fragments of the following four gene markers: DUSP6, EIF2S3, GRB2 and RNF4.

21. The method of claim 19, wherein step (d) amplifies the cDNA fragments of the following four gene markers: DUSP6, EIF2S3, MDM2 and NF1.

22. The method of claim 19, wherein step (d) amplifies the cDNA fragments of the following four gene markers: DUSP6, EIF2S3, MDM2, and RNF4.

23. The method of claim 19, wherein:
step (d) amplifies the cDNA fragments of at least five cancer gene markers selected from the group consisting of: DUSP6, EIF2S3, MDM2, GRB2, RNF4, NF1, MMD, and MCM4;
step (e) measures the quantity of the amplified cDNA fragments of the at least five cancer gene markers;
step (f) normalizes the measured quantity of the amplified cDNA fragments of each of the at least five cancer gene markers to a housekeeping gene to obtain a normalized quantity of the amplified cDNA fragments of each of the at least five cancer gene markers;
step (g) provides a logistic regression prediction model containing a positive coefficient for MDM2, GRB2, MCM4, NF1 and DUSP6 each and a negative coefficient for EIF2S3, RNF4, and MMD each;
step (h) applies the normalized quantity of the amplified cDNA fragments of each of the at least five cancer gene markers to the logistic regression prediction model to calculate the probability of cancer and/or cancer recurrence risk; and
step (i) determines the presence and/or severity of lung and/or colorectal cancer based on the calculated probability.

24. The method of claim 23, wherein step (d) amplifies the cDNA fragments of the following five gene markers: DUSP6, EIF2S3, GRB2, RNF4 and MMD.

25. The method of claim 23, wherein step (d) amplifies the cDNA fragments of the following five gene markers: DUSP6, EIF2S3, MDM2, RNF4, and NF1.

26. The method of claim 1, wherein at least one of the primers targets an exon-exon junction.

27. The method of claim 13, wherein at least one of the primers targets an exon-exon junction.

28. The method of claim 16, wherein at least one of the primers targets an exon-exon junction.

29. The method of claim 19, wherein at least one of the primers targets an exon-exon junction.