WO2011076142A1

WO2011076142A1 - Compositions and methods for microrna expession profiling in plasma of colorectal cancer

Info

Publication number: WO2011076142A1
Application number: PCT/CN2010/080233
Authority: WO
Inventors: Zhaoyong Li; Ying Wu; Hongguang Zhu; Jian Li; Yiping Ren
Original assignee: Fudan University
Priority date: 2009-12-24
Filing date: 2010-12-24
Publication date: 2011-06-30

Abstract

The present invention relates to compositions and methods for microRNA (miRNA) expression profiling in plasma of colorectal cancer. In particular, the invention relates to a diagnostic kit of molecular markers in blood for diagnosing colorectal cancer, monitoring the cancer therapy and/or treating colorectal cancer, the kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in plasma of colorectal cancer and healthy control plasma, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative for the presence of colorectal cancer. The invention further relates to corresponding methods using such nucleic acid expression signatures for identifying colorectal cancer as well as for preventing or treating such a condition. Finally, the invention is directed to a pharmaceutical composition for the prevention and/or treatment of colorectal cancer.

Description

COMPOSITIONS AND METHODS FOR MICRORNA EXPESSION PROFILING IN PLASMA OF COLORECTAL CANCER

FIELD OF THE INVENTION

The present invention relates to compositions and methods for microRNA expression profiling in plasma of colorectal cancer.

BACKGROUND OF THE INVENTION

Colorectal cancer (CRC) is the most significant human cancer with an incidence of -1041, 200 new cases worldwide in 2008. It is the third most common cancer and the fourth leading cause of cancer deaths in the world (Gryfe, R. et al. (1997) Curr Probl Cancer 21, 233-300; Petersen, G.M. et al. (1999) Cancer 86, 2540-2550). CRC is curable if diagnosed at an early stage of development. At this early stage, most patients have no phenotypic symptoms of the disease. Therefore, screening is necessary to detect CRC in its early stages. Evidence indicates that early colorectal cancer detection can significantly reduce the incidence and mortality of the disease (Anwar, R. (2006) Digestive and Liver Disease 34, 279-282).

Initially, CRC is characterized by the occurrence of a hyper-proliferative (dysplastic) epithelium in the colon, which first turns into inflammatory adenomatous polyps, then into adenomas, which are abnormal neoplasms (i.e. benign tumors) in the inner lining of the colon or rectum. Usually, only a small subset of the adenomas formed (occurring with an incidence of 60-70% by age 60) progress into malignant adenocarcinomas. More than 95% of the cases of CRC are manifested as adenocarcinomas (Muto, T. et al. (1975) Cancer 36, 2251-2270; Fearon, E.R. and Vogelstein, B. (1990) Cell 61, 759-767).

The current standard screening methods for CRC include colonoscopy and the fecal occult blood tests. Both tests, however, suffer from serious disadvantages. The colonoscopy test is effective, but many people are hesitant to have this procedure due to its high cost, high discomfort and its potential for more significant side effects. The fecal occult blood test, on the other hand, is a simple and cheap test, but is relatively inaccurate. However, no specific molecular markers have been identified so far that allow for a reliable diagnosis of CRC, preferably CRC manifested as an adenocarcinoma, and/or the progression of a benign adenoma into such a malignant tumor.

Therefore, the discovery of new biomarkers would be of utmost clinical importance, particularly if these markers enable a diagnosis at an early stage of tumor progression in order to allow early stage treatment of carcinomas while avoiding unnecessary surgical intervention. Ideally, such markers should enable the identification of a carcinoma at a stage where the presence of malignant cells is not yet detectable by in situ techniques or microscopic analysis of biopsy or resection material.

Many diagnostic assays are also hampered by the fact that they are typically based on the analysis of only a single molecular marker, which might affect detection reliability and/or accuracy. In addition, a single marker normally does not enable detailed predictions concerning latency stages, tumor progression, and the like. Thus, there is still a continuing need for the identification of alternative molecular markers and assay formats overcoming these limitations.

One approach to address this issue might be based on small regulatory RNA molecules, in particular on microRNAs (miRNAs) which, constitute an evolutionary conserved class of endogenously expressed small non-coding RNAs of 20-25 nucleotides (nt) in size that can mediate the expression of target mRNAs and thus - since their discovery about ten years ago - have been implicated with critical functions in cellular development, differentiation, proliferation, and apoptosis (Bartel, D.P. (2004) Cell 116, 281-297, Ambros,V. (2004) Nature 431, 350-355; He. L. et al. (2004) Nat Rev Genet 5, 522-531). Furthermore, miRNAs have advantages over mRNAs as cancer biomarkers, since they are very stable in vitro and long-lived in vivo (Lu, J. et al. (2005) Nature 435, 834-838; Lim, L.P. et al. (2005) Nature 433, 769-773).

MiRNAs are produced from primary transcripts that are processed to stem-loop structured precursors (pre-miRNAs) by the RNase III Drosha. After transport to the cytoplasm, another RNase III termed Dicer cleaves of the loop of the pre-miRNA hairpin to form a short double-stranded (ds) RNA, one strand of which is incorporated as mature miRNA into a miRNA-protein (miRNP). The miRNA guides the miRNPs to their target mRNAs where they exert their function (Bartel, D.P. (2004) Cell 23, 281- 292; He, L. and Hannon, G.J. (2004) Nat Rev Genet 5, 522-531).

Depending on the degree of complementarity between the miRNA and its target, miRNAs can guide different regulatory processes. Target mRNAs that are highly complementary to miRNAs are specifically cleaved by mechanisms identical to RNA interference (RNAi). Thus, in such scenario, the miRNAs function as short interfering RNAs (siRNAs). Target mRNAs with less complementarity to miRNAs are either directed to cellular degradation pathways or are translationally repressed without affecting the mRNA level. However, the mechanism of how miRNAs repress translation of their target mRNAs is still a matter of controversy. Emerging data available indicate that miRNAs can play roles in cancer as oncogenes or tumor suppressor genes, such as overexpressed mir- 17-92 in cancers, may function as oncogenes and promote cancer development by negatively regulating tumor suppressor genes and/or genes that control cell differentiation or apoptosis, as well as underexpressed let-7a, function as tumor suppressor genes and may inhibit cancers by regulating oncogenes and/or genes that control cell differentiation or apoptosis (Zhang, B. (2007) Dev Biol 302, 1-12), suggesting their contribution to cancer development and progression.

High-throughput miRNA quantification technologies, such as miRNA microarray, real-time RT-PCR-based TaqMan miRNA assay and so on, have provided powerful tools to study the global miRNA profile in whole cancer genome. Increasing evidence shows that many miRNAs are deregulated in human cancer including leukemia, lymphoma, glioblastoma, colon, lung, breast, prostate, thyroid, liver, and ovarian cancer and are differentially expressed in normal tissues and cancers (Zhang, L. (2008) Adv Exp Med Biol 622, 69-78). Therefore, miRNA profiling is used to create signatures for a variety of cancers, indicating that the profile will help further establish molecular diagnosis, prognosis and therapy. The aberrant expression of miRNAs in human cancer indicates the potential of these miRNAs as biomarkers and targets for molecular therapy. Among the many possible types of samples, blood is thought to be ideal for screening high risk individuals, leading to early detection, diagnosis, monitoring and efficient treatment of cancers- since blood can be collected easily in a minimally invasive manner. It has been demonstrated that tumor-derived miRNAs are present in human plasma or serum in a remarkably stable form that is protected from endogenous RNase activity. These tumor-derived miRNAs in serum or plasma are at levels sufficient to be measurable as biomarkers for cancer detection. Moreover, the levels of plasma and serum miRNAs correlate strongly, suggesting that either plasma or serum samples will be suitable for clinical applications using miRNAs as cancer diagnostic biomarkers (Mitchell, P.S. et al. (2008) Proc Natl Acad Sci USA 105, 10513-10518; Gilad, S. et al. (2008) PLoS ONE 3, e3148; Chen, X. et al. (2008) Cell Res 18, 997- 1006).

Several studies have reported miRNA expression profiling in plasma or serum of human colorectal cancer (Chen, X. (2008) Cell Res 18, 997-1006; Ng, E.K.O. (2009) Gut 58, 1375-1381 ; Huang, Z. 2009, Int J Cancer, published on line). More than 100 circulating miRNAs can be identified in the blood of healthy individuals (Mitchell, P.S. (2008) Proc Natl Acad Sci USA 105, 10513-10518) and this profile differs significantly from that of patients with colorectal cancer who have several tumor-specific miRNAs. Chen and colleagues demonstrated 69 miRNAs in the serum of patients with colorectal cancer, which were not present in the serum of healthy controls (Chen, X. (2008) Cell Res 18, 997-1006). Moreover, they identified a unique expression profile of 14 serum miRNAs for colorectal cancers that were not present in another cancer group (lung cancer). Ng et al's study suggested that miR-92 is significantly elevated in plasma of patients with CRC and can be a potential non-invasive molecular marker for CRC screening (E.K.O. (2009) Gut 58, 1375-1381). Furthermore, Huang and colleagues found that plasma miR-29a and miR-92a have strong potential as novel noninvasive biomarkers for early detection of CRC (Huang, Z. 2009, Int J Cancer, published on line). Nevertheless, another study found that hsa-miR-92a dramatically decreased in the plasma of acute leukemia patients and the ratio of miR-92a/miR-638 in plasma has strong potential as a novel biomarker for detection of leukemia (Tanaka, M. et al. (2009) PLoS ONE 4,e5532). However, lack of sufficient sensitivity and specificity determined by single miRNA is unsuitable as accurate diagnostic biomarkers in clinical use.

Thus, there remains a need for discovery of a set of diagnostic miRNA markers in plasma or serum of colorectal cancer patients. Establishment of blood-based miRNA profiles (fingerprints) by combination of multiple miRNA biomarkers will enable a non-invasive, rapid, accurate, and cost-saving identification of patients exhibiting colorectal cancer. In addition, there is also a continuing need for corresponding methods for early stage-colorectal cancer screening in high risk individuals, early detection of the cancer recurrence, and/or monitoring the cancer therapy. OBJECT AND SUMMARY OF THE INVENTION

It is an objective of the present invention to provide novel approaches for diagnosing colorectal cancer, monitoring the cancer therapy and/or treating colorectal cancer by determining a plurality of nucleic acid molecules in blood, each nucleic acid molecule encoding a microRNA (miRNA) sequence, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in plasma of colorectal cancer, analyzed as compared to healthy controls, and/or as compared to healthy individuals, hepatocellular carcinoma and lung cancer, wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative for the presence of colorectal cancer, wherein the nucleic acid expression signatures include tumor-related signatures and plasma- specific signatures.

Furthermore, it is an object of the invention to provide corresponding methods for identifying one or more nucleic acid expression signatures in blood exhibiting colorectal cancer. More specifically, it is an object of the invention to provide methods for differentiating colorectal cancer as compared to healthy control, and/or as compared to healthy individuals, hepatocellular carcinoma and lung cancer.

These objectives as well as others, which will become apparent from the ensuing description, are attained by the subject matter of the independent claims. Some of the preferred embodiments of the present invention are defined by the subject matter of the dependent claims.

In a first aspect, the present invention relates to a diagnostic kit of molecular markers in blood for identifying colorectal cancer, the kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in the target plasma as compared to healthy controls, and wherein the differentially expressed signatures are derived from tumor-related or plasma- specific signatures, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative for the presence of colorectal cancer.

The nucleic acid expression signature, as defined herein, may comprise at least thirty-five acid molecules, preferably at least twelve nucleic acid molecules, and particularly preferably at least six nucleic acid molecules.

In preferred embodiments, the nucleic acid expression signature comprises at least one nucleic acid molecule encoding a microRNA sequence whose expression is up-regulated in the one or more target plasma compared to the one or more healthy controls and at least one nucleic acid molecule encoding a microRNA sequence whose expression is down-regulated in the one or more target plasma compared to the one or more healthy controls.

In preferred embodiments, the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding tumor-related signatures: hsa-miR- 409-3p, hsa-miR-25, hsa-miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa- miR-19b, hsa-miR-451, hsa-miR-486-5p, hsa-miR-187*, hsa-miR-92a, hsa-miR-19a, hsa-miR-20b, hsa-miR-20a, hsa-miR-139-3p, hsa-miR-107, hsa-miR-17, hsa-miR-140- 3p, hsa-miR-30e, hsa-miR-185 and plasma-specific signatures: hsa-mir-671-3p, hsa- mir-16-2*, hsa-miR-30c-l*, hsa-miR-548c-5p, hsa-miR-16, hsa-miR-15a, hsa-miR-425, hsa-let-7i, hsa-miR-363, hsa-miR-15b, hsa-miR-101, hsa-miR-190b, hsa-miR-130a and internal stable controls: hsa-miR-1238 and hsa-miR-1228.

Particularly preferably, the expression of any one or more of the nucleic acid molecules encoding hsa-miR-409-3p and hsa-mir-671-3p is up-regulated and the expression of any one or more of the nucleic acid molecules encoding hsa-miR-25, hsa- miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451, hsa- miR-486-5p, hsa-miR-187*, hsa-miR-92a, hsa-miR-19a, hsa-miR-20b, hsa-miR-20a, hsa-miR-139-3p, hsa-miR-107, hsa-miR-17, hsa-miR-140-3p, hsa-miR-30e, hsa-miR- 185' hsa-mir-16-2*, hsa-miR-30c-l*, hsa-miR-548c-5p, hsa-miR-16, hsa-miR-15a, hsa-miR-425, hsa-let-7i, hsa-miR-363, hsa-miR-15b, hsa-miR-101, hsa-miR-190b, hsa- miR-130a is down-regulated; hsa-miR-1238 and hsa-miR-1228 is un-changed in the one or more target plasma compared to the one or more healthy controls.

In more preferred embodiments, the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding tumor-related signatures: hsa- miR-409-3p, hsa-miR-25, hsa-miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451 and plasma- specific signatures: hsa-mir-671-3p, hsa-mir- 16-2*, hsa-miR-30c-l*, hsa-miR-548c-5p.

Particularly preferably, the expression of any one or more of the nucleic acid molecules encoding hsa-miR-409-3p and hsa-mir-671-3p is up-regulated and the expression of any one or more of the nucleic acid molecules encoding hsa-miR-25, hsa- miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451, hsa- mir-16-2*, hsa-miR-30c-l* and hsa-miR-548c-5p is down-regulated in the one or more target plasma compared to the one or more healthy controls.

In further preferred embodiments, the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding tumor-related signatures: hsa-miR-409-3p, hsa-miR-25, hsa-miR-93, hsa-miR-96, and plasma- specific signatures: hsa-mir-671-3p, hsa-mir-16-2*.

Particularly preferably, the expression of any one or more of the nucleic acid molecules encoding hsa-miR-409-3p and hsa-mir-671-3p is up-regulated and the expression of any one or more of the nucleic acid molecules encoding hsa-miR-25, hsa- miR-93, hsa-miR-96 and hsa-mir-16-2* is down-regulated in the one or more target plasma compared to the one or more healthy controls.

In particularly preferred embodiments, the nucleic acid expression signature comprises any one or more nucleic acid combinations encoding hsa-miR-409-3p/hsa- miR-16-2* hsa-miR-409-3p/hsa-miR-96, hsa-miR-671-3p/hsa-miR-548c-5p, hsa-miR- 671 -3p hsa-miR- 16-2*, hsa-miR-671 -3p hsa-miR-30c- 1 *, hsa-miR-671 -3p/hsa-miR- 342-3p, hsa-miR-67 l-3p/hsa-miR-96, hsa-miR-67 l-3p hsa-miR-30 la, hsa-miR-671- 3p/hsa-miR-345, hsa-miR-409-3p/hsa-miR-345, hsa-miR-409/hsa-miR-331-3p, hsa- miR-671-3p hsa-miR-331-3p, hsa-miR-671-3p/ hsa-miR-25, hsa-miR-409-3p/ hsa-miR- 93 and hsa-miR-671-3p/ hsa-miR-93.

In a second aspect, the present invention relates to a diagnostic kit of molecular markers for discriminating colorectal cancer from healthy individuals, hepatocellular carcinoma and lung cancer, the kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in the target plasma and in one or more healthy individuals, hepatocellular carcinoma and lung cancer, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative for the presence of colorectal cancer.

The nucleic acid expression signature, as defined herein, may comprise at least twenty-three nucleic acid molecules, preferably at least eighteen nucleic acid molecules, and particularly preferably at least six nucleic acid molecules.

In preferred embodiments, the nucleic acid expression signature comprises at least one nucleic acid molecule encoding a microRNA sequence whose expression is up-regulated in the one or more target plasma compared to the one or more healthy individuals, hepatocellular carcinoma and lung cancer, and at least one nucleic acid molecule encoding a microRNA sequence whose expression is down-regulated in the one or more target plasma compared to the one or more healthy individuals, hepatocellular carcinoma and lung cancer.

In more preferred embodiments, the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding tumor-related signatures: hsa- miR-409-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-7, hsa-miR-196b, hsa-miR-93, hsa-miR-486-5p, hsa-miR-25, hsa-miR-92a, hsa-miR-19b; plasma-specific signatures: hsa-miR-671-3p and hsa-miR-16-2*, hsa-miR-92b, hsa-miR-129*, hsa-miR-563, hsa- miR-602, hsa-miR-1227, hsa-miR-196a and internal stable controls: hsa-miR-1238 and hsa-miR-1228.

Particularly preferably, the expression of any one or more of the nucleic acid molecules encoding hsa-miR-409-3p, hsa-mir-671-3p, hsa-miR-33b*, hsa-miR-92b, hsa-miR-149, hsa-miR-129*, hsa-miR-563, hsa-miR-129-3p, hsa-miR-634, hsa-let-7b*, hsa-miR-602 and hsa-miR-1227 is up-regulated and the expression of any one or more of the nucleic acid molecules encoding hsa-miR-16-2*, hsa-miR-7, hsa-miR-196a, hsa- miR-196b, hsa-miR-486-5p, hsa-miR-93, hsa-miR-25, hsa-miR-92a and hsa-miR-19b is down-regulated, hsa-miR-1238 and hsa-miR-1228 is un-changed in the one or more target plasma compared to the one or more healthy individuals, hepatocellular carcinoma and lung cancer.

In more preferred embodiments, the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding tumor-related signatures: hsa- miR-409-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-7 and plasma- specific signatures: hsa-miR-671-3p and hsa-miR-16-2*.

Particularly preferably, the expression of any one or more of the nucleic acid molecules encoding hsa-miR-409-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-671-3p is up-regulated and the expression of any one or more of the nucleic acid molecules encoding hsa-miR-16-2* and hsa-miR-7 is down-regulated in the one or more target plasma compared to the one or more healthy individuals, hepatocellular carcinoma and lung cancer.

In particularly preferred embodiments, the nucleic acid expression signature comprises any one or more nucleic acid combinations encoding hsa-miR-33b*/hsa- miR-196a, hsa-miR-92b/hsa-miR-196a, hsa-miR-563/hsa-miR-196a, hsa-miR- 1227/hsa-miR-196a, hsa-miR-129-3p/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-16-2*, hsa-miR-92b/hsa-miR- 16-2*, hsa-miR- 129*/hsa-miR- 196a, hsa-miR- 1227/hsa-miR- 16- 2*, hsa-miR- 129-3p/hsa-miR- 196a, hsa-miR-602/hsa-miR-196a, hsa-miR-33b*/hsa- miR-16-2* hsa-miR-563/hsa-miR-16-2*, hsa-miR- 129*/hsa-miR-7, hsa-miR-33b*/hsa- miR-7, hsa-miR-563/hsa-miR-7, hsa-miR-602/hsa-miR-16-2*, hsa-miR-129-3p/hsa- miR-7, hsa-miR-92b/hsa-miR-7, hsa-miR-1227 hsa-miR-7, hsa-miR-33b*/hsa-miR- 196b, hsa-miR-1227/hsa-miR-196b, hsa-miR-92b/hsa-miR-196b and hsa-miR-602/hsa- miR-7.

In a third aspect, the present invention relates to a method for identifying one or more target plasma exhibiting colorectal cancer, the method comprising: (a) determining in the one or more target plasma the expression levels of a plurality of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence; (b) determining the expression levels of the plurality of nucleic acid molecules in one or more healthy control plasma; and (c) identifying from the plurality of nucleic acid molecules one or more nucleic acid molecules that are differentially expressed in the target and control plasma by comparing the respective expression levels obtained in steps (a) and (b), wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature, as defined herein, that is indicative for the presence of colorectal cancer.

In preferred embodiments of the invention, the method comprising: (a) determining in the one or more target plasma the expression levels of a combination of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, and calculate with certain formula, then ; (b) determining the expression levels of the combination of nucleic acid molecules in healthy control plasma, and calculate with certain formula; and (c) identifying the difference of the combination in the one or more target and control plasma by comparing the respective calculation results obtained in steps (a) and (b), wherein the one or more differentially expressed combinations together represent a signature, as defined herein, that is indicative for the presence of colorectal cancer.

In more preferred embodiments of the invention, the method is for the further use of discriminating colorectal cancer from healthy individuals, hepatocellular carcinoma and lung cancer.

In a fourth aspect, the present invention relates to a method for monitoring treatment of colorectal cancer, the method comprising: (a) identifying in the one or more target plasma a nucleic acid expression signature by using a method, as defined herein; and (b) monitoring in blood the expression of one or more nucleic acid molecules encoding a microRNA sequence that is/are comprised in the nucleic acid expression signature in such way that the expression of a nucleic acid molecule whose expression in plasma is up-regulated before treatment but is down-regulated after treatment and the expression of a nucleic acid molecule whose expression in plasma is down-regulated before treatment but is up-regulated after treatment. In a fifth aspect, the present invention relates to a method for preventing or treating colorectal cancer, the method comprising: (a) identifying in plasma a nucleic acid expression signature by using a method, as defined herein; and (b) modifying in blood the expression of one or more nucleic acid molecules encoding a microRNA sequence that is/are comprised in the nucleic acid expression signature in such way that the expression of a nucleic acid molecule whose expression is up-regulated in plasma is down-regulated and the expression of a nucleic acid molecule whose expression is down-regulated in plasma is up-regulated.

In a sixth aspect, the present invention relates to a pharmaceutical composition for the prevention and/or treatment of colorectal cancer in blood, the composition comprising one or more nucleic acid molecules, each nucleic acid molecule encoding a sequence that is at least partially complementary to a microRNA sequence encoded by a nucleic acid molecule whose expression is up-regulated in plasma from colorectal cancer patients, as defined herein, and/or that corresponds to a microRNA sequence encoded by a nucleic acid molecule whose expression is down-regulated in plasma from colorectal cancer patients, as defined herein.

Finally, in a seventh aspect, the present invention relates to the use of said pharmaceutical composition for the manufacture of a medicament for the prevention and/or treatment of colorectal cancer.

Other embodiments of the present invention will become apparent from the detailed description hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a flow chart schematically illustrating the essential method steps for determining an expression signature according to the present invention for identifying one or more target plasma exhibiting colorectal cancer.

Figure 2 illustrates the human miRNAs comprised in particularly preferred expression signatures in the first aspect according to the present invention for identifying one or more target plasma exhibiting colorectal cancer. Also indicates the expression levels and accuracy (RUC) of these miRNAs in the patients with colorectal cancer as compared to healthy controls (i.e. an up- regulation or a down-regulation).

depicts the respective expression levels and ROC curve analysis for two up-regulated miRNAs (hsa-miR-409-3p and has-miR- 671-3p) in the progression of colorectal cancer selected from the group consisting of Dukes' A, B, C and D carcinomas. The respective data obtained on the microarrays were normalized against an internal stable control hsa-miR-1238. The data indicate that colorectal cancer can be detected at early Dukes' A stage by the miRNA signatures discovered in the present invention.

illustrates the human miRNAs comprised in particularly preferred expression signatures in the second aspect according to the present invention for further discriminating colorectal cancer from healthy controls, hepatocellular cancer and lung cancer. Also indicates the expression levels and accuracy (AUC) of these miRNAs in the patients with colorectal cancer as compared to healthy control, hepatocellular carcinoma and lung cancer (i.e. an up-regulation or a down-regulation). The top combination (hsa- miR-33b*/hsa-miR-196a) showed 85% accuracy (AUC=0.860) as diagnostic biomarkers in differentiating CRC from healthy individuals, hepatocellular carcinoma and lung cancer, depicts examples of ROC curve analysis for two tumor-related miRNAs (hsa-miR-93 and hsa-miR-25) detected by quantitative RT-PCR in the target plasma of colorectal cancer and control healthy plasma (0: healthy individuals; 1: colorectal cancer). The results indicate high sensitivity and specificity of these miRNAs as diagnostic biomarkers. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the unexpected finding that colorectal cancer can be reliably identified based on particular miRNA expression signatures in plasma with high sensitivity and specificity, wherein the expression signatures as defined herein typically comprises both up- and down-regulated human miRNAs. More specifically, said miRNA expression signatures - by analyzing the overall miRNA expression pattern and/or the respective individual miRNA expression level(s) in plasma - allow the detection of colorectal cancer at an early disease state and discriminating healthy individuals, hepatocellular carcinoma and lung cancer.

The present invention illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are to be considered non- limiting.

Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising of". If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group, which preferably consists only of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

The term "about" in the context of the present invention denotes an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of + 10%, and preferably + 5%.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

Further definitions of term will be given in the following in the context of which the terms are used.

The following terms or definitions are provided solely to aid in the understanding of the invention. These definitions should not be construed to have a scope less than understood by a person of ordinary skill in the art.

It is an objective of the present invention to provide novel approaches for diagnosing colorectal cancer, monitoring the cancer therapy and/or treating colorectal cancer by determining a plurality of nucleic acid molecules in blood, each nucleic acid molecule encoding a microRNA (miRNA) sequence, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in plasma of colorectal cancer, analyzed as compared to healthy individuals, hepatocellular carcinoma and lung cancer, wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative for the presence of colorectal cancer, wherein the nucleic acid expression signatures include tumor- related signatures and plasma-specific signatures.

The term "colorectal", as used herein, relates to the colon, the rectum and/or the appendix, i.e. the complete large intestine.

The term "cancer" (also referred to as "carcinoma"), as used herein, generally denotes any type of malignant neoplasm, that is, any morphological and/or physiological alterations (based on genetic re-programming) of target cells exhibiting or having a predisposition to develop characteristics of a carcinoma as compared to unaffected (healthy) wild- type control cells. Examples of such alterations may relate inter alia to cell size and shape (enlargement or reduction), cell proliferation (increase in cell number), cell differentiation (change in physiological state), apoptosis (programmed cell death) or cell survival. Hence, the term "colorectal cancer" refers to cancerous growths in the colon, rectum, and appendix.

The most common colorectal cancer (CRC) cell type is adenocarcinoma that accounts for about 95% of cases. Other types of CRC include inter alia lymphoma and squamous cell carcinoma.

Colorectal cancer may be classified according to the Dukes system (Dukes, C.E.

(1932) J. Pathol. Bacterwl. 35, 323-325), which identifies the following stages: Dukes A - a tumour confined to the intestinal wall; Dukes B - a tumor invading through the intestinal wall; Dukes C - a tumor also involving the lymph node(s); and Dukes D - a tumor with distant metastasis.

The term "plasma", as used herein, is the yellow liquid component of blood, in which the blood cells in whole blood would normally be suspended. It makes up about 55% of the total blood volume. It is mostly water (90% by volume) and contains dissolved proteins, glucose, clotting factors, mineral ions, hormones and carbon dioxide

(plasma being the main medium for excretory product transportation). Blood plasma is prepared by spinning a tube of fresh blood in a centrifuge until the blood cells fall to the bottom of the tube. The blood plasma is then poured or drawn off. Blood plasma has a density of approximately 1025 kg/m³, or 1.025 kg/1. Recent research showed that miRNA is stable in plasma. The term "plasma sample" refers to plasma taken from individuals being examined or from healthy control.

The term "patient", as used herein, refers to a human being at least supposed to have colorectal cancer; where as "target plasma", as used herein, refers to plasma collected from patients; The term "healthy individual" or "healthy control" typically denotes a healthy person not having characteristics of such a cancerous phenotype. And

"control plasma", as used herein, denotes plasma collected from healthy individuals.

However, in some applications, for example, when comparing different cancer types, the individual having the other cancer types and plasma collected from these individuals is typically considered the "control".

Typically, the plasma samples used are derived from biological specimens collected from the subjects to be diagnosed for the presence of colorectal cancer.

Furthermore, in order to corroborate the data obtained "comparative samples" may also be collected from subjects having a given known disease state. The biological samples may include body tissues and fluids, such as colorectal tissue, serum, blood cell, sputum, and urine. Furthermore, the biological sample may be obtained from individual have colorectal cancerous characteristics or suspected to be cancerous. Furthermore, the sample may be purified from the obtained body tissues and fluids if necessary, and then used as the biological sample. According to the present invention, the expression level of the nucleic acid markers of the present invention is determined in the subject-derived biological sample(s).

The sample used for detection in the in vitro methods of the present invention should generally be collected in a clinically acceptable manner, preferably in a way that nucleic acids (in particular RNA) or proteins are preserved. The samples to be analyzed are typically from blood. Furthermore, lung tissue and other types of sample can be used as well. Samples, in particular after initial processing may be pooled. However, also non-pooled samples may be used.

The term "microRNA" (or "miRNA"), as used herein, is given its ordinary meaning in the art (Bartel, D.P. (2004) Cell 23, 281-292; He, L. and Hannon, G.J. (2004) Nat Rev Genet 5, 522-531). Accordingly, a "microRNA" denotes an RNA molecule derived from a genomic locus that is processed from transcripts that can form local RNA precursor miRNA structures. The mature miRNA is usually 20, 21, 22, 23, 24, or 25 nucleotides in length, although other numbers of nucleotides may be present as well, for example 18, 19, 26 or 27 nucleotides.

The miRNA encoding sequence has the potential to pair with flanking genomic sequences, placing the mature miRNA within an imperfect RNA duplex (herein also referred to as stem-loop or hairpin structure or as pre-miRNA), which serves as an intermediate for miRNA processing from a longer precursor transcript. This processing typically occurs through the consecutive action of two specific endonucleases termed Drosha and Dicer, respectively. Drosha generates from the primary transcript (herein also denoted "pri-miRNA") a miRNA precursor (herein also denoted "pre-miRNA") that typically folds into a hairpin or stem-loop structure. From this miRNA precursor a miRNA duplex is excised by means of Dicer that comprises the mature miRNA at one arm of the hairpin or stem-loop structure and a similar- sized segment (commonly referred to miRNA*) at the other arm. The miRNA is then guided to its target mRNA to exert its function, whereas the miRNA* is degraded. In addition, miRNAs are typically derived from a segment of the genome that is distinct from predicted protein-coding regions.

The term "miRNA precursor" (or "precursor miRNA" or "pre-miRNA"), as used herein, refers to the portion of a miRNA primary transcript from which the mature miRNA is processed. Typically, the pre-miRNA folds into a stable hairpin (i.e. a duplex) or a stem-loop structure. The hairpin structures typically range from 50 to 80 nucleotides in length, preferably from 60 to 70 nucleotides (counting the miRNA residues, those pairing to the miRNA, and any intervening segment(s) but excluding more distal sequences).

The term "nucleic acid molecule encoding a microRNA sequence", as used herein, denotes any nucleic acid molecule coding for a microRNA (miRNA). Thus, the term does not only refer to mature miRNAs but also to the respective precursor miRNAs and primary miRNA transcripts as defined above. Furthermore, the present invention is not restricted to RNA molecules but also includes corresponding DNA molecules encoding a microRNA, e.g. DNA molecules generated by reverse transcribing a miRNA sequence. A nucleic acid molecule encoding a microRNA sequence according to the invention typically encodes a single miRNA sequence (i.e. an individual miRNA). However, it is also possible that such nucleic acid molecule encodes two or more miRNA sequences (i.e. two or more miRNAs), for example a transcriptional unit comprising two or more miRNA sequences under the control of common regulatory sequences such as a promoter or a transcriptional terminator.

The term "nucleic acid molecule encoding a microRNA sequence", as used herein, is also to be understood to include "sense nucleic acid molecules" (i.e. molecules whose nucleic acid sequence (5'— 3') matches or corresponds to the encoded miRNA (5'→ 3') sequence) and "anti-sense nucleic acid molecules" (i.e. molecules whose nucleic acid sequence is complementary to the encoded miRNA (5'— » 3') sequence or, in other words, matches the reverse complement (3'→ 5') of the encoded miRNA sequence). The term "complementary", as used herein, refers to the capability of an "anti-sense" nucleic acid molecule sequence of forming base pairs, preferably Watson-Crick base pairs, with the corresponding "sense" nucleic acid molecule sequence (having a sequence complementary to the anti-sense sequence).

Within the scope of the present invention, two nucleic acid molecules (i.e. the "sense" and the "anti-sense" molecule) may be perfectly complementary, that is, they do not contain any base mismatches and/or additional or missing nucleotides. Alternatively, the two molecules comprise one or more base mismatches or differ in their total numbers of nucleotides (due to additions or deletions). Preferably, the "complementary" nucleic acid molecule comprises at least ten contiguous nucleotides showing perfect complementarity with a sequence comprised in corresponding "sense" nucleic acid molecule.

Accordingly, the plurality of nucleic acid molecules encoding a miRNA sequence that are comprised in a diagnostic kit of the present invention may include one or more "sense nucleic acid molecules" and/or one or more "anti-sense nucleic acid molecules". In case, the diagnostic kit includes one or more "sense nucleic acid molecules" (i.e. the miRNA sequences as such), said molecules are to be considered to constitute the totality or at least a subset of differentially expressed miRNAs (i.e. molecular markers) being indicative for the presence of or the disposition to develop a particular condition, here lung cancer. On the other hand, in case a diagnostic kit includes one or more "anti-sense nucleic acid molecules" (i.e. sequences complementary to the miRNA sequences), said molecules may comprise inter alia probe molecules (for performing hybridization assays) and/or oligonucleotide primers (e.g., for reverse transcription or PCR applications) that are suitable for detecting and/or quantifying one or more particular (complementary) miRNA sequences in a given sample.

A plurality of nucleic acid molecules as defined within the present invention may comprise at least two, at least ten, at least 50, at least 100, at least 200, at least 500, at least 1.000, at least 10.000 or at least 100.000 nucleic acid molecules, each molecule encoding a miRNA sequence.

The term "differentially expressed", as used herein, denotes an altered expression level of a particular miRNA in the disease plasma as compared to the healthy controls, or as compared to other types of disease samples, which may be an up- regulation (i.e. an increased miRNA concentration in the plasma) or a down-regulation (i.e. a reduced or abolished miRNA concentration in the plasma). In other words, the nucleic acid molecule is activated to a higher or lower level in the disease plasma samples than in the control plasma.

Within the scope of the present invention, a nucleic acid molecule is to considered differentially expressed if the respective expression levels of this nucleic acid molecule in disease plasma samples and control samples typically differ by at least 5% or at least 10%, preferably by at least 20% or at least 25%, and most preferably by at least 30% or at least 50%. Thus, the latter values correspond to an at least 1.3-fold or at least 1.5-fold up-regulation of the expression level of a given nucleic acid molecule in the disease plasma samples compared to the control plasma samples or vice versa an at least 0.7-fold or at least 0.5-fold down-regulation of the expression level in the disease plasma samples, respectively.

The term "expression level", as used herein, refers to extent to which a particular miRNA sequence is transcribed from its genomic locus, that is, the concentration of a miRNA in the plasma sample to be analyzed.

As outlined above, the term "control plasma" typically denotes a plasma sample collected from (healthy) individual not having characteristics of a colorectal cancer phenotype. However, in some applications, for example, when comparing other cancer types, the plasma collected from the patients with other cancer types is typically considered the "control plasma".

The determining of expression levels typically follows established standard procedures well known in the art (Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, F.M. et al. (2001) Current Protocols in Molecular Biology. Wiley & Sons, Hoboken, NJ). Determination may occur at the RNA level, for example by Northern blot analysis using miRNA-specific probes, or at the DNA level following reverse transcription (and cloning) of the RNA population, for example by quantitative PCR or real-time PCR techniques. The term "determining", as used herein, includes the analysis of any nucleic acid molecules encoding a microRNA sequence as described above. However, due to the short half-life of pri- miRNAs and pre-mRNAs typically the concentration of only the mature miRNA is measured. In specific embodiments, the standard value of the expression levels obtained in several independent measurements of a given sample (for example, two, three, five or ten measurements) and/or several measurements within several samples or control samples are used for analysis. The standard value may be obtained by any method known in the art. For example, a range of mean + 2 SD (standard deviation) or mean ± 3 SD may be used as standard value.

The difference between the expression levels obtained for disease and control plasma may be normalized to the expression level of further control nucleic acids, e.g. housekeeping genes whose expression levels are known not to differ depending on the disease states of the individual from whom the sample was collected. Exemplary housekeeping genes include inter alia β-actin, glycerinaldehyde 3-phosphate dehydrogenase, and ribosomal protein PI . In preferred embodiments, the control nucleic acid is another miRNA known to be stably expressed during the various non-cancerous and (pre-)cancerous states of the individual from whom the sample was collected.

However, instead of determining in any experiment the expression levels for plasma sample it may also be possible to define based on experimental evidence and/or prior art data on or more cut-off values for a particular disease phenotype (i.e. a disease state). In such scenario, the respective expression levels for the plasma sample can be determined by using a stably expressed control miRNA for normalization. If the "normalized" expression levels calculated are higher than the respective cutoff value defined, then this finding would be indicative for an up- regulation of gene expression. Vice versa, if the "normalized" expression levels calculated are lower than the respective cutoff value defined, then this finding would be indicative for a down-regulation of gene expression.

In the context of the present invention, the term "identifying colorectal cancer and/or discriminating other cancer types" is intended to also encompass predictions and likelihood analysis (in the sense of "diagnosing"). The compositions and methods disclosed herein are intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria such as disease stages, and disease monitoring and surveillance for the disease. According to the present invention, an intermediate result for examining the condition of a subject may be provided. Such intermediate result may be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose that a subject suffers from the disease. Alternatively, the invention may be used to detect cancerous changes through plasma sample, and provide a doctor with useful information for diagnosis. Furthermore, the invention may also be used to discriminate between colorectal cancer and other cancer types including hepatocellular carcinoma and lung cancer.

Within the present invention, one or more differentially expressed nucleic acid molecules identified together represent a nucleic acid expression signature that is indicative for colorectal cancer through plasma sample. The term "expression signature", as used herein, denotes a set of nucleic acid molecules (e.g., miRNAs), wherein the expression level of the individual nucleic acid molecules differs between the plasma collected from colorectal cancer patient and the healthy control. Herein, a nucleic acid expression signature is also referred to as a set of markers and represents a minimum number of (different) nucleic acid molecules, each encoding a miRNA sequence that is capable for identifying a phenotypic state of an individual.

The nucleic acid expression signature, as defined herein, may comprise at least thirty-fivenucleic acid molecules, preferably at least twelve nucleic acid molecules, and particularly preferably at least six nucleic acid molecules.

In preferred embodiment, the nucleic acid expression signature comprises at least one nucleic acid molecule encoding a microRNA sequence whose expression is up-regulated in the one or more target plasma compared to the one or more healthy controls and at least one nucleic acid molecule encoding a microRNA sequence whose expression is down-regulated in the one or more target plasma compared to the one or more healthy control plasma.

The term "derived from tumor" or "tumor-related", as used herein, refers to signatures that differentially expressed in plasma from colorectal cancer patients and in control plasma and are also differentially expressed in colorectal cancer tissues cells and non-cancer tissue cells.

The colorectal cancer tissue cells, as used herein, refer to cancerous colorectal cells collected from dissections derived from the subjects to be diagnosed for the presence of colorectal cancer. The non-cancer tissue cells, as used herein, typically denotes a (healthy) wild- type cell not having characteristics of such a cancerous phenotype.

The term "plasma- specific", as used herein, refers to signatures that are that differentially expressed in plasma from colorectal cancer patients and in control plasma are not found significantly differentially expressed in colorectal cancer tissues cells and non-cancer tissue cells.

Typically, the nucleic acid molecules comprised in the nucleic acid expression signature are human sequences (hereinafter designated "hsa" (Homo sapiens).

In preferred embodiments, the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding tumor-related signatures: hsa-miR-409-3p (SEQ ID NO:l), hsa-miR-25 (SEQ ID NO:2), hsa-miR-93 (SEQ ID NO:3), hsa-miR-96 (SEQ ID NO:4), hsa-miR-301a (SEQ ID NO:5), hsa-miR- 342-3p (SEQ ID NO:6), hsa-miR-19b (SEQ ID NO:7), hsa-miR-451 (SEQ ID NO:8), hsa-miR-486-5p (SEQ ID NO:9), hsa-miR-187* (SEQ ID NO:10), hsa-miR- 92a (SEQ ID NO:l l), hsa-miR-19a (SEQ ID NO:12), hsa-miR-20b (SEQ ID N0:13), hsa-miR-20a (SEQ ID NO:14), hsa-miR-139-3p (SEQ ID N0:15), hsa- miR-107 (SEQ ID N0:16), hsa-miR-17 (SEQ ID N0:17), hsa-miR-140-3p (SEQ ID N0:18), hsa-miR-30e (SEQ ID NO:19), hsa-miR-185 (SEQ ID NO:20) and plasma- specific signatures: hsa-mir-671-3p (SEQ ID N0:21), hsa-mir-16-2* (SEQ ID NO:22), hsa-miR-30c-l* (SEQ ID NO:23), hsa-miR-548c-5p (SEQ ID NO:24), hsa-miR-16 (SEQ ID NO:25), hsa-miR-15a (SEQ ID NO:26), hsa-miR-425 (SEQ ID NO:27), hsa-let-7i (SEQ ID NO:28), hsa-miR-363 (SEQ ID NO:29), hsa-miR- 15b (SEQ ID NO:30), hsa-miR-101 (SEQ ID N0:31), hsa-miR-190b (SEQ ID NO:32) and hsa-miR-130a (SEQ ID NO:33), hsa-miR-331-3p (SEQ ID NO:46), hsa-miR-345 (SEQ ID NO:47).

For normalizing the expression levels in plasma obtained for the nucleic acid molecules encoding microRNA sequences that are comprised in the nucleic acid expression signature the miRNA hsa-miR-1238 (SEQ ID NO: 44) and hsa-miR- 1228 (SEQ ID NO: 45) may be preferably used, which is stably expressed in colorectal cancer plasma.

The nucleic acid sequences of the above-referenced miRNAs are listed in Table 1.

TABLE 1

miRNA Sequence (5'→ 3')

Tumor -related miRNA

hsa-miR-409-3p Gaauguugcucggugaaccccu

hsa-miR-25 Cauugcacuugucucggucuga

hsa-miR-93 Caaagugcuguucgugcagguag

hsa-miR-96 Uuuggcacuagcacauuuuugcu

hsa-miR-301a Cagugcaauaguauugucaaagc

hsa-miR-342-3p Ucucacacagaaaucgcacccgu

hsa-miR-19b Ugugcaaauccaugcaaaacuga

hsa-miR-451 Aaaccguuaccauuacugaguu hsa-miR-486-5p Uccuguacugagcugccccgag hsa-miR-187* Ggcuacaacacaggacccgggc hsa-miR-92a Uauugcacuugucccggccugu hsa-miR-19a Ugugcaaaucuaugcaaaacuga hsa-miR-20b Caaagugcucauagugcagguag hsa-miR-20a Uaaagugcuuauagugcagguag hsa-miR-139-3p Ggagacgcggcccuguuggagu hsa-miR-107 Agcagcauuguacagggcuauca hsa-miR-17 Caaagugcuuacagugcagguag hsa-miR-140-3p Uaccacaggguagaaccacgg hsa-miR-30e Uguaaacauccuugacuggaag hsa-miR-185 Uggagagaaaggcaguuccuga

Plasma-specific miRNA

hsa-miR-671-3p Uccgguucucagggcuccacc hsa-miR-16-2* Ccaauauuacugugcugcuuua hsa-miR-30c-l* Cugggagaggguuguuuacucc hsa-miR-548c-5p Aaaaguaauugcgguuuuugcc hsa-miR-16 Uagcagcacguaaauauuggcg hsa-miR-15a Uagcagcacauaaugguuugug hsa-miR-425 Aaugacacgaucacucccguuga hsa-let-7i Ugagguaguaguuugugcuguu hsa-miR-363 Aauugcacgguauccaucugua hsa-miR-15b Uagcagcacaucaugguuuaca hsa-miR-101 Uacaguacugugauaacugaa hsa-miR-190b Ugauauguuugauauuggguu hsa-miR-130a Cagugcaauguuaaaagggcau hsa-miR-331-3p Gccccugggccuauccuagaa hsa-miR-345 Gcugacuccuaguccagggcuc

Internal stable control

hsa-miR-1238 Cuuccucgucugucugcccc hsa-miR-1228 Ucacaccugccucgcccccc

All miRNA sequences disclosed herein have been deposited in the miRBase database (http://microrna.sanger.ac.uk/; see also Griffiths-Jones S. et al. (2008) Nucl. Acids Res. 36, D154-D158).

In more preferred embodiments, the expression of any one or more of the nucleic acid molecules encoding hsa-miR-409-3p and hsa-mir-671-3p is up- regulated and the expression of any one or more of the nucleic acid molecules encoding hsa-miR-25, hsa-miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451, hsa-miR-486-5p, hsa-miR-187*, hsa-miR-92a, hsa- miR-1 a, hsa-miR-20b, hsa-miR-20a, hsa-miR-139-3p, hsa-miR-107, hsa-miR-17, hsa-miR-140-3p, hsa-miR-30e, hsa-miR-185' hsa-mir-16-2*, hsa-miR-30c-l*, hsa- miR-548c-5p, hsa-miR-16, hsa-miR-15a, hsa-miR-425, hsa-let-7i, hsa-miR-363, hsa-miR-15b, hsa-miR-101, hsa-miR-190b, hsa-miR-130a, hsa-miR-331-3p and hsa-miR-345 is down-regulated; hsa-miR-1238 and hsa-miR-1228 is un-changed in the one or more target plasma compared to the one or more healthy controls.

The terms "any one or more of the plurality of nucleic acid molecules" or "any one or more of the plurality of nucleic acid molecules" as used herein, may relate to any subgroup of the plurality of nucleic acid molecules, e.g., any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten, and so forth nucleic acid molecules, each encoding a microRNA sequence that are comprised in the nucleic acid expression signature, as defined herein.

In particularly preferred embodiments, the nucleic acid expression signature comprises any one or more nucleic acid combinations encoding hsa-miR-409- 3p/hsa-miR-16-2*, hsa-miR-409-3p/hsa-miR-96, hsa-miR-671-3p/hsa-miR-548c-5p, hsa-miR-671-3p/hsa-miR-16-2^:|:, hsa-miR-671-3p hsa-miR-30c-l^:|:, hsa-miR-671- 3p/hsa-miR-342-3p, hsa-miR-671-3p/hsa-miR-96, hsa-miR-671-3p hsa-miR-301a, hsa-miR-671-3p/hsa-miR-345, hsa-miR-409-3p/hsa-miR-345, hsa-miR-409/hsa- miR-331-3p, hsa-miR-671-3p hsa-miR-331-3p, hsa-miR-671-3p/ hsa-miR-25, hsa- miR-409-3p/ hsa-miR-93 and hsa-miR-671-3p/ hsa-miR-93. The term "nucleic acid combinations", as used herein, refers to the usage of at least two nucleic acid expression levels as a whole. Preferably may use the relative changes or calculate results through a formulation as a whole.

In a second aspect, the present invention relates to a diagnostic kit of molecular markers for discriminating colorectal cancer from healthy controls and other cancer types, the kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in the target plasma and in one or more healthy control plasma as well as other cancer types, and wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative for the presence of colorectal cancer, and wherein the other cancer types include hepatocellular carcinoma and lung cancer.

The nucleic acid expression signature, as defined herein, may comprise at least twenty- threenucleic acid molecules, preferably at least eighteen nucleic acid molecules, and particularly preferably at least sixnucleic acid molecules.

In preferred embodiments, the nucleic acid expression signature comprises at least one nucleic acid molecule encoding a microRNA sequence whose expression is up-regulated in the one or more target plasma compared to the one or more healthy individuals, hepatocellular carcinoma and lung cancer, and at least one nucleic acid molecule encoding a microRNA sequence whose expression is down- regulated in the one or more target plasma compared to the one or more healthy individuals, hepatocellular carcinoma and lung cancer.

In more preferred embodiments, the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding tumor-related signatures: hsa-miR-409-3p (SEQ ID NO: 1), hsa-miR-129-3p (SEQ ID NO: 34), hsa-miR-33b* (SEQ ID NO: 35), hsa-miR-7 (SEQ ID NO: 36), hsa-miR-196b (SEQ ID NO: 37), hsa-miR-93 (SEQ ID NO: 3), hsa-miR-486-5p (SEQ ID NO: 9), hsa- miR-25 (SEQ ID NO: 2), hsa-miR-92a (SEQ ID NO: 11), hsa-miR-19b (SEQ ID NO: 7); plasma- specific signatures: hsa-miR-671-3p (SEQ ID NO: 21), hsa-miR- 16-2* (SEQ ID NO: 22), hsa-miR-92b (SEQ ID NO: 38), hsa-miR-129* (SEQ ID NO: 39), hsa-miR-563 (SEQ ID NO: 40), hsa-miR-602 (SEQ ID NO: 41), hsa- miR-1227 (SEQ ID NO: 42), hsa-miR-196a (SEQ ID NO: 43) and internal stable controls: hsa-miR-1238 (SEQ ID NO: 44) and hsa-miR-1228 (SEQ ID NO: 45).

The nucleic acid sequences of the above-referenced miRNAs are listed in

Table 2.

Table 2

All miRNA sequences disclosed herein have been deposited in the miRBase database (http://microrna.sanger.ac.uk/; see also Griffiths-Jones S. et al. (2008) Nucl. Acids Res. 36, D154-D158). In more preferred embodiments, the expression of any one or more of the nucleic acid molecules encoding hsa-miR-409-3p, hsa-mir-671-3p, hsa-miR-33b*, hsa-miR-92b, hsa-miR-149, hsa-miR-129*, hsa-miR-563, hsa-miR-129-3p, hsa- miR-634, hsa-let-7b*, hsa-miR-602 and hsa-miR-1227 is up-regulated and the expression of any one or more of the nucleic acid molecules encoding hsa-miR-16- 2*, hsa-miR-7, hsa-miR-196a, hsa-miR-196b, hsa-miR-486-5p, hsa-miR-93, hsa- miR-25, hsa-miR-92a and hsa-miR-19b is down-regulated, hsa-miR-1238 and hsa- miR-1228 is un-changed in the one or more target plasma compared to the one or more healthy individuals, hepatocellular carcinoma and lung cancer.

In more preferred embodiments, the nucleic acid expression signature comprises any one or more nucleic acid combinations encoding hsa-miR-33b*/hsa- miR-196a, hsa-miR-92b/hsa-miR-196a, hsa-miR-563/hsa-miR-196a, hsa-miR- 1227/hsa-miR- 196a, hsa-miR- 129-3p/hsa-miR- 16-2*, hsa-miR- 129*/hsa-miR- 16- 2*, hsa-miR-92b/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-196a, hsa-miR-1227/hsa- miR-16-2*, hsa-miR-129-3p/hsa-miR-196a, hsa-miR-602/hsa-miR-196a, hsa-miR- 33b*/hsa-miR-16-2*, hsa-miR-563/hsa-miR-16-2*, hsa-miR-129* hsa-miR-7, hsa- miR-33b*/hsa-miR-7, hsa-miR-563 hsa-miR-7, hsa-miR-602/hsa-miR-16-2*, hsa- miR-129-3p/hsa-miR-7, hsa-miR-92b/hsa-miR-7, hsa-miR- 1227/hsa-miR-7, hsa- miR-33b*/hsa-miR-196b, hsa-miR-1227/hsa-miR-196b, hsa-miR-92b/hsa-miR- 196b and hsa-miR-602/hsa-miR-7.

In a fourth aspect, the present invention relates to a method for monitoring treatment of colorectal cancer, the method comprising: (a) identifying in the one or more target plasma a nucleic acid expression signature by using a method, as defined herein; and (b) monitoring in blood the expression of one or more nucleic acid molecules encoding a microRNA sequence that is/are comprised in the nucleic acid expression signature in such way that the expression of a nucleic acid molecule whose expression in plasma is up-regulated before treatment but is down-regulated after treatment and the expression of a nucleic acid molecule whose expression in plasma is down-regulated before treatment but is up-regulated after treatment.

The term "modifying the expression of a nucleic acid molecule encoding a miRNA sequence", as used herein, denotes any manipulation of a particular nucleic acid molecule resulting in an altered expression level of said molecule, that is, the production of a different amount of corresponding miRNA as compared to the expression of the "wild-type" (i.e. the unmodified control). The term "different amount", as used herein, includes both a higher amount and a lower amount than determined in the unmodified control. In other words, a manipulation, as defined herein, may either up-regulate (i.e. activate) or down-regulate (i.e. inhibit) the expression (i.e. particularly transcription) of a nucleic acid molecule.

In a fifth aspect, the present invention relates to a method for preventing or treating colorectal cancer, the method comprising: (a) identifying in plasma a nucleic acid expression signature by using a method, as defined herein; and (b) modifying in blood the expression of one or more nucleic acid molecules encoding a microRNA sequence that is/are comprised in the nucleic acid expression signature in such way that the expression of a nucleic acid molecule whose expression is up- regulated in plasma is down-regulated and the expression of a nucleic acid molecule whose expression is down-regulated in plasma is up-regulated.

Within the present invention, expression of one or more nucleic acid molecules encoding a microRNA sequence comprised in the nucleic acid expression signature is modified in such way that the expression of a nucleic acid molecule whose expression is up-regulated in plasma is down-regulated and the expression of a nucleic acid molecule whose expression is down-regulated in plasma is up- regulated. In other words, the modification of expression of a particular nucleic acid molecule encoding a miRNA sequence occurs in an anti-cyclical pattern to the regulation of said molecule in plasma of cancer patients in order to interfere with the "excess activity" of an up-regulated molecule and/or to restore the "deficient activity" of a down-regulated molecule in plasma.

In a preferred embodiment of the inventive method, down-regulating the expression of a nucleic acid molecule comprises introducing into the patient a nucleic acid molecule encoding a sequence that is complementary to the microRNA sequence encoded by nucleic acid molecule to be down-regulated.

The term "introducing into blood", as used herein, refers to any manipulation allowing the transfer of one or more nucleic acid molecules into blood. Examples of such techniques include injection, digestion or any other techniques may be involved.

The term "complementary sequence", as used herein, is to be understood that the "complementary" nucleic acid molecule (herein also referred to as an "anti-sense nucleic acid molecule") introduced into blood is capable of forming base pairs, preferably Watson-Crick base pairs, with the up-regulated endogenous "sense" nucleic acid molecule.

Two nucleic acid molecules (i.e. the "sense" and the "anti-sense" molecule) may be perfectly complementary, that is, they do not contain any base mismatches and/or additional or missing nucleotides. In other embodiments, the two molecules comprise one or more base mismatches or differ in their total numbers of nucleotides (due to additions or deletions). In further embodiments, the "complementary" nucleic acid molecule comprises a stretch of at least ten contiguous nucleotides showing perfect complementarity with a sequence comprised in the up-regulated "sense" nucleic acid molecule.

The "complementary" nucleic acid molecule (i.e. the nucleic acid molecule encoding a nucleic acid sequence that is complementary to the microRNA sequence encoded by nucleic acid molecule to be down-regulated) may be a naturally occurring DNA- or RNA molecule or a synthetic nucleic acid molecule comprising in its sequence one or more modified nucleotides which may be of the same type or of one or more different types.

For example, it may be possible that such a nucleic acid molecule comprises at least one ribonucleotide backbone unit and at least one deoxyribonucleotide backbone unit. Furthermore, the nucleic acid molecule may contain one or more modifications of the RNA backbone into 2'-0-methyl group or 2'-0-mefhoxyefhyl group (also referred to as "2'-( -methylation"), which prevented nuclease degradation in the culture media and, importantly, also prevented endonucleolytic cleavage by the RNA-induced silencing complex nuclease, leading to irreversible inhibition of the miRNA. Another possible modification - which is functionally equivalent to 2'-0-methylation - involves locked nucleic acids (LNAs) representing nucleic acid analogs containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA-mimicking sugar conformation (Orom, U.A. et al. (2006) Gene 372, 137-141).

Another class of silencers of miRNA expression was recently developed. These chemically engineered oligonucleotides, named "antagomirs", represent single- stranded 23-nucleotide RNA molecules conjugated to cholesterol (Krutzfeldt, J. et al. (2005) Nature 438, 685-689). As an alternative to such chemically modified oligonucleotides, microRNA inhibitors that can be expressed in cells, as RNAs produced from transgenes, were generated as well. Termed "microRNA sponges", these competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest (Ebert, M.S. et al. (2007) Nat. Methods 4, 721-726).

In particularly preferred embodiments of the inventive method, the one or more nucleic acid molecules whose expression is to be down-regulated encode microRNA sequences selected from the group consisting of hsa-miR-25, hsa-miR- 93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451, hsa- miR-486-5p, hsa-miR-187*, hsa-miR-92a, hsa-miR-19a, hsa-miR-20b, hsa-miR- 20a, hsa-miR-139-3p, hsa-miR-107, hsa-miR-17, hsa-miR-140-3p, hsa-miR-30e, hsa-miR-185, hsa-miR-16-2*, hsa-miR-30c-l*, hsa-miR-548c-5p, hsa-miR-16, hsa-miR-15a , hsa-miR-425, hsa-let-7i , hsa-miR-363, hsa-miR-15b , hsa-miR-101, hsa-miR-190b, hsa-miR-130a, hsa-miR-7, hsa-miR-196b and hsa-miR-196a with respect to the expression signature, presumably indicative for colorectal cancer as defined above.

In a further preferred embodiment of the inventive method, up-regulating the expression of a nucleic acid molecule comprises introducing into blood a nucleic acid molecule encoding the microRNA sequence encoded by nucleic acid molecule to be up-regulated. In other words, the up-regulation of the expression of a nucleic acid molecule encoding a miRNA sequence is accomplished by introducing into the one or more cells another copy of said miRNA sequence (i.e. an additional "sense" nucleic acid molecule). The "sense" nucleic acid molecule to be introduced into blood may comprise the same modification as the "anti-sense" nucleic acid molecules described above.

In a particularly preferred embodiment, the one or more nucleic acid molecules whose expression is to be up-regulated encode microRNA sequences selected from the group consisting of hsa-miR-409-3p, hsa-miR-671-3p, hsa-miR- 129-3p, hsa-miR-33b*, hsa-miR-92b, hsa-miR-129*, hsa-miR-563, hsa-miR-602 and hsa-miR-1227 with respect to the expression signature, presumably indicative for colorectal cancer as defined above.

The "sense" and/or the "anti-sense" nucleic acid molecules to be introduced into blood in order to modify the expression of one or more nucleic acid molecules encoding a microRNA sequence that is/are comprised in the nucleic acid expression signature may be operably linked to a regulatory sequence in order to allow expression of the nucleotide sequence.

In order to unravel any potential implication of the miRNAs identified in the cancerous or pre-cancerous samples preliminary functional analyses may be performed with respect to the identification of mRNA target sequences to which the miRNAs may bind. Based on the finding that miRNAs may be involved in both tumor suppression and tumorigenesis (Esquela-Kerscher, A. and Slack, F.J (2006) supra; Calin, G.A. and Croce, CM. (2007) supra; Blenkiron, C. and Miska, E.A. (2007) supra) it is likely to speculate that mRNA target sites for such miRNAs include tumor suppressor genes as well as oncogenes.

A nucleic acid molecule is referred to as "capable of expressing a nucleic acid molecule" or capable "to allow expression of a nucleotide sequence" if it comprises sequence elements which contain information regarding to transcriptional and/or translational regulation, and such sequences are "operably linked" to the nucleotide sequence encoding the polypeptide. An operable linkage is a linkage in which the regulatory sequence elements and the sequence to be expressed (and/or the sequences to be expressed among each other) are connected in a way that enables gene expression.

The precise nature of the regulatory regions necessary for gene expression may vary among species, but in general these regions comprise a promoter which, in prokaryotes, contains both the promoter per se, i.e. DNA elements directing the initiation of transcription, as well as DNA elements which, when transcribed into RNA, will signal the initiation of translation. Such promoter regions normally include 5' non-coding sequences involved in initiation of transcription and translation, such as the -35/- 10 boxes and the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5'-capping elements in eukaryotes. These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native polypeptide to a specific compartment of a host cell. In addition, the 3' non-coding sequences may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. If, however, these termination sequences are not satisfactory functional in a particular host environment, then they may be substituted with signals functional in that environment.

Furthermore, the expression of the nucleic molecules, as defined herein, may also be influenced by the presence, e.g., of modified nucleotides (cf. the discussion above). For example, locked nucleic acid (LNA) monomers are thought to increase the functional half-life of miRNAs in vivo by enhancing the resistance to degradation and by stabilizing the miRNA-target duplex structure that is crucial for silencing activity (Naguibneva, I. et al. (2006) Biomed Pharmacother 60, 633-638).

Therefore, a nucleic acid molecule of the invention to be introduced into blood provided may include a regulatory sequence, preferably a promoter sequence, and optionally also a transcriptional termination sequence. The promoters may allow for either a constitutive or an inducible gene expression. Suitable promoters include inter alia the E. coli ZacUV5 and tet (tetracycline-responsive) promoters, the T7 promoter as well as the SV40 promoter or the CMV promoter.

The nucleic acid molecules of the invention may also be comprised in a vector or other cloning vehicles, such as plasmids, phagemids, phages, cosmids or artificial chromosomes. In a preferred embodiment, the nucleic acid molecule is comprised in a vector, particularly in an expression vector. Such an expression vector can include, aside from the regulatory sequences described above and a nucleic acid sequence encoding a genetic construct as defined in the invention, replication and control sequences derived from a species compatible with the host that is used for expression as well as selection markers conferring a selectable phenotype on host. Large numbers of suitable vectors such as pSUPER and pSUPERIOR are known in the art, and are commercially available.

In a sixth aspect, the present invention relates to a pharmaceutical composition for the prevention and/or treatment of colorectal cancer in blood, the composition comprising one or more nucleic acid molecules, each nucleic acid molecule encoding a sequence that is at least partially complementary to a microRNA sequence encoded by a nucleic acid molecule whose expression is up- regulated in plasma from colorectal cancer patients, as defined herein, and/or that corresponds to a microRNA sequence encoded by a nucleic acid molecule whose expression is down-regulated in plasma from colorectal cancer patients, as defined herein.

Within the scope of the present invention, suitable pharmaceutical compositions include inter alia those compositions suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), peritoneal and parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation. Administration may be local or systemic. Preferably, administration is accomplished via the oral or intravenous routes. The formulations may also be packaged in discrete dosage units.

Pharmaceutical compositions according to the present invention include any pharmaceutical dosage forms established in the art, such as inter alia capsules, microcapsules, cachets, pills, tablets, powders, pellets, multi-p articulate formulations (e.g., beads, granules or crystals), aerosols, sprays, foams, solutions, dispersions, tinctures, syrups, elixirs, suspensions, water-in-oil emulsions such as ointments, and oil-in water emulsions such as creams, lotions, and balms.

The ("sense" and "anti-sense") nucleic acid molecules described above can be formulated into pharmaceutical compositions using pharmacologically acceptable ingredients as well as established methods of preparation (Gennaro, A.L. and Gennaro, A.R. (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA; Crowder, T.M. et al. (2003 ) A Guide to Pharmaceutical Particulate Science. Interpharm/CRC, Boca Raton, FL; Niazi, S.K. (2004) Handbook of Pharmaceutical Manufacturing Formulations, CRC Press, Boca Raton, FL).

In order to prepare the pharmaceutical compositions, pharmaceutically inert inorganic or organic excipients (i.e. carriers) can be used. To prepare e.g. pills, tablets, capsules or granules, for example, lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid polyols, natural and hardened oils may be used. Suitable excipients for the production of solutions, suspensions, emulsions, aerosol mixtures or powders for reconstitution into solutions or aerosol mixtures prior to use include water, alcohols, glycerol, polyols, and suitable mixtures thereof as well as vegetable oils.

The pharmaceutical composition may also contain additives, such as, for example, fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, and furthermore solvents or solubilizers or agents for achieving a depot effect. The latter is to be understood that the nucleic acid molecules may be incorporated into slow or sustained release or targeted delivery systems, such as liposomes, nanoparticles, and microcapsules.

To target most tissues within the body, clinically feasible noninvasive strategies are required for directing such pharmaceutical compositions, as defined herein, into cells. In the past years, several approaches have achieved impressive therapeutic benefit following intravenous injection into mice and primates using reasonable doses of siRNAs without apparent limiting toxicities.

One approach involves covalently coupling the passenger strand (miRNA* strand) of the miRNA to cholesterol or derivatives/conjugates thereof to facilitate uptake through ubiquitously expressed cell-surface LDL receptors (Soutschek, J. et al. (2004) Nature 432, 173-178). Alternatively, unconjugated, PBS-formulated locked-nucleic-acid-modified oligonucleotides (LNA-antimiR) may be used for systemic delivery (Elmen, J. et al. (2008) Nature 452, 896-899). Another strategy for delivering miRNAs involves encapsulating the miRNAs into specialized liposomes formed using polyethylene glycol to reduce uptake by scavenger cells and enhance time spent in the circulation. These specialized nucleic acid particles (stable nucleic acid-lipid particles or SNALPs) delivered miRNAs effectively to the liver (and not to other organs (Zimmermann, T.S. et al. (2006) Nature 441, 111- 114). Recently, a new class of lipid-like delivery molecules, termed lipidoids (synthesis scheme based upon the conjugate addition of alkylacrylates or alkyl- acrylamides to primary or secondary amines) has been described as delivery agents for RNAi therapeutics (Akinc, A. et al. (2008) Nat Biotechnol 26, 561-569).

A further targeting strategy involves the mixing of miRNAs with a fusion protein composed of a targeting antibody fragment linked to protamine, the basic protein that nucleates DNA in sperm and binds miRNAs by charge (Song, E. et al. (2005) Nat. Biotechnol. 23, 709-717). Multiple modifications or variations of the above basic delivery approaches have recently been developed. These techniques are known in the art and reviewed, e.g., in de Fougerolles, A. et al. (2007) Nat. Rev. Drug Discov 6, 443-453; Kim, D.H. and Rossi, JJ. (2007) Nat Genet 8, 173-184).

The invention is further described by the figures and the following examples, which are solely for the purpose of illustrating specific embodiments of this invention, and are not to be construed as limiting the scope of the invention in any way.

EXAMPLES

Example 1: Tissue sample collection and preparation

Sixty-nine colorectal cancer tissues and 69 matched normal colorectal tissue specimens were taken during surgery. Surgical specimens were snap-frozen in liquid nitrogen at or immediately after collection. Samples were stored at -80°C.

Patient data (age, sex, imaging data, therapy, other medical conditions, family history, and the like) were derived from the hospital databases for matching the various samples collected. Pathologic follow-up (for example, histological analysis via hematoxylin and eosin (H&E) staining) was used for evidently determining the disease state (i.e. control, pre -cancerous stage (e.g., colorectal adenoma), primary malignancy (e.g., colorectal carcinoma) of a given sample as well as to ensure a consistent classification of the specimens.

Laser-capture micro-dissection was optionally performed for each cancerous sample in order to specifically isolate tumor cell populations (about 200.000 cells). In brief, a transparent transfer film is applied to the surface of a tissue section or specimen. Under a microscope, the thin tissue section is viewed through the glass slide on which it is mounted and clusters of cells are identified for isolation. When the cells of choice are in the center of the field of view, a near IR laser diode integral with the microscope optics is activated. The pulsed laser beam activates a spot on the transfer film, fusing the film with the underlying cells of choice. The transfer film with the bonded cells is then lifted off the thin tissue section (Emmert- Buck, M.R. et al. (1996). Science 274, 998-1001; Espina, V. et al. (2007) Expert Rev Mol. Diagn 7, 647-657). The preparation of the cryostat sections and the capturing step using a laser capture microspope (Arcturus VeritasTM Laser Capture Microdissection Instrument (Molecular Devices, Inc., Sunnyvale, CA, USA) were performed essentially according to the instructions of the manufacturer.

Total RNA was extracted from the tissue sections by using mirVana miRNA i+solation kit according to the instructions from the manufacturer (Ambion, Austin, TX). The concentration was quantified by NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Waltham, MA). The quality control of RNA was performed by a 2100 Bioanalyzer using the RNA 6000 Pico LabChip kit (Agilent Technologies, Santa Clara, CA). Example 2: Analysis of the miRNA expression profile in the tissue samples

A qualitative analysis of the miRNAs (differentially) expressed in a particular sample may optionally be performed using the Agilent miRNA microarray platform (Agilent Technologies, Santa Clara, CA, USA). The microarray contains probes for 723 human miRNAs from the Sanger database v.10.1. Total RNA (100 ng) derived from each of 138 LCM-selected colorectal samples were used as inputs for labeling via Cy3 incorporation. Microarray slides were scanned by XDR Scan (PMTIOO, PMT5). The labeling and hybridization were performed according to the protocols in the Agilent miRNA microarray system.

For the data analysis, the raw data obtained for single-color (CY3) hybridization were normalized by applying a Quantile method and using GeneSpring GX10 software (Agilent Technologies, Santa Clara, CA, USA) known in the art. Unpaired t-test (p value <0.01) after Fisher test (F-test) was used to identify differentially expressed miRNAs between colorectal cancer and matched normal control tissues.

Independent experiments on 138 tissue specimens were performed for each measurement and the miRNA expression level determined represents the mean value of the respective individual data obtained.

Example 3: Plasma sample collection and preparation

The principal method steps for identifying one or more nucleic acid expression signatures in the target plasma exhibiting colorectal cancer are shown in Figure 1.

114 blood specimens from the cancer patients and healthy individuals were collected at Zhongshan and Huashan Hospitals in Shanghai between 2008 and 2009. Baseline characteristics of the blood specimens used in the invention are shown in Table 3. All of the samples from the patients were procured before surgery. Patient data (age, sex, imaging data, therapy, other medical conditions, family history, and the like) were derived from the hospital databases. Tumor histopathology was classified according to the World Health Organization Classification of Tumor system by three pathologists independently. Table 3

Baseline characteristics of blood specimens

Peripheral blood (2 ml) was drawn into EDTA tubes. Within two hours, the tubes were subjected to centrifuge at 820g for 10 min. Then, 1-ml aliquots of the plasma was transferred to 1.5-ml tubes and centrifuged at 16,000g for 10 min to pellet any remaining cellular debris. Subsequently, the supernatant was transferred to fresh tubes and stored them at -80 °C.

Total RNA was extracted from the plasma by using mirVana PARIS miRNA Isolation kit according to the instructions from the manufacturer (Ambion, Austin, TX). The concentration was quantified by NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Waltham, MA). The quality control of RNA was performed by a 2100 Bioanalyzer using the RNA 6000 Pico LabChip kit (Agilent Technologies, Santa Clara, CA).

Example 4: Analysis of the miRNA expression profile in plasma samples

A qualitative analysis of the miRNAs (differentially) expressed in a particular plasma sample may optionally be performed using the Agilent miRNA microarray platform (Agilent Technologies, Santa Clara, CA, USA). The microarray contains probes for 723 human miRNAs from the Sanger database v.10.1. Total RNA (100 ng) derived from each of 114 plasma samples were used as inputs for labeling via Cy3 incorporation. Microarray slides were scanned by XDR Scan (PMT100, PMT5). The labeling and hybridization were performed according to the protocols in the Agilent miRNA microarray system.

For the data analysis, the raw data obtained for single-color (CY3) hybridization were normalized by applying a stable internal miRNA control (has-miR-1238). Unpaired t-test after Fisher test (F-test) was used to identify differentially expressed miRNAs between colorectal cancer patients vs. healthy individuals, and/or colorectal cancer patients vs. controls (healthy individuals, hepatocellular carcinoma and lung cancer), respectively.

For the specificity and sensitivity of the individual miRNA as diagnostic biomarkers, MedCalc software was used to perform receiver operating characteristic (ROC) curve analysis of the individual miRNA in the colorectal cancer vs. healthy individuals vs. and/or controls (healthy individuals, hepatocellular carcinoma and lung cancer), respectively. 95% confidence interval was used to determine the significance.

For assessing whether a particular miRNA is differentially expressed in primary liver cancer or metastatic liver cancer as compared to healthy individuals or controls the following criteria were used:

i) p-value (probability value) of < = 0.01 with fold change >2 ii) AUC (accuracy as a diagnostic biomarker) AUC of > 0.700

In case, the two criteria were fulfilled, the miRNA was considered to be differentially expressed in colorectal cancer patients as compared to healthy individuals, and/or controls, respectively.

Independent experiments on 114 plasma samples were performed for each measurement and the miRNA expression level determined represents the mean value of the respective individual data obtained.

The experimental data in the differential miRNA expression analysis between CRC vs. healthy controls are summarized in Tables 4-6 below. Table 4 lists the miRNAs exhibiting significantly differential expressions in both tissue and plasma of CRC patients as compared to control tissues and healthy control plasma, respectively. Table 5 summarizes the miRNAs exhibiting a differential expression only in plasma of colorectal cancer patients as compared to healthy individuals, whereas Table 6 lists the ^' best combinations of the miRNA signatures in plasma of CRC patients. In the column "t"_. denotes the colorectal cancer tissue, and "n" denotes match normal control tissue, whereas "p" denotes the patients and "h" denotes healthy controls. Particularly preferred miRNAs (SEQ ID NO: 1 to SEQ ID NO: 8 in Table 4; SEQ ID NO: 21 to SEQ ID NO: 24 in Table 5, respectively) are shown in bold.

TABLE 4

Tumor-related miRNA signatures in plasma of colorectal cancer

TABLE 5

Plasma-specific miRNA signatures for colorectal cancer

TABLE 6

Combined miRNA signatures in plasma of colorectal cancer

Combination t-test geometric mean Fold AUC p-value healthy patients patients/healthy hsa-miR-409-3p/hsa-miR-16-2* 3.6E-06 0 8 66.8 0.866 hsa-miR-409-3p/hsa-miR-590-3p 2.9E-05 1 34 67.4 0.860 hsa-miR-409-3p/hsa-miR-96 5.6E-07 0 14 66.2 0.853 hsa-miR-409-3p/hsa-miR-550* 6.5E-07 1 47 33.7 0.850 hsa-miR-67 l-3p/hsa-miR-548c-5p 4.5E-05 0 8 26.1 0.848 hsa-miR-67 l-3p/hsa-miR-16-2* 3.8E-05 0 7 44.0 0.848 hsa-miR-671 -3p hsa-miR-30c- 1 * 4.6E-05 0 3 20.0 0.840 hsa-miR-67 l-3p hsa-miR-590-3p 4.4E-05 1 26 44.4 0.839 hsa-miR-67 l-3p/hsa-miR-550* 1.9E-05 2 36 22.2 0.824 hsa-miR-67 l-3p hsa-miR- 183 6.3E-05 1 22 37.7 0.824 hsa-miR-67 l-3p hsa-miR-342-3p 6.2E-05 0 2 22.2 0.808 hsa-miR-67 l-3p hsa-miR-96 4.3E-05 0 11 43.6 0.805 hsa-miR-67 l-3p hsa-miR-30 la 7.7E-05 0 3 36.4 0.805 The expression data on the preferred expression signatures in the second aspect for discriminating colorectal cancer from healthy individuals, hepatocellular carcinoma and lung cancer are summarized in Table 7-9 below. Table 7 lists tumor-related miRNA signatures for colorectal cancer; Table 8 shows plasma-specific miRNA signatures, whereas Table 9 displays the best combinations of the miRNA signatures for colorectal cancer. The top combination (hsa- miR-33b* hsa-rniR-196a) had 85% accuracy as diagnostic biomarkers in differentiating CRC from healthy individuals, hepatocellular carcinoma and lung cancer. In the column "t" denotes the colorectal cancer tissue, and "n" denotes match normal control tissue, whereas column "CRC" denotes the colorectal cancer plasma, and "control" denotes plasma derived from healthy individuals, hepatocellular carcinoma and lung cancer patients. Particularly preferred miRNAs (SEQ ID NO: 1; SEQ ID NO: 34 to SEQ ID NO: 37; SEQ ID NO: 3 in Table 7, SEQ ID NO:21, SEQ ID NO:38 to SEQ ID NO:42 in Table 8, SEQ ID NO:35 and SEQ ID NO:43 in Table 9, respectively) are shown in bold.

Table 7

Tumor-related miRNA signatures for colorectal cancer in discriminating healthy control.

hepatocellular carcinoma and lung cancers

TABLE 8

Plasma-specific miRNA signatures for colorectal cancer in discriminating healthy control, hepatocellular carcinoma and lung cancers

Table 9

Combinations of miRNA signatures for colorectal cancer in discriminating healthy controls, hepatocellular carcinoma and lung cancers

Combination t-test Fold ROC analysis

p-value CRC/ Sensitivity Specificity AUC 95% CI control

hsa-miR-33b*/hsa-miR-196a 5.5E-07 13.8 84 81 0.860 0.782 to

0.919 hsa-miR-92b hsa-miR- 196a 7.7E-07 12.8 81 88 0.852 0.772 to

0.912 hsa-miR-563/hsa-miR- 196a 2.4E-06 1 1.3 81 83 0.844 0.763 to

0.906 hsa-miR- 1227/hsa-miR- 1 6a 5.9E-08 12.1 81 84 0.844 0.762 to

0.906 hsa-miR- 129-3p/hsa-miR- 16- 2.1 E-08 12.7 97 63 0.843 0.762 to 2* 0.905 hsa-miR- 129*/hsa-miR- 16-2* 6.7E-09 1 1.5 94 64 0.842 0.761 to

0.904 hsa-miR-92b/hsa-miR-16-2* 1.6E-08 13.7 87 68 0.840 0.759 to

0.903 hsa-miR- 129*/hsa-miR- 196a 3.8E-06 10.8 84 75 0.838 0.756 to

0.901 hsa-miR- 1227/hsa-miR- 16-2* 3.4E-08 13.0 90 65 0.838 0.756 to

0.901 hsa-miR- 129-3p/hsa-miR- 196a 2.4E-08 1 1.8 84 78 0.836 0.754 to

0.899 hsa-miR-602/hsa-miR- 196a 2.8E-08 1 1.5 74 86 0.83 1 0.748 to

0.895 hsa-miR-33b*/hsa-miR-16-2* 2.6E-06 14.8 84 69 0.829 0.746 to

0.894 hsa-miR-563/hsa-miR-16-2* 4.5E-08 12.1 81 71 0.826 0.742 to 0.891 sa-miR-129*/hsa-mi^'R-7 1.6E-06 7.5 87 70 0.825 0.74 J to

0.891 hsa-miR-33b*/hsa-miR-7 1.4E-07 9.6 90 69 0.824 0.740 to

0.890 hsa-miR-563/hsa-miR-7 9.3E-07 7.9 77 76 0.822 0.738 to

0.888 hsa-miR-602/hsa-miR- 16-2* 4.6E-08 12.3 87 69 0.822 0.738 to

0.888 hsa-miR- 129-3p/hsa-miR-7 3.9E-06 8.2 81 74 0.821 0.736 to

0.887 hsa-miR-92b/hsa-miR-7 1.9E-06 8.9 94 60 0.818 0.733 to

0.885 hsa-miR- 1227/hsa-miR-7 5.6E-06 8.4 74 79 0.816 0.731 to

0.883 hsa-miR-33b*/hsa-miR-196b 4.9E-05 8.0 68 85 0.81 1 0.726 to

0.879 hsa-miR- 1227/hsa-miR- 196b 6.0E-06 7.0 77 74 0.810 0.724 to

0.878 hsa-miR-92b/hsa-miR-l 96b 3.9E-05 7.4 87 63 0.805 0.719 to

0.874 hsa-miR-602/hsa-miR-7 5.8E-06 8.0 97 54 0.804 0.718 to

0.874

Example 5: Verification of the microarray data

For verifying (and/or quantifying) the miRNA expression data acquired on microarrays, an established quantitative RT-PCR employing a TaqMan MicroRNA assay (Applied Biosystems, Foster City, CA, USA) was used according to the manufacturer's instructions. Briefly, reverse transcription (RT) was performed with Taqman microRNA RT Kits according to the instruction from Applied Biosystem. lOng total RNA was reverse-transcripted in 15ul RT solution mix that contains IX Reverse Transcription Buffer, IX RT primer, InM dNTP, 4U RNase Inhibitor and 50U Multi Scribe Reverse Transcriptase. Then the RT solutions were performed by using the thermal program of 16°C, 30min; 42°C, 30min; 85°C, 5min on the PCR machine (Thermal cycler alpha engine, Bio-rad). Quantitative PCR was performed with TaqMan Universal PCR Master Mix kit and and Taqman microRNA assays kits according to the instruction from Applied Biosystem. 2ul RT products were PCR amplified in IX TaqMan Universal PCR Master Mix, No AmpErase UNG, IX TaqMan MicroRNA Assay mix. Each reaction was duplicated in triple. The real-time PCR was performed in Roch Light Cycling 480 machine with the program of 96°C, 5min initial heating; then 45 or 50 cycles of 95°C, 15s; 60°C, 60s. Cp value was calculated with 2nd derivative method in LC480 software. Then miRNAs were absolutely quantified with the standard samples CP values. For the CP value for each miRNA was normalized one internal stable control hsa-miR- 1228.

The experimental data on platform comparison with 8 miRNA combinations with 7 miRNAs from plasma samples of 54 healthy individuals and 54 colorectal cancer patients are summarized in Table 10. Overall, the fold change between the patients and healthy controls acquired on the arrays was greater than that found in quantitative RT-PCR. The regulations (i.e. an up-regulation or a down-regulation) acquired on the arrays correlated well with that obtained from quantitative RT-PCR. The results demonstrate that the miRNA signatures discovered on Agilent miRNA microarrays are highly reliable.

TABLE 10

5 Platform comparison of the expression profiles in plasma of colorectal cancer patients and healthy individuals

The results obtained demonstrate a global highly specific regulation of miRNA 10 expression in colorectal cancer. Thus, the respective subsets of miRNAs specified herein represent unique miRNA expression signatures for expression profiling of colorectal cancer that do not only allow the identification of a cancerogenous state as such but also enables the discrimination of hepatocellular carcinoma and lung cancer

The identification of the miRNA expression signatures of the present invention 15 provides a unique molecular marker that allows screening, detection, diagnosing colorectal cancer in blood. Furthermore, the expression signatures can be used to monitor the therapy response and guide the treatment decision in colorectal cancer patients. Additionally, the expression signatures may be also used for development of anti-colorectal cancer drugs.

The present invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by embodiments and optional features, modifications and variations of the inventions embodied therein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

Diagnostic kit of molecular markers in blood for identifying one or more target plasma exhibiting colorectal cancer, the kit comprising a plurality of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence, wherein one or more of the plurality of nucleic acid molecules are differentially expressed in the target plasma and in one or more control plasma, and

wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature that is indicative for the presence of colorectal cancer.

The kit of claim 1, wherein the nucleic acid expression signature may comprise at least thirty-fivenucleic acid molecules, preferably at least twelve nucleic acid molecules, and particularly preferably at least sixnucleic acid molecules.

The kit of claim 1 or 2, wherein the nucleic acid expression signature comprises at least one nucleic acid molecule encoding a microRNA sequence whose expression is up-regulated in the one or more target plasma compared to the one or more control plasma and at least one nucleic acid molecule encoding a microRNA sequence whose expression is down-regulated in the one or more target plasma compared to the one or more control plasma.

The kit of any of claims 1 to 3, wherein the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding tumor- related signatures: hsa-miR-409-3p, hsa-miR-25, hsa-miR-93, hsa-miR-96, hsa- miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451, hsa-miR-486-5p, hsa- miR-187*, hsa-miR-92a, hsa-miR-19a, hsa-miR-20b, hsa-miR-20a, hsa-miR- 139-3p, hsa-miR-107, hsa-miR-17, hsa-miR-140-3p, hsa-miR-30e, hsa-miR-185 and plasma-specific signatures: hsa-mir-671-3p, hsa-mir-16-2*, hsa-miR-SOc-l*, hsa-miR-548c-5p, hsa-miR-16, hsa-miR-15a, hsa-miR-425, hsa-let-7i, hsa-miR- 363, hsa-miR-15b, hsa-miR-101, hsa-miR-190b, hsa-miR-130a, hsa-miR-331- 3p, hsa-miR-345 and internal stable controls: hsa-miR-1238 and hsa-miR-1228. 5. The kit of any of claims 4 , wherein the expression of any one or more of the nucleic acid molecules encoding hsa-miR-409-3p and hsa-mir-671-3p is up- regulated and the expression of any one or more of the nucleic acid molecules encoding hsa-miR-25, hsa-miR-93, hsa-miR-96, hsa-miR-301a, hsa-miR-342-3p, hsa-miR-19b, hsa-miR-451, hsa-miR-486-5p, hsa-miR-187*, hsa-miR-92a, hsa- miR-19a, hsa-miR-20b, hsa-miR-20a, hsa-miR-139-3p, hsa-miR-107, hsa-miR-

17, hsa-miR-140-3p, hsa-miR-30e, hsa-miR-185' hsa-mir-16-2*, hsa-miR-30c- 1*, hsa-miR-548c-5p, hsa-miR-16, hsa-miR-15a, hsa-miR-425, hsa-let-7i, hsa- miR-363, hsa-miR-15b, hsa-miR-101, hsa-miR-190b, hsa-miR-130a, hsa-miR- 331-3p and hsa-miR-345 is down-regulated; hsa-miR-1238 and hsa-miR-1228 is un-changed in the one or more target plasma compared to the one or more healthy controls.

6. The kit of any of claim 1 to 3, wherein the nucleic acid expression signature comprises any one or more nucleic acid combinations encoding hsa-miR-409- 3p/hsa-miR-16-2*, hsa-miR-409-3p/hsa-miR-96, hsa-miR-67 l-3p/hsa-miR-

548c-5p, hsa-miR-671 -3p/hsa-miR- 16-2*, hsa-miR-67 l-3p/hsa-miR-30c- 1 *, hsa-miR-671-3p/hsa-miR-342-3p, hsa-miR-671-3p/hsa-miR-96, hsa-miR-671- 3p/hsa-miR-301 a, hsa-miR-671 -3p/hsa-miR-345, hsa-miR-409-3p/hsa-miR-345, hsa-miR-409/hsa-miR-331-3p, hsa-miR-671-3p/hsa-miR-331-3p, hsa-miR-671- 3p/ hsa-miR-25, hsa-miR-409-3p/ hsa-miR-93 and hsa-miR-67 l-3p/ hsa-miR-93.

7. The kit of any of claims 1 to 6, for the further use of discriminating colorectal cancer from healthy individuals, hepatocellular carcinoma and lung cancer.

8. The kit of claim 7, wherein the nucleic acid expression signature may comprise at least twenty-threenucleic acid molecules, preferably at least eighteennucleic acid molecules, and particularly preferably at least sixnucleic acid molecules.

The kit of any of claims 1 to 8, wherein the nucleic acid expression signature comprises any one or more of the nucleic acid molecules encoding tumor- related signatures: hsa-miR-409-3p, hsa-miR-129-3p, hsa-miR-33b*, hsa-miR-7, hsa-miR-196b, hsa-miR-93, hsa-miR-486-5p, hsa-miR-25, hsa-miR-92a, hsa- miR-19b; plasma-specific signatures: hsa-miR-671-3p and hsa-miR-16-2*, hsa- miR-92b, hsa-miR-129*, hsa-miR-563, hsa-miR-602, hsa-miR-1227, hsa-miR- 196a and internal stable controls: hsa-miR-1238 and hsa-miR-1228.

The kit of any of claims 9, wherein the expression of any one or more of the nucleic acid molecules encoding hsa-miR-409-3p, hsa-mir-671-3p, hsa-miR- 33b*, hsa-miR-92b, hsa-miR-149, hsa-miR-129*, hsa-miR-563, hsa-miR-129- 3p, hsa-miR-634, hsa-let-7b*, hsa-miR-602 and hsa-miR-1227 is up-regulated and the expression of any one or more of the nucleic acid molecules encoding hsa-miR-16-2*, hsa-miR-7, hsa-miR-196a, hsa-miR-196b, hsa-miR-486-5p, hsa-miR-93, hsa-miR-25, hsa-miR-92a and hsa-miR-19b is down-regulated, hsa-miR-1238 and hsa-miR-1228 is un-changed in the one or more target plasma compared to the one or more healthy individuals, hepatocellular carcinoma and lung cancer.

The kit of any of claim 7 to 10, wherein the nucleic acid expression signature comprises any one or more nucleic acid combinations encoding hsa-miR- 33b*/hsa-miR- 196a, hsa-miR-92b/hsa-miR- 196a, hsa-miR-563/hsa-miR- 196a, hsa-miR-1227/hsa-miR-196a, hsa-miR-129-3p hsa-miR-16-2*, hsa-miR- 129*/hsa-miR-16-2*, hsa-miR-92b/hsa-miR-16-2*, hsa-miR-129*/hsa-miR- 196a, hsa-miR-1227/hsa-miR-16-2*, hsa-miR-129-3p/hsa-miR-196a, hsa-miR- 602/hsa-miR-196a, hsa-miR-33b*/hsa-miR-16-2*, hsa-miR-563/hsa-miR-16-2*, hsa-miR-129*/hsa-miR-7, hsa-imR-33b*/hsa-miR-7, hsa-miR-563/hsa-miR-7, hsa-miR-602/hsa-miR- 16-2*, hsa-miR- 129-3p/hsa-miR-7, hsa-miR-92b/hsa- miR-7, hsa-miR- 1227/hsa-miR-7, hsa-miR-33b*/hsa-miR-196b, hsa-miR- 1227/hsa-miR-196b, hsa-miR-92b/hsa-miR-196b and hsa-miR-602/hsa-miR-7.

Method for identifying one or more target plasma exhibiting colorectal cancer, the method comprising:

(a) determining in the one or more target plasma the expression levels of a plurality of nucleic acid molecules, each nucleic acid molecule encoding a microRNA sequence;

(b) determining the expression levels of the plurality of nucleic acid molecules in one or more healthy control plasma; and

(c) identifying from the plurality of nucleic acid molecules one or more nucleic acid molecules that are differentially expressed in the target and control plasma by comparing the respective expression levels obtained in steps (a) and (b), wherein the one or more differentially expressed nucleic acid molecules together represent a nucleic acid expression signature, as defined in any of 1 to 11, that is indicative for the presence of colorectal cancer.

The method of claim 12, for the further use of discriminating colorectal cancer from healthy individuals, hepatocellular carcinoma and lung cancer.

Method for monitoring treatment of colorectal cancer, the method comprising:

(a) identifying in the one or more target plasma a nucleic acid expression signature by using a method, as defined herein; and

(b) monitoring in blood the expression of one or more nucleic acid molecules encoding a microRNA sequence that is/are comprised in the nucleic acid expression signature in such way that the expression of a nucleic acid molecule whose expression in plasma is up-regulated before treatment but is down-regulated after treatment and the expression of a nucleic acid molecule whose expression in plasma is down-regulated before treatment but is up-regulated after treatment.

Method for preventing or treating colorectal cancer, the method comprising:

(a) identifying a nucleic acid expression signature in blood by using a method, as defined claim 12 or 13, and

(b) modifying in blood the expression of one or more nucleic acid molecules encoding a microRNA sequence that is/are comprised in the nucleic acid expression signature in such way that the expression of a nucleic acid molecule whose expression is up-regulated in blood is down-regulated and the expression of a nucleic acid molecule whose expression is down-regulated in blood is up-regulated.

Pharmaceutical composition for the prevention and/or treatment of colorectal cancer in blood, the composition comprising one or more nucleic acid molecules, each nucleic acid molecule encoding a sequence that is at least partially complementary to a microRNA sequence encoded by a nucleic acid molecule whose expression is up-regulated in plasma from colorectal cancer patients, as defined herein, and/or that corresponds to a microRNA sequence encoded by a nucleic acid molecule whose expression is down-regulated in plasma from colorectal cancer patients, as defined in any of claim 1 to 13.

17. Use of the pharmaceutical composition of 16 for the manufacture of a

medicament for the prevention and/or treatment of colorectal cancer.