CN106148508B - Methods and kits for colorectal cancer prognosis - Google Patents

Abstract

The present invention relates to methods and kits for colorectal cancer prognosis, especially methods comprising: a) obtaining a peripheral blood sample and extracting total RNA from said blood sample, b) combining said total RNA with at least one contacting an agent specific for at least one NK cell gene with no more than 25 agents specific for 25 NK cell genes, c) determining the expression level of the at least one NK cell gene and up to 25 NK cell genes to obtain The expression profile of the patient, d) analyzing the expression profile of the patient with the NK cell gene expression profile of a patient previously clinically classified as having a good prognosis and the NK cell gene expression profile of a patient previously classified as having a poor prognosis, wherein- The patient is determined to have a poor prognosis if the patient's expression profile is clustered with an expression profile from a patient previously clinically classified as having a poor prognosis, and - if the patient's expression profile is associated with a good prognosis from a previous clinical classification The expression profiles of patients with good prognosis are clustered, then it is determined that the patient has a good prognosis.

Description

Methods and kits for colorectal cancer prognosis

This application is a divisional application of an invention patent application with an application date of June 8, 2010, an application number of 201080067290.1, and an invention title of "method and kit for colorectal cancer prognosis".

Field of Invention

The present invention relates to the prognosis of colorectal cancer, and in particular to methods and kits for the prognosis of such cancer.

Background technique

Colorectal cancer (CRC), also known as colon cancer or colorectal cancer, is the fifth most common form of cancer in the United States, the fourth most common cancer in China and the third leading cause of cancer-related death in Europe. Early detection of CRC remains a major challenge to public health. In fact, CRC is often curable, especially when diagnosed at an early stage. Several screening strategies have been implemented in different countries. Routine CRC screening tests include fecal occult blood test (FOBT), sigmoidoscopy, colonoscopy, double barium gas contrast, or digital rectal examination. They all have advantages and limitations, but compliance remains lower than expected, mainly due to logistics or patient discomfort.

The search for blood markers for early detection of CRC has been in focus for several years, especially because of its convenience. Meanwhile, the feasibility of blood-based tests is supported by a very small number of studies showing that genetic biomarkers in blood can distinguish CRC patients from controls. These studies are based on flow cytometry, a technique for counting and examining microscopic particles, such as cells, by suspending them in a liquid stream and passing them through an electronic detection device.

The inventors have found that differentially expressed genes are mainly associated with immune cell activation and trafficking. In particular, they demonstrate that natural killer cells (NK cells) represent an important biomarker in peripheral blood samples. Instead of using classical flow cytometry, they identified differentially expressed genes in whole blood. Determining gene expression levels by analyzing transcripts in whole blood is uncommon, as it is generally recognized by those skilled in the art that specific information is obtained without specific purification steps when diluted in complex RNA mixtures (total RNA) it is hard. The advantage of this method is that it also avoids the step of purifying RNA.

Accordingly, the present invention relates to a method for determining the prognosis of colorectal cancer in a peripheral blood sample from a patient, the method comprising:

a) obtaining said peripheral blood sample and extracting total RNA from said blood sample,

b) contacting the total RNA with at least one agent specific for at least one NK cell gene and no more than 25 agents specific for 25 NK cell genes,

c) determining the expression level of said at least one NK cell gene and up to 25 NK cell genes to obtain an expression profile of said patient,

d) analyzing the expression profile of said patient with the NK cell gene expression profile of a patient previously clinically classified as having a good prognosis and the NK cell gene expression profile of a patient previously classified as having a poor prognosis, wherein

- determining the patient has a poor prognosis if the patient's expression profile is clustered with an expression profile from a patient previously clinically classified as having a poor prognosis, and

- A patient is determined to have a good prognosis if the patient's expression profile is clustered with an expression profile from a patient previously clinically classified as having a good prognosis.

Specifically, in the above step b), the total RNA is contacted with at least one reagent specific for at least one NK cell gene and no more than 25 reagents specific for 25 NK cell genes, the NK cell genes including The nucleic acid sequences shown in SEQ ID NOs: 1-12, wherein the at least one agent is specific for at least one NK cell gene selected from the group consisting of:

(i) a KLRB1 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 1,

(ii) a KLRC2 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 2, 3 or 4,

(iii) a KLRC3 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 5, 6 or 7,

(iv) a KLRD1 gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 8, 9, 10, 11 or 12, and

(v) a KLRK1 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 13, and

The expression level of the at least one NK cell gene is determined in step c) to obtain an expression profile of the patient.

As detailed in the experimental data, the expression level of at least one of the above genes is informative enough to predict CRC risk.

In one embodiment, the total RNA is contacted in step b) with an agent specific for a combination of at least 5 NK cell genes and no more than 25 NK cell genes, wherein the agent comprises at least the following NK Cell gene-specific reagents:

(i) a KLRB1 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 1,

(ii) a KLRC2 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 2, 3 or 4,

(iii) a KLRC3 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 5, 6 or 7,

(iv) a KLRD1 gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 8, 9, 10, 11 or 12, and

(v) KLRK1 comprising, for example, the full-length sequence shown in SEQ ID NO: 13,

The expression levels of at least the 4 NK cell genes are determined in step c) to obtain the expression profile of the patient.

In addition, in step b), the total RNA can be contacted with at least one agent specific for at least one target cell gene and no more than five agents specific for five target cell genes, the target cell gene Include the nucleic acid sequences shown in SEQ ID NOs: 12-24, wherein the at least one agent is specific for at least one target cell gene selected from the group consisting of:

(i) a GZMB gene comprising, for example, the full-length sequence shown in SEQ ID NO: 14, 15, 16 or 17,

(ii) a CD247 gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 18, 19 or 20,

(iii) an RRAS2 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 21 or 22, and

(iv) the SH2D1B gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 23 or 24, and

(v) an LCK gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 25, 26, 27, 28, 29 or 30, and

In step c), the expression level of the at least one target cell gene is determined to obtain the expression profile of the patient; while in one embodiment, the total RNA is contacted with an agent specific for the combination of the five target cell genes, wherein the reagents are specific for the following target cell genes:

(i) a GZMB gene comprising, for example, the full-length sequence shown in SEQ ID NO: 14, 15, 16 or 17,

(ii) a CD247 gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 18, 19 or 20,

(iii) an RRAS2 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 21 or 22, and

(iv) the SH2D1B gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 23 or 24, and

(v) an LCK gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 25, 26, 27, 28, 29 or 30, and

In step c), the expression levels of the at least 5 cellular genes are determined to obtain the expression profile of the patient.

In another embodiment, in step b), the total RNA is further contacted with at least one agent specific for at least one target cell gene and no more than 100 agents specific for 100 target cell genes, The target cell gene includes the nucleic acid sequences shown in SEQ ID NOs: 25-59, wherein the at least one reagent is specific for at least one target cell gene selected from the group consisting of:

(i) the MRPS6 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 31, 32 or 33,

(ii) a SPRY4 gene comprising, for example, the full-length sequence shown in SEQ ID NO:34,

(iii) a NEAT1 gene comprising, for example, the full-length sequence shown in SEQ ID NO:35,

(iv) a CYBB gene comprising, for example, the full-length sequence shown in SEQ ID NO:36,

(v) a DUSP2 gene comprising, for example, the full-length sequence shown in SEQ ID NO:37,

(vi) a PDEAD gene comprising, for example, the full-length sequence shown in SEQ ID NO: 38 or 39,

(vii) a SH2D2A gene comprising, for example, the full-length sequence shown in SEQ ID NO: 40, 41 or 42,

(viii) an INSR gene comprising, for example, the full-length sequence shown in SEQ ID NO: 43 or 44,

(ix) an ITGAM gene comprising, for example, the full-length sequence shown in SEQ ID NO:45,

(x) a VCAN gene comprising, for example, the full-length sequence shown in SEQ ID NO: 46, 47, 48 or 49,

(xi) a CD163 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 50 or 51,

(xii) a P2RY10 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 52 or 53,

(xii) a CD226 gene comprising, for example, the full-length sequence shown in SEQ ID NO:54,

(xiii) the MRPL10 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 55 or 56,

(xiv) an ITPRIPL2 gene comprising, for example, the full-length sequence shown in SEQ ID NO:57,

(xv) a CD2 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 58, and

(xvi) the NUDT16 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 59, and

The expression level of the at least one target cell gene is determined in step c) to obtain an expression profile of the patient.

In particular, in step b) the total RNA is contacted with an agent specific for at least 17 target cell genes and a combination of no more than 100 target cell genes, wherein the agent comprises at least the following target cell genes specific The reagents:

(i) the MRPS6 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 31, 32 or 33,

(ii) a SPRY4 gene comprising, for example, the full-length sequence shown in SEQ ID NO:34,

(iii) a NEAT1 gene comprising, for example, the full-length sequence shown in SEQ ID NO:35,

(iv) a CYBB gene comprising, for example, the full-length sequence shown in SEQ ID NO:36,

(v) a DUSP2 gene comprising, for example, the full-length sequence shown in SEQ ID NO:37,

(vi) a PDEAD gene comprising, for example, the full-length sequence shown in SEQ ID NO: 38 or 39,

(vii) a SH2D2A gene comprising, for example, the full-length sequence shown in SEQ ID NO: 40, 41 or 42,

(viii) an INSR gene comprising, for example, the full-length sequence shown in SEQ ID NO: 43 or 44,

(ix) an ITGAM gene comprising, for example, the full-length sequence shown in SEQ ID NO:45,

(x) a VCAN gene comprising, for example, the full-length sequence shown in SEQ ID NO: 46, 47, 48 or 49,

(xi) a CD163 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 50 or 51,

(xii) a P2RY10 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 52 or 53,

(xii) a CD226 gene comprising, for example, the full-length sequence shown in SEQ ID NO:54,

(xiii) the MRPL10 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 55 or 56,

(xiv) an ITPRIPL2 gene comprising, for example, the full-length sequence shown in SEQ ID NO:57,

(xv) a CD2 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 58, and

(xvi) the NUDT16 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 59, and

The expression levels of the at least 17 target cell genes are determined in step c) to obtain an expression profile of the patient.

More precisely, in the method described above, the at least one specific reagent described in step b) includes at least one hybridization probe, specifically at least one hybridization probe and at least one primer, and more specifically It includes at least one hybridization probe and two primers.

Total RNA includes transfer RNA (tRNA), messenger RNA (mRNA) (eg, mRNA transcribed from a target gene and from any other gene), and ribosomal RNA.

It is intended to illustrate that extraction of total RNA can be accomplished by a step of lysing cells present in a blood sample to release nucleic acid contained in the patient's cells. By way of example, lysis methods as described in patent applications WO00/05338 (which relate to mixed magnetic and mechanical lysis), WO 99/53304 (which relates to electrolysis), WO 99/15321 (which relates to mechanical lysis) can be used. Those skilled in the art can use other well-known lysis methods, such as heat shock or osmotic shock or chemical lysis using chaotropic agents such as guanidine salts (US Pat. No. 5,234,809). It is also possible to provide additional steps for isolating nucleic acids from other cellular components released during the lysis step. This often makes it possible to concentrate nucleic acids. By way of example, magnetic particles coated with oligonucleotides, optionally by adsorption or covalent action (see US Pat. No. 4,672,040 and US Pat. No. 5,750,338 in this regard) can be used, and can thus be purified by washing steps Nucleic acids bound to these magnetic particles. This nucleic acid purification step is particularly advantageous if subsequent amplification of the nucleic acid is desired. Particularly advantageous embodiments of these magnetic particles are described in patent applications WO-A-97/45202 and WO-A-99/35500.

The term "specific agent" refers to an agent that, when brought into contact with a biological material as defined above, binds to a material specific for the target gene. It is intended to illustrate that when the specific agent and the biological material are of nuclear origin, contacting the specific agent with the biological material may allow the specific agent to hybridize to the material specific for the target gene. The term "hybridization" refers to a process during which, under appropriate conditions, two nucleotide fragments bind with stable and specific hydrogen bonds to form a double-stranded complex. These hydrogen bonds are between complementary adenine (A) and thymine (T) (or uracil (U)) bases (this is called an A-T bond), or between complementary guanine (G) and cytosine (C) Formation between bases (this is called a G-C bond). Hybridization of two nucleotide fragments can be complete (referred to as complementary nucleotide fragments or sequences), ie the double-stranded complex obtained during this hybridization process contains only A-T and C-G bonds. This hybridization can be partial (referred to as a sufficiently complementary nucleotide fragment or sequence), i.e. the resulting double-stranded complex contains both A-T and C-G bonds that make double-stranded formation possible, as well as those that are not bound to complementary bases base. Hybridization between two nucleotide fragments depends on the working conditions used, especially the stringency. Stringency is specifically defined as a function of the base composition of two nucleotide fragments and is also defined by the degree of mismatch between the two nucleotide fragments. Stringency also depends on reaction parameters, such as the concentration and type of ions present in the hybridization solution, the nature and concentration of the denaturant, and/or the hybridization temperature. All of these data are well known and appropriate conditions can be determined by those skilled in the art. Generally, depending on the length of the nucleotide fragments to be hybridized, the hybridization temperature is about 20°C to 70°C, especially 35°C to 65°C, in a salt solution at a concentration of about 0.5-1 M. A sequence, or fragment of nucleotides, or oligonucleotide, or polynucleotide, is a series of nucleotide motifs assembled together by phosphate linkages, characterized by an informative native nucleic acid sequence capable of interacting with nucleoside Acid fragments hybridize, the series possibly containing monomers with different structures and possibly obtained from natural nucleic acid molecules and/or by genetic recombination and/or by chemical synthesis. Motifs are derivatives of monomers, which may be the natural nucleotides of nucleic acids, the constituent elements of which are sugars, phosphate groups, and nitrogenous bases; in DNA, the sugar is deoxy-2-ribose , in RNA, the sugar is ribose; according to whether DNA and RNA are involved, the nitrogenous base is selected from adenine, guanine, uracil, cytosine and thymine; alternatively, the monomer is three constituent elements At least one of the modified nucleotides; by way of example, modifications can occur at the base level (with modified bases such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino -5-deoxyuridine, diamino-2,6-purine, bromo-5-deoxyuridine, or any other modified base capable of hybridization), or occurs at the sugar level (e.g. at least one deoxyribose is replaced by a poly amides (P.E. Nielsen et al., Science, 254, 1497-1500 (1991)[3])), or alternatively at the level of the phosphate group (e.g. replaced by esters, specifically selected from diphosphates, alkylphosphates and acyl groups phosphate and phosphorothioate).

According to a specific embodiment of the present invention, the specific reagent includes at least one hybridization probe, or at least one hybridization probe and at least one primer specific for the target gene, or at least one hybridization probe and two pairs of Target gene-specific primers.

For the purposes of the present invention, the term "amplification primer" refers to a fragment of nucleotides comprising 5-100 nucleotides, preferably 15-30 nucleotides, which enables the initiation of an enzymatic polymerization such as an enzymatic amplification reaction . The term "enzymatic amplification reaction" refers to a process by which multiple copies of nucleotide fragments are produced by the action of at least one enzyme. Such amplification reactions are well known to those skilled in the art, with particular reference to the following techniques: PCR (polymerase chain reaction) (as described in US Pat. No. 4,683,195, US Pat. No. 4,683,202 and US Pat. No. 4,800,159), LCR (ligase chain reaction) (eg disclosed in patent application EP0201 184), RCR (repair chain reaction) (described in patent application WO 90/01069), 3SR (autonomous sequence replication) (patent application WO 90/ 06995), NASBA (Nucleic Acid Sequence Based Amplification) (Patent Application WO 91/02818), TMA (Transcription-Mediated Amplification) (US Patent No. 5,399,491) and RT-PCR.

When the enzymatic amplification is PCR, the specific reagents include at least two target gene-specific amplification primers that allow amplification of target gene-specific material. The material specific for the target gene then preferably comprises complementary DNA obtained by reverse transcription of messenger RNA from the target gene (referred to as target gene-specific cDNA) or complementary RNA obtained by transcribing cDNA specific for the target gene (referred to as target gene-specific cDNA). cRNA). When the enzymatic amplification is completed after the reverse transcription reaction, it is called RT-PCR.

The term "hybridization probe" refers to a fragment of nucleotides comprising at least 5 nucleotides, such as 5-100 nucleotides, in particular 10-75 nucleotides, such as 15-35 nucleotides and 60-70 nucleotides that have hybridization specificity under given conditions to form hybridization complexes with materials specific for the target gene. In the present invention, the material specific to the target gene may be a nucleotide sequence contained in a messenger RNA derived from the target gene (referred to as target gene-specific mRNA), a nucleotide sequence contained in a messenger RNA obtained by reverse transcribing the messenger RNA A nucleotide sequence in complementary DNA (referred to as target gene-specific cDNA), or otherwise a nucleotide sequence contained in complementary RNA obtained by transcribing the cDNA as described above (referred to as target gene-specific cRNA) ). Hybridization probes may contain labels for their detection. The term "detection" refers to direct detection, eg, counting methods, or indirect detection, by detection methods using tags. Numerous detection methods exist for the detection of nucleic acids (see, e.g., Kricka et al., Clinical Chemistry, 1999, no 45(4), pp. 453-458, or Keller GH et al., DNA Probes, 2nd Ed., Stockton Press, 1993, Parts 5 and 6, pp. 173-249). The term "tag" refers to a tracer capable of producing a detectable signal. A non-limiting list of these tracers includes enzymes (which produce a detectable signal by, for example, colorimetry, fluorescence or luminescence) such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, Glucose-6-phosphate dehydrogenase; chromophores, such as fluorescent, luminescent or dye compounds; electron-dense groups, which can be detected by electron microscopy or by their electronic properties such as conductivity by amperometric or voltammetric detectable by optical methods such as diffraction, surface plasmon resonance or contact angle changes, or by physical methods such as atomic force spectroscopy, tunneling, etc.; radioactive molecules such as ³² P, ³⁵ S or ^125I .

For the purposes of the present invention, hybridization probes may be "detection" probes. In this case, the "detection" probe is labeled with a label. The detection probe may be in particular a "molecular beacon" detection probe as described by Tyagi and Kramer (Nature biotech, 1996, 14:303-308). These "molecular beacons" become fluorescent agents during hybridization. They have a stem-loop structure and contain fluorophores and "quencher" groups. Binding of the specific loop sequence to its complementary target nucleic acid sequence causes the stem to unfold and emit a fluorescent signal during excitation at the appropriate wavelength. The detection probes may be in particular "reporter probes" comprising "color coded barcodes" according to NanoString ^™ technology.

For detection of the hybridization reaction, either directly (particularly by incorporating a tag into the target sequence) or indirectly (particularly using a detection probe as defined above) labeled target sequences can be used. In particular, a step of labeling and/or cleavage of the target sequence may be performed prior to the hybridization step, eg using labelled deoxyribonucleoside triphosphates during an enzymatic amplification reaction. The cleavage can be specifically completed by the action of imidazole or manganese chloride. The target sequence can also be labeled after the amplification step by hybridization to a detection probe according to the sandwich hybridization technique described, for example, in document WO 91/19812. Another specific preferred method for labelling nucleic acids is described in application FR 2780059.

According to a preferred embodiment of the present invention, the detection probe comprises a fluorophore and a quencher. According to an even more preferred embodiment of the present invention, the hybridization probe comprises a FAM (6-carboxyfluorescein) or ROX (6-carboxy-X-rhodamine) fluorophore at its 5' end and a quencher at its 3' end agent (Dabsyl).

Hybridization probes can also be "capture" probes. In this case, the "capture" probe is or may be immobilized on the solid substrate by any suitable method, either directly or indirectly, for example by covalent or adsorption. As solid substrates synthetic or natural materials (optionally chemically modified) can be used, in particular polysaccharides such as cellulose-based materials such as paper, cellulose derivatives such as cellulose acetate and nitrocellulose or dextran , polymers, copolymers (especially based on styrenic monomers), natural fibers such as cotton, and synthetic fibers such as nylon; inorganic materials such as silica, quartz, glass or ceramics; latex; magnetic particles; metal derivatives , gel, etc. Solid substrates may be in the form of microtiter plates, membranes (as described in application WO-A-94/12670) or particles. It is also possible to immobilize several different capture probes on the substrate, each specific for one target gene. Specifically, a biochip can be used as a substrate on which a large number of probes can be immobilized. The term "biochip" refers to a small volume of solid substrate to which a large number of capture probes can be attached at predetermined locations. The concept of biochips (or DNA chips) dates back to the early 1990s. It is based on multidisciplinary technology, integrating microelectronics, nucleic acid chemistry, image analysis and information technology. How it works is based on the fundamentals of molecular biology: the phenomenon of hybridization, the pairing of two DNA and/or RNA sequences through base complementarity. The biochip approach is based on the use of capture probes attached to a solid substrate to which a sample of target nucleotide fragments labeled with a fluorescent dye, either directly or indirectly, acts. Capture probes are specifically positioned on the substrate or chip, and each hybridization gives a specific piece of information about the target nucleotide fragment. The information obtained is cumulative, making it possible, for example, to quantify the expression level of one or more target genes. Then to analyze the expression of the target gene, a substrate can be prepared containing a number of probes corresponding to all or part of the target gene transcribed into mRNA. For the purposes of the present invention, the term "low density substrate" refers to a substrate containing less than 50 probes. For the purposes of the present invention, the term "medium density substrate" refers to a substrate comprising from 50 probes to 10,000 probes. For the purposes of the present invention, the term "high density substrate" refers to a substrate containing more than 10,000 probes.

The cDNA or cRNA specific for the target gene to be analyzed is then hybridized to, for example, a specific capture probe. After hybridization, the substrate or chip is washed and the labeled cDNA or cRNA/capture antibody complex is revealed by a high affinity ligand bound to, for example, a fluorescent dye-type tag. Fluorescence is read with eg a scanner and analyzed by information technology. By way of illustration, mention may be made of DNA chips developed by Affymetrix for molecular diagnostics ("Accessing Genetic Information with High-Density DNA arrays", M. Chee et al., Science, 1996, 274, 610-614. "Light -generated oligonucleotide arrays for rapid DNA sequence analysis", A. Caviani Pease et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 5022-5026). In this technique, capture probes are generally small, about 25 nucleotides. In publications G. Ramsay, Nature Biotechnology, 1998, No. 16, pp. 40-44; F. Ginot, Human Mutation, 1997, No. 10, pp. 1-10; J. Cheng et al., Moleculardiagnosis, 1996, No. 1(3), pp. 183-200; T. Livache et al., Nucleic Acids Research, 1994, No. 22(15), pp. 2915-2921; J. Cheng et al., Nature Biotechnology, 1998, No. 16, Additional examples of biochips are given on pages 541-546 or in US Pat. No. 4,981,783, US Pat. No. 5,700,637, US Pat. No. 5,445,934, US Pat. No. 5,744,305, and US Pat. No. 5,807,522. The main feature of the solid substrate should be to maintain the hybridization characteristics of the capture probes to the target nucleotide fragments while producing minimal background noise for the detection method. The immobilization of probes on substrates can be divided into three main types.

First, there is a first technique, which consists in depositing pre-synthesized probes. Attachment of the probes is done by direct transfer, either by micropipette or microdot or by inkjet device. This technique allows the ligation of probes ranging in size from a few bases (5-10) to relatively large 60 bases (printing) to hundreds of bases (microdeposition).

The printing method is a variation on the method used by inkjet printers. It is based on propelling very small liquid spheres (volume < 1 nl) at a rate that can reach 4000 drops/sec. The printing method does not involve any contact between the system releasing the liquid and the surface on which the liquid is deposited.

Microdeposition is characterized by attaching long probes ranging from tens to hundreds of bases to the surface of a glass slide. These probes are typically extracted from databases and are in the form of amplified and purified products. This technology makes it possible to produce chips called microarrays that carry about 10,000 DNA spots on a surface area of slightly less than 4 square centimeters, called the recognition area. However, the use of nylon membranes (called "macroarrays") should not be forgotten, which carry amplified (generally by PCR) products of 0.5 mm to 1 mm diameter at a density of up to 25 points/cm 2 . Many laboratories use this very flexible technique. In the present invention, the biochip is considered to include the latter technology. However, as in the case of patent applications WO-A-00/71750 and FR00/14896, a certain volume of sample can be deposited at the bottom of each well of a microtiter plate, or according to another patent application FR 00/14691, a A certain number of droplets separated from each other are deposited on the bottom of the same sterile petri dish.

A second technique for attaching probes to substrates or chips is called in situ synthesis. This technique results in the creation of short probes directly on the chip surface. It is based on in situ oligonucleotide synthesis (see in particular, patent applications WO 89/10977 and WO 90/03382) and on oligonucleotide synthesizer methods. It consists in moving the reaction chamber along the glass surface in which the nucleotide extension reaction takes place.

Finally, a third technique is called photolithography, a method developed by Affymetrix for biochips. It is also synthesized in situ. Photolithography technology is derived from microprocessor technology. The chip surface is modified by attaching photolabile (photoactivatable) chemical groups. Once illuminated, these groups are able to react with the 3' end of the oligonucleotide. By protecting this surface with a light shield of defined shape, it is possible to selectively illuminate and thus activate regions where one of the four nucleotides or other nucleotides is desired to be attached. The sequential use of different light shields makes it possible to alternate protection/reaction cycles and thus produce oligonucleotide probes on the order of tens of square micrometers (μm ² ) spots. This resolution makes it possible to generate up to hundreds of thousands of points on a surface area of several square centimeters (cm ² ). The photolithographic technique has the advantage of batch parallelism, which makes it possible to generate N-mer chips in only 4 by N cycles. All of these techniques can be used in the present invention. According to a preferred embodiment of the present invention, the at least one specific reagent of step b) as defined above comprises at least one hybridization probe, which is preferably immobilized on a substrate. This substrate is preferably a low, high or medium density substrate as defined above.

In order to increase the amount of target genetic material, the enzymatic amplification reaction steps as defined above can be performed before these hybridization steps on substrates containing a large number of probes.

The determination of the target gene expression level in step c) can be accomplished by any protocol known to those skilled in the art. In general, the expression of a target gene can be analyzed by detecting mRNA (messenger RNA) transcribed from the target gene at a given time.

The present invention preferably relates to determining the expression level of a target gene by detecting mRNA derived from the target gene according to any protocol known to those skilled in the art. According to a specific embodiment of the invention, by detecting several different mRNAs, each derived from one target gene, the expression levels of several target genes can be determined simultaneously.

When the specific reagent includes at least one amplification primer, it is possible to determine the expression level of the target gene in the following manner: 1) Extraction of total RNA (comprising transfer RNA (tRNA), ribosomal RNA (rRNA) and messenger RNA) from whole blood ( mRNA)), a reverse transcription step is performed to obtain the complementary DNA (or cDNA) of the mRNA. It is intended to illustrate that this reverse transcription reaction can be carried out using a reverse transcriptase enzyme capable of obtaining complementary DNA fragments from RNA fragments. Specifically, reverse transcriptases from AMV (Avian Myeloblastoma Virus) or MMLV (Moroney Murine Leukemia Virus) can be used. When more specifically only the cDNA of the mRNA needs to be obtained, this reverse transcription step is carried out in the presence of a fragment of nucleotides containing only thymine bases (poly-T), which by polymerizing with the mRNA The A sequences are complementary to hybridize to form a poly-T-poly-A complex, which then serves as the initiation site for the reverse transcription reaction by reverse transcriptase. Then, cDNA complementary to mRNA derived from the target gene (target gene specific cDNA) and cDNA complementary to mRNA derived from genes other than the target gene (non-target gene specific cDNA) are obtained. 2) The amplification primer specific for the target gene is brought into contact with the cDNA specific for the target gene and the cDNA specific for the non-target gene. Target gene-specific amplification primers hybridize to the target gene-specific cDNA and specifically amplify a predetermined region of known length of the cDNA (which is derived from mRNA from the target gene). Non-target gene-specific cDNAs are not amplified, thereby obtaining a large number of target gene-specific cDNAs. For the purposes of the present invention, "target gene-specific cDNA" or "cDNA derived from mRNA from the target gene" may be referred to without distinction. This step can be carried out in particular by means of an amplification reaction of the PCR type or by any other amplification technique as defined above. For PCR, it is also possible to simultaneously amplify several different cDNAs (each specific for a different target gene) by using several pairs of different amplification primers (each specific for one target gene): called multiplex amplification . 3) Determine the expression of the target gene by detecting and quantifying the target gene-specific cDNA obtained in the above step 2). This detection can be performed after electrophoretic migration of the cDNA specific for the target gene according to its size. The gels and media used for migration can contain ethidium bromide, so that after a given migration time period, when the gel is placed on a UV (ultraviolet) stage, the target gene-specific gene is directly detected by the light signal emitted. cDNA. The greater the amount of target gene-specific cDNA, the stronger the light signal. These electrophoresis techniques are well known to those skilled in the art. Target gene-specific cDNA can also be detected and quantified using the range of quantification obtained by performing amplification reactions to saturation. To account for differences in enzyme efficiency that may be observed at different steps (reverse transcription, PCR, etc.), the different groups can be normalized by simultaneously determining the expression of "housekeeping" genes whose expression is similar in different groups of patients Expression of target genes in patients. Any variation between experiments is corrected for by obtaining the ratio of target gene expression to housekeeping gene expression, ie by obtaining the ratio of the amount of cDNA specific for the target gene to the amount of cDNA specific for the housekeeping gene. Those skilled in the art may specifically refer to the following publications: Bustin S A, J Mol Endocrinol, 2002, 29: 23-39; Giulietti A Methods, 2001, 25: 386-401.

When the specific reagent comprises at least one hybridization probe, it is possible to determine the expression of the target gene in the following way: 1) After extraction of total RNA from whole blood, a reverse transcription step is performed as described above, thereby obtaining a cDNA complementary to the mRNA of the target gene (target gene-specific cDNA) and cDNA complementary to mRNA derived from a gene other than the target gene (non-target gene-specific cDNA). 2) all cDNAs are brought into contact with a substrate on which a capture probe specific for the target gene whose expression needs to be analyzed is immobilized, thereby performing a hybridization reaction between the target gene-specific cDNA and the capture probe, and cDNA that is not specific for the target gene does not hybridize to the capture probe. Hybridization reactions can be carried out on solid substrates comprising all of the materials indicated above. According to a preferred embodiment of the present invention, the hybridization probes are immobilized on the substrate. Preferably, the substrate is a low, high or medium density substrate as defined above. A step consisting of enzymatic amplification of target gene-specific cDNA as defined above can be performed prior to the hybridization reaction, thereby obtaining a large amount of target gene-specific cDNA and increasing the hybridization of target gene-specific cDNA with capture probes specific for the target gene possibility. It is also possible to perform the step of labeling and/or cleaving the target gene-specific cDNA as described above before the hybridization reaction, eg using labelled deoxyribonucleoside triphosphates for the amplification reaction. Specifically, the cleavage can be accomplished by the action of imidazole and manganese chloride. The target gene-specific cDNA can also be labeled after the amplification step, eg by hybridization with labeled probes according to the sandwich hybridization technique described in document WO-A-91/19812. Other preferred specific methods for labelling and/or cleaving nucleic acids are described in applications WO 99/65926, WO 01/44507, WO 01/44506, WO 02/090584, WO 02/090319. 3) A step consisting of a detection hybridization reaction is then carried out. The detection can be performed by contacting a substrate (on which a capture probe specific for the target gene is hybridized with a cDNA specific for the target gene) with a labeled "detection" probe, and detecting the signal emitted by the label. When the cDNA specific for the target gene has been previously labeled with the tag, the signal emitted by the tag is directly detected.

When the at least one specific reagent in step b) comprises at least one hybridization probe, the expression of the target gene can also be determined in the following manner: 1) After extraction of total RNA from whole blood, reverse transcription is performed as described above step, thereby obtaining the cDNA of the mRNA of the biological material. The polymerization of the complementary RNA of the cDNA is then carried out using T7 polymerase, which functions under the control of the promoter and makes it possible to obtain the complementary RNA from the DNA template. The cRNA of the cDNA of the mRNA specific for the target gene and the cRNA of the cDNA of the non-target gene-specific mRNA are then obtained. 2) all cRNAs are brought into contact with a substrate on which a capture probe specific for the target gene whose expression needs to be analyzed is immobilized, so as to carry out a hybridization reaction between the target gene specific cRNA and the capture probe, and Non-target gene-specific cRNAs do not hybridize to the capture probe. When the expression of several target genes needs to be analyzed simultaneously, several different capture probes can be immobilized on the substrate, each probe specific for one target gene. It is also possible to carry out the step of labeling and/or cleaving the target gene-specific cRNA as described above before the hybridization reaction. 3) A step consisting of a detection hybridization reaction is then carried out. Detection can be accomplished by contacting a substrate (on which a capture probe specific for the target gene is hybridized to cRNA specific for the target gene) with a label-labeled "detection" probe, and detecting the signal emitted by the label. When the target gene-specific cRNA has been previously labeled with the tag, the signal emitted by the tag is directly detected. The use of cRNA is particularly advantageous when using a biochip-like substrate on which a large number of probes are hybridized.

The present invention also includes a kit for prognosing colorectal cancer in a peripheral blood sample from a patient, the kit comprising at least one reagent specific for at least one NK cell gene and no more than 25 for 25 An NK cell gene-specific reagent, the NK cell gene comprises at least the nucleic acid sequences shown in SEQ ID NOs: 1-13, wherein the at least one reagent is specific to at least one NK cell gene selected from the following:

(i) a KLRB1 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 1,

(ii) a KLRC2 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 2, 3 or 4,

(iii) a KLRC3 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 5, 6 or 7,

(iv) a KLRD1 gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 8, 9, 10, 11 or 12, and

(v) KLRK1 gene comprising, for example, the full-length sequence shown in SEQ ID NO:13.

In one embodiment, the kit includes a combination of reagents specific for the following NK cell genes:

(i) a KLRB1 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 1,

(ii) a KLRC2 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 2, 3 or 4,

(iii) a KLRC3 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 5, 6 or 7,

(iv) a KLRD1 gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 8, 9, 10, 11 or 12, and

(v) KLRK1 gene comprising, for example, the full-length sequence shown in SEQ ID NO:13.

In such embodiments, the specific agent can target a combination of several NK cell genes but no more than 25 NK genes.

Additionally, the kit may include at least one reagent specific for at least one target cell gene and no more than 5 target cell genes selected from the group consisting of:

(i) a GZMB gene comprising, for example, the full-length sequence shown in SEQ ID NO: 14, 15, 16 or 17,

(ii) a CD247 gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 18, 19 or 20,

(iii) an RRAS2 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 21 or 22, and

(iv) the SH2D1B gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 23 or 24, and

(v) an LCK gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 25, 26, 27, 28, 29 or 30.

Specifically, it includes 5 reagents specific for the following target cell genes:

(i) a GZMB gene comprising, for example, the full-length sequence shown in SEQ ID NO: 14, 15, 16 or 17,

(ii) a CD247 gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 18, 19 or 20,

(iii) an RRAS2 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 21 or 22, and

(iv) the SH2D1B gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 23 or 24, and

(v) an LCK gene comprising, for example, the full-length sequence set forth in SEQ ID NO: 25, 26, 27, 28, 29 or 30.

In such an embodiment, the specific agent can target, for example, a combination of several target cell genes described above but no more than 5 target cell genes.

In another embodiment, a kit such as defined above may comprise at least one reagent specific for at least one target cell gene and up to 100 reagents specific for 100 target cell genes, the at least one target cell gene Selected from:

(i) the MRPS6 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 31, 32 or 33,

(ii) a SPRY4 gene comprising, for example, the full-length sequence shown in SEQ ID NO:34,

(iii) a NEAT1 gene comprising, for example, the full-length sequence shown in SEQ ID NO:35,

(iv) a CYBB gene comprising, for example, the full-length sequence shown in SEQ ID NO:36,

(v) a DUSP2 gene comprising, for example, the full-length sequence shown in SEQ ID NO:37,

(vi) a PDEAD gene comprising, for example, the full-length sequence shown in SEQ ID NO: 38 or 39,

(vii) a SH2D2A gene comprising, for example, the full-length sequence shown in SEQ ID NO: 40, 41 or 42,

(viii) an INSR gene comprising, for example, the full-length sequence shown in SEQ ID NO: 43 or 44,

(ix) an ITGAM gene comprising, for example, the full-length sequence shown in SEQ ID NO:45,

(x) a VCAN gene comprising, for example, the full-length sequence shown in SEQ ID NO: 46, 47, 48 or 49,

(xi) a CD163 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 50 or 51,

(xii) a P2RY10 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 52 or 53,

(xii) a CD226 gene comprising, for example, the full-length sequence shown in SEQ ID NO:54,

(xiii) the MRPL10 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 55 or 56,

(xiv) an ITPRIPL2 gene comprising, for example, the full-length sequence shown in SEQ ID NO:57,

(xv) a CD2 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 58, and

(xvi) NUDT16 gene comprising, eg, the full-length sequence shown in SEQ ID NO:59.

And specifically, it includes 17 reagents specific for the following 17 target cell genes:

(i) the MRPS6 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 31, 32 or 33,

(ii) a SPRY4 gene comprising, for example, the full-length sequence shown in SEQ ID NO:34,

(iii) a NEAT1 gene comprising, for example, the full-length sequence shown in SEQ ID NO:35,

(iv) a CYBB gene comprising, for example, the full-length sequence shown in SEQ ID NO:36,

(v) a DUSP2 gene comprising, for example, the full-length sequence shown in SEQ ID NO:37,

(vi) a PDEAD gene comprising, for example, the full-length sequence shown in SEQ ID NO: 38 or 39,

(vii) a SH2D2A gene comprising, for example, the full-length sequence shown in SEQ ID NO: 40, 41 or 42,

(viii) an INSR gene comprising, for example, the full-length sequence shown in SEQ ID NO: 43 or 44,

(ix) an ITGAM gene comprising, for example, the full-length sequence shown in SEQ ID NO:45,

(x) a VCAN gene comprising, for example, the full-length sequence shown in SEQ ID NO: 46, 47, 48 or 49,

(xi) a CD163 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 50 or 51,

(xii) a P2RY10 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 52 or 53,

(xii) a CD226 gene comprising, for example, the full-length sequence shown in SEQ ID NO:54,

(xiii) the MRPL10 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 55 or 56,

(xiv) an ITPRIPL2 gene comprising, for example, the full-length sequence shown in SEQ ID NO:57,

(xv) a CD2 gene comprising, for example, the full-length sequence shown in SEQ ID NO: 58, and

(xvi) NUDT16 gene comprising, eg, the full-length sequence shown in SEQ ID NO:59.

In such embodiments, the specific agent may target, for example, a combination of several target cell genes described above but no more than 100 target cell genes.

As explained above, the at least one specific reagent includes at least one hybridization probe, specifically at least one hybridization probe and at least one primer, and more specifically at least one hybridization probe and two primers .

Finally, the present invention relates to the use of at least one agent specific for at least one NK cell gene and no more than 25 agents specific for 25 NK cell genes in the preparation of a composition for use in a patient-derived Prognosis of colorectal cancer in the biological sample, the NK cell gene comprises the nucleic acid sequence shown in SEQ ID NO:1-12, wherein the at least one reagent pair comprises any one selected from the group consisting of SEQ ID NO:1-12. The nucleic acid sequence shown is specific for at least one NK cell gene;

In particular the use of an agent specific for a combination of at least 5 NK cell genes and no more than 25 NK cell genes in the preparation of a composition for the prognosis of colorectal cancer in a biological sample from a patient, wherein the agent is specific for at least 5 NK cell genes comprising nucleic acid sequences selected from the group consisting of nucleotide sequences shown in SEQ ID NOs: 1, 2-4, 5-7 and 8-12, respectively;

In particular the use of an agent specific for a combination of 10 target cell genes in the preparation of a composition for prognosing colorectal cancer in a biological sample from a patient, wherein the pair of agents comprises a pair of agents each selected from the group consisting of SEQ ID NOs: IDNO: 1, 2-4, 5-7, 8-12, 13, 14-17, 18-20, 21-22, 23-24 and 25-30 are specific for the target cell gene of the nucleic acid sequence; and

More particularly the use of an agent specific for a combination of 10 target cell genes and no more than 100 target genes in the preparation of a composition for the prognosis of colorectal cancer in a biological sample from a patient, wherein the pair of reagents comprises a pair of reagents selected from the group consisting of SEQ ID NOs: 1, 2-4, 5-7, 8-12, 13, 14-17, 18-20, 21-22, 23-24, 25-30, 31, respectively -33, 34, 35, 36, 37, 38-39, 4-42, 43-44, 45, 46-49, 50-51, 52-53, 54, 55-56, 57, 58 and 59 The nucleic acid sequence of the target cell gene is specific;

Wherein, the at least one specific reagent includes at least one hybridization probe, at least one hybridization probe and at least one primer, or at least one hybridization probe and two primers.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph of NK cell scores in blood samples from colonoscopy negative controls (CNC) and colorectal cancer (CRC) patients, and the distribution of CRC samples by cancer stage. Circles represent CNC; boxes, positive triangles, inverted triangles and diamonds represent stage I, II, III and IV CRC, respectively.

Example

I) Materials and Methods

1. Patient and Sample Collection

This study has been approved by the local clinical research ethics committee. Written informed consent was obtained from all participants.

For the CRC group, 119 colorectal cancer patients were consecutively recruited for this study between July 2006 and March 2008 at the Department of Colorectal Surgery, Fudan University Cancer Hospital (FUCH), China. Tumors were staged according to the tumor-lymph node-metastasis system recommended by the International Union Against Cancer (UICC). No patients received preoperative radiation or chemotherapy. Patients with hereditary colorectal cancer or inflammatory bowel disease (Crohn's disease or ulcerative colitis) were excluded from this study. For each patient, at least one week after colonoscopy and before surgery, 2.5 ml of peripheral blood was collected into PAXgene ^™ blood RNA tubes (PreAnalytiX GmbH, Hombrechtikon, CH) and processed according to the manufacturer's instructions. For the control group, 101 FOBT-test-positive participants who had been confirmed by colonoscopy without any symptoms of polyps or colorectal cancer were enrolled from community hospitals in the Shanghai area. Peripheral blood samples were collected into PAXgene tubes one week prior to colonoscopy. Detailed characteristics of all participants included in this study are given in Table 1.

Table 1: Patient Characteristics

2. RNA extraction and microarray experiments

Total RNA was extracted with the PAXgene ^™ Blood RNA System (PreAnalytix) following the manufacturer's instructions. The amount of total RNA was measured by spectrophotometer at optical density at 260 nm using RNA 6000 Nano on BioAnalyzer Agilent 2100 (Agilent Technologies, Palo Alto, CA, USA) Kit assessment quality. Only samples with RNA integrity values of 7 to 10 were analyzed. 50 ng of total RNA was then reverse transcribed and linearly amplified into single strands using the WT-Ovation ^™ RNA Amplification System of Ribo-SPIA ^™ Technology (NuGEN Technologies Inc., San Carlos, CA, USA) according to the manufacturer's standard protocol cDNA, and the product was purified using the QIAquick ^™ PCR purification kit (QIAGENGmbH, Hilden, Germany). 2 mg of the amplified and purified cDNA was subsequently fragmented with RQ1 RNase-free DNase (Promega Corp., Fitchburg, WI, USA) and passed through terminal transferase (Roche Diagnostics Corp.) with biotinylated deoxynucleoside triphosphates. ., Indianapoli, IN, USA) and DNA labeling reagent (Affymetrix Inc., Santa Clara, CA, USA) labeled. Labeled cDNA was hybridized onto a HG U133Plus 2.0 Array (Affymetrix) in a hybridization oven 640 (Agilent Technologies) at 50°C for 18 minutes at 60 rpm. The HG U133Plus 2.0 Array contains 54,675 probe sets representing approximately 39,000 best characterized human genes. After hybridization, EukGE-WS2v4 was used according to the Affymetrix protocol Microarrays were washed and stained with FluidicsStation 450 (Affymetrix). use Microarrays were scanned by Scanner 3000 (Affymetrix).

3. Analysis of Microarray Data

Quality control analysis was done according to standard Affymetrix quality control parameter recommendations. All our experiments met minimum quality requirements based on evaluation criteria. Affymetrix expression microarrays were preprocessed with background correction, quantile normalization and median smoothing induction by RMA (Robust Multi-Chip Averaging) [1]. Probe sets with extreme signal intensities (below 50 or above 2×10 ¹⁴ ) were filtered out. To reduce the possibility of batch effects, the normalization algorithm CamBat ¹¹ was applied to the filtered expression data. The CamBat method (http://statistics.byu.edu/johnson/ComBat/) applies a parametric or nonparametric empirical Bayu framework to adjust for batch effects in a given dataset. Differentially expressed genes (DEGs) ¹² were determined by microarray significance analysis (SAM) at a false discovery rate (FDR) equal to 0.05. Preprocessing and statistical steps were performed using the R environment with the Bioconductor library. Gene Ontology and Canonical Pathway analyses were performed using Ingenuity Pathway Analysis software version 8.5 (Ingenuity Systems, Redwood City, CA, USA).

II) Results

1. Characteristics of colorectal cancer and control patient populations

Clinical and demographic variables of 119 colorectal cancer (CRC) patients and 101 colonoscopy negative controls (CNC) are summarized in Table 1. For CRC, the diagnosis of colorectal cancer was confirmed by a pathologist after colonoscopy. Controls were selected from FOBT-positive patients enrolled in a community hospital who were ultimately negative by colonoscopy at Fudan University Cancer Hospital (FUCH). Age and gender were well balanced between the CRC and CNC groups.

2. Identification of genes whose expression in peripheral blood differs in colorectal cancer patients and colonoscopy negative controls

The inventors searched for the differentially expressed genes (DEGs) with the highest differences between the two groups in 119 CRCs and 101 CNCs, considering the CRC group (stages I, II, III and IV) as a whole. After appropriate preprocessing, 20169 probe sets were retained for DEG analysis. At FDR equal to 0.05 and fold change (FC) greater than 1.2, 327 DEGs were identified using SAM.

Of these 327 DEGs, 195 (59.6%) and 132 (40.36%) were found to be expressed at higher and lower levels in CRC samples, respectively. T-test P-values ranged from 1.43× ^10-25 to 1.51× ^10-01 , 18 DEGs had T-test P-values less than 6.27× ^10-15 and all corresponded to well-annotated genes: MRPS6, SPRY4, NEAT1, CYBB, DUSP2, PDE4D, SH2D2A, G(1-2)NSR, ITGAM, VCAN, CD163, P2RY10, CD226, MRPL10, ITPRIPL2, CD2 and NUDT16 (Table 2). The highest fold change (FC) values were 1.83 (higher level of NEAT1 in CRC) and 1.71 (lower level of HBG2 in CRC), while 26 (8%) of 327 DEGs had higher than 1.40 FC value.

For example, for SPRY4 (higher expression level in CRC ranked first, T-test P value 4.04 x 10-23, FC 1.79) and ^MRPS6 (lower expression level in CRC ranked first, T-test P value 1.43 x ^10-25 , FC 1.27) observed results. Such examples represent genes that are significantly differentially expressed between CRC and CNC patients. For SPRY4, very similar hybridization signal values were observed for the 101 CNCs, while the CRC values were more varied but had a significantly (p-value 4.04 x 10&lt;&quot; ²³ &gt;) increased mean (FC 1.78) compared to CNC. For MRPS6, both populations showed similar dispersion, with CRC having a significantly (p-value 1.43 x ^10-25 ) reduced mean (FC 1.27).

Among the top 18 DEGs, 4 membranous leukocyte markers were observed, showing different expression levels in peripheral blood of CRC patients compared to CNC: lower levels of CD2 and CD226, which are produced by T cells and mainly by NK, respectively Cellular expression; higher levels of CD163 and CD11B (ITGAM), which are mainly expressed by monocytes and many leukocytes involved in the innate immune system, respectively. Also interesting was the lower expression of granzyme B, encoded by the GZMB gene, in cytotoxic T lymphocytes and natural killer (NK) cells, in CRC samples. Other genes like INSR, SPRY4, DUSP2, PDE4D and ITPRIPL2 were reported as part of different signaling pathways, and SH2D2A was reported as T cell specific. VCAN has been reported to be expressed in monocytes, and its higher expression levels in CRC samples, along with CD163 and ITGAM, were associated with some activation of circulating monocytes in peripheral blood in these patients compared to CNC.

The 327 DEGs have been analyzed by using Ingenuity Pathway Analysis (IPA), which returns 321 localized IDs suitable for interpretation of relevant Bio Fuctions and canonical pathways. For Physiological System Development and Function, a high score was observed for Immune Cell Trafficking (p-value from 1.44×10 ⁻¹² to 1.57×10 ⁻⁰² , including 50 molecules), which covered Activation, migration, accumulation, influx, chemotaxis, cell spreading, cell motility, chemoattraction, priming, and adhesion of various immune cells have been described. Interestingly for the classical pathway, Natural Killer Cell Signaling was the item with the lowest P value (2.55 x 10-05), which included 10 genes: ^CD247 , KLRB1, KLRC2, KLRC3, KLRD1 , KLRK1, LCK, PRKCH, RRAS2 and SH2D1D. The involvement of five NK cell-specific membrane receptors (KLRB1, KLRC2, KLRC3, KLRD1, KLRK1) very strongly suggests specific NK cell component differences in gene expression levels in the peripheral blood of CRC patients. All NK cell gene expression was decreased in CRC. The results are summarized in Tables 2 and 3 below.

Table 2: Top 18 genes (DEGs) differentially expressed between colorectal cancer (CRC) and colonoscopy-negative control (CNC) patient samples; gene description, T-test P-value and fold change related information

*From NetAffx ^TM and from Ingenuity Pathway Gene description for version 8.5

Table 3: NK cell scores: selected genes, T-test P-values and fold change related information

*From NetAffx ^TM and from Ingenuity Pathway Gene description for version 8.5

For these 10 NK cell-related genes, lower expression levels were observed in the CRC group, suggesting a decreased number of circulating NK cells, or efflux of such cells to other organ/tissue compartments, especially tumor sites. It is also notable to observe lower levels of GZMB expression, representing a major event occurring at cytotoxic levels in CRC patients.

The top-ranked classical pathways involve T Cell Receptor Signaling, Communication between Innate and Adaptive Immune Cells, and iCOS-iCOSL Signaling in T helper cells ( iCOS-iCOSL Signaling in THelper Cells) with P-values of 9.08×10 ⁻⁰⁵ , 2.85×10 ⁻⁰⁴ and 5.78×10 ⁻⁰⁴ , respectively.

Interestingly, low NK cell scores below the top quartile were observed for 51 of the 119 CRC patient samples, compared to only 4 of the 101 CNC patient samples. Using such a straightforward cutoff, the performance of this discrimination can be expressed as a sensitivity of 43% and a specificity of 96%. Additionally, when differentiating CRC patients according to tumor TNM stage (stage I, II, III or IV), we observed that this NK cell score gradually decreased in CRC patients from stage I to stage IV (Figure 1). Statistically significant differences were observed mainly between CNC and stage II, III and IV CRC, and between I CRC and II-III and IV CRC.

This study shows the potential of the transcriptome to discover biomarkers in peripheral blood and provides new insights into immune responses in colorectal cancer. In addition to preparing possible alternatives/complements to existing screening modalities, these results show that expression analysis of genes associated with e.g. NK cells makes it possible to differentiate patients with colorectal cancer, opening the door to personalized medicine .

references

1. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, ScherfU, Speed TP. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 2003;4:249-64)

2. Johnson WE, Li C, Rabinovic A. Adjusting batch effects in microarrayexpression data using empirical Bayes methods. Biostatistics 2007;8:118-27.

3. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 2001;98:5116-21.

4.Team RDC.R:A Language and Environment for StatisticalComputing.Vienna,Austria,2009.

Claims (13)

1. Use of a combination of reagents specific for the following at least 5 NK cell genes in the preparation of a kit for determining the prognosis of colorectal cancer in a peripheral blood sample from a patient by the following method, wherein the at least 5 NK cells Genes include: (i) a KLRB1 gene comprising the full-length sequence as shown in SEQ ID NO: 1, (ii) a KLRC2 gene comprising the full-length sequence shown in SEQ ID NO: 2, 3 or 4, (iii) a KLRC3 gene comprising the full-length sequence shown in SEQ ID NO: 5, 6 or 7, (iv) a KLRD1 gene comprising the full-length sequence as set forth in SEQ ID NO: 8, 9, 10, 11 or 12, and (v) a KLRK1 gene comprising the full-length sequence shown in SEQ ID NO: 13; wherein the method includes: a) obtaining said peripheral blood sample and extracting total RNA from said peripheral blood sample, b) contacting the total RNA with an agent specific for at least 5 NK cell genes, c) determining the expression levels of said at least 5 NK cell genes to obtain an expression profile of said patient, d) analyzing the expression profile of said patient with the NK cell gene expression profile of a patient previously clinically classified as having a good prognosis and the NK cell gene expression profile of a patient previously classified as having a poor prognosis, wherein - determining the patient has a poor prognosis if the patient's expression profile is clustered with an expression profile from a patient previously clinically classified as having a poor prognosis, and - A patient is determined to have a good prognosis if the patient's expression profile is clustered with an expression profile from a patient previously clinically classified as having a good prognosis. 2. The purposes of claim 1, wherein said combination of reagents further comprises other reagents specific for at least one target cell gene selected from the group consisting of: (i) a GZMB gene comprising the full-length sequence shown in SEQ ID NO: 14, 15, 16 or 17, (ii) the CD247 gene comprising the full-length sequence as set forth in SEQ ID NO: 18, 19 or 20, (iii) an RRAS2 gene comprising the full-length sequence shown in SEQ ID NO: 21 or 22, (iv) the SH2D1B gene comprising the full-length sequence as set forth in SEQ ID NO: 23 or 24, and (v) an LCK gene comprising the full-length sequence set forth in SEQ ID NO: 25, 26, 27, 28, 29 or 30. 3. The purposes of claim 2, wherein the combination of the reagents further comprises other reagents specific for the following at least 5 target cell genes: (i) a GZMB gene comprising the full-length sequence shown in SEQ ID NO: 14, 15, 16 or 17, (ii) the CD247 gene comprising the full-length sequence as set forth in SEQ ID NO: 18, 19 or 20, (iii) an RRAS2 gene comprising the full-length sequence shown in SEQ ID NO: 21 or 22, (iv) the SH2D1B gene comprising the full-length sequence as set forth in SEQ ID NO: 23 or 24, and (v) an LCK gene comprising the full-length sequence set forth in SEQ ID NO: 25, 26, 27, 28, 29 or 30. 4. The purposes of claim 3, wherein the combination of reagents further comprises other reagents specific for at least one target cell gene selected from the group consisting of: (i) an MRPS6 gene comprising the full-length sequence shown in SEQ ID NO: 31, 32 or 33, (ii) a SPRY4 gene comprising the full-length sequence as shown in SEQ ID NO:34, (iii) a NEAT1 gene comprising the full-length sequence as shown in SEQ ID NO:35, (iv) a CYBB gene comprising the full-length sequence as shown in SEQ ID NO:36, (v) a DUSP2 gene comprising the full-length sequence shown in SEQ ID NO:37, (vi) a PDEAD gene comprising the full-length sequence shown in SEQ ID NO: 38 or 39, (vii) SH2D2A gene comprising the full-length sequence as shown in SEQ ID NO: 40, 41 or 42, (viii) an INSR gene comprising the full-length sequence shown in SEQ ID NO: 43 or 44, (ix) an ITGAM gene comprising the full-length sequence as shown in SEQ ID NO:45, (x) a VCAN gene comprising the full-length sequence shown in SEQ ID NO: 46, 47, 48 or 49, (xi) a CD163 gene comprising the full-length sequence as shown in SEQ ID NO: 50 or 51, (xii) a P2RY10 gene comprising the full-length sequence shown in SEQ ID NO: 52 or 53, (xii) the CD226 gene comprising the full-length sequence as shown in SEQ ID NO:54, (xiii) the MRPL10 gene comprising the full-length sequence shown in SEQ ID NO: 55 or 56, (xiv) the ITPRIPL2 gene comprising the full-length sequence as shown in SEQ ID NO:57, (xv) a CD2 gene comprising the full-length sequence shown in SEQ ID NO:58, and (xvi) NUDT16 gene comprising the full-length sequence as shown in SEQ ID NO:59. 5. The purposes of claim 4, wherein the combination of reagents further comprises other reagents specific for at least 17 target cell genes below: (i) an MRPS6 gene comprising the full-length sequence shown in SEQ ID NO: 31, 32 or 33, (ii) a SPRY4 gene comprising the full-length sequence as shown in SEQ ID NO:34, (iii) a NEAT1 gene comprising the full-length sequence as shown in SEQ ID NO:35, (iv) a CYBB gene comprising the full-length sequence as shown in SEQ ID NO:36, (v) a DUSP2 gene comprising the full-length sequence shown in SEQ ID NO:37, (vi) a PDEAD gene comprising the full-length sequence shown in SEQ ID NO: 38 or 39, (vii) SH2D2A gene comprising the full-length sequence as shown in SEQ ID NO: 40, 41 or 42, (viii) an INSR gene comprising the full-length sequence shown in SEQ ID NO: 43 or 44, (ix) an ITGAM gene comprising the full-length sequence as shown in SEQ ID NO:45, (x) a VCAN gene comprising the full-length sequence shown in SEQ ID NO: 46, 47, 48 or 49, (xi) a CD163 gene comprising the full-length sequence as shown in SEQ ID NO: 50 or 51, (xii) a P2RY10 gene comprising the full-length sequence shown in SEQ ID NO: 52 or 53, (xii) the CD226 gene comprising the full-length sequence as shown in SEQ ID NO:54, (xiii) the MRPL10 gene comprising the full-length sequence shown in SEQ ID NO: 55 or 56, (xiv) the ITPRIPL2 gene comprising the full-length sequence as shown in SEQ ID NO:57, (xv) a CD2 gene comprising the full-length sequence shown in SEQ ID NO:58, and (xvi) NUDT16 gene comprising the full-length sequence as shown in SEQ ID NO:59. 6. The use of any one of claims 1-5, wherein the specific reagent described in step b) comprises at least one hybridization probe. 7. The use of claim 6, wherein the specific reagents described in step b) comprise at least one hybridization probe and at least one primer. 8. The use of claim 7, wherein the specific reagent in step b) comprises at least one hybridization probe and two primers. 9. A kit for prognosing colorectal cancer in a peripheral blood sample from a patient, the kit comprising a combination of reagents specific for at least 5 NK cell genes below, wherein the at least 5 NK cell genes include : (i) a KLRB1 gene comprising the full-length sequence as shown in SEQ ID NO: 1, (ii) a KLRC2 gene comprising the full-length sequence shown in SEQ ID NO: 2, 3 or 4, (iii) a KLRC3 gene comprising the full-length sequence shown in SEQ ID NO: 5, 6 or 7, (iv) a KLRD1 gene comprising the full-length sequence as set forth in SEQ ID NO: 8, 9, 10, 11 or 12, and (v) KLRK1 gene comprising the full-length sequence as shown in SEQ ID NO:13. 10. The test kit of claim 9, wherein the test kit further comprises other reagents specific for at least one target cell gene selected from the group consisting of: (i) a GZMB gene comprising the full-length sequence shown in SEQ ID NO: 14, 15, 16 or 17, (ii) the CD247 gene comprising the full-length sequence as set forth in SEQ ID NO: 18, 19 or 20, (iii) an RRAS2 gene comprising the full-length sequence shown in SEQ ID NO: 21 or 22, (iv) the SH2D1B gene comprising the full-length sequence as set forth in SEQ ID NO: 23 or 24, and (v) an LCK gene comprising the full-length sequence set forth in SEQ ID NO: 25, 26, 27, 28, 29 or 30. 11. The test kit of claim 10, further comprising other reagents specific to the following at least 5 target cell genes in the combination of reagents: (i) a GZMB gene comprising the full-length sequence shown in SEQ ID NO: 14, 15, 16 or 17, (ii) the CD247 gene comprising the full-length sequence as set forth in SEQ ID NO: 18, 19 or 20, (iii) an RRAS2 gene comprising the full-length sequence shown in SEQ ID NO: 21 or 22, (iv) the SH2D1B gene comprising the full-length sequence as set forth in SEQ ID NO: 23 or 24, and (v) an LCK gene comprising the full-length sequence set forth in SEQ ID NO: 25, 26, 27, 28, 29 or 30. 12. The test kit of claim 11, wherein said combination of reagents further comprises other reagents specific for at least one target cell gene selected from the group consisting of: (i) an MRPS6 gene comprising the full-length sequence shown in SEQ ID NO: 31, 32 or 33, (ii) a SPRY4 gene comprising the full-length sequence as shown in SEQ ID NO:34, (iii) a NEAT1 gene comprising the full-length sequence as shown in SEQ ID NO:35, (iv) a CYBB gene comprising the full-length sequence as shown in SEQ ID NO:36, (v) a DUSP2 gene comprising the full-length sequence shown in SEQ ID NO:37, (vi) a PDEAD gene comprising the full-length sequence shown in SEQ ID NO: 38 or 39, (vii) SH2D2A gene comprising the full-length sequence as shown in SEQ ID NO: 40, 41 or 42, (viii) an INSR gene comprising the full-length sequence shown in SEQ ID NO: 43 or 44, (ix) an ITGAM gene comprising the full-length sequence as shown in SEQ ID NO:45, (x) a VCAN gene comprising the full-length sequence shown in SEQ ID NO: 46, 47, 48 or 49, (xi) a CD163 gene comprising the full-length sequence as shown in SEQ ID NO: 50 or 51, (xii) a P2RY10 gene comprising the full-length sequence shown in SEQ ID NO: 52 or 53, (xii) the CD226 gene comprising the full-length sequence as shown in SEQ ID NO:54, (xiii) the MRPL10 gene comprising the full-length sequence shown in SEQ ID NO: 55 or 56, (xiv) the ITPRIPL2 gene comprising the full-length sequence as shown in SEQ ID NO:57, (xv) a CD2 gene comprising the full-length sequence shown in SEQ ID NO:58, and (xvi) NUDT16 gene comprising the full-length sequence as shown in SEQ ID NO:59. 13. The test kit of claim 12, wherein said combination of reagents further comprises other reagents specific for the following at least 17 target cell genes: (i) an MRPS6 gene comprising the full-length sequence shown in SEQ ID NO: 31, 32 or 33, (ii) a SPRY4 gene comprising the full-length sequence as shown in SEQ ID NO:34, (iii) a NEAT1 gene comprising the full-length sequence as shown in SEQ ID NO:35, (iv) a CYBB gene comprising the full-length sequence as shown in SEQ ID NO:36, (v) a DUSP2 gene comprising the full-length sequence shown in SEQ ID NO:37, (vi) a PDEAD gene comprising the full-length sequence shown in SEQ ID NO: 38 or 39, (vii) SH2D2A gene comprising the full-length sequence as shown in SEQ ID NO: 40, 41 or 42, (viii) an INSR gene comprising the full-length sequence shown in SEQ ID NO: 43 or 44, (ix) an ITGAM gene comprising the full-length sequence as shown in SEQ ID NO:45, (x) a VCAN gene comprising the full-length sequence shown in SEQ ID NO: 46, 47, 48 or 49, (xi) a CD163 gene comprising the full-length sequence as shown in SEQ ID NO: 50 or 51, (xii) a P2RY10 gene comprising the full-length sequence shown in SEQ ID NO: 52 or 53, (xii) the CD226 gene comprising the full-length sequence as shown in SEQ ID NO:54, (xiii) the MRPL10 gene comprising the full-length sequence shown in SEQ ID NO: 55 or 56, (xiv) the ITPRIPL2 gene comprising the full-length sequence as shown in SEQ ID NO:57, (xv) a CD2 gene comprising the full-length sequence shown in SEQ ID NO:58, and (xvi) NUDT16 gene comprising the full-length sequence as shown in SEQ ID NO:59.