WO2009080437A1

WO2009080437A1 - Micro-rna based drug resistance analysis method

Info

Publication number: WO2009080437A1
Application number: PCT/EP2008/066239
Authority: WO
Inventors: Lars Kongsbak; Ralf SØKILDE; Thomas Litman; Søren Møller
Original assignee: Exiqon A/S
Priority date: 2007-12-21
Filing date: 2008-11-26
Publication date: 2009-07-02
Also published as: WO2009080437A9

Abstract

The invention relates to the analysis of samples, such as cancer samples, isolated from patients, to determine or predict the phenotype of the patient or disease, such as cancer, with respect to the resistance or susceptibility of the patient or disease to treatment, such as cancer treatment. The analysis is based on determination of the abundance of microRNAs which are associated with resistance against the treatment, such as extreme drug resistance (EDR).

Description

MICRO-RNA BASED DRUG RESISTANCE ANALYSIS METHOD

FIELD OF THE INVENTION

The invention relates to the analysis of samples, such as disease samples, such as cancer samples, isolated from patients to determine or predict the phenotype of the patient or disease, such as cancer, with respect to the resistance or susceptibility of the patient or disease to treatment, such as cancer treatment. The analysis is based on determination of the abundance of microRNAs which are associated with resistance against the treatment, such as extreme drug resistance (EDR).

BACKGROUND TO THE INVENTION

MicroRNAs (miRNAs) have rapidly emerged as an important class of short endogenous RNAs that act as post-transcriptional regulators of gene expression by base-pairing with their target mRNAs.

microRNAs are differentially expressed in human cancers and a series of recent publication show that microRNA classify human cancers; in some cases improvement over mRNA classification is observed. Indeed, in a series of publications during recent years, it has become clear that microRNAs are extensively involved in cancer pathogenesis, and microRNA has been shown to be differentially expressed in a number of cancers (Breast cancer: Iorio et al Cancer Res 2005; 65: 7065. Lung cancer: Yanaihara et al Cell Science 2006; 9: 189-198. Chronic lymphocytic leukaemia (CLL) : GaNn et al PNAS, 2004 101(32) : 11755-11760. Colon cancer: Cummins et al PNAS 2006, 103 (10) :3687-3692. Prostate cancer: Volinia et al PNAS 2006; 103: 2257). Indeed, in a landmark paper Lu et al (Nature 2005; 435:834-838) demonstrated that differential expression of microRNA in multiple cancers types, and that signatures based on approximately 200 microRNAs improve classification of poorly differentiated cancers over mRNA profiles.

The expected complexity of the "microRNA'nome" is far smaller than the human transcriptome with the total number of microRNAs being approximately limited to between 800 and 1000. Therefore, a microRNA cancer signature can be predicted to include from 5 - 20 microRNAs, suggesting that microRNA based theranostics will be of limited complexity and far more robust than mRNA profiles.

PCT/EP2007/061210 and US 11/975,644, both hereby incorporated by reference, report on novel microRNAs which are associated with cancer, and detection probes based on the complement of the novel miRNA sequences, including LNA detection probes, for diagnostic use: Including the use of miRNA profiling for therapy outcome prediction, such as a prediction of the responsiveness of the cancer to chemotherapy and/or radiotherapy and/or the suitability of said cancer to hormone treatment, and such as the suitability of said cancer for removal by invasive surgery. In one embodiment, the therapy out come predication may be the prediction of the suitability of the treatment of the cancer to combined adjuvant therapy. The therapy may be herceptin, which is frequently used for the treatment of oestrogen receptor positive cancers (such as breast cancer).

PCT/DK2005/000838, and US application 11/324,177, both hereby incorporated by reference, discloses methods for the detection of microRNAs (miRNAs) using oligonucleotides which comprise nucleotide analogues, such as locked nucletic acids (LNAs).

WO2005/098029, hereby incorporated by reference, discloses a method using oligonucleotides for the detection, quantification, monitoring of expression of miRNA. It is suggested that the method can be used for determining the differences between nucleic acid samples from e.g. a cancer patient.

The Sanger Institute publishes known miRNA sequences in the miRBase database (http://microrna.sanqer.ac.uk/sequences/index.shtml). To date there are 533 human miRNAs present in the miRBase database.

The Extreme Drug Resistance (EDR®, Oncotech Inc.) assay is an in vitro test that measures the ability of pharmaceutical agents and other chemotherapies to stop cancer cells from dividing and growing - it has been reported that the assay identifies patients that will not respond to a particular cancer treatment with over 99% accuracy and is used to exclude agents unlikely to provide a therapeutic benefit in the treatment of cancer in an individual patient, as well as in providing information which can be used to select those agents which are likely to be clinically effective, resulting in improved response rates and prolonged survival of cancer patients.

The EDR® assay method involves the isolation of fresh viable tumor tissue which is minced and digested with enzymes to disaggregate the tumor cells. The cells are then placed in soft agar to encourage cell proliferation before being exposed to the chemotherapeutic agents, typically for a period of five days and at an elevated dosage, during the latter period of drug exposure tritiated thymidine is added as a measure of cell proliferation. By comparing the level of label incorporated into drug treated and untreated controls, the degree of cell proliferation under the drug treatment is determined, and thereby the resistance phenotype of the cancer cells.

Despite the effectiveness of the EDR® assay in identifying drug resistance tumours, the assay requires a relatively large amount of tumour tissue, and takes about 7 days to perform. There is therefore a need for improved drug resistance assays, which use less cancer tissue and may be performed in a shorter time period.

The correlation of specific gene (mRNA) or protein expression or DNA copy number and the drug resistance phenotype has been used to assay drug resistance of individual tumour samples:

US 2004/0214203 reports on methods for prognosis, diagnosis, staging and disease progression in human cancer patients related to expression of levels of a plurality of genes that are differentially expressed in chemotherapeutic drug resistant and drug sensitive tumour cells.

US 2006/0160114 reports on methods for prognosis, diagnosis, staging and disease progression in human cancer patients related to expression of levels of one or a plurality of genes or genetic loci that are differentially deleted, amplified, expressed or amplified and over-expressed in chemotherapeutic drug resistant tumor cells.

RELATED APPLICATIONS

The following applications are hereby incorporated by reference PCT/EP2007/061210, US 11/975,644, and US provisional applications US 60/853,410 and US 60/900,081.

SUMMARY OF THE INVENTION

The invention is based upon the discovery that microRNA profiling of cancer cells or tissues may be used as an efficient, effective and rapid indicator of a drug resistance or drug susceptibility phenotype of cancer cells.

This discovery illustrates that microRNA profiling may be used as a method of predicting the phenotype of a subject suffering from a disease, or or the phenotype of a sample or cell(s) obtained from said patient, which may, suitably consist or comprise of a sample of diseased tissue or cells, such as a cancer sample (or cell(s)), with respect to the resistance or susceptibility of the subject or sample or cell(s) to one or more treatments of the disease. The invention provides for a method for determining whether a disease or state of disease shows resistance to, or susceptibility to, at least one treatment of said disease, such as administration of at least one therapeutic compound, said method comprising the steps of

a. Isolating or obtaining a sample of tissue from a subject; b. assaying the abundance of at least one microRNA present in said sample,

wherein over or under abundance of said at least one microRNA is correlated to the resistance or susceptibility of the disease or state of disease to said administration of at least one treatment of said disease.

In one preferred embodiment, the disease is cancer, and the at least one treatment is a cancer treatment, such as at least one chemotherapeutic drug. In one embodiment, the sample referred to in step a) is a sample of said cancer.

Therefore, the invention provides for a method for determining whether a cancer shows resistance (or is resistant to) or susceptibility to at least one cancer treatment, such as at least one chemotherapeutic drug, said method comprising the steps of:

a. isolating or obtaining a sample of said cancer from said subject; b. assaying the abundance of at least one microRNA present in said sample;

wherein over or under abundance of said at least one microRNA is correlated to the either resistance or susceptibility of the cancer to said at least one cancer treatment.

Therefore the invention provides for a method for determining whether a cancer shows or is resistant to, or is susceptible to at least one cancer treatment, said method comprising the step of assaying the abundance of at least one microRNA present in said cancer, wherein over or under abundance of said at least one microRNA is correlated to the resistance or susceptibility of the cancer to said at least one cancer treatment.

As the above methods may form part of a prognostic or diagnostic method, the invention provides for a method for the prognosis or diagnosis of at least one disease, such as cancer treatment in a subject suffering from said disease, such as cancer, said method comprising the steps of:

a. isolating or obtaining a sample from said subject; b. assaying the abundance of at least one microRNA present in said sample, wherein over or under abundance of said at least one microRNA is correlated to the resistance or susceptibility of the disease to said at least one disease treatment;

c. based on the results obtained in b. determining either:

a. the likely prognosis for the subject if said at least one disease treatment were to be administered (prognosis).

b. Deciding on the appropriate form of treatment, such as cancer treatment, for the patient.

Suitably the sample isolated or obtained from the subject is or comprises diseased tissue or cells, such as is a cancer sample. The disease is preferably a cell hyperproliferative disease such as cancer.

The invention further provides for the use of one (or more) detection probe(s) which, (independently) comprises a contiguous nucleobases sequence which is complementary to a microRNA sequence (or is capable of specifically hybridising to said microRNA), for the detection of the abundance of the microRNA in a disease sample (e.g. isolated from a patient), in order to determine whether the disease sample (such as the disease present in the patient) has a disease treatment resistant or disease treatment susceptible phenotype, wherein over or under abundance of said at least one microRNA is correlated to the resistance or susceptibility of the disease to at least one disease treatment.

The invention further provides for a method of treatment of a disease, such as cancer, in a subject suffering from said disease, said method comprising the steps of performing the method according to one of the above embodiment to identify one or more disease treatments, such as cancer treatments, which have a positive prognosis in treatment of said disease, and subsequently administering said one or more disease treatments which have a positive prognosis to said subject.

The invention further provides for a method for identifying one or more microRNAs which are indicators of either the resistance or the susceptibility of a disease, such as cancer, to at least one treatment, such as cancer treatment, said method comprising the steps of:

a. isolating a microRNA containing fraction from i) at least one disease sample, such as cancer sample which is identified as showing resistance to said at least one disease treatment; and

ii) at least one disease sample, such as cancer sample which is identified as being sensitive to said at least one disease treatment;

b. comparing the abundance of each member of a population of independent microRNAs in the fractions obtained in i) and ii) of step a) to identify those microRNAs whose abundance is either correlated with the resistant of the disease to the at least one disease, or the susceptibility of the disease to the at least one disease treatment.

In the above method, the method may be performed on a population of samples, such as disease or cancer samples, which may represent a disease or cancer samples isolated from a population of subjects suffering from the disease, such as cancer patients, which, suitably, are (or were) suffering from the disease, such as a particular form of cancer, such as those referred to herein.

In one embodiment, the invention provides a method for identifying one or a plurality of microRNAs having a pattern of expression that is different in a tumor cell sensitive to at least one cancer treatment compared to the expression pattern in a cancer cell resistant to the at least one cancer treatment, the method comprising the steps of:

a) obtaining a population of cancer samples which comprises individual members (independent cancer samples) which exhibit a) resistance, such as extreme resistance and/or intermediate resistance, to said at least one cancer treatment, and b) susceptibility to said at least one cancer treatment

b) assaying microRNA expression in each of the individual members of the population of cancer samples populations of cancer treatment sensitive and cancer treatment resistant cells; and

c) identifying at least one microRNA having an expression pattern that is different in the cancer treatment resistant cells than in the cancer treatment sensitive cells. FIGURES

Figure 1 provides a list of cancer types and the therapeutic agents which are commonly used to treat the cancer type which are typically amenable to the EDR® assay, and therefore may be suitable cancer types and cancer treatments according to the present invention.

Figure 2 is a diagram representing the steps involved in performing the classical EDR® assay.

Figure 3 shows the frequency of single and multiple drug resistance (EDR) of various colon cancer samples.

Figure 4 shows a Venn diagram illustrating the correlation of specific miRNAs with the EDR status of these chemotherapeutic agents in various colon cancer samples.

Figure 5 shows the frequency of single and multiple LDR status of various colon cancer samples.

Figure 6 shows a summary of the groups of EDR, LDR and IDR status of the colon samples.

Figure 7 shows LDA plot based on the 40 most significant miRs (p<0.05) after standard deviation (SD) filtering showing near-perfect separation between EDR to irinotecan (red/light grey) and EDR to Oxaliplatin (blue/dark grey) for formalin fixed, paraffin embedded (FFPE) specimens.

Figure 8 shows unsupervised hierarchical clustering based on the 33 miRNAs that vary most between EDR-Oxaliplatin and EDR-Irinotecan samples (p<0.05, logR>0.2) for FFPE specimens.

Figure 9 shows LDA plot based on the 37 most significant miRNAs (p<0.05) after SD filtering. Perfect separation between EDR to irinotecan (red/light grey) and EDR to 5-FU (blue/dark grey) for FFPE specimens.

Figure 10 shows unsupervised hierarchical clustering based on the 34 miRNAs that vary most between EDR-5-FU and EDR-Irinotecan samples (p<0.05, logR>0.2) for FFPE specimens. Figure 11 shows LDA plot based on the 33 most significant miRs (p<0.01) after SD filtering. Perfect separation between EDR to Oxaliplatin (red/light grey) and EDR to 5-FU (blue/dark grey) for FFPE specimens.

Figure 12 shows unsupervised hierarchical clustering based on the 33 miRNAs that vary most between EDR-5-FU and EDR-Oxalilpatin samples (p<0.01) for FFPE specimens.

Figure 13 shows LDA plot based on the 28 most significant miRNAs (p<0.05). Perfect separation between EDR to irinotecan (red/light grey) and EDR to Oxaliplatin (blue/dark grey) for fresh frozen specimens.

Figure 14 shows unsupervised hierarchical clustering based on the 28 miRNAs that vary most between EDR-Oxaliplatin and EDR-Irinotecan samples (p<0.05) for fresh frozen specimens.

DESCRIPTION OF INVENTION

Definitions

In the present context "ligand" means something, which binds. Ligands may comprise biotin and functional groups such as: aromatic groups (such as benzene, pyridine, naphtalene, anthracene, and phenanthrene), heteroaromatic groups (such as thiophene, furan, tetrahydrofuran, pyridine, dioxane, and pyrimidine), carboxylic acids, carboxylic acid esters, carboxylic acid halides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulphides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, Ci-C_2O alkyl groups optionally interrupted or terminated with one or more heteroatoms such as oxygen atoms, nitrogen atoms, and/or sulphur atoms, optionally containing aromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as poly-β-alanine, polyglycine, polylysine, peptides, oligo/polysaccharides, oligo/polyphosphates, toxins, antibiotics, cell poisons, and steroids, and also "affinity ligands", i.e. functional groups or biomolecules that have a specific affinity for sites on particular proteins, antibodies, poly- and oligosaccharides, and other biomolecules. The singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. The term "a nucleic acid molecule" includes a plurality of nucleic acid molecules.

Sample" refers to a sample of cells, or tissue or fluid isolated from an organism or organisms, including but not limited to, for example, skin, plasma, serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, blood cells, organs, tumors, and also to samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, recombinant cells and cell components).

The terms "Detection probes" or "detection probe" or "detection probe sequence" refer to an oligonucleotide or oligonucleotide analogue, which oligonucleotide or oligonucleotide analogue comprises a recognition sequence complementary to a nucleotide target, such as an RNA (or DNA) target sequence. It is preferable that the detection probe(s) are oligonucleotides, preferably where said recognition sequence is substituted with high-affinity nucleotide analogues, e.g. LNA, to increase the sensitivity and specificity of conventional oligonucleotides, such as DNA oligonucleotides, for hybridization to short target sequences, e.g. mature miRNAs, stem-loop precursor miRNAs.

The terms "miRNA" and "microRNA" refer to about 18-25 nt non-coding RNAs derived from endogenous genes. They are processed from longer (ca 75 nt) hairpin-like precursors termed pre-miRNAs. MicroRNAs assemble in complexes termed miRNPs and recognize their targets by antisense complementarity. If the microRNAs match 100% their target, i.e. the complementarity is complete, the target mRNA is cleaved, and the miRNA acts like a siRNA. If the match is incomplete, i.e. the complementarity is partial, then the translation of the target mRNA is blocked.

The terms "microRNA precursor" or "miRNA precursor" or "pre-miRNA" or "premature miRNA" refer to polynucleotide sequences (approximately 70 nucleotides in length) that form hairpin- like structures having a loop region and a stem region. The stem region includes a duplex cre-ated by the pairing of opposite ends of the pre-miRNA polynucleotide sequence. The loop region connects the two halves of the stem region. The pre-miRNAs are transcribed as mono- or poly-cistronic, long, primary precursor transcripts (pri-miRNAs) that are then cleaved into individual pre-miRNAs by a nuclear RNAse Ill-like enzyme. Subsequently pre-miRNA hairpins are exported to the cytoplasm where they are processed by a second RNAse Ill-like enzyme into miRNAs. The target nucleic acid may be present in a premature miRNA sequence. The fragments from the opposing arm, called the miRNA* (or "miRNA-star") sequences (Lau et al, Science (2001) 294:858-862) are found in libraries of cloned miRNAs but typically at much lower frequency than are the miRNAs. For example, in an effort that identified over 3400 clones representing 80 C. elegans miRNAs, only 38 clones representing 14 miRNAs* were found. This approximately 100-fold difference in cloning frequency indicates that the miRNA:ιτιiRNA* duplex is generally short lived compared to the miRNA single strand (Bartel et al, Cell (2004) 116:281-297). The target nucleic acid may be present in a miRNA* sequence.

The "miRNA precursor loop sequence" or "loop sequence of the miRNA precursor" or "loop region" of an miRNA precursor is the portion of an miRNA precursor that is not present in the stem region and that is not retained in the mature miRNA (or its complement) upon cleavage by a RNAse Ill-like enzyme.

The "miRNA precursor stem sequence" or "stem sequence of the miRNA precursor" or "stem region" of an miRNA precursor is the portion of an miRNA precursor created by the pairing of opposite ends of the pre-miRNA polynucleotide sequence, and including the portion of the miRNA precursor that will be retained in the "mature miRNA."

The term "Recognition sequence" refers to a nucleotide sequence that is complementary to a region within the target nucleotide sequence essential for sequence-specific hybridization between the target nucleotide sequence and the recognition sequence. Typically the recognition seqeunce comprises or consists of a contiguous nucleobase sequence which corresponds to a contiguous nucleotide seqeunce present in the miRNA target.

The terms "corresponding to" and "corresponds to" refer to the comparison between the nucleobase sequence of the compound of the invention, and the equivalent nucleotide sequence or the reverse complement thereof. Nucleobases sequences which "correspond to" a miRNA target, therefore have between 8 and 30 contiguous nucleobases which form a sequence which is found with i) either the one or more of the miRNA(s), or ii) the reverse complement thereof. Nucleotide analogues are compared directly to their equivalent or corresponding natural nucleotides. Sequences which form the reverse complement of a target miRNA are referred to as the complement sequence of the miRNA. In a preferable embodiment, the term complementary refers to fully or perfectly complementary. In such an embodiment, "corresponding" means identical to or complementary to the designated nucleotide or nucleobase sequence. A corresponding or complementary oligonucleotide or detection probe referred to herein, is not necessarily physically derived from any existing or natural sequence but may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription or a combination thereof. The term 'natural allelic variants' and the term 'allelic variants' encompasses both variants which although have a slightly different sequence (such as a homologue, fragment or variant), originate from the same chromosomal position, or the same position on an allelic chromosome, as the non-coding RNAs, and precursors thereof herein listed. The term 'natural allelic variants' and the term 'allelic variants' also encompasses mature non-coding RNAs encompasses, which may be differentially processed by the processing enzymes, as this may lead to variants of the same microRNAs having different lengths e.g. shortened by 1 or 2 nucleotides, despite originating from the same allelic chromosome position.

The term "label" as used herein refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetric, X- ray diffraction or absorption, magnetism, enzymatic activity, and the like.

As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" refer to primers, probes, oligomer fragments to be detected, oligomer controls and unlabeled blocking oligomers and shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D- ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide which is an N glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases, and in one embodiment, nucleobases (a collective term used to describe both nucleotides and nucleotide analogues, such as LNA). There is no intended distinction in length between the term "nucleic acid", "polynucleotide" and "oligonucleotide", and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single stranded RNA. The oligonucleotide is comprised of a sequence of approximately at least 3 nucleotides, preferably at least about 6 nucleotides, and more preferably at least about 8 - 30 nucleotides corresponding to a region of the designated target nucleotide sequence.

The terms "oligonucleotide" or "nucleic acid" intend a polynucleotide of genomic DNA or RNA, cDNA, semi synthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature; and (3) is not found in nature. Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5'-phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbour in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have a 5' and 3' ends. When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, the 3' end of one oligonucleotide points toward the 5' end of the other; the former may be called the "upstream" oligonucleotide and the latter the "downstream" oligonucleotide.

By the term "SBC nucleobases" is meant "Selective Binding Complementary" nucleobases, i.e. modified nucleobases that can make stable hydrogen bonds to their complementary nucleobases, but are unable to make stable hydrogen bonds to other SBC nucleobases. As an example, the SBC nucleobase A', can make a stable hydrogen bonded pair with its complementary unmodified nucleobase, T. Likewise, the SBC nucleobase T' can make a stable hydrogen bonded pair with its complementary unmodified nucleobase, A. However, the SBC nucleobases A' and T' will form an unstable hydrogen bonded pair as compared to the base pairs A'-T and A-T'. Likewise, a SBC nucleobase of C is designated C and can make a stable hydrogen bonded pair with its complementary unmodified nucleobase G, and a SBC nucleobase of G is designated G' and can make a stable hydrogen bonded pair with its complementary unmodified nucleobase C, yet C and G' will form an unstable hydrogen bonded pair as compared to the base pairs C-G and C-G'. A stable hydrogen bonded pair is obtained when 2 or more hydrogen bonds are formed e.g. the pair between A' and T, A and T', C and G', and C and G. An unstable hydrogen bonded pair is obtained when 1 or no hydrogen bonds is formed e.g. the pair between A' and T', and C and G'. Especially interesting SBC nucleobases are 2,6-diaminopurine (A', also called D) together with 2-thio- uracil (U', also called ^2SU)(2-thio-4-oxo-pyrimidine) and 2-thio-thymine (T', also called ^2ST)(2- thio-4-oxo-5-methyl-pyrimidine). Figure 4 in PCT Publication No. WO 2004/024314 illustrates that the pairs A-^2ST and D-T have 2 or more than 2 hydrogen bonds whereas the D-^2ST pair forms a single (unstable) hydrogen bond. Likewise the SBC nucleobases pyrrolo-[2,3- d]pyrimidine-2(3H)-one (C, also called PyrroloPyr) and hypoxanthine (G', also called I)(6- oxo-purine) are shown in Figure 4 in PCT Publication No. WO 2004/024314 where the pairs PyrroloPyr-G and C-I have 2 hydrogen bonds each whereas the PyrroloPyr-I pair forms a single hydrogen bond.

"SBC LNA oligomer" refers to a "LNA oligomer" containing at least one LNA monomer where the nucleobase is a "SBC nucleobase". By "LNA monomer with an SBC nucleobase" is meant a "SBC LNA monomer". Generally speaking SBC LNA oligomers include oligomers that besides the SBC LNA monomer(s) contain other modified or naturally occurring nucleotides or nucleosides. By "SBC monomer" is meant a non-LNA monomer with a SBC nucleobase. By "isosequential oligonucleotide" is meant an oligonucleotide with the same sequence in a Watson-Crick sense as the corresponding modified oligonucleotide e.g. the sequences agTtcATg is equal to agTscD^2SUg where s is equal to the SBC DNA monomer 2-thio-t or 2- thio-u, D is equal to the SBC LNA monomer LNA-D and ^2SU is equal to the SBC LNA monomer LNA ^2SU.

The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in "antiparallel association." Bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention include, for example, inosine and 7-deazaguanine. Complementarity may not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, percent concentration of cytosine and guanine bases in the oligonucleotide, ionic strength, and incidence of mismatched base pairs.

Stability of a nucleic acid duplex is measured by the melting temperature, or "T_m". The T_m of a particular nucleic acid duplex under specified conditions is the temperature at which half of the duplexes have disassociated.

The term "internal reference marker", refers to a genetic sequence, such as a DNA or RNA sequence, whose abundance does not differ significantly across samples, such as between a diseased and a comparative non-disease cell. The internal reference marker in one embodiment may be a non-coding RNA, or a mRNA. Typically, house keeping genes or their respective RNA species, which show constitutive expression in most cell types, such as in the cell types where the cancer sample was isolated or obtained, are selected as internal reference markers.

The term "nucleobase" covers the naturally occurring nucleobases adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) as well as non-naturally occurring nucleobases such as xanthine, diaminopurine, 8-oxo-N⁵-methyladenine, 7-deazaxanthine, 7-deazaguanine,

N⁴,N⁴-ethanocytosin, N⁵,N⁵-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C³-C⁵)-alkynyl- cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4- triazolopyridin, isocytosine, isoguanine, inosine and the "non-naturally occurring" nucleobases described in Benner et al., U.S. Patent No. 5,432,272 and Susan M. Freier and Karl-Heinz Altmann, Nucleic Acid Research, 25: 4429-4443, 1997. The term "nucleobase" thus includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Further naturally and non naturally occurring nucleobases include those disclosed in U.S. Patent No. 3,687,808; in chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993; in Englisch, et al., Angewandte Chemie, International Edition, 30: 613-722, 1991 (see, especially pages 622 and 623, and in the Concise Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed., John Wiley & Sons, pages 858-859, 1990, Cook, Anti-Cancer DrugDesign 6: 585-607, 1991, each of which are hereby incorporated by reference in their entirety).

The term "nucleoside base" or "nucleobase analogue" is further intended to include heterocyclic compounds that can serve as like nucleosidic bases including certain "universal bases" that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases. Especially mentioned as a universal base is 3-nitropyrrole or a 5-nitroindole. Other preferred compounds include pyrene and pyridyloxazole derivatives, pyrenyl, pyrenylmethylglycerol derivatives and the like. Other preferred universal bases include, pyrrole, diazole or triazole derivatives, including those universal bases known in the art.

By "oligonucleotide," "oligomer," or "oligo" is meant a successive chain of monomers (e.g., glycosides of heterocyclic bases) connected via internucleoside linkages. The linkage between two successive monomers in the oligo consist of 2 to 4, desirably 3, groups/atoms selected from -CH₂-, -O-, -S-, -NR^H-, >C=O, >C=NR^H, >C=S, -Si(R")₂-, -SO-, -S(O)₂-, -P(O)₂-, -PO(BH₃)-, -P(O,S)-, -P(S)₂-, -PO(R")-, -PO(OCH₃)-, and -PO(NHR^H)-, where R^H is selected from hydrogen and Ci-₄-alkyl, and R" is selected from Ci-₆-alkyl and phenyl. Illustrative examples of such linkages are -CH₂-CH₂-CH₂-_, -CH₂-CO-CH₂-_, -CH₂-CHOH-CH₂-_, -0-CH₂-O-, -0-CH₂-CH₂-, -0-CH₂-CH= (including R⁵ when used as a linkage to a succeeding monomer), -CH₂-CH₂-O-, -NR^H-CH₂-CH₂-, -CH₂-CH₂-NR^H-, -CH₂-NR^H-CH₂-, -O-CH₂-CH₂-NR^H-, -NR^H-CO-O-, -NR^H-CO-NR^H-, -NR^H-CS-NR^H-, -NR^H-C( = NR^H)-NR^H-, -NR^H-CO-CH₂-NR^H-, -0-C0- 0-, -0-CO-CH₂-O-, -0-CH₂-CO-O-, -CH₂-CO-NR^H-, -O-CO-NR^H-, -NR^H-CO-CH₂-, -0-CH₂-CO- NR^H-, -O-CH₂-CH₂-NR^H-, -CH = N-O-, -CH₂-NR^H-O-, -CH₂-O-N= (including R⁵ when used as a linkage to a succeeding monomer), -CH₂-O-NR^H-, -CO-NR^H-CH₂-, -CH₂-NR^H-O-, -CH₂-NR^H-CO- , -O-NR^H-CH₂-, -O-NR^H-, -0-CH₂-S-, -S-CH₂-O-, -CH₂-CH₂-S-, -0-CH₂-CH₂-S-, -S-CH₂-CH = (including R⁵ when used as a linkage to a succeeding monomer), -S-CH₂-CH₂-, -S-CH₂-CH₂-O- , -S-CH₂-CH₂-S-, -CH₂-S-CH₂-, -CH₂-SO-CH₂-, -CH₂-SO₂-CH₂-, -0-S0-0-, -0-S(O)₂-O-, -O- S(O)₂-CH₂-, -O-S(O)₂-NR^H-, -NR^H-S(O)₂-CH₂-, -0-S(O)₂-CH₂-, -0-P(O)₂-O-, -O-P(O,S)-O-, -O- P(S)₂-O-, -S-P(O)₂-O-, -S-P(O,S)-O-, -S-P(S)₂-O-, -0-P(O)₂-S-, -O-P(O,S)-S-, -0-P(S)₂-S-, -S-P(O)₂-S-, -S-P(CS)-S-, -S-P(S)₂-S-, -O-PO(R")-O-, -O-PO(OCH₃)-O-, -O-PO(OCH₂CH₃)-O- , -0-PO(OCH₂CH₂S-R)-O-, -O-PO(BH₃)-O-, -O-PO(NHR^N)-O-, -O-P(O)₂-NR^H-, -NR^H-P(O)₂-O-, - O-P(O,NR^H)-O-, -CH₂-P(O)₂-O-, -0-P(O)₂-CH₂-, and -O-Si(R")₂-O-; among which -CH₂-CO- NR^H-, -CH₂-NR^H-O-, -S-CH₂-O-, -0-P(O)₂-O-, -0-P(CS)-O-, -0-P(S)₂-O-, -NR^H-P(O)₂-O-, -O- P(O,NR^H)-O-, -O-PO(R")-O-, -O-PO(CH₃)-O-, and -O-PO(NHR^N)-O-, where R^H is selected form hydrogen and Ci-₄-alkyl, and R" is selected from Ci-₆-alkyl and phenyl, are especially desirable. Further illustrative examples are given in Mesmaeker et. al., Current Opinion in Structural Biology 1995, 5, 343-355 and Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol 25, pp 4429-4443. The left-hand side of the internucleoside linkage is bound to the 5-membered ring as substituent P^* at the 3'-position, whereas the right-hand side is bound to the 5'-position of a preceding monomer.

By "LNA" or "LNA monomer" (e.g., an LNA nucleoside or LNA nucleotide) or an LNA oligomer (e.g., an oligonucleotide or nucleic acid) is meant a nucleoside or nucleotide analogue that includes at least one LNA monomer. LNA monomers as disclosed in PCT Publication WO

99/14226 are in general particularly desirable modified nucleic acids for incorporation into an oligonucleotide of the invention. Additionally, the nucleic acids may be modified at either the 3' and/or 5' end by any type of modification known in the art. For example, either or both ends may be capped with a protecting group, attached to a flexible linking group, attached to a reactive group to aid in attachment to the substrate surface, etc. Desirable LNA monomers and their method of synthesis also are disclosed in US 6,043,060, US 6,268,490, PCT Publications WO 01/07455, WO 01/00641, WO 98/39352, WO 00/56746, WO 00/56748 and WO 00/66604 as well as in the following papers: Morita et al., Bioorg. Med. Chem. Lett. 12(l) :73-76, 2002; Hakansson et al., Bioorg. Med. Chem. Lett. ll(7) :935-938, 2001; Koshkin et al., J. Org. Chem. 66(25) :8504-8512, 2001; Kvaerno et al., J. Org. Chem. 66(16) :5498-5503, 2001; Hakansson et al., J. Org. Chem. 65(17) :5161-5166, 2000; Kvaerno et al., J. Org. Chem. 65(17) :5167-5176, 2000; Pfundheller et al., Nucleosides Nucleotides 18(9) :2017-2030, 1999; and Kumar et al., Bioorg. Med. Chem. Lett. 8(16) :2219-2222, 1998.

Preferred LNA monomers, also referred to as "oxy-LNA" are LNA monomers which include bicyclic compounds as disclosed in PCT Publication WO 03/020739 wherein the bridge between R⁴ and R² as shown in formula (I) below together designate -CH₂-O- or -CH₂-CH₂-O-.

By "LNA modified oligonucleotide" or "LNA substituted oligonucleotide" is meant a oligonucleotide comprising at least one LNA monomer of formula (I), described infra, having the below described illustrative examples of modifications:

wherein X is selected from -O-, -S-, -N(R^N)-, -C(R⁵R^5*)-, -0-C(R⁷R^7*)-, -C(R⁵R^5*)-O-, -S- C(R⁷R^7*)-, -C(R⁵R^5*)-S-, -N(R^N*)-C(R⁷R^7*)-, -C(R⁵R^5*)-N(R^N*)-, and -C(R⁵R^5*)-C(R⁷R^7*). B is selected from a modified base as discussed above e.g. an optionally substituted carbocyclic aryl such as optionally substituted pyrene or optionally substituted pyrenylmethylglycerol, or an optionally substituted heteroalicylic or optionally substituted heteroaromatic such as optionally substituted pyridyloxazole, optionally substituted pyrrole, optionally substituted diazole or optionally substituted triazole moieties; hydrogen, hydroxy, optionally substituted Ci-₄-alkoxy, optionally substituted Ci-₄-alkyl, optionally substituted Ci_-4- acyloxy, nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.

P designates the radical position for an internucleoside linkage to a succeeding monomer, or a 5'-terminal group, such internucleoside linkage or 5'-terminal group optionally including the substituent R⁵. One of the substituents R², R^2*, R³, and R^3* is a group P* which designates an internucleoside linkage to a preceding monomer, or a 2'/3'-terminal group. The substituents of R^1*, R^4*, R⁵, R^5*, R⁵, R^5*, R⁷, R^7*, R^N, and the ones of R², R^2*, R³, and R^3* not designating P^* each designates a biradical comprising about 1-8 groups/atoms selected from -C(R^aR^b)-, - C(R^a)=C(R^a)-, -C(R^a) = N-, -C(R^a)-O, -O-, -Si(R^a)₂-, -C(R^a)-S, -S-, -SO₂-, -C(R^a)-N(R^b)-, - N(R^a)-, and >C=Q, wherein Q is selected from -O-, -S-, and -N(R^a)-, and R^a and R^b each is independently selected from hydrogen, optionally substituted Ci-i₂-alkyl, optionally substituted C₂_i₂-alkenyl, optionally substituted C₂_i₂-alkynyl, hydroxy, Ci_i₂-alkoxy, C_2-I2- alkenyloxy, carboxy, Ci_i₂-alkoxycarbonyl, Ci_i₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, hetero-aryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci-₆-alkyl)amino, carbamoyl, mono- and di(Ci-₆-alkyl)-amino-carbonyl, amino-Ci-₆-alkyl-aminocarbonyl, mono- and di(Ci-₆-alkyl)amino-Ci-₆-alkyl-aminocarbonyl, Ci-₆-alkyl-carbonylamino, carbamido, Ci-₆-alkanoyloxy, sulphono, Ci-₆-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci-₆-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents R^a and R^b together may designate optionally substituted methylene (=CH₂), and wherein two non-geminal or geminal substituents selected from R^a, R^b, and any of the substituents R^1*, R², R^2*, R³, R^3*, R^4*, R⁵, R^5*, R⁵ and R^5*, R⁷, and R^7* which are present and not involved in P, P^* or the biradical(s) together may form an associated biradical selected from biradicals of the same kind as defined before; the pair(s) of non-geminal substituents thereby forming a mono- or bicyclic entity together with (i) the atoms to which said non-geminal substituents are bound and (ii) any intervening atoms.

Each of the substituents R^1*, R², R^2*, R³, R^4*, R⁵, R^5*, R⁵ and R^5*, R⁷, and R^7* which are present and not involved in P, P^* or the biradical(s), is independently selected from hydrogen, optionally substituted Ci_i₂-alkyl, optionally substituted C₂_i₂-alkenyl, optionally substituted C₂-i₂-alkynyl, hydroxy, Ci_i₂-alkoxy, C₂_i₂-alkenyloxy, carboxy, Ci_i₂-alkoxycarbonyl, Ci_-I2- alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di-(Ci-₆- alkyl)amino, carbamoyl, mono- and di(Ci-₆-alkyl)-amino-carbonyl, amino-Ci-₆-alkyl- aminocarbonyl, mono- and di(Ci-₆-alkyl)amino-Ci-₆-alkyl-aminocarbonyl, Ci-₆-alkyl- carbonylamino, carbamido, Ci-₆-alkanoyloxy, sulphono, Ci-₆-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci-₆-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene, or together may form a spiro biradical consisting of a 1-5 carbon atom(s) alkylene chain which is optionally interrupted and/or terminated by one or more heteroatoms/groups selected from -O-, -S-, and -(NR^N)- where R^N is selected from hydrogen and Ci-₄-alkyl, and where two adjacent (non- geminal) substituents may designate an additional bond resulting in a double bond; and R^N*, when present and not involved in a biradical, is selected from hydrogen and Ci-₄-alkyl; and basic salts and acid addition salts thereof.

Exemplary 5', 3', and/or 2' terminal groups include -H, -OH, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., phenyl or benzyl), alkyl (e.g., methyl or ethyl), alkoxy (e.g., methoxy), acyl (e.g. acetyl or benzoyl), aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamino, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, amidino, amino, carbamoyl, sulfamoyl, alkene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trityl(triphenylmethyl)), linkers (e.g., a linker containing an amine, ethylene glycol, quinone such as anthraquinone), detectable labels (e.g., radiolabels or fluorescent labels), and biotin.

It is understood that references herein to a nucleic acid unit, nucleic acid residue, LNA monomer, or similar term are inclusive of both individual nucleoside units and nucleotide units and nucleoside units and nucleotide units within an oligonucleotide.

A "modified base" or other similar terms refer to a composition (e.g., a non-naturally occurring nucleobase or nucleosidic base), which can pair with a natural base (e.g., adenine, guanine, cytosine, uracil, and/or thymine) and/or can pair with a non-naturally occurring nucleobase or nucleosidic base. Desirably, the modified base provides a T_m differential of 15, 12, 10, 8, 6, 4, or 2⁰C or less as described herein. Exemplary modified bases are described in EP 1 072 679 and WO 97/12896. The term "chemical moiety" refers to a part of a molecule. "Modified by a chemical moiety" thus refer to a modification of the standard molecular structure by inclusion of an unusual chemical structure. The attachment of said structure can be covalent or non-covalent.

The term "inclusion of a chemical moiety" in an oligonucleotide probe thus refers to attachment of a molecular structure. Such as chemical moiety include but are not limited to covalently and/or non-covalently bound minor groove binders (MGB) and/or intercalating nucleic acids (INA) selected from a group consisting of asymmetric cyanine dyes, DAPI, SYBR Green I, SYBR Green II, SYBR Gold, PicoGreen, thiazole orange, Hoechst 33342, Ethidium Bromide, l-O-(l-pyrenylmethyl)glycerol and Hoechst 33258. Other chemical moieties include the modified nucleobases, nucleosidic bases or LNA modified oligonucleotides.

"Oligonucleotide analogue" refers to a nucleic acid binding molecule capable of recognizing a particular target nucleotide sequence. A particular oligonucleotide analogue is peptide nucleic acid (PNA) in which the sugar phosphate backbone of an oligonucleotide is replaced by a protein like backbone. In PNA, nucleobases are attached to the uncharged polyamide backbone yielding a chimeric pseudopeptide-nucleic acid structure, which is homomorphous to nucleic acid forms.

"High affinity nucleotide analogue" or "affinity-enhancing nucleotide analogue" refers to a non-naturally occurring nucleotide analogue that increases the "binding affinity" of an oligonucleotide probe to its complementary recognition sequence when substituted with at least one such high-affinity nucleotide analogue.

As used herein, a probe with an increased "binding affinity" for a recognition sequence compared to a probe which comprises the same sequence but does not comprise a stabilizing nucleotide, refers to a probe for which the association constant (K₃) of the probe recognition segment is higher than the association constant of the complementary strands of a double- stranded molecule. In another preferred embodiment, the association constant of the probe recognition segment is higher than the dissociation constant (K_d) of the complementary strand of the recognition sequence in the target sequence in a double stranded molecule.

Monomers are referred to as being "complementary" if they contain nucleobases that can form hydrogen bonds according to Watson-Crick base-pairing rules (e.g. G with C, A with T or A with U) or other hydrogen bonding motifs such as for example diaminopurine with T, 5- methyl C with G, 2-thiothymidine with A, inosine with C, pseudoisocytosine with G, etc. The term "succeeding monomer" relates to the neighbouring monomer in the 5'-terminal direction and the "preceding monomer" relates to the neighbouring monomer in the 3'- terminal direction.

Suitably, the "target" or "target nucleic acid" or "target ribonucleic acid" refers to any relevant nucleic acid of a single specific sequence, e. g., a biological nucleic acid, e. g., derived from a subject or human being. Within the context of the oligonucleotides and detection probes used in the invention to detect miRNAs, the "target" is a human miRNA or precursor thereof, or in one embodiment, a molecule which retains the genetic sequence information contained therein - such as all or (a sufficient) part of the seqeunce of nucleobases or reverse complement thereof.

"Target sequence" refers to a specific nucleic acid sequence (or corresponding nuceltobase seqeunce) within any target nucleic acid.

The term "stringent conditions", as used herein, is the "stringency" which occurs within a range from about T_m-5° C. (5° C. below the melting temperature (T_m) of the probe) to about 20° C. to 25° C. below T_m. As will be understood by those skilled in the art, the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences. Hybridization techniques are generally described in Nucleic Acid Hybridization, A Practical Approach, Ed. Hames, B. D. and Higgins, S. J., IRL Press, 1985; Gall and Pardue, Proc. Natl. Acad. ScL, USA 63: 378-383, 1969; and John, et al. Nature 223: 582-587, 1969.

In one embodiment the term "specifically hybridise" is determined by whether the oligonucleotide or compound of the invention hybridises to the target nucleic acid sequence under stringent conditions.

The terms "contiguous sequence of nucleobases" and "contiguous nucleobase sequence" are used interchangeably.

Whilst it is recognised that the methods and uses according to the present invention may be used for determining whether a disease or state of disease shows resistance to or susceptibility to a treatment, the preferred embodiment refers to the disease being cancer. In the specific embodiments which refer to cancer, herein, where appropriate the embodiments may also be used to refer to a disease or disease state in general.

Determining cancer resistance/susceptibility phenotypes in vitro The invention provides for a method for determining whether a cancer, or a sample thereof, or a cancer cell or a population of cancer cells, shows resistance (or is resistant to) or susceptibility to at least one cancer treatment, such as at least one chemotherapeutic drug, said method comprising the steps of:

a. isolating or obtaining a sample of said cancer or a sample thereof, or a cancer cell or a population of cancer cells, such as from said subject; b. assaying the abundance of at least one microRNA present in said sample;

wherein over or under abundance of said at least one microRNA is correlated to the either resistance or susceptibility of the cancer, or the sample thereof, or the cancer cell or the population of cancer cells, to said at least one cancer treatment.

The method for determining whether a cancer (or cancer sample) shows resistance or is susceptible to at least one cancer treatment, according to the invention, is suitably an in vitro method, although, in one optional embodiment, it may comprise the step of obtaining the cancer sample from the subject.

In this regard the determination of the cancer (or cancer sample) resistance or susceptibility to the cancer treatment is an in vitro determination, which may, subsequent to the method be used to predict whether a given form of cancer treatment may be effective in treating the cancer in the subject, and therefore to assist the medical practitioner in selecting an appropriate form of cancer treatment for the subject.

As has been reported with respect to the EDR® assay, it is recognised that the determination of a cancer's resistance or susceptibility phenotype with respect to a cancer treatment may not be a 100% accurate determination of the in vivo performance of the cancer treatment. In this respect, the EDR® assay was highly effective for determining a negative predictive value was above 99% (Kern and Weisenthal, 1990 - J. Nat. Cancer. Inst. 82: 582 - 588) - i.e. over 99% of the tumours which were found to be resistant to treatment in the EDR® in vitro assay, were also found to be resistant in vivo. The accuracy of the positive predictive value was found to be 52% - which was significantly higher than the overall response of 29%.

The present inventors have found that the profile of the (relative) abundance of independent microRNAs in a cancer sample can be used to predict the likely performance of cancer cells derived from that sample in an EDR® assay, and as such, assaying the (relative) abundance of miRNAs (i.e. miRNA profiling) in the cancer sample provides an assay that requires considerably less tissue, and an efficient and quick assay for determining the in vitro resistance (negative predictive value) or susceptibility (positive predictive value) phenotype of the cancer sample.

Negative predictive value (NPV): The EDR assay will be high in the discrimination of drugs that will not work - higher drug concentration than in vivo

Positive predictive value (PPV): The EDR assay will have medium value in finding drugs that work as in vitro sensitivity do not always translate into in vivo sensitivity as other factors play a role.

Therefore in one aspect the determination is a determination of a predictive value of the effect of the cancer treatment on the cancer, or a sample thereof, or a cancer cell or a population of cancer cells.

Therefore in one aspect the determination is a predictive determination of a cancer treatment resistant, or cancer treatment susceptible phenotype, such as the EDR, IDR and LDR phenotypes referred to herein.

In one embodiment, the abundance of the at least one microRNA is a relative abundance of the microRNA present in said sample as compared to

i) the abundance of the at least one microRNA present in at least one further sample, such as at least one further cancer sample or a population of such further samples, with a known treatment resistance phenotype, such as known cancer resistance phenotype; and/or

ii) or an internal control, such as the internal reference markers referred to herein; and/or

iii) and/or the abundance of a genetic marker associated with the disease, suitably the mRNA or protein level of such a genetic marker.

In one embodiment, the abundance of the at least one microRNA is a relative abundance of the microRNA present in said cancer sample as compared to the abundance of the at least one microRNA present in at least one further cancer sample, or a population of cancer samples, with a known cancer treatment resistance phenotype. In one embodiment the at least one further (e.g. cancer) sample has a characterised drug resistance phenotype, such as extreme (EDR) or intermediate drug resistance (IDR) phenotype with respect to said at least one (e.g. cancer) treatment.

In one embodiment the relative abundance of the at least one microRNA is compared to the abundance of the at least one microRNA present in at least one further (cancer) sample with a drug susceptibility phenotype, such as a low drug resistance (LDR) phenotype with respect to said at least one (cancer) treatment.

In one embodiment the abundance of more than one microRNA correlated to the resistance of the disease or cancer to said at least one disease or cancer treatment is assessed.

In one embodiment, the resistance to more than one (e.g. cancer) treatment is determined.

In one embodiment, the present invention provides a method for identifying microRNAs (and microRNA expression patterns) that are predictive of the clinical effectiveness of drug treatment therapies, such as anticancer drug treatment therapies.

In one embodiment, the relative abundance of the at least one microRNA is compared to the abundance of the at least one microRNA present in a population of at least two further samples.

In one embodiment, the population of at least two further samples comprises both i) one or more members which exhibit a characterised susceptibility phenotype, and ii) one or more members which exhibit a characterised resistance phenotype, with respect to the at least one treatment.

In one non-limiting aspect that population of at least two further samples comprises more than 2 independent samples (i.e. individual members of the population), such as at least 5 members, at least 10 members, at least 20 members, at least 40 members or at least 50 members. In one embodiment, between 2 and 1000 members, such as between 10 and 500 members. In one embodiment between 50 and 200 members. Suitably, in one embodiment, the population contains between 10 - 90%, such as between 20 - 80%, such as between 30 - 70% of samples which exhibit resistance to the disease treatment. Suitably, in one embodiment, the population contains between 10 - 90%, such as between 20 - 80%, such as between 30 - 70% of samples which exhibit susceptibility to the disease treatment. In one embodiment, the abundance of the at least one microRNA is a relative abundance of the microRNA present in said sample as compared to the abundance of the at least one internal reference marker present in said sample, such as a non-coding RNA, such as a non- coding RNA selected form the group consisting of U6B, SNORD7, SNORD24, SNORD38B, SNORD43, SNORD44, SNORD48, SNORD49A, SNORD66, RNU19, 5.8S rRNA, and 5S rRNA. In one aspect, mRNA reference markers, such as those commonly used internal controls in gene expression analysis in molecular biology may also be used, for example GADPH, histones, actin mRNA levels, or equivalents, are often used as controls. A list of potential "housekeeping genes / reference genes/miRs can be found at

335.

It should be recognised that the use of further (control) samples, or populations thereof, or internal reference markers, are not necessarily mutually exclusive. Indeed, it is considered that there is a benefit in assessing the relative abundance to both. In this respect, an analysis of the abundance of miRNA target in the further samples (with known resistance or susceptibility phenotype) with one or more suitable internal control markers, allows for the correlation of the abundance of the miRNA, relative to the one or more suitable internal control markers (such as in the sample isolated or obtained from the subject) to the disease treatment resistance or disease treatment susceptibility. Once the one or more internal reference markers have been validated, it is therefore, in one aspect, no longer required to compare the abundance of miRNA in with the abundance in the at least one further sample (control samples), or populations thereof.

The Subject

The subject is typically a human being who is suffering from a disease or a disease state, or is likely to develop a disease or disease state.

In a preferable embodiment, the subject is typically a human being who has cancer. The patient may be male or female, although this may depend on the type of tissue/cancer being investigated (e.g. ovarian cancer effects only women).

Whilst the sample may be normal tissue from the subject, it is recognised that in a preferred embodiment, the sample is or comprises diseased tissue or cells, such as cancer tissue or cells.

The cancer (test) sample is typically obtained from the subject by biopsy or tissue sampling. The Cancer

In one embodiment, the cancer may be selected from the group consisting of: breast cancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer, cancer in the stomach, head and neck cancer, ovary cancer, testicular cancer, cervix cancer, liver cancer, thyroid cancer, epithelial cancer, urothelial cancer, nasopharyngeal cancer, myelodysplastic cancer, leukemias such as chronic lymphocytic leukaemia (CLL), acute lymphocytic leukaemia (ALL), chronic myeloid leukaemia (CML), acute myeloid leukaemia (AML), prolymphocytic leukaemia and erythroleukemia, lymphomas such as Hodgkin's lymphoma and non-Hodgkin's lymphoma such as follicular lymphoma and Burkitt's lymphoma, blastomas such as glioblastoma, neuroblastoma and retinoblastoma, and adenomas such as pituitary adenoma.

In one embodiment, the cancer is a carcinoma. The carcinoma is typically selected from the group consisting of malignant melanoma, basal cell carcinoma, ovarian carcinoma, breast carcinoma, thyroid papillary carcinoma, hepatocellular carcinoma, non-small cell lung cancer, renal cell carcinoma, bladder carcinoma, recurrent superficial bladder cancer, stomach carcinoma, prostatic carcinoma, pancreatic carcinoma, lung carcinoma, cervical carcinoma, cervical dysplasia, laryngeal papillomatosis, colon carcinoma, colorectal carcinoma and carcinoid tumors. More typically, said carcinoma is selected from the group consisting of malignant melanoma, non-small cell lung cancer, breast carcinoma, colon carcinoma and renal cell carcinoma. The malignant melanoma is typically selected from the group consisting of superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral melanoma, amelanotic melanoma and desmoplastic melanoma.

Alternatively, the cancer may suitably be a sarcoma. The sarcoma is typically in the form selected from the group consisting of osteosarcoma, Ewing's sarcoma, chondrosarcoma, malignant fibrous histiocytoma, fibrosarcoma and Kaposi's sarcoma.

The cancer may, suitably comprise neoplastic cells. In one embodiment the cancer is in the form of a tumour, suitably a malignant tumour.

In one embodiment, the cancer may be selected from the group consisting of: brain, breast, colorectal, endometrial, kidney (e.g. renal-cell), lung (e.g. non-small-cell), melanoma, ovarian, pancreatic, sarcoma, stomach or an unknown primary cancer. Figure 1 illustrates a selection of cancer treatments which are used to treat each of these forms of cancer, and therefore provide examples of the cancer treatment according to the invention. In one embodiment the cancer is colon cancer. In one embodiment the cancer is breast cancer. In one embodiment the cancer may be a haematological neoplasm, such as leukemia, lymphoma or multiple myeloma.

The sample

In a preferred embodiment, the sample is a cancer sample.

In the method for determining whether a cancer is resistant to a cancer treatment, the cancer used in the method is in the form of a cancer sample or biopsy obtained from a patient.

Whilst the sample obtained from the subject required for the traditional EDR® assay is typically at least 2gms, the sample isolated from the subject used in the present invention may be less than 2gms, such as less than 1.5gms, less than lgm, less than 0.5gm, less than 0.2gm, less than O.lgm, less than O.Olgm, less than 0.05gm.

In one embodiment the weight of the sample is at least O.OOOlgm, such as at least O.OOlgm.

It should be noted that the use of molecular detection methods such as micro-arrays and especially PCR to detect and quantify the target miRNAs (and internal controls) will allow for considerably smaller samples to be used that are used in the traditional EDR® assay.

In one embodiment the sample is in the form of a body fluid such as blood or lymph fluid, which may undergo a step of enrichment for, e.g. cancer cells.

Preparation of the Cancer Sample

Suitable methods of tumour tissue handling are widely known in the art, such as those provided in US 2006/0160114, US 2004/0214203, and will depend on the type and amount of tissue available.

In one embodiment the sample used is obtained directly from the patient, and is used without the removal of non-cancerous cells (once isolated from the subject's body). In this regards, an advantage of the present method is the ability to isolate small quantities of sample, thereby reducing the risk of sample contamination by surrounding healthy tissues, or non-cancerous tissues which may be intricately associated with the cancer - for example vascular tissue or dead cells. However, in one embodiment, the methods of the invention which refer to obtaining or isolating the sample, may comprise the steps of:

a) obtaining the sample from a subject and/or

b) Separating the cancer cells from non-cancer cells present in the sample obtained from the subject - i.e. enriching the sample for cancer cells or removing non-cancer cells from the sample.

Thereby the sample is enriched for the cancer cells prior to analysis of the at least one miRNA present in the cancer (sample).

Numerous methods are known in the art for enriching cancer samples for cancer cells - and commercial kits are available for this purpose - for example the Panomics Cancer cell isolation kit (Panomics Inc); Veridex Cellsearch system for isolation of circulating tumor cells from for example blood samples; Lacer capture microdissection (LCM) or equivalent technologies for isolation of cell populations from heterogonous tissue specimens.

Further methods of separating the cancer cells from non-cancer cells are provided in US 2004/0214203, hereby incorporated by reference. Such a method may comprise the following steps which may be incorporated into the method(s) according to the invention :

a) separating living cancer cells from dead cells, vascular endothelial cells and living cancer cells in the mixed population of cells from a cancer sample, by

a. contacting the mixed population of cells with a vital stain or fluorescent dye; b. contacting the mixed population of cells with a detectably-labelled immunological reagent that specifically binds to cancer cells; and c. selecting the cells in the mixed population that are not stained with the vital stain and that bind the immunological reagent.

The 'at least one' Cancer Treatment

A cancer treatment refers to conventionally used cancer treatments, such as drug therapy, including chemotherapy, and, in one embodiment, radiotherapy.

A broad variety of chemotherapeutic agents are used in the treatment of human cancer these include the plant alkaloids vincristine, vinblastine, vindesine, and VM-26; the antibiotics actinomycin-D, doxorubicin, daunorubicin, mithramycin, mitomycin C and bleomycin; the antimetabolites methotrexate, 5-fluorouracil, 5-fluorodeoxyuridine, 6-mercaptopurine, 6- thioguanine, cytosine arabinoside, 5-aza-cytidine and hydroxyurea; the alkylating agents cyclophosphamide, melphalan, busulfan, CCNU, MeCCNU, BCNU, streptozotocin, chlorambucil, bis-diamminedichloroplatinum, azetidinylbenzoquinone; and the miscellaneous agents dacarbazine, mAMSA and mitoxantrone. In one embodiment the chemotherapeutic agent according to the invention is selected from the group of the above agents.

Suitable chemotherapeutic drugs are provided in the following table. The first column is an indicator of which type of cancers the drugs are commonly used in, 1 = brain, 2 = breast, 3 = colorectal, 4 = endometrial, 5 = kidney, 6 = lung, 7 = melanoma, 8 = ovarian, 9 = pancreatic, 10 = sarcoma, 11 = stomach, 12 = unknown primary. Synonyms and chemical names also provided as is the CAS registry number.

In one embodiment the at least one cancer treatment is or comprises one or more chemotherapeutic drugs, such as one or more chemotherapeutic drugs selected from the group consisting of: Gemcitabine, Vinblastine, Temozolomide, Navelbine, Oxaliplatin, Vincristine, Fluorouracil, Floxuridine, Cyclophosphamide, Mitomycin C, Carboplatin, Ifosfamide, Etoposide, Taxol, Doxorubicin, Cisplatin, Carmustine, Capecitabine, 5 FU, 5 FU + Leucovorin, Topotecan, Taxotere, Irinotecan, 5 FU + Irinotecan, Alpha Interferon, Doxil, Interferon, Interferon + Vinblastine, Interleukin 2, Alpha Interferon, Taxotere + Navelbine, Cisplatin + Gemcitabine, and Doxil.

In one embodiment the at least one cancer treatment is or comprises one or more chemotherapeutic drugs, such as one or more chemotherapeutic drugs selected from the group consisting of: 5FU (optionally +Leucovorin (FULEU)), Irinotecan (SN38), Oxaliplatin (Oxapl) and Topotecan (topo).

In one embodiment the at least one cancer treatment is or comprises at least one chemotherapeutic drug selected from the group consisting of: anti-metabolites, such as azathioprine or mercaptopurine; plant alkaloids and terpeoids, such as vinca alkaloids and taxanes; thymidylate synthase inhibitors, such as 5-fluoro uracil (5-FU) and citrovorin, topoisomerase (TSI) acting drugs, such as SN-38 (Irinotecan) and camptothecin, alylating agents such as Oxaliplatin, monoclonal antibodies, such as Herceptin (Trastuzumab), Avastin (Bevacizumab), Erbitux (Cetuximab), Rituxan (Rituximab) , (anti-)hormonal treatments such as Tamoxifen, and the armoatase inhibitor letrozole.

In one embodiment the at least one cancer treatment is or comprises combined adjuvant therapy.

In one embodiment the at least one cancer treatment is or comprises herceptin, which is frequently used for the treatment of oestrogen receptor positive cancers (such as breast cancer).

It will be recognised that the method according to the invention may refer to more than one cancer treatment - in this regards the miRNA profile of cancer samples may be indicative of the cancer treatment resistance phenotype of more than one cancer treatment. Indeed, as illustrated in the examples the miRNA profile of the cancer sample can be indicative of the resistance phenotype to several cancer treatments, and as such may be used to predict which cancer treatments are likely to exhibit a EDR, IDR or LDR phenotype in the EDR® assay. In this way the method according to the invention can be utilised by the medical practitioner in their decision as to the most appropriate form of therapy, and not necessarily whether only one treatment is likely to be of benefit or not. The term at least one, therefore may refer to at least two, two, at least three, three, at least four, four, at least five, five, at least six, six, at least seven, seven, at least eight, eight, at least 9, nine, at least 10, or 10, for example.

Therefore the at least one cancer treatment may comprise of radiotherapy and chemotherapy, such as one or more of the chemotherapeutic drugs referred to herein, such as one or more of the following: 5-FU (optionally in combination with Leucovorin), Capecitabine, Floxuridine, Fluorouracil (optionally in combination with Irinotecan), Irinotecan, Oxaliplatin, Topotecan, And Leucovorin.

In one embodiment, the at least one cancer treatment is selected from the group consisting of 5-FU (optionally in combination with Leucovorin), Capecitabine, Floxuridine, Fluorouracil (optionally in combination with Irinotecan), Irinotecah, Oxaliplatin, Topotecan, And Leucovorin.

In one embodiment, the at least one cancer treatment is selected from the group consisting of: 5-FU (5'fluoro Uracil), Leucovorin, Oxaliplatin, and Inninitecan.

In one embodiment the at least one cancer treatment is a monoclonal antibody treatment, such as Bevacizumab or Cetuximab.

In one embodiment the at least one cancer treatment is a Taxol, such as Paclitaxel.

In one embodiment, the chemotherapeutic cancer treatments include Irinotecan, Topotecan, Oxalipatin, and/or 5'FU (optionally in combination with Leucovorin). Figure 3 shows the frequency of single and multiple drug resistance (EDR - see next section) of various colon cancer samples. Figure 4 shows a Venn diagram illustrating the correlation of specific miRNAs with the EDR status of these chemotherapeutic agents in various colon cancer samples. Figure 5 shows a Venn diagram illustrating the correlation of specific miRNAs with the LDR status of these chemotherapeutic agents in the same various colon cancer samples.

Resistance to cancer treatment - The EDR® Assay

It is recognised that the degree of resistance of a cancer to a cancer treatment may not be complete, i.e. the cancer may be partially susceptible to the cancer treatment, i.e. show a reduced rate of cell proliferation in the presence of the cancer treatment, as compared to an untreated control. The use of the EDR® assay for determining resistance or susceptibility to radiation treatment (radiotherapy) is disclosed in US 6,008,007 and US 6,261,795, which are hereby incorporated by reference.

The use of the EDR® assay for determining resistance or susceptibility to chemotherapeutic drug treatments is disclosed in Kern and Weisenthal (1990, J. Nat Cancer Inst 82: 582-588), US 2006/0160114 and US 2004/0214203, which are all hereby incorporated by reference.

The degree of resistance to a cancer treatment may be determined using the EDR® assay, as described in the above mentioned references and Holloway et al., 2002, Gynecologic Oncology 87, 8-16, and Meht et al., 2001, Breast Cancer Research and Treatment 66, 225- 237), both hereby incorporated by reference. Preferably, cancer (samples) which shows an extreme resistance are referred to as having a growth rate greater than 1 standard deviation above the median. In one embodiment, cancer (samples) which show an intermediate drug resistance have a growth rate greater than the median, but of less than 1 standard deviation. In one embodiment, which may be the same embodiment, cancer (samples) which show a low drug resistance have a tumor cell growth less than the median growth.

A method for determination of PCI: After incorporation with tritiated thymidine the cells are harvested onto glass fiber filters, and the cells are lysed with deionized water. The incorporated radioactivity in the filter-trapped macromolecular DNA is measured as counts per minute (CPM). Positive control (supralethal drug-exposed) and negative control (media- exposed) cultures are performed with each assay. Results are reported as percent cell inhibition (PCI) compared with media-exposed control cultures after subtraction of positive control CPM. The population median PCI and standard deviation (SD) are determined. For a tumor specimen the in vitro responses to individual drugs is scored as EDR when PCI is greater than or equal to ISD below the median, IDR when PCI is between the median and ISD below the median, or LDR if the PCI is above the median.

Both extreme drug resistance and intermediate drug resistance are therefore characteristics of cancer cells which are able to proliferate in vitro in the presence of, or after exposure to, the drug treatment, such as determined by the EDR® assay.

The EDR® assay method involves the isolation of fresh viable tumor tissue which is minced and digested with enzymes to disaggregate the tumor cells. The cells are then placed in soft agar to encourage cell proliferation before being exposed to the cancer treatment such as chemotherapeutic agents, typically for a period of five days and at an elevated dosage, during the latter period of drug exposure (such as the final two days) tritiated thymidine is added as a measure of cell proliferation. By comparing the level of label incorporated into drug treated and untreated controls, the degree of cell proliferation under the drug treatment is determined, and thereby the resistance phenotype of the cancer cells.

It is recognised that the dosage of treatment used in the EDR® assay will vary depending upon the agent used, and as such, in one embodiment, the dosage level may be selected by determining the effective concentration or dose of the therapeutic agent which is sufficient to prevent significant incorporation of the radio label into the cells in control samples which have a susceptible phenotype, or in one embodiment, healthy cells which are obtained from the same tissue type as the disease sample was obtained or isolated from.

The dosage level of the cancer treatment used to determine the degree of resistance may, in one embodiment, between 5 - 80 times greater, such as between 10 and 50 times greater, than the appropriate in vivo dose of the treatment agent.

microRNAs

The term "microRNA" or "miRNA", in the context of the present invention, means an RNA oligonucleotide consisting of between 18 to 25 nucleotides. In functional terms miRNAs are typically regulatory endogenous RNA molecules.

The terms "target microRNA" or "target miRNA" or "miRNA target" refer to a microRNA which comprises the reverse complement of the contiguous nucleobase sequence of the oligonucleotide or detection probes referred to herein.

The Sanger Institute publishes known miRNA sequences in the miRBase database

(http://microrna.sanqer.ac.uk/sequences/index.shtml). To date there are 533 human miRNAs present in the miRBase database. miRBase release 10.0 is hereby incorporated by reference, including all the miRNA mature and pre-mature sequences disclosed therein.

In one embodiment, the at least one microRNA is a oncomiR (i.e a microRNA whose aberrant (i.e. over or under expression) is associated with a cancer phenotype. Novel miRNAs which are associated with cancer are disclosed in PCT/EP2007/061210, hereby incorporated by reference. In particular table 5 in PCT/EP2007/061210 disclose specific mature miRNA sequences, which in the following may be referred to with an "454_hsa" extension. As an example "miR_2147" may be referred to as "454_hsa_miR_2147".

Further onco-miRs include the hsa-miR-93 family, such as the following human microRNAs: miR-93, miR-106b, miR-20b, miR-20a, miR-106a, miR-17, miR-18a, and miR-18b. The present inventors have found that multiple members of the hsa-miR-93 family are upregulated in cells with a high resistance to 5-FU and/or leucoverin. Targets of the miR-93 family include the E2F transcription factor which is an activator of cell cycle progression.

In one embodiment, the at least one miRNA include miRPIus_28431 and/or hsa-miR-129-5p, which, for example, have, in the present work, been correlated to oxaliplatin resistance.

In one embodiment, the at least one miRNA include hsa-miR-148, hsa-miR-148* and/or hsa- miR-203, which have been associated, for example, with SN-38 (topoisomerase 1 inhibitor) resistance.

The following table provides a list of miRNAs which the present inventors have found are associated with an EDR status of various colon cancers in relation to the shown treatments:

Drug 5FU + 5FU + Irinotecan hsa- Oxaliplatin Topotecan

Leucoverin Irinotecan hsa- hsa-let-7i* hsa-let-7d* miR-1 let-7i* hsa-miR-106a hsa-miR-17 hsa-miR-106b hsa-miR-105 hsa-miR-106b hsa-miR-193b hsa-miR-106b* hsa-miR-130b* hsa-miR-106b hsa-miR-125a- hsa-miR-19b hsa-miR-128a hsa-miR-132* hsa-miR-106b*

3p hsa-miR- hsa-miR-19b hsa-miR-130b hsa-miR-181c hsa-miR-125b-

141* 1* hsa-miR-17 hsa-miR-20a hsa-miR-137 hsa-miR-188-5p hsa-miR-128a hsa-miR-20b hsa-miR-141* hsa-miR-205 hsa-miR-130b hsa-miR-17* hsa-miR-30e hsa-miR-142-3p hsa-miR-216a hsa-miR-137 hsa-miR-181c hsa-miR-30e hsa-miR-142-5p hsa-miR-302b* hsa-miR-142- hsa-miR-18a hsa-miR-339-5p hsa-miR-142-5p hsa-miR-302d 3p hsa-miR-18b hsa-miR-362-3p hsa-miR-146a hsa-miR-425* hsa-miR-142- hsa-miR-18b hsa-miR-373* hsa-miR-148b hsa-miR-523 5p hsa-miR-142-

hsa-miR-19a hsa-miR-374a hsa-miR-25 hsa-miR-553 hsa-miR-148b hsa-miR-19b hsa-miR-423-5p hsa-miR-301b hsa-miR-592 hsa-miR-150 hsa-miR-19b hsa-miR-525-5p hsa-miR-302a* hsa-miR-614 hsa-miR-151- hsa-miR-20a hsa-miR-548c- hsa-miR-324-5p hsa-miR-617 3p hsa-miR-20b 3p hsa-miR- hsa-miR-339-5p hsa-miR-662 hsa-miR-186

590-5p hsa-miR-33a hsa-miR-99a* hsa-miR-187 hsa-miR-617 hsa-miR-18a hsa-miR-302d ROS hsa_miR 1 hsa-miR-633 hsa-miR-345 745 hsa-miR-18b hsa-miR-32* hsa-miR-671- hsa-miR-362-5p hsa-miR-25 hsa-miR-373 3p hsa-miR-92a hsa-miR-381 hsa-miR-302b* hsa-miR-493 hsa-miR-92a hsa-miR-485-5p hsa-miR-345 hsa-miR-570 ROC_hcy_miR_l hsa-miR-496 hsa-miR-361- hsa-miR-577 961 hsa-miR-500 5p hsa-miR-

ROC_hsa_miR_l 448

627 hsa-miR-92a ROC_hsa_miR_l hsa-miR-525-3p hsa-miR-453 hsa-miR-92a 637 hsa-miR-532-5p hsa-miR-525- hsa-miR-92b ROS_hsa_miR_l hsa-miR-568 5p hsa-miR-93 748 hsa-miR-572 hsa-miR-532-

ROS_hsa_miR_l 5p

780 hsa-miR-568 hsa-miR-93 hsa-miR-585 hsa-miR-570

ROC_hsa_miR_l hsa-miR-652 hsa-miR-572

659

ROS_hsa_miR_l hsa-miR-652 hsa-miR-634

733

ROS_hsa_miR_l hsa-miR-885-5p hsa-miR-652

751 hsa-miR-92a hsa-miR-663

ROS_hsa_miR_l

788 hsa-miR-92a hsa-miR-885- hsa-miR-93 5p hsa-miR-92a

ROC_hsa_miR_16 hsa-miR-92a

22

ROC_hsa_miR_16 hsa-miR-93

27

ROC_hsa_miR_16 hsa-miR-93

66

ROC_hsa_miR_19 ROC_hsa_miR

26 1662

ROS_hsa_miR_17 ROS_hsa_miR_

14 1732

The sequences of the above miRNA (Mature) sequences not present in miRBase (Release 10) are as follows:

ROC_ _hsa_ miR_ _1627 aaaagcugaguugagaggg

ROC_ _hsa_ miR_ _1666 cagagaggaccacuauggcggg

ROC_ _hsa_ miR_ _1926 ucgaccggaccucgaccggcuc

ROS_ _hsa_ miR_ _.1714 cagagcuuagcugauuggugaaca

ROS_ _hsa_ miR_ _1732 ggcuucuuuacagugcugccuu

It is according to one important aspect of the present invention, possible to determine the EDR status of cancers and in particular colorectal cancer based on the tumor's miRNA profile,such as described in example 2. Thus, in some embodiments it is possible to determine miRNAs that are most differentially expressed in cancer samples with EDR towards at least two different treatments, such as at least two different chemotherapeutic agents. In a further embodiment a selection of miRNAs, that are most differentially expressed in cancer samples with EDR towards at least two different treatments may be used to determine whether a patient sample has EDR towards one of the at least two different treatments. In some embodiments the cancer specimens are fresh frozen and in some embodiments they are formalin fixed, paraffin embedded specimens. In some particular embodiments the cancers of which the EDR status may be determined are resistant to 5-FU/Leucovorin, Oxaliplatin, or Irinotecan. Accordingly the present invention enables the identification of tumors that are EDR to 5-FU/Leucovorin, Oxaliplatin, or Irinotecan, based solely on the tumor cell's microRNA profiles.

In one embodiment, the at least one miRNA, which is most differentially expressed in samples with an EDR phenotype in respect to Irinotecan and samples with an EDR phenotype with respect to Oxaliplatin is selected from the list consisting of hsa-miR-133a, 454_hsa_miR_2147, hsa-miR-520c, hsa-miR-591, 454_hsa_miR_2364, hsa-miR-200b*, 454_hsa_miR_1975, hsa-miR-617, hsa-miR-629, hsa-miR-502-3p, hsa-miR-525-3p, hsa- miR-425*, hsa-miR-15b*, hsa-miR-29a*, hsa-miR-594, hsa-miR-124a, hsa-miR-585, hsa- miR-106b, hsa-miR-98, hsa-miR-609, 454_hsa_miR_2039, 454_hsa_miR_2010, hsa-miR- 518e, hsa-miR-193a-5p, hsa-miR-15a, ROC_hcy_miR_1965, hsa-miR-195, ROC_hcy_miR_1961, hsa-miR-346, hsa-let-7i, and 454_hsa_miR_1973. In one embodiment the irinotecan resistance and/or oxaliplatin resistance is associated with colorectal cancer.

In one embodiment, the at least one miRNA, which is most differentially expressed in samples with an EDR phenotype in respect to 5FU and samples with an EDR phenotype with respect to Irinotecan is selected from the list consisting of hsa-miR-122, hsa-miR-206, 454_hsa_miR_1994, hsa-miR-595, hsa-miR-147, hsa-miR-32*, 454_hsa_miR-2056, hsa- miR-297, 454_hsa_miR-2370, ROC_hsa_miR_1674, hsa-miR-574-5p, 454_hsa_miR-2069, 454_hsa_miR-2116, hsa-miR-603, hsa-miR-105, hsa-miR-329, hsa-miR-133a, hsa-miR-187, hsa-miR-544, hsa-miR-374a, ROS_hsa_miR_1778, hsa-miR-126*, hsa-miR-216a, hsa-miR- 577, hsa-miR-502-5p, hsa-miR-887, hsa-miR-361-3p, hsa-miR-672, hsa-miR-363*, ROC_hsa_miR_1622, 454_hsa_miR-2123, hsa-miR-542-5p, hsa-miR-422b, and 454_hsa_miR-2068. In one embodiment the Irinotecan resistance and/or 5FU resistance is associated with colorectal cancer.

In one embodiment, the at least one miRNA, which is most differentially expressed in samples with an EDR phenotype in respect to 5FU and samples with an EDR phenotype with respect to Oxaliplatin is selected from the list consisting of has-miR-184, 454_hsa_miR-2010, 454_hsa_miR-2092, 454_hsa_miR-2134, 454_hsa_miR-2039, hsa-miR-594, hsa-miR-512- 5p, 454_hsa_miR-2068, 454_hsa_miR-1628, hsa-miR-370, 454_hsa_miR-2159, hsa-miR- 617, 454_hsa_miR-2136, hsa-miR-370, hsa-miR-200b*, 454_hsa_miR-2119, 454_hsa_miR- 1989, hsa-miR-193a-5p, hsa-miR-574-3p, hsa-miR-497, 454_hsa_miR-2033, 454_hsa_miR- 2156, hsa-miR-672, hsa-miR-181c, 454_hsa_miR-2152, hsa-miR-768-5p, hsa-miR-768-3p, hsa-miR-668, and hsa-miR-363*. In one embodiment the oxaliplatin resistance and/or 5FU resistance is associated with colorectal cancer.

In one embodiment, the at least one miRNA, which is most differentially expressed in samples with an EDR phenotype in respect to Oxaliplatin and samples with an EDR phenotype with respect to Irinotecan is selected from the list consisting of hsa-miR-146a, hsa-miR-148a, hsa-miR-29c, hsa-miR-16, hsa-miR-27b, hsa-miR-101, hsa-miR-26b, hsa-miR-99a*, hsa- miR-140-5p, ROS_hsa_miR_0876, hsa-miR-92b, hsa-miR-203, ROS_hsa_miR_1759, hsa- miR-18a, hsa-miR-608, hsa-miR-92a, hsa-miR-1, hsa-miR-330-3p, hsa-miR-584, hsa-miR- 149, hsa-miR-301b, hsa-miR-381, hsa-miR-124a, hsa-miR-612, hsa-miR-141*, hsa-miR- 652, hsa-miR-302b, and hsa-miR-639. In one embodiment the oxaliplatin resistance and/or irinotecan resistance is associated with colorectal cancer.

Determining microRNA correlation to the resistance or susceptibility of the cancer

Isolating a RNA/ microRNA fraction

Whilst it is envisaged that the term 'isolating' a microRNA fraction may involve the separation of a microRNA containing fraction for the sample, it is also envisaged that in some cases the microRNA abundance is measured in situ within the sample - for example using in situ hybridisation, which is an effective means for determining microRNA's within tissues. An advantage of using in situ hybridisation is that the precise location of the miRNAs within the sample may be determined, thereby allowing for cellular heterogeneity within the tissue sample to be accounted for.

For the detection of the abundance of multiple microRNAs in a sample, it may be an advantage to isolate a microRNA fraction from the cancer sample - thereby enabling a wide range of molecular tools which may be used for detecting the abundance of specific microRNAs present in a sample. Suitable microRNA fraction isolation protocols are widely available, such as the mirVana™ miRNA Isolation Kit (Ambion) or the miRNeasy Kit (Qiagen).

The use of total RNA fractions for analysis and quantification of microRNA expression is routine in the art, and as such in one embodiment the microRNA fraction may be a total RNA fraction, which may for example be obtained using a standard Trizol extraction.

Control Markers

The control marker may be a genetic marker which is correlated to, or an indication of the disease or disease condition. For example, with respect to cancer, the control marker may be an oncogene or mRNA or protein product deriving therefore. The control marker may be a mutation in the genetic code which is associated to the disease, or an mRNA or an aberrant mRNA (such as an alternatively spliced mRNA), or a protein product derived (encoded) therefrom, whose presence (or absence) or abundance, is correlated to the disease or disease condition, such as cancer.

Therefore, in one embodiment of the method of the invention a further step, step c), is performed, either prior to, concurrent to, or subsequent to step b), wherein step c) comprises the assaying the presence or absence or the abundance of at least one control marker of said disease, such as a genetic mutation associated with said disease, or a the expression levels of a mRNA or protein which is associated with said disease.

With respect to cancer, the control marker may be, for example, selected from the group consisting of CD31, BAX, BCL-2, EGFR, ER receptor, HER2, Ki-67, MDR-I, p53, PR receptor, Thrombospondin 1, Thymidylate Synthase, and VEGV.

The Control Sample(s)

The control samples may, suitably be the at least one further samples referred to herein - and as such a preferred control sample reflects a population of characterised disease samples, which are characterised with respect to their disease resistance/susceptibility phenotype, for example by the EDR® assay.

However, it is considered that in addition to or in place of the at least one further sample, other control samples may also be utilised.

The control sample or sample(s) may be a sample taken previously, e.g. a sample or collection of samples of the same type of cancer/tumor, taken from one or more patients whose cancers have a defined phenotype to the at least one cancer treatment.

In one embodiment, the control sample may be taken from healthy tissue, for example tissue taken adjacent to the cancer, such as within 1 or 2 cm diameter from the external edge of said cancer. Alternatively the control sample may be taken from an equivalent position in the patients body, for example in the case of breast cancer, tissue may be taken from the breast which is not cancerous.

In one preferable embodiment, the control sample may also be obtained from a different patient, e.g. it may be a control sample, or a collection of control samples, representing different types of cancer, for example those listed herein (i.e. cancer reference samples). Comparison of the test sample data with data obtained from such cancer reference samples may for example allow for the characterisation of the test cancer to a specific type and/or stage of cancer.

In one embodiment, at least one control sample is obtained, and a second population of nucleic acids from the at least one control sample is, in addition to the test sample, presented and hybridised against at least one detection probe.

In one embodiment, the control sample may be obtained from a non tumorous tissue, such as from tissue adjacent to said putative tumor, and/or from an equivalent position elsewhere in the body.

In one embodiment, the at least one control sample may be obtained from a non tumorous tissue selected from the group consisting; brain tissue, breast tissue, colorectal tissue, endometrial tissue, kidney tissue, lung tissue, ovarian tissue, pancreatic tissue, stomach tissue.

In one embodiment, the at least one control sample may be obtained from a cancerous tissues may include brain cancer, breast cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g. renal-cell), lung cancer (e.g. non-small-cell), melanoma, ovarian cancer, pancreatic cancer, sarcoma, stomach cancer or unknown primary cancer.

In one embodiment the at least one control sample or samples may be a haematological sample, such as a haematological neoplasm, such as leukemia, lymphoma or multiple myeloma.

The RNA/ miRNA (enriched) fraction

In one embodiment, the miRNA may remain within the test sample, such as remain in the cells of the biopsy or tissue sample, for example for in situ hybridisation. The cells may still be living, or they may be dead. The cells may also be prepared for in situ hybridisation using methods known in the art, e.g. they may be treated with an agent to improve permeability of the cells; the cells may also be fixed or partially fixed.

However the miRNA fraction may be isolated from the cancer (or control) sample.

Suitably the fraction isolates miRNA the cells of the sample, and preferably enriches the miRNAs present in the sample as compared with the concentration of other soluble cellular components such as DNA or protein.

The enrichment may be of the total RNA of the samples - i.e. a total RNA fraction.

The miRNA fraction suitably contains a population of nucleic acids, which comprises a population of miRNAs - the population of miRNAs is preferably representative of the miRNAs present in the sample.

In one embodiment, the miRNA fraction may be derived from the miRNAs present in the sample - i.e. retain the biological information regarding the miRNA sequences and abundance thereof in the sample.

The miRNA fraction preferably comprises small RNAs such as those less than 100 bases in length.

In one embodiment the miRNA fraction may also comprise other RNA fractions such as mRNA, and/or in siRNAs and/or piRNAs.

In one embodiment, the miRNA fraction comprises snRNAs. The miRNA fraction may also comprise other nucleic acids, for example the miRNA fraction may be part of a total nucleic acid fraction which also comprises DNA, such as genomic and/or mitochondrial DNA. The miRNA fraction may be purified. Care should be taken during miRNA extraction to ensure at least a proportion of the miRNA are retained during the extraction. Suitably, specific protocols for obtaining RNA fractions comprising or enriched with small RNAs, such as miRNAs may be used. The fraction may undergo further purification to obtain an enriched miRNA fraction. This can be achieved, for example, by removing mRNAs by use of affinity purification, e.g. using an oligodT column.

In one embodiment the miRNA fraction is used directly in the hybridisation with the at least one detection probe.

The miRNA fraction may comprise the target molecule, e.g. the miRNA fraction obtained from a test sample, the presence of the target molecule within the miRNA fraction may indicate a particular phenotype. Alternatively the miRNA fraction may not comprise the target molecule, e.g. the miRNA fraction obtained from a test sample, the absence of the target (complementary) molecule within the RNA fraction may indicate a particular phenotype.

In one embodiment, prior to (or even during) said hybridisation, the miRNA fraction may be used as a template to prepare a complement of the miRNA present in the fraction, said compliment may be synthesised by template directed assembly of nucleoside, nucleotide and/or nucleotide analogue monomers, to produce, for example an oligonucleotides, such as a DNA oligonucleotide. The complement may be further copied and replicated. The compliment may represent the entire template miRNA molecule, or may represent a population of fragments of template molecules, such as fragments than, preferably in average, retain at least 8 consecutive nucleoside units of said miRNA template, such as at least 12 of said units or at least 14 of said units. It is preferred that at least 8 consecutive nucleoside units of said complementary target, such as at least 12 of said units or at least 14 of said units of said complementary target are retained. When the complementary target is a precursor miRNA, or a molecule derived therefore, if is preferred that at least part of the loop structure of the precursor molecule is retained, as this will allow independent detection over the mature form of the miRNA, or molecule derived there from.

Therefore, in one embodiment the miRNA fraction itself is not used in the hybridisation, but a population of molecules, such as population of oligonucleotides which are derived from said RNA fraction, and retain sequence information contained within said miRNA fraction, are used. It is envisaged that the population of molecules derived from said miRNA fraction may be further manipulated or purified prior to the hybridisation step - for example they may be labelled, or a sub-fraction may be purified there from. The target molecule (complementary target) may therefore be derived from miRNA, but may actually comprise an alternative oligo backbone, for example DNA. The target molecule may, therefore also be a complement to the original miRNA molecule, or part of the original RNA molecule from which it is derived.

In one embodiment, the miRNA fraction is analysed and the population of target miRNAs and optionally control nucleic acids are determined. For example the miRNA fraction, or a nucleic acid fraction derived there from may be undergo quantitative analysis for specific target and control sequences, for example using oligonucleotide based sequencing, such as oligonucleotide micro-array hybridization. The data from the quantitative analysis may then be used in a virtual hybridisation with a detection probe sequence.

Assaying the abundance of at least one microRNA

The assessment of the abundance of a microRNA may be determination or estimation of an absolute abundance, but it may, in one embodiment be a comparative assessment as compared to the abundance in previously determined value or values (e.g. obtained by analysis of the at least one microRNA(s) present in control cancer samples of known drug resistance phenotype (e.g. EDR, IDR or LDR), or an equivalent reference sample, or samples.

In one embodiment, the abundance of the at least one microRNA is a relative abundance of the microRNA present in said cancer sample as compared to the abundance of the at least one microRNA present in at least one control sample, such as at least one further cancer sample, or a population of cancer samples, with a known cancer treatment resistance phenotype.

In one embodiment, the abundance of the at least one microRNA is a relative abundance of the microRNA present in said cancer sample as compared to the abundance of the at least one microRNA present in at least one further cancer sample, or a population of cancer samples, with a known cancer treatment resistance phenotype.

It is recognised that, depending on the specific microRNA (and in one embodiment the cancer treatment) an increased (relative) abundance of a microRNA present in the sample may be indicative of a drug resistance phenotype (such as EDR or IDR), or a drug susceptible phenotype. Alternatively, it is also recognised that a decreased (relative) abundance of a microRNA present in the sample may be indicative of a drug resistance phenotype (such as EDR or IDR), or a drug susceptible phenotype. In some cases, it will be advantageous to assess the (relative) abundance of more than one microRNA, and therefore a more accurate method may be obtained by correlating the pattern of (relative) abundance of a population of microRNAs between samples. This may be particular poignant when assessing the drug resistance status of a sample with respect to numerous possible cancer treatments. Therefore, in a preferred embodiment, the method of the invention comprises the determination of the abundance, such as the relative abundance of more than one microRNA, such a population of microRNAs, suitably by the use of a corresponding population of detection probes.

In one embodiment, the abundance of population of individual microRNAs is determined. Therefore, in one embodiment the abundance of at least two microRNAs is determined, such as the abundance of at least three, such as at least four, such as at least five, such as at least six, such as at least seven, such as at least eight, such as at least nine, such as at least 10 microRNAs is determined - wherein the over or under abundance of the miRNAs are correlated to either the resistance or susceptibility of the disease or state of disease to the administration of the individual members of the population of miRNAs. In one embodiment the population comprises between 5 and 60 individual microRNAs whose abundance is correlated to either the resistance or susceptibility of the disease, such as between 10 - 40 or 15 - 40 of such members.

With respect to the population of microRNAs, it is recognised that the abundance of other miRNAs, and other molecular markers, which are not correlated to the resistance/susceptibility phenotype of the disease, may also be analysed, and as such in one embodiment, the total population of miRNAs analysed may be higher, such as up to 500, or 1000, or even more, and as such a majority or even all of the known human microRNAs may be analysed - for example using the arrays of detection probes referred to herein.

In one embodiment, the abundance of at least 2, such as at least five, such as at least 10, independent microRNAs are determined, such as the microRNAs independently selected from those referred to herein. In one embodiment, at least 60 independent microRNAs are determined.

In one embodiment, the abundance of each of the independent members of a population of microRNAs is determined, such as a population comprising microRNAs referred to or selected from those referred to in any one of the preceding claims, wherein the over or under abundance of each of the independent members of a population of microRNAs may be correlated to the resistance of the cancer to one or more of the cancer treatments referred to herein. In one embodiment, the method of the invention comprises hybridising the microRNA fraction to a population of detection probes, wherein said population of detection probes comprises independent members which correspond to each of, or a proportion of, such as at least 25%, 50% or 75%, of the independent members of a population of microRNAs, such as the populations (groups) of microRNAs referred to herein.

The determination of the abundance of the miRNA therefore may typically involve the collection of a signal caused by the hybridisation of one, or more (for example in the case of PCR based methods) detection probes (or oligonucleotides) from an assay performed on the cancer sample, or a microRNA enriched fraction obtained therefrom. This signal is typically compared to a control.

When assessing relative abundance, the control to which the abundance (signal) from the at least one microRNA may be compared may be an internal control, such as a housekeeping gene and/or the median abundance in a population of further samples. The control may be in the form of data obtained by a separate analysis, such as a previous analysis of the microRNA abundance in a population of further samples, such as the data generated by the method for identifying one or more microRNAs which are indicators of the susceptibility or resistance of a disease to a disease treatment, as referred to herein.

An internal control - i.e. the relative abundance of the miRNA is measured proportional to the abundance of a molecular marker present in the sample or microRNA fraction. This may for example be a RNA species such as a miRNA or an mRNA whose abundance is independent of the application of a cancer treatment.

The abundance of the at least one microRNA present in a non-cancerous tissue - such as healthy tissue obtained from the same origin as the cancer sample, for example this may be healthy equivalent tissue or tissue adjacent to the cancer, and may be collected from the subject.

The average abundance of microRNA in a population of cancer samples of the same type (such as tissue of origin, or histology types) - such as the population of cancer samples referred to herein - i.e. compared to the median abundance in cancer samples of the same type (see the examples - which utilise a collection of colon cancer samples). In this regards, access to collections of cancer tissue samples are available from Oncotech Inc.

As shown in the examples, when the method according to the invention is performed on a population of cancer samples which comprise both cancer treatment resistant and cancer treatment sensitive phenotypes, the relative abundance of multiple microRNA(s) between the samples may be used to identify the drug resistant and drug sensitive members of the population of cancer samples, both with a single cancer treatment, but also multiple cancer treatments.

The analysis of the abundance of multiple (independent) microRNAs in the sample thereby provides the medical practitioner with highly valuable information when selecting the appropriate cancer treatment out of numerous possible alternatives.

Detection of the abundance of microRNA

The detection of microRNAs in the cancer sample, or a microRNA enriched fraction obtained therefrom typically involves the use of hybridisation technologies, where a compound (such as an oligonucleotide) which has a complementary nucleobase sequence to a corresponding nucleotide seqeunce present in the microRNA target (or an equivalent complementary structure), is used to specifically hybridise to the target microRNA.

The signal

In one embodiment the target is labelled with a signal. In this respect the population of miRNA, present in the miRNA (enriched) fraction are labelled with a signal which can be detected. The hybridisation or the target molecules to the detection probe, which may be fixed to a solid surface, and subsequent removal of the remaining nucleic acids, including the remaining miRNA, from the population, and therefore allows the determination of the level of signal from those labelled target which is bound to the detection probe. This may be appropriate when screening immobilised probes, such as arrays of detection probes.

In one embodiment the detection probe is labelled with a signal. This may be appropriate, for example, when performing in situ hybridisation and northern blotting, where the miRNA present in the miRNA fraction (population of nucleic acids) are immobilised.

It is also envisaged that both population of miRNAs and detection probes are labelled. For example they may be labelled with fluorescent probes, such as pairs of FRET probes (Fluorescence resonance energy transfer), so that when hybridisation occurs, the FRET pair is formed, which causes a shift in the wavelength of fluorescent light emited. It is also envisaged that pairs of detection probes may be used designed to hybridise to adjacent regions of the target molecule, and each detection probe carrying one half of a FRET pair, so that when the probes hybridise to their respective positions on the target, the FRET pair is formed, allowing the shift in fluorescence to be detected.

Therefore, it is also envisaged that neither the population of nucleic acid molecules nor the detection probe need be immobilised.

Once the appropriate target miRNA sequences have been selected, probes, such as the preferred LNA substituted detection probes are preferably chemically synthesized using commercially available methods and equipment as described in the art (Tetrahedron 54: 3607-30, 1998). For example, the solid phase phosphoramidite method can be used to produce short LNA probes (Caruthers, et al., Cold Spring Harbor Symp. Quant. Biol. 47:411- 418, 1982, Adams, et al., J. Am. Chem. Soc. 105: 661 (1983).

Detection probes, such as LNA-containing-probes, can be labelled during synthesis. The flexibility of the phosphoramidite synthesis approach furthermore facilitates the easy production of detection probes carrying all commercially available linkers, fluorophores and labelling-molecules available for this standard chemistry. Detection probes, such as LNA- modified probes, may also be labelled by enzymatic reactions e.g. by kinasing using T4 polynucleotide kinase and gamma-³²P-ATP or by using terminal deoxynucleotidyl transferase (TDT) and any given digoxygenin-conjugated nucleotide triphosphate (dNTP) or dideoxynucleotide triphosphate (ddNTP).

Detection probes according to the invention can comprise single labels or a plurality of labels. In one aspect, the plurality of labels comprise a pair of labels which interact with each other either to produce a signal or to produce a change in a signal when hybridization of the detection probe to a target sequence occurs.

In another aspect, the detection probe comprises a fluorophore moiety and a quencher moiety, positioned in such a way that the hybridized state of the probe can be distinguished from the unhybridized state of the probe by an increase in the fluorescent signal from the nucleotide. In one aspect, the detection probe comprises, in addition to the recognition element, first and second complementary sequences, which specifically hybridize to each other, when the probe is not hybridized to a recognition sequence in a target molecule, bringing the quencher molecule in sufficient proximity to said reporter molecule to quench fluorescence of the reporter molecule. Hybridization of the target molecule distances the quencher from the reporter molecule and results in a signal, which is proportional to the amount of hybridization. In the present context, the term "label" means a reporter group, which is detectable either by itself or as a part of a detection series. Examples of functional parts of reporter groups are biotin, digoxigenin, fluorescent groups (groups which are able to absorb electromagnetic radiation, e.g. light or X-rays, of a certain wavelength, and which subsequently reemits the energy absorbed as radiation of longer wavelength; illustrative examples are DANSYL (5- dimethylamino)-l-naphthalenesulfonyl), DOXYL (N-oxyl-4,4-dimethyloxazolidine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, acridines, coumarins, Cy3 and Cy5 (trademarks for Biological Detection Systems, Inc.), erythrosine, coumaric acid, umbelliferone, Texas red, rhodamine, tetramethyl rhodamine, Rox, 7-nitrobenzo-2-oxa-l-diazole (NBD), pyrene, fluorescein, Europium, Ruthenium, Samarium, and other rare earth metals), radio isotopic labels, chemiluminescence labels (labels that are detectable via the emission of light during a chemical reaction), spin labels (a free radical (e.g. substituted organic nitroxides) or other paramagnetic probes (e.g. Cu²⁺, Mg²⁺) bound to a biological molecule being detectable by the use of electron spin resonance spectroscopy). Especially interesting examples are biotin, fluorescein, Texas Red, rhodamine, dinitrophenyl, digoxigenin, Ruthenium, Europium, Cy5, Cy3, etc.

Detection /Oligonucleotide Probes

In one embodiment, the determination of the abundance of the at least one microRNA is performed using at least one detection probe which comprises a complementary nucleobase sequence to at least 6 contiguous nucleotides present in said at least one microRNA.

In a preferred aspect, the complementary nucleobase sequence is complementary to a contiguous nucleotide sequence present in said at least on microRNA (target).

In one embodiment, the complementary nucleobase sequence is complementary to the entire contiguous nucleotide sequence present in said at least on microRNA (target).

In one embodiment, the complementary nucleobase sequence consists of between 8 and 25 contiguous nucleobases, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 25 contiguous nucleobases, or between 12 and 20 nucleobases, such as between 8 and 14 contiguous nucleobases. In one embodiment the nucleobase sequence consists of between 15 and 23 contiguous nucleobases, and may for example be 15 or 23 contiguous nucleobases. In one embodiment, the complementary nucleobase sequence consists of nucleotide analogues inserted between the nucleoside analogues with regular spacing over part or the entire nucleobases sequence.

In one embodiment, the regular spacing is a nucleotide analogue at every second, third or fourth nucleobase position, or combination thereof.

In one embodiment, the complementary nucleobase sequence comprises LNA nucleotide analogues.

In one embodiment, all the nucleotide analogues present in the complementary nucleobase sequence are LNA nucleotide analogues.

Detection probe and recognition sequence

The detection probe is preferably in the form of an oligonucleotide, or, such as in the case of PCT a set oligonucleotides, which are used to amplify the microRNA sequence present in the sample, or a molecule derived therefrom.

The oligonucleotide or detection probe referred to herein may include a plurality of nucleotide analogue monomers. The oligonucleotide or detection probe hybridizes to a miRNA or miRNA precursor. In one embodiment, the nucleotide analogue is LNA, such as alpha and/or xylo LNA monomers. In one embodiment, the oligonucleotide probe hybridizes to the loop sequence of a miRNA precursor, e.g., to 5 nucleotides of the miRNA precursor loop sequence or to the center of the miRNA precursor loop sequence. The oligonucleotide probe may or may not also hybridize to the stem sequence of the miRNA precursor. The oligonucleotide probe may have a number of nucleotide analogue monomers corresponding to 20% to 40% of the probe oligonucleotides. The probes may also have a spacing between nucleotide analogue monomers such that two of the plurality of nucleotide analogue monomers are disposed 3 or 4 nucleotides apart, or a combination thereof. Alternatively, each nucleotide analogue monomer in a probe may be spaced 3 or 4 nucleotides from the closest nucleotide analogue monomer. Typically, when nucleotide analogue monomers are spaced apart, only naturally-occurring nucleotides are disposed between the nucleotide analogue monomers. Alternatively, two, three, four, or more nucleotide analogue monomers may be disposed adjacent to one another. The adjacent nucleotide analogue monomers may or may not be disposed at the 3' or 5' end of the oligonucleotide probe or so that one of the nucleotide analogue monomers hybridizes to the center of the loop sequence of the miRNA precursor. The probe may include none or at most one mismatched base, deletion, or addition. Desirably, the probe hybridizes to the miRNA or precursor thereof under stringent conditions or high stringency conditions. Desirably, the melting point of the duplex formed between the probe and the miRNA precursor is at least 1° C higher, e.g., at least 5°C, than the melting point of the duplex formed between the miRNA precursor and a nucleic acid sequence not having a nucleotide analogue monomer, or any modified backbone. The probe may include at least 70% DNA; at least 10% nucleotide analogue monomers; and/or at most 30% nucleotide analogue monomers.

The probe may further include a 5' or 3' amino group and/or a 5' or 3' label, e.g., a fluorescent (such as fluorescein) label, a radioactive label, or a label that is a complex including an enzyme (such as a complex containing digoxigenin (DIG). The probe is for example 8 nucleotides to 30 nucleotides long, e.g., 12 nucleotides long or 15 nucleotides long. Other potential modifications of probes are described herein.

The probe when hybridized to the miRNA or precursor thereof may or may not provide a substrate for RNAse H. Preferably, the probes of the invention exhibit increased binding affinity for the target sequence by at least two-fold, e.g., at least 5-fold or 10-fold, compared to probes of the same sequence without nucleotide analogue monomers, under the same conditions for hybridization, e.g., stringent conditions or high stringency conditions.

In one aspect, oligonucleotide probes may be designed as reverse complementary sequences to the loop-region of the pre-miRNA. In this embodiment, a stretch of 25 nucleotides are identified centered around the loop-region and a capture probe is designed for this 25-mer sequence using the same design rules as for capture probes for the mature miRNAs. This design process takes into account predictions of T_m of the capture probe, self-hybridization of the capture probe to it-self and intra-molecular secondary structures and the difference between T_m and self-hybridization T_m. Further criteria to the capture probe design, includes that, in one embodiment, LNA-residues are not allowed in the 3'-end to enhance synthesis yield. Inter-probe comparison of capture probes against different miRNAs ensure that capture probes are designed against regions of the miRNAs that differ the most from other miRNAs in order to optimize the discrimination between different miRNAs.

Each detection probe comprises a recognition sequence consisting of nucleobases or equivalent molecular entities. The recognition sequence of the diagnostic probe according to the invention corresponds to the target nucleotide sequence or sequences as referred to herein, and typically comprises of a contiguous sequence which corresponds to a contiguous nucleotide sequence present in the microRNA target sequence. The length of the contiguous nucleobase sequence may be as short as 6 nucleotides, such as 6 or 7 nucleobases, or may represent a nucleobase sequence which corresponds to the majority or even full length of the microRNA target. In one embodiment, the detection probe or probes are capable of specifically hybridising to the precursor form of the miRNA, but are not capable of specifically hybridising to the mature form of the miRNA. Suitable detection probes are routinely designed and made utilising the sequence information available, e.g. by selecting a detection probe which at least partially hybridises to the loop structure which is cleaved during miRNA processing. It should be noted that several mature miRNAs may originate from more than one precursor, hence by designing specific probes for a particular precursor, highly specific detection probes for use in the invention may be used.

It will be understood that whilst in the preferred embodiment the target sequence are the miRNA or pre-miRNA precursors themselves, in one embodiment, the target sequence may be a further nucleotide or nucleobase sequence which retains the sequence information from the corresponding miRNA/pre-miRNA.

The detection element of the detection probes according to the invention may be single or double labelled (e.g. by comprising a label at each end of the probe, or an internal position). In one aspect, the detection probe comprises two labels capable of interacting with each other to produce a signal or to modify a signal, such that a signal or a change in a signal may be detected when the probe hybridizes to a target sequence. A particular aspect is when the two labels comprise a quencher and a reporter molecule.

A particular detection aspect of the invention referred to as a "molecular beacon with a stem region" is when the recognition segment is flanked by first and second complementary hairpin-forming sequences which may anneal to form a hairpin. A reporter label is attached to the end of one complementary sequence and a quenching moiety is attached to the end of the other complementary sequence. The stem formed when the first and second complementary sequences are hybridized (i.e., when the probe recognition segment is not hybridized to its target) keeps these two labels in close proximity to each other, causing a signal produced by the reporter to be quenched by fluorescence resonance energy transfer (FRET). The proximity of the two labels is reduced when the probe is hybridized to a target sequence and the change in proximity produces a change in the interaction between the labels. Hybridization of the probe thus results in a signal (e.g. fluorescence) being produced by the reporter molecule, which can be detected and/or quantified.

Preferably, the compound of the invention, such as the detection probes of the invention, are modified in order to increase the binding affinity of the probes for the target sequence by at least two-fold compared to probes of the same sequence without the modification, under the same conditions for hybridization or stringent hybridization conditions. The preferred modifications include, but are not limited to, inclusion of nucleobases, nucleosidic bases or nucleotides that have been modified by a chemical moiety or replaced by an analogue to increase the binding affinity. The preferred modifications may also include attachment of duplex-stabilizing agents e.g., such as minor-groove-binders (MGB) or intercalating nucleic acids (INA). Additionally, the preferred modifications may also include addition of non- discriminatory bases e.g., such as 5-nitroindole, which are capable of stabilizing duplex formation regardless of the nucleobase at the opposing position on the target strand. Finally, multi-probes composed of a non-sugar-phosphate backbone, e.g. such as PNA, that are capable of binding sequence specifically to a target sequence are also considered as a modification. All the different binding affinity-increasing modifications mentioned above will in the following be referred to as "the stabilizing modification(s)", and the tagging probes and the detection probes will in the following also be referred to as "modified oligonucleotide". More preferably the binding affinity of the modified oligonucleotide is at least about 3-fold, 4- fold, 5-fold, or 20-fold higher than the binding of a probe of the same sequence but without the stabilizing modification(s).

Most preferably, the stabilizing modification(s) is inclusion of one or more LNA nucleotide analogs. Probes from 8 to 30 nucleotides according to the invention may comprise from 1 to 8 stabilizing nucleotides, such as LNA nucleotides. When at least two LNA nucleotides are included, these may be consecutive or separated by one or more non-LNA nucleotides. In one aspect, LNA nucleotides are alpha-L-LNA and/or xylo LNA nucleotides as disclosed in PCT Publications No. WO 2000/66604 and WO 2000/56748..

In a preferable embodiment, each detection probe preferably comprises affinity enhancing nucleobase analogues, and wherein the recognition sequences exhibit a combination of high melting temperatures and low self-complementarity scores, said melting temperatures being the melting temperature of the duplex between the recognition sequence and its complementary DNA or RNA sequence.

This design provides for probes which are highly specific for their target sequences but which at the same time exhibits a very low risk of self-annealing (as evidenced by a low self- complementarity score) - self-annealing is, due to the presence of affinity enhancing nucleobases (such as LNA monomers) a problem which is more serious than when using conventional deoxyribonucleotide probes.

In one embodiment the recognition sequences exhibit a melting temperature (or a measure of melting temperature) corresponding to at least 5°C higher than a melting temperature or a measure of melting temperature of the self-complementarity score under conditions where the probe hybridizes specifically to its complementary target sequence (alternatively, one can quantify the "risk of self-annealing" feature by requiring that the melting temperature of the probe-target duplex must be at least 5°C higher than the melting temperature of duplexes between the probes or the probes internally).

In a preferred embodiment all of the detection probes include recognition sequences which exhibit a melting temperature or a measure of melting temperature corresponding to at least 5°C higher than a melting temperature or a measure of melting temperature of the self- complementarity score under conditions where the probe hybridizes specifically to its complementary target sequence.

However, it is preferred that this temperature difference is higher, such as at least 10⁰C, such as at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, and at least 50⁰C higher than a melting temperature or measure of melting temperature of the self-complementarity score.

In one embodiment, the affinity-enhancing nucleobase analogues are regularly spaced between the nucleobases in said detection probes. One reason for this is that the time needed for adding each nucleobase or analogue during synthesis of the probes of the invention is dependent on whether or not a nucleobase analogue is added. By using the

"regular spacing strategy" considerable production benefits are achieved. Specifically for LNA nucleobases, the required coupling times for incorporating LNA amidites during synthesis may exceed that required for incorporating DNA amidites. Hence, in cases involving simultaneous parallel synthesis of multiple oligonucleotides on the same instrument, it is advantageous if the nucleotide analogues such as LNA are spaced evenly in the same pattern as derived from the 3'-end, to allow reduced cumulative coupling times for the synthesis. The affinity enhancing nucleobase analogues are conveniently regularly spaced as every 2^nd, every 3^rd, every 4^th or every 5^th nucleobase in the recognition sequence, and preferably as every 3^rd nucleobase. Therefore, the affinity enhancing nucleobase analogues may be spaced at a mixture of, for example every 2^nd, every 3^rd, every 4^th nucleobase.

The presence of the affinity enhancing nucleobases in the recognition sequence preferably confers an increase in the binding affinity between a probe and its complementary target nucleotide sequence relative to the binding affinity exhibited by a corresponding probe, which only include nucleobases. Since LNA nucleobases/monomers have this ability, it is preferred that the affinity enhancing nucleobase analogues are LNA nucleobases.

In some embodiments, the 3' and 5' nucleobases are not substituted by affinity enhancing nucleobase analogues. As detailed herein, one huge advantage of such probes for use in the method of the invention is their short lengths which surprisingly provides for high target specificity and advantages in detecting small RNAs and detecting nucleic acids in samples not normally suitable for hybridization detection strategies. It is, however, preferred that the probes comprise a recognition sequence is at least a 6-mer, such as at least a 7-mer, at least an 8-mer, at least a 9-mer, at least a 10-mer, at least an 11-mer, at least a 12-mer, at least a 13-mer, at least a 14-mer, at least a 15-mer, at least a 16-mer, at least a 17-mer, at least an 18-mer, at least a 19-mer, at least a 20-mer, at least a 21-mer, at least a 22-mer, at least a 23-mer, and at least a 24-mer. On the other hand, the recognition sequence is preferably at most a 25-mer, such as at most a 24-mer, at most a 23-mer, at most a 22-mer, at most a 21-mer, at most a 20-mer, at most a 19-mer, at most an 18-mer, at most a 17-mer, at most a 16- mer, at most a 15-mer, at most a 14-mer, at most a 13-mer, at most a 12-mer, at most an 11-mer, at most a 10-mer, at most a 9-mer, at most an 8-mer, at most a 7-mer, and at most a 6-mer. In one embodiment, such as for use in arrays, such as micro-arrays, the preferred length is between 15mer-23mer, including collections of detection probes which may comprise or consist of 15mer, 16, 17, 18, 19, 20, 21, 22 and 23mers, or mixtures thereof.

In a preferred embodiment the number of nucleoside analogue corresponds to from 20 to 40% of the oligonucleotide of the invention.

In a preferred embodiment the probe sequences are substituted with a nucleoside analogue with regular spacing between the substitutions

In another preferred embodiment the probe sequences are substituted with a nucleoside analogue with irregular spacing between the substitutions

In a preferred embodiment the nucleoside analogue is LNA.

In a further preferred embodiment the detection probe sequences comprise a photochemically active group, a thermochemically active group, a chelating group, a reporter group, or a ligand that facilitates the direct of indirect detection of the probe or the immobilisation of the oligonucleotide probe onto a solid support.

In a further preferred embodiment: (a) the photochemically active group, the thermochemically active group, the chelating group, the reporter group, or the ligand includes a spacer (K), said spacer comprising a chemically cleavable group; or

(b) the photochemically active group, the thermochemically active group, the chelating group, the reporter group, or the ligand is attached via the biradical of at least one of the LNA(s) of the oligonucleotide.

Methods for defining and preparing probes and probe collections are disclosed in PCT/DK2005/000838.

In another aspect, the invention features detection probes whose sequences have been furthermore modified by Selectively Binding Complementary (SBC) nucleobases, i.e. modified nucleobases that can make stable hydrogen bonds to their complementary nucleobases, but are unable to make stable hydrogen bonds to other SBC nucleobases. Such SBC monomer substitutions are especially useful when highly self-complementary detection probe sequences are employed. As an example, the SBC nucleobase A', can make a stable hydrogen bonded pair with its complementary unmodified nucleobase, T. Likewise, the SBC nucleobase T' can make a stable hydrogen bonded pair with its complementary unmodified nucleobase, A. However, the SBC nucleobases A' and T' will form an unstable hydrogen bonded pair as compared to the base pairs A'-T and A-T'. Likewise, a SBC nucleobase of C is designated C and can make a stable hydrogen bonded pair with its complementary unmodified nucleobase G, and a SBC nucleobase of G is designated G' and can make a stable hydrogen bonded pair with its complementary unmodified nucleobase C, yet C and G' will form an unstable hydrogen bonded pair as compared to the base pairs C-G and C-G'. A stable hydrogen bonded pair is obtained when 2 or more hydrogen bonds are formed e.g. the pair between A' and T, A and T', C and G', and C and G. An unstable hydrogen bonded pair is obtained when 1 or no hydrogen bonds is formed e.g. the pair between A' and T', and C and G'. Especially interesting SBC nucleobases are 2,6-diaminopurine (A', also called D) together with 2-thio-uracil (U', also called 2SU)(2-thio-4-oxo-pyrimidine) and 2-thio-thymine (T', also called 2ST)(2-thio-4-oxo-5-methyl-pyrimidine).

In another aspect, the detection probe sequences of the invention are covalently bonded to a solid support by reaction of a nucleoside phosphoramidite with an activated solid support, and subsequent reaction of a nucleoside phosphoramide with an activated nucleotide or nucleic acid bound to the solid support. In some embodiments, the solid support or the detection probe sequences bound to the solid support are activated by illumination, a photogenerated acid, or electric current. In other embodiments the detection probe sequences contain a spacer, e.g. a randomized nucleotide sequence or a non-base sequence, such as hexaethylene glycol, between the reactive group and the recognition sequence. Such covalently bonded detection probe sequence populations are highly useful for large-scale detection and expression profiling of mature miRNAs and stem-loop precursor miRNAs.

Collection of probes of the invention

In one embodiment a collection of (detection) probes according to the present invention comprises at least 10 detection probes, 15 detection probes, such as at least 20, at least 25, at least 50, at least 75, at least 100, at least 200, at least 500, at least 1000, and at least 2000 members.

In one embodiment, the collection of probes according to the present invention consists of no more than 500 detection probes, such as no more than 200 detection probes, such as no more than 100 detection probes, such as no more than 75 detection probes, such as no more than 50 detection probes, such as no more that 50 detection probes, such as no more than 25 detection probes, such as no more than 20 detection probes.

In one embodiment, the collection of probes according to the present invention has between 3 and 100 detection probes, such as between 5 and 50 detection probes, such as between 10 and 25 detection probes.

In one embodiment, the affinity-enhancing nucleobase analogues are regularly spaced between the nucleobases in at least 80% of the members of said collection, such as in at least 90% or at least 95% of said collection (in one embodiment, all members of the collection contains regularly spaced affinity-enhancing nucleobase analogues).

In one embodiment of the collection of probes, all members contain affinity enhancing nucleobase analogues with the same regular spacing in the recognition sequences.

Also for production purposes, it is an advantage that a majority of the probes in a collection are of the same length. In preferred embodiments, the collection of probes of the invention is one wherein at least 80% of the members comprise recognition sequences of the same length, such as at least 90% or at least 95%.

As discussed above, it is advantageous, in order to avoid self-annealing, that at least one of the nucleobases in the recognition sequence is substituted with its corresponding selectively binding complementary (SBC) nucleobase. Typically, the nucleobases in the sequence are selected from ribonucleotides and deoxyribonucleotides, preferably deoxyribonucleotides. It is preferred that the recognition sequence consists of affinity enhancing nucleobase analogues together with either ribonucleotides or deoxyribonucleotides.

In certain embodiments, each member of a collection is covalently bonded to a solid support. Such a solid support may be selected from a bead, a microarray, a chip, a strip, a chromatographic matrix, a microtiter plate, a fiber or any other convenient solid support generally accepted in the art.

The collection may be so constituted that at least 90% (such as at least 95%) of the recognition sequences exhibit a melting temperature or a measure of melting temperature corresponding to at least 5°C higher than a melting temperature or a measure of melting temperature of the self-complementarity score under conditions where the probe hybridizes specifically to its complementary target sequence (or that at least the same percentages of probes exhibit a melting temperature of the probe-target duplex of at least 5°C more than the melting temperature of duplexes between the probes or the probes internally).

As also detailed herein, each detection probe in a collection of the invention may include a detection moiety and/or a ligand, optionally placed in the recognition sequence but also placed outside the recognition sequence. The detection probe may thus include a photochemically active group, a thermochemically active group, a chelating group, a reporter group, or a ligand that facilitates the direct of indirect detection of the probe or the immobilisation of the oligonucleotide probe onto a solid support.

Arrays

The collection of detection probes may be in the form of an array or micro-array to which (optionally labeled) miRNA fraction is applied. As described herein a miRNA fraction obtained from one or more control samples may be applied to the same array (for example using a different label for the control fraction), or an equivalent array, so that the relative abundance of miRNAs present in each of the fractions may be obtained.

Arrays may be in the form of micro-arrays, bioarrays, biochips, biochip arrays etc - these are defined in US 2006/0160114. Hybridisation

Hybridisation refers to the bonding of two complementary single stranded nucleic acid polymers (such as oligonucleotides), such as RNA, DNA or polymers comprising or consisting of nucleotide analogues (such as LNA oligonucleotides). Hybridisation is highly specific, and may be controlled by regulation of the concentration of salts and temperature. Hybridisation occurs between complementary sequences, but may also occur between sequences which comprise some mismatches. The probes used in the methods of the present invention may, therefore be 100% complementary to the target molecule. Alternatively, in one embodiment the detection probes may comprise one or two mismatches. Typically a single mismatch will not unduly affect the specificity of binding, however two or more mismatches per 8 nucleotide/nucleotide residues usually prevents specific binding of the detection probe to the target species. The position of the mismatch may also be of importance, and as such the use of mismatches may be used to determine the specificity and strength of binding to target RNAs, or to allow binding to more than one allelic variant of mutation of a target species.

In one embodiment, the detection probe consists of no more than 1 mismatch with the miRNA target.

In one embodiment, the detection probe consists of no more than 1 mismatch per 8 nucleotide/nucleotide analogue bases.

In one embodiment, hybridisation may also occur between a single stranded target molecule, such as a miRNA and a probe which comprises a complementary surface to the said target molecule, in this respect, it is the ability of the probe to form the specific bonding pattern with the target which is important.

Suitable methods for hybridisation include RNA in-situ hybridisation, dot blot hybridisation, reverse dot blot hybridisation, northern blot analysis, RNA protection assays, or expression profiling by microarrays. Such methods are standard in the art.

In one embodiment, the detection probe is capable of binding to the target non coding RNA sequence under stringent conditions, or under high stringency conditions.

Exiqon (Denmark) provide microarrays suitable for use in the methods of the invention (microRNA Expression Profiling with miRCURY™ LNA Array). The detection probe, such as each member of a collection of detection probes, may be bound (such as conjugated) to a bead. Luminex (Texas, USA) provides multiplex technology to allow the use of multiple detection probes to be used in a single hybridisation experiment.. See also Panomics QuantigenePlexTM (http://www.panomics.com/pdf/qgplexbrochure.pdf).

Suitable techniques for performing in situ hybridisation are disclosed in PCT/DK2005/000838

PCR Hybridisation

Whilst it is recognised that many of the short non-coding RNAs which are targets for the detection probes are too short to be detected by amplification by standard PCR, methods of amplifying such short RNAs are disclosed in WO2005/098029. Therefore, the hybridisation may occur during PCR, such as RT-PCT or quantitative PCR (q-PCR).

EXAMPLES

Example 1 :

Methods

Tissue

140 fresh frozen colorectal cancer samples (provided from Oncotech Incs. Biobank), were used for the study. The samples were characterized with respect to their drug resistance profile by the EDR® assay. In addition, the following metadata are available: age and sex of the donor, histology of the tumor, site of metastasis, prior treatment, pathology description, and - for some of the samples: tumor markers (genetic markers), including CD31, BAX, BCL- 2, EGFR, ER receptor, HER2, Ki-67, MDR-I, p53, PR receptor, Thrombospondin 1, Thymidylate Synthase, and VEGV.

EDR® assay

The EDR® assay was performed on the cancer samples by Oncotech Inc. using their commercially available service. For each tumor sample, cells were analyzed for their EDR status to the following drugs: 5FU+LEUCOVORIN (FULEU), IRINOTECAN (SN38),

OXALIPLATIN (OXAPL) and TOPOTECAN (TOPO). The status of EDR, IDR or LDR was calculated based on the method descried in US2006/0160114 (Example 1) : The EDR assay is an agarose-based culture system, using tritiated thymidine incorporation to define in vitro drug response. This assay is predictive of clinical response (Kern et al., 1990, "Highly specific prediction of antineoplastic resistance with an in vitro assay using suprapharmacologic drug exposures," J. Nat. Cancer Inst. 82: 582-588). Tumors are cut with scissors into pieces of 2 mm or smaller in a Petri dish containing 5 ml_ of complete medium. The resultant slurries are mixed with complete media containing 0.03% DNAase (2650 Kunitz units/ml_) and 0.14% collagenase I (both enzymes obtained from Sigma Chemical Co., St. Louis, Mo.), placed into 50 ml_ Erlenmeyer flasks with stirring, and incubated for 90 min at 37. degree. C. under a humidified 5% CO. sub.2 atmosphere. After enzymatic dispersion into a near single cell suspension, tumor cells are filtered through nylon mesh, and washed in complete media. A portion of the cell suspension is used for cytospin slide preparation and stained with Wright-Giemsa for examination by a medical pathologist in parallel with Hematoxylin-Eosin stained tissue sections to confirm the diagnosis and to determine the tumor cell count and viability. Tumor cells are then suspended in soft agarose (0.13%) and plated at 20,000-50,000 cells per well onto an agarose underlayer (0.4%) in 24-well plates. Tumor cells are incubated under standard culture conditions for 4 days in the presence or absence of the cancer treatment, which is typically dosed at between 5 and 80 times the in vivo dosage. For example 2.45 .mu.M paclitaxel may be used. Cells are pulsed with tritiated thymidine (New Life Science Products, Boston, Mass.) at 5 .mu.Ci per well for the last 48 hours of the culture period. After labeling, cell culture plates are heated to 96. degree. C. to liquify the agarose, and the cells are harvested with a micro-harvester (Brandel, Gaithersburg, Md.) onto glass fiber filters. The radioactivity trapped on the filters is counted with an LS-6500 scintillation Counter (Beckman, Fullerton, Calif.). Untreated cells served as a negative control. In the positive (background) control group, cells are treated with a supratoxic dose of Cisplatin (33 .mu.M), which causes 100% cell death. Detectable radioactivity for this group is considered non-specific background related to debris trapping of tritiated thymidine on the filter. After subtracting background control values, percent cell inhibition (PCI) of proliferation is determined by comparing thymidine incorporation by the treatment group with incorporation by the negative control group: PCI = 100%. times. [1-(CPM treatment group/CPM control group)]. The determinations of cancer treatment effects on tumor proliferation is performed in duplicate. With respect to paclitaxel : Specimens were classified as EDR to the cancer treatment if the PCI was 19% or less; Specimens were classified as LDR to the cancer treatment if the PCI was 43% or greater: A PCI of above 19% but below 43% is indicative of an intermediate drug resistance (IDR). RNA extraction

The RNA extraction was performed at Oncotech (Tustin, CA) by a standard Trizol extraction method. A RNA reference pool consisting of RNA from all colon samples was made and aliquots of the reference stock were stored at -8O⁰C for later use in the hybridization.

RNA labeling and hybridization

The instructions detailed in the "miRCURY™ LNA microRNA, Hy3™/Hy5™ Power labeling kit" were followed (Exiqon, Denmark). All kit reagents were thawed on ice for 15 min, vortexed and spun down for 10 min.

For hybridization, the 12-chamber TECAN HS4800Pro hybridization station was used. 25 μl_ 2x hybridization buffer was added to each sample, vortexed and spun.

Samples were incubated at 95 ⁰C for 3 min before injection into the hybridization chamber, which was primed with lOOul of Ix Hybridization buffer. 50 μl of the target preparation was injected into the Hybridization station and incubated at 56 ⁰C for 16 hours (overnight). For the analysis of the colon cancer samples, the miRCURY™ LNA microRNA array version 9.2 was used.

The slides were washed at 60 ⁰C for 1 min with Buffer A twice, at 23 ⁰C for 1 min with Buffer B twice, at 23 ⁰C for 1 min with Buffer C twice, at 23 ⁰C for 30 sec with Buffer C once. The slides were dried for 5 min.

Slides were scanned using the DNA Microarray Scanner (Agilent) at 100% PMT setting.

Image analysis and data preprocessing

Image analysis and spot identification was done using Imagene 7.0.0 software (Biodiscovery). Within slide normalized was done with a Lowess smooth fitting using Genesight 4.1.6 Lite edition software (Biodiscovery).

Ratios of tumor/reference pool were calculated and Iog2 transformed from the Lowess normalized data sets. Raw values (Lowess normalized values) were further treated and median scaled for comparison between samples. Results

Array expression results were combined with data provided by Oncotech. The patients were divided into groups based on their EDR status to the drugs, see table Ia -table Id .

The Iog2 ratios between the common reference and the sample channel for each patient were used in the statistical evaluation of the data set.

Significant difference between the LDR and the EDR patient groups for each drug were evaluated in the MeV software package (Tigr) using the Welch approximation t-test with a p- value cutoff of 0.01. See table 2 for group mean Iog2 ratios, standard deviation and p-values.

Table Ia: EDR status for 5FU + Table Ib: EDR status for Oxaliplatin

Leucoverin

5FU+LEUCOVORIN (FULEU) OXALIPLATIN (OXAPL)

EDR IDR LDR NA EDR IDR LDR NA

1 14 11 43 17 2 4 1

2 15 12 44 18 6 28 3

3 16 13 45

19 7 29 5

4 26 21 46

5 27 22 50 20 8 30 51

6 58 23 51 42 9 47

7 60 24 84 14 10 81

8 66 25 124 15 41 109

9 67 31 16 48 110

10 68 32 11 49 122

17 76 33 12 83 123

18 77 34

13 135 58

19 78 35

20 79 36 43 136 66

28 82 37 44 26 67

29 86 38 45 27 76

30 103 39 46 60 77

41 104 40 68 78

42 106 52 82 79

47 107 53 86 103

48 108 54

132 104

49 118 55

133 106

81 119 56

83 120 57 134 107

109 132 59 25 108

110 133 62 85 118

122 134 63 128 119

123 64 129 120

135 65 130 21

136 69 131 22

70

- 137 23

/JA1

72 84 24

73 31

74 32

127

138

139

140

50

124

Table Ic: EDR status for Irinotecan. Table Id: EDR status for Topotecan.

IRINOTECAN (SN38) TOPOTECAN (TOPO)

EDR IDR LDR NA EDR IDR LDR NA

7 1 4 13 5 3 4 1

12 2 5 46 7 6 31 2

16 3 8 12 8 32 10

17 6 10 14 9 33 26

21 9 14 17 11 34 44

22 11 31 18 13 35 46

23 15 32 21 15 36 54

24 18 33 22 16 37 57

25 19 34 23 19 38 63

26 20 35 24 20 39 66

27 51 36 25 40 52 79

28 57 37 27 43 53 84

29 66 38 28 45 55 101

30 80 39 29 51 56 104

41 81 40 30 59 58 106

42 85 43 41 68 60 113

47 111 44 42 70 62 115

48 112 45 47 72 64 124

49 113 52 48 77 65 131

50 114 53 49 81 69 136

59 115 54 50 83 71

67 116 55 67 91 73

86 117 56 80 94 74

125 118 58 85 108 75

126 119 60 86 112 76

127 120 62 110 114 78

137 122 63 111 116 82

123 64 119 117 87

124 65 125 120 88

134 68 126 122 89

135 69 134 123 90

136 70 137 127 92

71 135 93

72 95

73 96

74 97

75 98

76 99

77 100

78 102

79 103

82 107

83 109

84 118

87 128

88 129

References of statistic methods used (and those which maybe used to determine correlation between miRNA abundance and the resistance/susceptibility phenotype) include: Pan, W. (2002). A comparative review of statistical methods for discovering differentially expressed genes in replicated microarray experiments. Bioinformatics 18: 546-554. Dudoit, S., Y. H. Yang, M.J. Callow, and T. Speed (2000). Statistical methods for identifying differentially expressed genes in replicated cDNA microarray experiments. Technical report 2000 Statistics Department, University of California, Berkeley. Welch B. L. (1947). The generalization of 'students' problem when several different population variances are involved. Biometπka 34: 28-35.

Table 2a: Significant miRs correlating with 5-FU + Leucoverin treatment, with a p-value below < 0.01

LDR group EDR group

Gene ID Gene name mean LDR group SD mean EDR gorup SD P-value

14270 hsa-mιR-493 -0.12087378 0.13748857 -0.23858322 0.106658444 2.93E-04

28737 hsa-mιR-106a -0.25965062 0.7517461 0.57990074 1.0910594 4.15E-04

10983 hsa-mιR-18a -0.04170616 0.8583468 0.6970638 0.9175219 4.18E-04

28736 hsa-mιR-17 -0.21722881 0.7927852 0.5705118 1.052369 6.32E-04

11046 hsa-mιR-302d 0.03991232 0.21820264 0.167608 0.133205 6.56E-04

19588 hsa-mιR-17* 0.05164243 0.42490336 0.47200006 0.57181734 7.62E-04

13141 hsa-mιR-18b -0.052204568 0.9031924 0.65412796 0.92020637 8.14E-04

17605 hsa-mιR-17 -0.17557734 0.7533504 0.57213145 1.0368525 8.94E-04

17842 hsa-mιR-141* 0.17953378 0.24016319 0.34830788 0.21991517 0.00108844

28306 hsa-mιR-18b -0.08910116 0.31882086 0.25962916 0.5111618 0.00133796

10998 hsa-mιR-19b -0.21664423 0.718152 0.39072233 0.8648763 0.00144276

4890 hsa-mιR-19b -0.25110206 0.8295765 0.45287785 1.0037196 0.00144605

32789 hsa-mιR-92a -0.13529636 0.54783964 0.42371508 0.8264743 0.00151315 ROC_hsa_mιR_ 29878 659 0.06723125 0.1792964 0.16400538 0.11434289 0.00162669

19582 hsa-mιR-106b -0.028644452 0.6250246 0.56323 0.8935187 0.00196973

30744 hsa-mιR-93 -0.025904056 0.63506645 0.49398676 0.7961723 0.00270272

29075 hsa-mιR-92a -0.09721776 0.5457547 0.41435128 0.8313235 0.00358436

11085 hsa-mιR-373 -0.107961446 0.24108706 0.03884338 0.2150072 0.00361006

11008 hsa-mιR-20a -0.18985485 0.806732 0.64018184 1.3670176 0.00361265

10973 hsa-mιR-181c 0.17394622 0.18051714 0.26625818 0.13045354 0.00504357 ROS_hsa_mιR_ _1 17941 751 -0.14569074 0.22778334 -0.011749594 0.20605512 0.00526174 ROS hsa miR _1 17844 733 -0.07725674 0.1915418 0.039923687 0.18295868 0.00528945

30687 hsa-mιR-93 -0.06439966 0.68153197 0.44623673 0.8485056 0.00536037

11009 hsa-mιR-20b -0.062297516 0.716613 0.6441098 1.2282075 0.00551093

17814 hsa-mιR-570 0.053167455 0.12594776 -0.02559247 0.122627184 0.00580999

17942 hsa-mιR-125a-3p 0.14667146 0.28624678 0.3351377 0.31168753 0.00635329

10999 hsa-mιR-20a -0.16656806 1.0596209 0.69216275 1.4882951 0.00643683

32790 hsa-mιR-577 -0.037342325 0.084247775 0.009910884 0.050149333 0.00717543

17815 hsa-mιR-32* 0.15732768 0.45056084 0.40403193 0.3939907 0.00763652 ROS hsa miR 17920 788 -0.040444426 0.11375556 -0.11041333 0.10292392 0.00835223

10997 hsa-mιR-19a -0.04032427 0.5635257 0.35684824 0.7110541 0.00922297

13169 hsa-let-7d* -0.029959101 0.18393375 0.0772111 0.17641649 0.00965058

17718 hsa-mιR-92b -0.06361756 0.54466707 0.30408487 0.6595294 0.00980337 Table 2b: Significant miRs correlating with Irinotecan treatment, with a p-value below < 0.01

LDR group LDR group EDR group

Gene ID Gene name mean SD mean EDR gorup SD P-value

10916 hsa-mιR-1 0.77763534 0.33991203 0.56926954 0.1802114 1.59E-04

19582 hsa-mιR-106b -0.06426275 0.5268652 0.5079194 0.8482499 0.00290585

17854 hsa-mιR-106b* 0.12352065 0.24824806 0.44979736 0.46377042 0.00180838

33902 hsa-mιR-128a -0.075571015 0.22517243 0.14625849 0.3114326 0.00203365

10936 hsa-mιR-130b -7.08E-04 0.26540765 0.25848272 0.39810115 0.00409097

10944 hsa-mιR-137 0.15217371 0.12242772 0.09018547 0.07625988 0.00411212

17842 hsa-mιR-141* 0.17790957 0.25485557 0.40134606 0.21073446 5.29E-05

10947 hsa-mιR-142-3p 0.021595888 1.2176237 -0.9775752 1.0569683 2.15E-04

19015 hsa-mιR-142-5p 0.0365269 1.0423985 -0.58619034 0.7501718 0.00170724

30532 hsa-mιR-142-5p -0.013959016 1.1323816 -0.64802456 0.7702658 0.00226742

10952 hsa-mιR-146a 0.2166745 1.1216568 -0.68924385 0.69501364 7.67E-06

19585 hsa-mιR-148b 0.073782355 0.31212014 0.5450871 0.6668622 0.0016786

11029 hsa-mιR-25 -0.16037025 0.38535827 0.31024858 0.5674029 4.10E-04

17886 hsa-mιR-301b 0.05283704 0.25964192 0.4127879 0.513555 0.00184484

11042 hsa-mιR-302a* -0.09248377 0.18790115 0.033270925 0.16025402 0.00220347

11057 hsa-mιR-324-5p 0.008601506 0.34405145 0.27492774 0.44930562 0.00917226

19597 hsa-mιR-339-5p 0.14005728 0.14347395 0.25753444 0.13209043 3.89E-04

11062 hsa-mιR-33a 0.011741081 0.27766842 0.20240559 0.31401855 0.00898758

11070 hsa-mιR-345 -0.14135224 0.24566658 0.1708913 0.3496936 1.86E-04

14279 hsa-mιR-362-5p 0.02983355 0.22976543 0.16127475 0.2080551 0.00959146

14306 hsa-mιR-381 -0.26551214 0.34544072 -0.049256727 0.34855416 0.00897883

11119 hsa-mιR-485-5p 0.15451413 0.18737216 0.30052584 0.2196321 0.0043463

11128 hsa-mιR-496 0.28177723 0.1252025 0.21704341 0.07304293 0.00231952

17875 hsa-mιR-500 -0.06861512 0.34561023 0.22242683 0.39444116 0.00184423

11174 hsa-mιR-525-3p 0.29140633 0.17058447 0.1674787 0.12900077 2.78E-04

17624 hsa-mιR-532-5p 0.30059958 0.27331957 0.5442324 0.37290162 0.00427869

0.11842240

17661 hsa-mιR-568 0.31007457 4 0.24259353 0.08594044 0.00281934

17551 hsa-mιR-572 -0.057059955 0.22110763 -0.22778186 0.22829108 0.0018803

17546 hsa-mιR-585 0.30439517 0.19869798 0.2028082 0.121461965 0.00293174

17281 hsa-mιR-652 0.046010256 0.11321534 0.111277856 0.08553584 0.00336064

28903 hsa-mιR-652 -0.19018562 0.21743625 0.03479345 0.31444812 0.0018987

17830 hsa-mιR-885-5p -0.08926926 0.35408795 0.16398287 0.4073686 0.00747845

29075 hsa-mιR-92a -0.08083457 0.5815456 0.2875719 0.5559019 0.00690151

32789 hsa-mιR-92a -0.12011061 0.601878 0.2759601 0.6033213 0.00599536

30687 hsa-mιR-93 -0.16678078 0.5964371 0.53713727 0.9110124 8.54E-04

ROC hsa miR 1

17945 622 -0.06684745 0.36790675 0.24644719 0.3950778 0.00100009

R0C_hsa_mιR_l

17865 627 -0.046196476 0.51313365 0.29407766 0.3590124 4.48E-04

ROC hsa miR 1

17867 666 0.056875255 0.4736647 -0.22033007 0.40087768 0.00552184

R0C_hsa_mιR_l

28454 926 -0.15733054 0.41037786 0.09879063 0.3929407 0.0068177

ROS hsa miR 1

17956 714 -0.003149068 0.1821998 0.11993551 0.14826827 0.0011785

R0S_hsa_mιR_l

17912 732 0.07306917 0.15672445 0.14596099 0.095977604 0.00688182

ROS hsa miR 1

17843 763 0.28040224 0.2681863 0.40189588 0.14460008 0.00474961 Table 2c: Significant miRs correlating with Oxliplatin treatment, with a p-value below < 0.01

LDR group EDR group

Gene ID Gene name mean LDR group SD mean EDR gorup SD P-value

17913 hsa-let-7ι* -0.054620873 0.0927563 -0.13173288 0.06427804 0.00209029

5250 hsa-mιR-105 0.077790774 0.15251537 0.18752326 0.07664361 5.32E-04

17908 hsa-mιR-130b* -0.029038746 0.09946102 -0.13437077 0.10299589 0.00548267

17808 hsa-mιR-132* 0.012678962 0.26620218 0.19422795 0.2051231 0.0062188

10973 hsa-mιR-181c 0.17685251 0.18048239 0.26255184 0.078478046 0.00360798

19589 hsa-mιR-188-5p 0.5969036 0.12941146 0.5060377 0.10087383 0.00538896

11006 hsa-mιR-205 -0.5776098 1.5289184 -1.6959511 1.2920785 0.00658798

11016 hsa-mιR-216a 0.039982833 0.09003997 -0.052337337 0.08440989 0.0034873

5930 hsa-mιR-302b* -0.029620422 0.106272355 -0.12564869 0.07667894 8.95E-04

11046 hsa-mιR-302d 0.03731503 0.22956164 0.21275383 0.115645245 1.64E-04

11108 hsa-mιR-425* 0.29698193 0.1538196 0.18558416 0.12540725 0.00577403

11173 hsa-mιR-523 0.25475132 0.10178255 0.15280531 0.11837648 0.00592835

17271 hsa-mιR-553 -0.059226505 0.1147735 -0.17760274 0.09536556 0.00217518

17312 hsa-mιR-592 0.6338867 0.35662082 0.42283052 0.25020498 0.00948382

27552 hsa-mιR-614 -0.045604423 0.10303213 -0.14483261 0.06939303 0.00142172

17552 hsa-mιR-617 0.16690554 0.35422328 -0.04486118 0.21749824 0.00401383

17507 hsa-mιR-662 -0.012053206 0.2709278 0.10643449 0.117346816 0.00683747

17852 hsa-mιR-99a* 0.06580131 0.33480698 0.37718573 0.33720094 0.00387817 ROS_hsa_mιR_17 17948 45 0.052548118 0.10509228 -0.034151092 0.06654849 0.00100892

Table 2d: Significant miRs correlating with Topotecan treatment, with a p-value below < 0.01

LDR group EDR group EDR gorup

Gene ID Gene name mean LDR group SD mean SD P-value

17913 hsa-let-7ι* -0.051925175 0.09785022 -0.11770348 0.0856046 0.00972364

19582 hsa-mιR-106b -0.06472175 0.47542363 0.49327573 0.77082795 7.29E-04

17854 hsa-mιR-106b* 0.11964023 0.25674516 0.40998656 0.4464785 0.00192647

17876 hsa-mιR-125b-l* 0.00682102 0.6493672 -0.34895742 0.5504748 0.0096425

33902 hsa-mιR-128a -0.07979879 0.20174992 0.09359044 0.311721 0.00811005

10936 hsa-mιR-130b 0.011829599 0.2777323 0.23575217 0.3963281 0.00807084

10944 hsa-mιR-137 0.16710702 0.11640862 0.09010733 0.09209577 0.00164048

10947 hsa-mιR-142-3p 0.15048276 1.258835 -0.64093196 1.0029911 0.0023211

19015 hsa-mιR-142-5p 0.11120251 1.0060761 -0.4742673 0.8885971 0.0073585

30532 hsa-mιR-142-5p 0.06874051 1.1090007 -0.5393403 0.9222733 0.00886881

10952 hsa-mιR-146a 0.23495117 1.138152 -0.4556541 0.6252534 6.17E-04

19585 hsa-mιR-148b 0.056911256 0.31465897 0.46272087 0.64627415 0.00227395

19587 hsa-mιR-150 0.40618795 0.2829017 0.2548239 0.15524131 0.0023325

17463 hsa-mιR-151-3p 0.051719647 0.36667547 0.49889556 0.84518033 0.00830546

10979 hsa-mιR-186 0.29665703 0.19004565 0.4531655 0.28137675 0.00858121

27535 hsa-mιR-187 -0.037033882 0.103537545 -0.112759225 0.08967753 0.00402328

10983 hsa-mιR-18a -0.071471214 0.5945133 0.44430032 0.891108 0.00626809

13141 hsa-mιR-18b -0.10231642 0.6147151 0.37961945 0.87195826 0.00953676

11029 hsa-mιR-25 -0.19505678 0.35924444 0.27585298 0.5594567 1.29E-04

5930 hsa-mιR-302b* -0.010361828 0.09245244 -0.09514926 0.10481537 0.0020344

11070 hsa-mιR-345 -0.12965858 0.25746018 0.1101012 0.3368612 0.00133451

14301 hsa-mιR-361-5p -0.004867047 0.4731807 0.30608046 0.48729348 0.00607797

11113 hsa-mιR-448 -0.11124658 0.07903573 -0.17288023 0.0785673 0.00761498

29421 hsa-mιR-453 -0.04414194 0.12620634 -0.13808687 0.08739536 0.0021681

11175 hsa-mιR-525-5p 0.23020154 0.5540268 -0.084468335 0.47502622 0.00777341

17624 hsa-mιR-532-5p 0.28661186 0.27860296 0.5429465 0.34280527 8.78E-04

17661 hsa-mιR-568 0.3240692 0.10477289 0.25474277 0.09391494 0.00270043

17814 hsa-mιR-570 0.05402727 0.122755006 -0.028221449 0.12850313 0.00642089

17551 hsa-mιR-572 -0.06802761 0.21898311 -0.20857853 0.21421659 0.00559569

17398 hsa-mιR-634 0.39476794 0.22987647 0.24424042 0.16010681 7.57E-04

28903 hsa-mιR-652 -0.18413326 0.2275585 0.005065979 0.29814655 0.00418991

17558 hsa-mιR-663 0.047168132 0.15274826 -0.08093758 0.18945104 0.00234824

17830 hsa-mιR-885-5p -0.075139865 0.36784968 0.17925335 0.41572967 0.00671865

29075 hsa-mιR-92a -0.11909539 0.527634 0.30074516 0.61070246 0.00269791

32789 hsa-mιR-92a -0.16587105 0.53762436 0.28691927 0.7006634 0.00318757

30687 hsa-mιR-93 -0.13994372 0.5768414 0.46723315 0.8902837 0.0014385

30744 hsa-mιR-93 -0.029199323 0.49266592 0.4361644 0.81353176 0.00687483 ROC_hsa_mιR_162 17945 2 -0.06373311 0.3531004 0.1829678 0.41716635 0.00791671 ROS hsa miR 173 17912 2 0.05313304 0.15043297 0.13923706 0.11258309 0.0044271

Example 2: Increasing evidence suggests that microRNAs play a key role in the initiation and progression of cancer, and therefore, may comprise a novel class of molecular biomarkers with prognostic and predictive potential.

Drug resistance is a major impediment to the successful chemotherapeutic treatment of cancer. Current tests assay the ex vivo growth of a tumor in the presence of chemotherapeutic drugs but new molecular tests are needed. We have analyzed 210 colorectal cancer specimens - both fresh frozen and FFPE - that had been previously tested in the Extreme Drug Resistance assay, a clinically validated drug resistance test. Each sample's microRNA expression pattern was assayed on an LNA (Locked Nucleic Acid) enhanced microarray platform, which allows for very sensitive and specific detection of small RNA targets like microRNAs.

This study demonstrates that the microRNA expression pattern of a tumor biopsy reflects the drug resistance status of the cancer. Our analysis enabled identification of tumors that were resistant to 5-FU/Leucovorin, Oxaliplatin, or Irinotecan, based solely on the tumor cell's microRNA profiles. Thus, we propose that a molecular Drug Resistance (mDR) assay, based on a tumor's microRNA profile can potentially provide a basis for prediction of drug response. Two advantages of such an mDR assay are that it requires very little amount of input RNA (down to 100 ng), and its fast (one day) response time.

METHODS

Samples

From Oncotech's tissue bank 210 colon cancer samples were collected from both fresh frozen (n = 140) and formalin fixed, paraffin embedded (FFPE) sections (n =70). The samples were all characterized with respect to their resistance status, i.e. low (LDR), intermediate (IDR) or extreme (EDR) drug resistance towards a panel of chemotherapeutic drugs, including 5- FU/Leucovorin, Irinotecan, and Oxaliplatin, as explained in Example 1.

In addition, immunohistochemical data for thymidilate synthase expression, P-glycoprotein expression, p53 mutation status, and DNA ploidity, were available for a number of the tumors.

RNA isolation

Fresh frozen samples: Total RNA was isolated by guanidinum isothiocyanate/phenol : chloroform. Formalin fixed, paraffin embedded (FFPE) samples: Total RNA was isolated by using the miRNEasy FFPE kit from Qiagen.

Microarray profiling

0.5 μg total RNA was analyzed for miRNA expression on miRCURY™ LNA Discovery arrays containing Tm normalized capture probes for miRNAs annotated in miRBase v 10.0 as well as human miRPIus™ sequences not yet annotated in miRBase and miRNAs discovered by 454 high throughput sequencing (PCT/EP2007/061210), as well as the corresponding pre- miRNAs. See table 3 for mature sequences of the detected miRNAs.

Table 3 : Mature sequences for the miRNAs detected in the miRCURY™ LNA Discovery array.

Mature miR name Mature sequence

454 hsa miR 1628 UUUCCGGCUCGCGUGGGUGUGU

454 hsa miR 1643 UUGGGGAAACGGCCGCUGAGUG

454 hsa miR 1966 CGAGCCUGGUGAUAGCUGGUUGUCCAAG

454 hsa miR 1967 GAGGCUGAGGCGAGAGGU

454 hsa miR 1968 AUUUGUGGCCGAGUGUAACAACC

454 hsa miR 1969 UCCCUUCGUGGUCGCC

454 hsa miR 1970 GCAGGGGAAGAAGCCUUCCGU

454 hsa miR 1971 ACUUUGAGAGUUAGAAAUGGUUACU

454 hsa miR 1972 AACCAAUGAUGUAAUGAUUCUGCC

454 hsa miR 1973 AUGAUUCUGUGACGCCAGCU

454 hsa miR 1974 GAAAGCUGAGCGUGAACGUGGU

454 hsa miR 1975 UAGCAGCACAUAAUGGUUUGAAU

454 hsa miR 1976 UGUUUAGACGGGCUCACAU

454 hsa miR 1977 GGAAAUUUGAGACCAGCAAGUACU

454 hsa miR 1978 AUUGUAUAUCAGCAUGGGGAUUAUU

454 hsa miR 1979 UUGGAGGCGUGGGUU

454 hsa miR 1980 AACGUGCAGCGGCUGAAGGAGU

454 hsa miR 1981 AAUUGUGAGGCUUGAGUGU

454 hsa miR 1982 UGAUAGGAUUGACAUGGAGCAC

454 hsa miR 1983 AACGGCCGCGGUACCCUAAC

454 hsa miR 1984 UCUUGCCAAGAGAAUUAAUGUGCGU

454 hsa miR 1985 AAUCUGAGUGAGAGGUUAGUUGCU

454 hsa miR 1986 AUUAAAAAUUUCGGUUGGG

454 hsa miR 1987 GAUAGCUGCCAGUGACAGGAGUAGU

454 hsa miR 1989 UGGCGCGGAGCGGAGCGG

454 hsa miR 1990 AGGCGGCGGAGGGGCG

454 hsa miR 1991 AUGCGUGCGAGAAGUCAGUGG

454 hsa miR 1992 UGUGUUUGAGAGCAACGCCAUUGCCU

454 hsa miR 1993 GCAUGAGUGGUUCAGUGGU

454 hsa miR 1994 AAGGAAUGAGUUAGCUUUG 454 hsa miR 1995 ACAAGUGAAUACACUGAGGC

454 hsa miR 1996 CUCGCGCCCGCGUCGCGGCAGC

454 hsa miR 1997 GUGGUGUUGAGGAAAGCAGAC

454 hsa miR 1998 GUUUUUGUGACUGCCUUGAGU

454 hsa miR 1999 AAGGCACAGCUGGAAAUGAUGGUG

454 hsa miR 2000 AAACUAGGCGGCUAUGGUAU

454 hsa miR 2001 GUGGUGGUCGUACGCUGUG

454 hsa miR 2002 GCUGAGUGAAGCAUUGGACUGU

454 hsa miR 2003 AGGUUCACAUGGAAAAGGUU

454 hsa miR 2004 CCAUUGUGUAGCAAGAUGUCAU

454 hsa miR 2005 GGAAAUAGUUUGUGACAAACUGGG

454 hsa miR 2006 CUCCUCUUCGUCUCCUCGGUC

454 hsa miR 2008 GCCGCGAGUGGGAGCGGGAGCG

454 hsa miR 2009 GCUUGACUGAGUGUGGCUGGACGUG

454 hsa miR 2010 AGUCGGUGCCUGAGGUUGC

454 hsa miR 2011 AGUGAUGAGGAUGUGCUGAU

454 hsa miR 2012 AUUGAGUGGGGCUCAGGAUU

454 hsa miR 2013 GCAUGGGUGGUUCAGUGU

454 hsa miR 2014 AACUGUUAUAUUAUGAUUGUGAC

454 hsa miR 2015 UUGCUGUGAUGACUAUCUUAGGAC

454 hsa miR 2016 AGAAUUGAGUGAUCUCAUGGAU

454 hsa miR 2017 AAAACGAUCUUUCAGAUUUAGAGU

454 hsa miR 2018 GCGGUCGGGCGGCGGCG

454 hsa miR 2019 AUGAGAACUUGAGCGACAGAGU

454 hsa miR 2020 AUUGGUCGUGGUUGUAGU

454 hsa miR 2021 GAUGAGAGAACAGUGGGUACUUC

454 hsa miR 2022 CAAGGUGUAGCCCAUGAGGUGGC

454 hsa miR 2023 AGCACCAGCCUAGGAAGAGGGU

454 hsa miR 2024 ACUUUAACACUGCUGUGGAAGGC

454 hsa miR 2025 AUUUGACAAGAGUAUGCCAGGUGU

454 hsa miR 2026 CUGGGAGCUUGAAAGGAG

454 hsa miR 2027 AUUUGGGCAGGUUGAAAGAAUUU

454 hsa miR 2028 GUGCUGGAGGCCAGGCUGAGGCCC

454 hsa miR 2029 GCUGUUGGUGGAGAAGGU

454 hsa miR 2030 AUGGCAGUUGGAGAGAAAGAAC

454 hsa miR 2031 GCAGAUAAACUCAUGCCAGAGAACU

454 hsa miR 2032 AGUGCCAGGUGGGGAGG

454 hsa miR 2033 GACUCUUAGCGGUGGAUC

454 hsa miR 2034 CUGUUGCGGGACCCGGGGUGU

454 hsa miR 2035 UUAAAGCUGCCAUUUGUUACU

454 hsa miR 2036 GAGCAGAGGCGAUAGUUGAAGU

454 hsa miR 2037 GAUGACAUGGGCUUUGGUCUUUUU

454 hsa miR 2038 AGAAAGGGCCUUGUGUUU

454 hsa miR 2039 ACUCGGCGUGGCGUCGGUCGUGGUAG

454 hsa miR 2040 GUGUUUAGUGAGUAUUUGUU

454 hsa miR 2041 ACUGCUGCUGCUGCUUGGCC

454 hsa miR 2042 UGCAGAGUGGGGUUUUGCAGUCCUU 454 hsa miR 2043 AUGACCACCAAACCCAGGAGC

454 hsa miR 2044 CAGAGUCUGUAGAAGAGGCG

454 hsa miR 2045 CCUUGACUUCUGCCAGAGU

454 hsa miR 2046 AUCACUGACUGAUCAAGUAGAGGU

454 hsa miR 2047 AAGGAU U GG ACAGGG U UAGAU U

454 hsa miR 2048 AGGCCAAGGCUGCGGGGUU

454 hsa miR 2049 AACGGGAGGCGCUAGCCAUGG

454 hsa miR 2050 ACCUUUCAGUGCAGUUUCUUUU

454 hsa miR 2051 AUUUUGGGUGGAAGAGGCAU

454 hsa miR 2052 GGAAUGAGGAGCUUUGAC

454 hsa miR 2054 AGUAAGGUCAGCUAAAUAAGCU

454 hsa miR 2055 GAAUUGACGGAAGGGAC

454 hsa miR 2056 UUGUGUCUUGUGUCUUUU

454 hsa miR 2057 UCCCUGGUGGUCUAGU

454 hsa miR 2058 UGAGGCUGUAACUGAGAGAAAGAUU

454 hsa miR 2059 UUAGGGCCCUGGCUCCAUCUCCU

454 hsa miR 2060 GAGUGUGAGUGGACGCGUGAGUGU

454 hsa miR 2061 AUUGAGUCUGGCAGUCCCUGUU

454 hsa miR 2062 AAAAUGUUUAGACGGGCUCAC

454 hsa miR 2063 GAAUGUUUAUGGCACCUGAC

454 hsa miR 2065 ACAGUAGGGCCUUUGGAGUGAU

454 hsa miR 2066 CUUGAAGUCUGGUGAUGCUGCCAUU

454 hsa miR 2067 AGAUGAGCUGAAGGG

454 hsa miR 2068 AAAAAGGGAGCCAAGAAG

454 hsa miR 2069 AGUUUUGUGUGUUGGCUGCUCC

454 hsa miR 2070 GGUUUAGUGAGCAGAGUU

454 hsa miR 2071 AAGACGAGAAGACCCUAUGGAGCUU

454 hsa miR 2072 AGCAGGGUGCAGGCUUGGAGUC

454 hsa miR 2073 AUGUUGGGUUGUUACAGAGU

454 hsa miR 2074 ACUGAGUUGACUGUUCCCUU

454 hsa miR 2075 ACUGGGCAGUGACAAGCACGAU

454 hsa miR 2076 AGAAAGCGUGAGUGUCCAGAGCCU

454 hsa miR 2077 GCUUGAGGGCAGUUGGUGCGG

454 hsa miR 2079 UGGAGAUGGCUGGCAGAAUGGUUCU

454 hsa miR 2080 AAGUAGAAGCCUCAGGGAAG

454 hsa miR 2081 AUGGUUCUGGACAGUGGAUU

454 hsa miR 2082 GAUGAGUCAGGCUAGGCU

454 hsa miR 2083 AUGUGAGAGCAGCAGAGGCGGU

454 hsa miR 2084 GUUUUAAGGACUUAAGGGUAU

454 hsa miR 2086 AGAAAGCCAGGAGCUGUGAUU

454 hsa miR 2087 AUUCUAAGUCAGUCAGUCAUC

454 hsa miR 2088 UUCUCUGUUUUGGCCAUGUGUGU

454 hsa miR 2090 UUCUAAGCCAGUUUCUGUCUGAU

454 hsa miR 2091 GGUUUUGACAUGUCACUGUU

454 hsa miR 2092 CUCGCCCGUGGUCUCUCGUCUU

454 hsa miR 2093 AACACGCAUACGGUUAAGGCAUUGC

454 hsa miR 2094 UGAAACAGCAUCUGAUCUUGAACUU 454 hsa miR 2095 GCGUAAAGAGUGUUUUAGAUCACCC

454 hsa miR 2096 GUCCGUUUCCUGUCAGAGUGAUCC

454 hsa miR 2097 CCAUCGGUGAUCCCAGUGACAAGU

454 hsa miR 2098 GAAAUGUUGAGUGUUUACCCUGU

454 hsa miR 2099 GAAGAAACAGCUCAUGAGGCU

454 hsa miR 2100 AUGAACCACCAGUCCAAGAAUCU

454 hsa miR 2101 GGAAGGUUGGGGGGUGU

454 hsa miR 2102 UUCUAGGUUGUGGCAUUU

454 hsa miR 2104 AGAAUACAGCAGAAUUGGCCUC

454 hsa miR 2105 CUGAUGGAGAGAAGAAGGCAU

454 hsa miR 2107 AUGAGGUGGCAAGAAAUGGGCU

454 hsa miR 2108 AAUUUUGACAGAUGCUCAAGGCUGU

454 hsa miR 2109 AAAAGCUGGGUUGAGAAG

454 hsa miR 2110 AUUGAUGGUUAAGCUCAGCUUUU

454 hsa miR 2111 AUGGAUUGAGAUGUGAUCAAAGGC

454 hsa miR 21 13 AUUUGGCAUUUGGAAGAUAGGUU

454 hsa miR 2114 ACUGCUGAGGAACUGUCACUUGU

454 hsa miR 2115 AGGUGAGGGGCAGGACCUGAAGGU

454 hsa miR 2116 AGUGUCUGUGUGUGCUUGCU

454 hsa miR 2117 ACGAAGAUGGCGACCGUAAC

454 hsa miR 2118 AACUGGAAUGGCGGCAAGGUCCU

454 hsa miR 2119 AGUUGAGUCAGGGCCUGUGUG

454 hsa miR 2120 GCCGCCCGGGGCCAUGGCG

454 hsa miR 2121 AAGUCUAAGUCUAACAUUCGGUGU

454 hsa miR 2122 CGGCGGGAGCCCGGGG

454 hsa miR 2123 ACGUGGAUGGCGUGGAGGUGC

454 hsa miR 2125 AUUUCUGCAGUCAGGUGAGAC

454 hsa miR 2126 AAGAAUG ACCGCU GAAGAACG U

454 hsa miR 2127 AAAUAUGAGCCACUGGGUGU

454 hsa miR 2128 AAGGUCCUCUGAGGCAGCAGGCU

454 hsa miR 2129 GUCGAGGUCUUUGGUGGGUUG

454 hsa miR 2130 GGUUUCGGGUUUGAAGGCAGC

454 hsa miR 2131 GCUGGUGAGUGCAGGCUGCUUC

454 hsa miR 2132 GACAGAGGUGGCAUCAAGCU

454 hsa miR 2134 UCGUGUCGCGUGGGGGGCGG

454 hsa miR 2135 GGGAUGCCAGGCAAGUGAGCAGGUC

454 hsa miR 2136 AGGGCCGUCAGGACACGGGAGGGUU

454 hsa miR 2138 AACUAGCUAGGGGUUCG

454 hsa miR 2139 GGGUUUGGGGGAUGUCAGAGGGC

454 hsa miR 2140 UGAAGGGAGAUGUGAAGAAGCC

454 hsa miR 2141 AGAAAGUCCAAGUGUUCAGG

454 hsa miR 2142 UGAACGGGUAUUUUACUG

454 hsa miR 2143 CUGAGACAUGCACUUCUGGUU

454 hsa miR 2144 AAUUGAGGAUGUGUGAGGUUU

454 hsa miR 2145 AGUUUCUAUGAGUGUAUACCAUUU

454 hsa miR 2146 AGGUGGAGAUCAAGCCCGAGAUGAU

454 hsa miR 2147 GAAGACAGCAAUAACCACAGU 454 hsa miR 2148 GUUUCUGUUGAGUGUGGGUUUAGU

454 hsa miR 2149 AUCGCCGUGGAGUGGGAGAGC

454 hsa miR 2150 GGAUGAAGUGCACUGAGGCUCUU

454 hsa miR 2151 UCCUGUUGGCCGAGUGGAGACUGGUGU

454 hsa miR 2152 GUGGAGUCUGUCAUCGAGGUGCGU

454 hsa miR 2153 UUGCAGCUGCCUGGGAGUGACUUC

454 hsa miR 2154 UGCAGCCAGCGUCCCAUGCUCG

454 hsa miR 2156 ACGGCCAAGCAGAAAAUGUUUU

454 hsa miR 2157 GAUUAGGGUGCUUAGCUGUUAACU

454 hsa miR 2158 CCUUCGAGGCGGCUGAGACCC

454 hsa miR 2159 AGGUGGUGGCAGCUGGAGGGACC

454 hsa miR 2160 AGAAGAAGGACGGCAAGAAGCGC

454 hsa miR 2161 AAAAGCAAAUGUUGGGUGAACGG

454 hsa miR 2162 GGCGGAGGGGCCGCGGGCC

454 hsa miR 2164 UCUCUCCAGGUGACAGAAAGGGCU

454 hsa miR 2165 CGUUCUUGCUCUGCCUCGGUC

454 hsa miR 2166 AUGCCAAGAGGGCCAGUGUCUU

454 hsa miR 2167 UUUUGUGUGUGUGUUUGUUUUU

454 hsa miR 2168 AUGAGGAACACUGACUUUAUUAAGC

454 hsa miR 2169 GGGUCGGAGUUAGCUCAAGCGGUU

454 hsa miR 2170 GGAGGUUCAGAGUUGGAAG

454 hsa miR 2171 GAGAGUCUCAGGAAAGAAAGGUC

454 hsa miR 2364 ACCCGUCCCGUUCGUCCCCGG

454 hsa miR 2365 ACCGGGUGCUGUAGGCUUU

454 hsa miR 2366 ACGCGGGUGAUGCGAACUGGAGUCUGAGC

454 hsa miR 2370 AGAGCAGUGUGUGUUGCCUGG

454 hsa miR 2372 AGGACGGUGGCCAUGGAAG

454 hsa miR 2374 AGUUGGUGGAGUGAUUUGUCU

454 hsa miR 2378 AGAACGCGGUCUGAGUGGU

454 hsa miR 2379 AUGAGGAUGGAUAGCAAGG

454 hsa miR 2380 CGGAGCUGGGGAUUGUGGGU

454 hsa miR 2381 CGGCGGCUCCAGGGACCUGGCG

454 hsa miR 2382 CGGCGGGGACGGCGAUUGGU

454 hsa miR 2383 CGGGGAUCGCCGAGGGCCGGUCGGCCGCC

454 hsa miR 2385 CUGCCCAGUGCUCUGAAUG

454 hsa miR 2387 CUGCCCUGGCCCGAGGGACCGACU

454 hsa miR 2388 CUGGAGGAGCUGGCCUGU

454 hsa miR 2389 CUUGGCACCUAGCAAGCACUC

454 hsa miR 2392 GAGGAAGGUGGGGAUGC

454 hsa miR 2393 GAGUGAGAGGGAGAGAACGCGGUCUGAGUG

454 hsa miR 2394 GAUGCCUGGGAGUUGCGAUCU

454 hsa miR 2396 GCAUGGGUGGUUCAGUGG

454 hsa miR 2399 GCAUUGGUGGUUCAGUGGU

454 hsa miR 2400 GCGUUGGUGGUAUAGUGGU

454 hsa miR 2403 GCUGUGGCUGUGACUGGCG

454 hsa miR 2405 GGGGAUGUAGCUCAGUGGU

454 hsa miR 2406 GGUUCCCUCAGACCUGGU

hsa-miR-95 UUCAACGGGUAUUUAUUGAGCA hsa-miR-96 UUUGGCACUAGCACAUUUUUGCU hsa-miR-96* AAUCAUGUGCAGUGCCAAUAUG hsa-miR-98 UGAGGUAGUAAGUUGUAUUGUU hsa-miR-99a AACCCGUAGAUCCGAUCUUGUG hsa-miR-99a* CAAGCUCGCUUCUAUGGGUCUG hsa-miR-99b CACCCGUAGAACCGACCUUGCG hsa-miR-99b* CAAGCUCGUGUCUGUGGGUCCG

ROC hsa miR 1622 UCGAGGAGCUCACAGUCUAGAC

ROC hsa miR 1626 CAGUGCAAUGAUAUUGUCAAAGCA

ROC hsa miR 1627 AAAAGCUGAGUUGAGAGGG

ROC hsa miR 1633 UUAAUAUGUACUGACAAAGCGU

ROC hsa miR 1634 AUGUUGGGAGCGGGCAGGUUGG

ROC hsa miR 1636 CGUGGGCCUGAUGUGGUGCUGG

ROC hsa miR 1637 AGUACCACGUGUCAGGGCCACA

ROC hsa miR 1644 CUGUAUGCCCUCACCGCUCAGC

ROC hsa miR 1646 GCGGCGGCGGCGGAGGCUGCUG

ROC hsa miR 1647 CGGCGGCGGCGGCGGCGGCUGU

ROC hsa miR 1648 UUGUGACAGAUUGAUAACUGAA

ROC hsa miR 1649 AUUGACACUUCUGUGAGUAGAG

ROC hsa miR 1651 UCUAGUAAGAGUGGCAGUCGAA

ROC hsa miR 1653 UGGGGCGGAGCUUCCGGAGGCC

ROC hsa miR 1654 CCAUGGAUCUCCAGGUGGGUCA

ROC hsa miR 1658 ACCAGGAGGCUGAGGCCCCUCA

ROC hsa miR 1659 CGGGCAGCUCAGUACAGGAU ROC hsa miR 1662 UUUGAAAGGCUAUUUCUUGGUC

ROC hsa miR 1662 UUUGAAAGGCUAUUUCUUGGUC

ROC hsa miR 1664 AAAGCAUGCUCCAGUGGCGCA

ROC hsa miR 1665 CGGCUCUGGGUCUGUGGGGAGC

ROC hsa miR 1666 CAGAGAGGACCACUAUGGCGGG

ROC hsa miR 1667 AUUGCCAUCCCCUAUGGACCAG

ROC hsa miR 1674 UGAGUGUGUGUGUGUGAGUGG

ROC hsa miR 1678 CUAUCUGUCCAUCUCUGUGCUG

ROC hsa miR 1681 GCGACCCAUACUUGGUUUCAGA

ROC hsa miR 1684 CCUGGAAACACUGAGGUUGUGU

ROC hsa miR 1686 GUGUUGAAACAAUCUCUACUGA

ROC hsa miR 1687 AUGGAUUUCUUUGUGAAUCACC

ROC hsa miR 1688 AAGACGGGAGGAAAGAAGGGAA

ROC hsa miR 1917 UUGGGGAAACGGCCGCUGAGUGA

ROC hsa miR 1919 GCGGCGGCGGCGGAGGCU

ROC hsa miR 1926 UCGACCGGACCUCGACCGGCUC

ROC hsa miR 1928 AAAGCAUGCUCCAGUGGCGC

ROC hsa miR 1930 AAGGCAGGGCCCCCGCUCCCCGG

ROC hsa miR 1958 CGGCGGGGACGGCGAUUGGUCC

ROS hsa miR 0246 AGCUGGUAAAAUGGAACCAAAU

ROS hsa miR 0876 UACCACAGGGUAGAACCACGGA

ROS hsa miR 1652 UAAUUUUAUGUAUAAGCUAGUC

ROS hsa miR 1697 CUGUACAGCCUCCUAGCUUUCC

ROS hsa miR 1699 GAUAACUAUACAAUCUAUUGCCUUC

ROS hsa miR 1707 ACCAAUAUUACUGUGCUGCUU

ROS hsa miR 1708 ACUGCCCUAAGUGCUCCUUCUGGC

ROS hsa miR 1711 CUGCAUUAUGAGCACUUAAAGU

ROS hsa miR 1712 CAACACCAGUCGAUGGGCUGUC

ROS hsa miR 1714 CAGAGCUUAGCUGAUUGGUGAACA

ROS hsa miR 1732 GGCUUCUUUACAGUGCUGCCUU

ROS hsa miR 1732 GGCUUCUUUACAGUGCUGCCUU ROS hsa miR 1733 AGCUUCUUUACAGUGCUGCCUUGU

ROS hsa miR 1734 UACUGCAAUGUAAGCACUUCUU

ROS hsa miR 1742 GGAUAUCAUCAUAUACUGUAAGU

ROS hsa miR 1745 GAAGUUCUGUUAUACACUCA

ROS hsa miR 1746 GUCAUUUUUGUGAUGUUGCAG

ROS hsa miR 1748 ACCAUCGACCGUUGAGUGGACC

ROS hsa miR 1751 CCCAAAGGUGAAUUUUUUGGGAA

ROS hsa miR 1755 CCAGUGGAGAUGCUGUUAC

ROS hsa miR 1759 AGUGGUUCUUAACAGUUCAACA

ROS hsa miR 1761 ACCUGGCAUACAAUGUAGAUUUCU

ROS hsa miR 1763 CGUGUAUUUGACAAGCUGAGUUG

ROS hsa miR 1764 GCUCUGACUUUAUUGCACUAC

ROS hsa miR 1765 UGGAGGCAGGGCCUUUGUGAAG

ROS hsa miR 1776 CCUCAAAUGUGGAGCACUAUUC

ROS hsa miR 1778 AGCGAGGUUGCCCUUUGUAUAU

ROS hsa miR 1780 AAUGACACGAUCACUCCCGUUGAGU

ROS hsa miR 1784 CCAAACCACACUGUGGUGUUAG

ROS hsa miR 1785 GAACAUCACAGCAAGUCUGUGC

ROS hsa miR 1788 GGGUAUUGUUUCCGCUGCCAG

ROS hsa miR 1789 AAUGUGUAGCAAAAGACAGAAU

ROS hsa miR 1791 GACAACUAUGGAUGAGCUCUCA

ROS hsa miR 1792 AUCAAGGAUCUUAAACUUUGC

ROS hsa miR 1795 UUUGAUAAGCUGACAUGGGACA

ROS hsa miR 1799 AAAGGAAAGUGUAUCCUAAAAG

ROS hsa miR 1800 ACAACCCUAGGAGAGGGUGCCA

RNA labeling and hybridization was performed as described above. All hybridizations were made against a common reference pool.

Data analysis Image analysis and data processing was carried out as described in Example 1. miRNAs with high discriminatory power between treatments were identified by Welsh T-test (p<0.01 or p<0.05). Both unsupervised hierarchical clustering and supervised LDA (linear discriminant analysis) was performed for classifying the samples according to EDR status as described in Wit E and McClure J. "Statistics for microarrays. Design, analysis, and inference". Wiley, West Sussex, England, 2004.

Results

Array expression results were combined with EDR status data provided by Oncotech.

The Iog2 ratios between the common reference and the sample channel for each patient (i.e. the Hy3 over Hy5, wherein Hy5 is the common reference consisiting of a mixture of all ratios) were used in the statistical evaluation of the data set.

Significant difference between two patient groups with EDR for two different drugs were evaluated in the MeV software package (Tigr) using the Welch approximation t-test with a p- value cutoff of 0.01 or 0.05.

Figures show LDA plots and unsupervised hierarchial clustering for all comparisons based on the miRs that vary most between samples with EDR to one drug and samples with EDR to the other.

Figure 7 shows LDA plot based on the 40 most significant miRs (p<0.05) after standard deviation (SD) filtering showing near-perfect separation between EDR to irinotecan (red/light grey) and EDR to Oxaliplatin (blue/dark grey) for FFPE specimens.

We are able to separate colon cancer samples with Extreme Drug Resistance (EDR) against the chemotherapeutic agents 5-FU/Leucovorin, Irinotecan and Oxaliplatin based on the expression profiles of a relatively few miRNAs.

Claims

1. A method for determining whether a disease, or state of disease, shows resistance to, or susceptibility to, at least one treatment of said disease, such as administration of at least one therapeutic compound, said method comprising the steps of

wherein over or under abundance of said at least one microRNA is correlated to the resistance or susceptibility of the disease, or state of disease, to said administration of at least one treatment of said disease.

2. The method according to claim 1, wherein the disease is cancer, wherein the at least one treatment is a cancer treatment, such as at least one chemotherapeutic drug or radiotherapy, and wherein the sample referred to in step a) is a sample of said cancer.

3. The method according to claim 1 or 2, wherein the abundance of the at least one microRNA is a relative abundance of the microRNA present in said sample as compared to the abundance of the at least one microRNA present in at least one further sample, such as at least one further cancer sample, or a population of such further samples, with a known treatment resistance phenotype, such as a known cancer resistance phenotype.

4. The method according to claim 3, wherein the at least one further sample has a characterised treatment resistance phenotype, such as extreme (EDR) or intermediate drug resistance (IDR) phenotype with respect to said at least one treatment.

5. The method according to claim 3 or 4, wherein the relative abundance of the at least one microRNA is compared to the abundance of the at least one microRNA present in at least one further sample, such as at least one further cancer sample, with a characterised susceptibility phenotype, such as a low drug resistance (LDR) phenotype, with respect to said at least one treatment.

6. The method according to any one of claims 3 - 5, wherein the relative abundance of the at least one microRNA is compared to the abundance of the at least one microRNA present in a population of at least one further samples.

7. The method according to claim 6, wherein the population of at least one further samples comprises both i) one or more members which exhibit a characterised susceptibility phenotype, and ii) one or more members which exhibit a characterised resistance phenotype, with respect to the at least one treatment.

8. The method according to any one of claims 1 - 7, wherein the abundance of the at least one microRNA is a relative abundance of the microRNA present in said sample as compared to the abundance of the at least one internal reference marker present in said sample, such as a non-coding RNA, such as a non-coding RNA selected form the group consisting of U6B, SNORD7, SNORD24, SNORD38B, SNORD43, SNORD44, SNORD48, SNORD49A, SNORD66, RNU19, 5.8S rRNA, and 5S rRNA.

9. The method according to any one of claims 1 - 8, wherein the abundance of more than one microRNA is correlated to the resistance or susceptibility of the disease or state of disease, such as cancer, to said at least one treatment of said disease is assessed.

10. The method according to any one of claims 1 - 9, wherein the resistance to more than one treatment of said disease is determined.

11. The method according to any one of claims 1 - 10, wherein a further step, step c), is performed, either prior to, concurrent to, or subsequent to step b), wherein step c) comprises the assaying of the presence or absence or the abundance of at least one genetic marker of said disease, such as a genetic mutation associated with said disease, or the expression levels of a mRNA or protein which is associated with said disease.

12. The method according to any one of claims 1- 11, wherein the disease is cancer, and the at least one treatment is at least one cancer treatment, such as at least one cancer treatment selected from at least one chemotherapeutic drug and/ or radiotherapy.

13. The method according to claim 12, wherein the at least one chemotherapeutic drug is selected from the group consisting of: Gemcitabine, Vinblastine, Temozolomide,

Navelbine, Oxaliplatin, Vincristine, Fluorouracil, Floxuridine, Cyclophosphamide, Mitomycin C, Carboplatin, Ifosfamide, Etoposide, Taxol, Doxorubicin, Cisplatin, Carmustine, Capecitabine, 5 FU, 5 FU + Leucovorin, Topotecan, Taxotere, Irinotecan, 5 FU + Irinotecan, Alpha Interferon, Doxil, Interferon, Interferon + Vinblastine, Interleukin 2, Alpha Interferon, Taxotere + Navelbine, Cisplatin + Gemcitabine, and Doxil.

14. The method according to claim 12, wherein the at least one chemotherapeutic drug is selected from the group consisting of: anti-metabolites, such as azathioprine or mercaptopurine; plant alkaloids and terpeoids, such as vinca alkaloids and taxanes; thymidylate synthase inhibitors, such as 5-fluoro uracil (5-FU) and citrovorin, topoisomerase (TSI) acting drugs, such as SN-38 (Irinotecan) and camptothecin, alylating agents such as Oxaliplatin, monoclonal antibodies, such as Herceptin (Trastuzumab), Avastin (Bevacizumab), Erbitux (Cetuximab), Rituxan (Rituximab) , (anti- hormonal treatments such as Tamoxifen, and the armoatase inhibitor letrozole, ...

15. The method according to any one of claims 1 - 14, wherein the at least one microRNA is selected from the group consisting of: hsa-let-7d*, hsa-miR-106a, hsa-miR-106b, hsa- miR-125a-3p, hsa-miR-141*, hsa-miR-17, hsa-miR-17*, hsa-miR-181c, hsa-miR-18a, hsa-miR-18b , hsa-miR-19a , hsa-miR-19b, hsa-miR-20a, hsa-miR-20b, hsa-miR-302d, hsa-miR-32*, hsa-miR-373, hsa-miR-493, hsa-miR-570, hsa-miR-577, hsa-miR-92a, hsa-miR-92b, hsa-miR-93, ROC_hsa_miR_1659, ROS_hsa_miR_1733, ROS_hsa_miR_1751, and ROS_hsa_miR_1788; hsa-miR-17, hsa-miR-193b, hsa-miR-

19b, hsa-miR-20a, hsa-miR-20b, hsa-miR-30e, hsa-miR-339-5p, hsa-miR-362-3p, hsa- miR-373*, hsa-miR-374a, hsa-miR-423-5p, hsa-miR-525-5p, hsa-miR-548c-3p, hsa-miR- 590-5p, hsa-miR-617, hsa-miR-633, hsa-miR-671-3p, hsa-miR-92a, ROC_hcy_miR_1961, ROC_hsa_miR_1627, ROC_hsa_miR_1637, ROS_hsa_miR_1748, and ROS_hsa_miR_1780; hsa-miR-1, hsa-miR-106b, hsa-miR-106b*, hsa-miR-128a, hsa-miR-130b, hsa-miR-137, hsa-miR-141*, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR- 146a, hsa-miR-148b, hsa-miR-25, hsa-miR-301b, hsa-miR-302a*, hsa-miR-324-5p, hsa- miR-339-5p, hsa-miR-33a, hsa-miR-345, hsa-miR-362-5p, hsa-miR-381, hsa-miR-485- 5p, hsa-miR-496, hsa-miR-500, hsa-miR-525-3p, hsa-miR-532-5p, hsa-miR-568, hsa- miR-572, hsa-miR-585, hsa-miR-652, hsa-miR-885-5p, hsa-miR-92a, hsa-miR-93,

ROC_hsa_miR_1622, ROC_hsa_miR_1627, ROC_hsa_miR_1666, ROC_hsa_miR_1926, and ROS_hsa_miR_1714; hsa- let-7i*, hsa-miR-105, hsa-miR-130b*, hsa-miR-132*, hsa-miR-181c, hsa-miR-188-5p, hsa-miR-205, hsa-miR-216a, hsa-miR-302b*, hsa-miR- 302d, hsa-miR-425*, hsa-miR-523, hsa-miR-553, hsa-miR-592, hsa-miR-614, hsa-miR- 617, hsa-miR-662, hsa-miR-99a*; and ROS_hsa_miR_1745, hsa-miR-106b, hsa-miR-

106b*, hsa-miR-125b-l*, hsa-miR-128a, hsa-miR-130b, hsa-miR-137, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-146a, hsa-miR-148b, hsa-miR-150, hsa-miR-151-3p, hsa-miR- 186, hsa-miR-187, hsa-miR-18a, hsa-miR-18b, hsa-miR-25, hsa-miR-302b*, hsa-miR- 345, hsa-miR-361-5p, hsa-miR-448, hsa-miR-453, hsa-miR-525-5p, hsa-miR-532-5p, hsa-miR-568, hsa-miR-570, hsa-miR-572, hsa-miR-634, hsa-miR-652, hsa-miR-663, hsa-miR-885-5p, hsa-miR-92a, hsa-miR-93, ROC_hsa_miR 1662, and ROS hsa miR 1732.

16. The method according to claim 15, wherein the at least one microRNA is selected from the group consisting of: hsa-let-7d*, hsa-miR-106a, hsa-miR-106b, hsa-miR-125a-3p, hsa-miR-141*, hsa-miR-17, hsa-miR-17*, hsa-miR-181c, hsa-miR-18a, hsa-miR-18b , hsa-miR-19a , hsa-miR-19b, hsa-miR-20a, hsa-miR-20b, hsa-miR-302d, hsa-miR-32*, hsa-miR-373, hsa-miR-493, hsa-miR-570, hsa-miR-577, hsa-miR-92a, hsa-miR-92b, hsa-miR-93, ROC_hsa_miR_1659, ROS_hsa_miR_1733, ROS_hsa_miR_1751, and ROS_hsa_miR_1788.

17. The method according to claim 16, wherein the cancer treatment is 5-FU, optionally in combination with Leucoverin.

18. The method according to claim 17, wherein the at least one microRNA is selected from the group consisting of: hsa-miR-17, hsa-miR-193b, hsa-miR-19b, hsa-miR-20a, hsa- miR-20b, hsa-miR-30e, hsa-miR-339-5p, hsa-miR-362-3p, hsa-miR-373*, hsa-miR-374a, hsa-miR-423-5p, hsa-miR-525-5p, hsa-miR-548c-3p, hsa-miR-590-5p, hsa-miR-617, hsa-miR-633, hsa-miR-671-3p, hsa-miR-92a, ROC_hcy_miR_1961, ROC_hsa_miR_1627, ROC_hsa_miR_1637, ROS_hsa_miR_1748, and ROS_hsa_miR_1780.

19. The method according to claim 18, wherein the cancer treatment is 5-FU, optionally in combination with Irinotecan.

20. The method according to claim 15, wherein the at least one microRNA is selected from the group consisting of: hsa-miR-1, hsa-miR-106b, hsa-miR-106b*, hsa-miR-128a, hsa- miR-130b, hsa-miR-137, hsa-miR-141*, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-

146a, hsa-miR-148b, hsa-miR-25, hsa-miR-301b, hsa-miR-302a*, hsa-miR-324-5p, hsa- miR-339-5p, hsa-miR-33a, hsa-miR-345, hsa-miR-362-5p, hsa-miR-381, hsa-miR-485- 5p, hsa-miR-496, hsa-miR-500, hsa-miR-525-3p, hsa-miR-532-5p, hsa-miR-568, hsa- miR-572, hsa-miR-585, hsa-miR-652, hsa-miR-885-5p, hsa-miR-92a, hsa-miR-93, ROC_hsa_miR_1622, ROC_hsa_miR_1627, ROC_hsa_miR_1666, ROC_hsa_miR_1926,

ROS_hsa_miR_1714.

21. The method according to claim 20, wherein the cancer treatment is Irinotecan.

22. The method according to claim 15, wherein the at least one microRNA is selected from the group consisting of: hsa- let-7i*, hsa-miR-105, hsa-miR-130b*, hsa-miR-132*, hsa- miR-181c, hsa-miR-188-5p, hsa-miR-205, hsa-miR-216a, hsa-miR-302b*, hsa-miR-

302d, hsa-miR-425*, hsa-miR-523, hsa-miR-553, hsa-miR-592, hsa-miR-614, hsa-miR- 617, hsa-miR-662, hsa-miR-99a*, and ROS_hsa_miR_1745.

23. The method according to claim 22, wherein the cancer treatment is Oxaliplatin.

24. The method according to claim 15, wherein the at least one microRNA is selected from the group consisting of: hsa-let-7i*, hsa-miR-106b, hsa-miR-106b*, hsa-miR-125b-l*, hsa-miR-128a, hsa-miR-130b, hsa-miR-137, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR- 146a, hsa-miR-148b, hsa-miR-150, hsa-miR-151-3p, hsa-miR-186, hsa-miR-187, hsa- miR-18a, hsa-miR-18b, hsa-miR-25, hsa-miR-302b*, hsa-miR-345, hsa-miR-361-5p, hsa-miR-448, hsa-miR-453, hsa-miR-525-5p, hsa-miR-532-5p, hsa-miR-568, hsa-miR- 570, hsa-miR-572, hsa-miR-634, hsa-miR-652, hsa-miR-663, hsa-miR-885-5p, hsa-miR- 92a, hsa-miR-93, ROC_hsa_miR 1662, and ROS_hsa_miR_1732.

25. The method according to claim 24, wherein the cancer treatment is Topotecan.

26. The method according to any one of claims 1 - 25, wherein the cancer type is selected from the group consisting of: brain cancer, breast cancer, colorectal cancer, endometrial cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, sarcoma, stomach cancer, unknown primary cancer, leukaemia, lymphoma and multiple melanoma.

27. The method according to any one of claims 1 - 26 wherein step b) comprises the in situ detection of the at least one microRNA within the cancer sample.

28. The method according to any one of claims 1 - 26, wherein step b) comprises the isolation of a microRNA comprising fraction, such as a total RNA fraction, from the sample.

29. The method according to claim 27 or 28, wherein the determination of the abundance of the at least one microRNA is performed using at least one detection probe which comprises a complementary nucleobase sequence to at least 6 contiguous nucleotides present in said at least one microRNA.

30. The method according to claim 29, wherein the complementary nucleobase sequence is complementary to a contiguous nucleotide sequence present in said at least on microRNA.

31. The method according to claim 30, wherein the complementary nucleobase sequence is complementary to the entire contiguous nucleotide sequence present in said at least on microRNA.

32. The method according to any one of claims 19 - 31, wherein the complementary nucleobase sequence consists of between 8 and 25 contiguous nucleobases, such as between 15 and 23 contiguous nucleobases.

33. The method according to any one of claims 29 - 32 wherein the complementary nucleobase sequence consists of nucleotide analogues inserted between the nucleoside analogues with regular spacing over part or the entire nucleobases sequence.

34. The method according to claim 33, wherein the regular spacing is a nucleotide analogue at every second, third or fourth nucleobase position, or combination thereof.

35. The method according to any one of claims 29 - 34 wherein the complementary nucleobase sequence comprises LNA nucleotide analogues.

36. The method according to any one of claims 29 - 35 wherein all the nucleotide analogues present in the complementary nucleobase sequence are LNA nucleotide analogues.

37. The method according to any one of claims 1 - 36, wherein the abundance of at least 2, such as at least five, such as at least 10, independent microRNAs are determined, such as the microRNAs independently selected from those referred to in any one of the preceding claims.

38. The method according to any one of claims 1 - 37, wherein the abundance of each of the independent members of a population of microRNAs is determined, such as a population comprising microRNAs referred to or selected from those referred to in any one of the preceding claims, wherein the over or under abundance of each of the independent members of a population of microRNAs may be correlated to the resistance or susceptibility of the disease or disease sample or cell(s) to one or more of the disease treatments according to any one of the preceding claims.

39. The method according to claim 38, wherein said method comprises hybridising the microRNA fraction referred to in claim 28 to a population of detection probes, such as the detection probes as described in any one of claims 29 - 36, wherein said population of detection probes comprises independent members which correspond to each of the independent members of a population of microRNAs.

40. The method according to claim 39, wherein the population of detection probes is in the form of an array, such as a micro-array, or a multiplexed qPCR assay, such as a Luminex assay, or a northern blot, such as a dot blot.

41. The method according to claim 1 - 40, wherein the abundance of the at least one microRNAs is performed using the polymerase chain reaction (PCR), wherein the detection probes are in the form of PCR primers.

42. The use of one or more detection probes which comprises a contiguous nucleobases sequence which are complementary to one or more microRNAs, for the detection of the abundance of the microRNA in a sample, such as a disease sample or cell(s) isolated from a subject, in order to determine whether the disease present in the patient has a disease treatment resistant or a disease treatment susceptible phenotype, wherein over or under abundance of said at least one microRNA is correlated to the resistance, or susceptibility of the disease to said disease treatment.

43. The use according to claim 42, wherein the detection probes, which may optionally be in the form of a collection of detection probes, are defined as those according to any one of claims 29 - 36.

44. A method of treatment of disease, in a subject suffering from said disease, said method comprising the steps of performing the method according to claims 1 or 41 to identify one or more disease treatments which have a positive prognosis in treatment of said disease, and subsequently administering said one or more disease treatments which have a positive prognosis to said subject.

45. A method for identifying one or more microRNAs which are indicators of the resistance of a disease to a disease treatment, and/or the susceptibility of a disease to a disease treatment, such as a chemotherapeutic drug, said method comprising the steps of:

a. isolating a microRNA containing fraction from

i) a disease sample which is identified as showing resistance to said disease treatment; and, ii) a disease sample which is identified as being sensitive to said disease treatment; b. comparing the abundance of one or more specific microRNAs in the fractions obtained in i) and ii) of step a) to identify those microRNAs which are correlated with the resistant and/or susceptibility of the disease to the disease treatment.

47. The method or use according to any one of claims 42 - 47, wherein the disease is cancer, the disease sample is a cancer sample or cell(s), and wherein the disease treatment is cancer treatment.