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WO2018127786A1 - Compositions et méthodes permettant de déterminer un plan d'action thérapeutique - Google Patents

Compositions et méthodes permettant de déterminer un plan d'action thérapeutique Download PDF

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Publication number
WO2018127786A1
WO2018127786A1 PCT/IB2018/000042 IB2018000042W WO2018127786A1 WO 2018127786 A1 WO2018127786 A1 WO 2018127786A1 IB 2018000042 W IB2018000042 W IB 2018000042W WO 2018127786 A1 WO2018127786 A1 WO 2018127786A1
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Prior art keywords
inhibitor
genes
cms
colorectal cancer
sample
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PCT/IB2018/000042
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English (en)
Inventor
Ragnhild A. Lothe
Jarle BRUUN
Peter Wold EIDE
Anita Sveen
Arild NESBAKKEN
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Oslo Universitetssykehus Hf
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Publication of WO2018127786A1 publication Critical patent/WO2018127786A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to compositions, systems, and methods for determining a treatment course of action.
  • the present invention relates to compositions, systems, and methods for utilizing gene expression profiles to determine drug sensitivity in colorectal cancer.
  • Colorectal cancer is a major, worldwide health burden with a high incidence and mortality rate, representing the fourth most common cause of cancer deaths (1).
  • CRC Colorectal cancer
  • Treatment decisions are primarily based on cancer stage and tumor location, and the repertoire of targeted treatments, as well as the extent of stratified treatment based on prognostic and predictive factors is limited (4, 5).
  • CRC is a heterogeneous disease also at the molecular level (3) and this is a clinical challenge, both with respect to precise interpretation of prognostic and predictive markers, and as a result of the potential to confer primary or secondary resistance to targeted treatment (6, 7).
  • MSI microsatellite instability
  • CMS consensus molecular subtypes
  • CMS1 includes most of the MSI+ tumors and tumors with BRAF mutations, and is a de-differentiated, immunogenic and inflammatory subtype.
  • CMS2 is the largest, and a canonical CRC subtype with epithelial characteristics and WNT activation.
  • CMS3 is also epithelial, but is specifically characterized by metabolic de-regulation.
  • CMS4 is a mesenchymal subtype with activation of epithelial-to-mesenchymal transition (EMT), has a high level of stromal infiltration and is associated with a poor patient survival.
  • EMT epithelial-to-mesenchymal transition
  • CMS represents a consensus of several gene expression based classifications of CRC, put forth by the international CRC Subtyping Consortium (12); however, independent validation is pending. Furthermore, there is great potential clinical benefit in exploring CMS classification as a basis for molecularly stratified treatment of CRC, with identification of subtype-specific prognostic associations and drug vulnerabilities.
  • Immortalized cancer cell lines are valuable in vitro models for efficient, large-scale analyses of drug sensitivities.
  • CRC in particular, cell lines have repeatedly been shown to recapitulate the molecular properties of primary tumors (13-16).
  • the tumor microenvironment has a strong potential influence on drug response
  • three-dimensional cultures (organoids) of CRC cells derived from patients' tumors have demonstrated good correlations between specific drug sensitivities and genomic alterations of the targeted pathways (17).
  • Infiltration of immune and stromal cells in tumors also has an important influence on gene expression measured in bulk tumor tissue samples (18).
  • the tumor microenvironment is an important constituent of CMS classification (12), and in vitro explorations of the subtypes therefore require a cancer cell-specific classification approach.
  • Customized therapies for CRC are needed.
  • the present invention relates to compositions, systems, and methods for determining a treatment course of action.
  • the present invention relates to compositions, systems, and methods for utilizing gene expression profiles for stratified patient treatment in colorectal cancer, based on subtype-specific drug sensitivities.
  • CRC colorectal cancer
  • a method for determining a treatment course of action in a subject diagnosed with colorectal cancer comprising: a) identifying a consensus molecular subtype (CMS) of a colorectal cancer sample; and b) determining a treatment course of action based on the CMS classification.
  • the methods comprise the step of stratifying a colorectal cancer sample from the subject by determining a consensus molecular subtype (CMS) classification selected from the group consisting of CMS1, CMS2, CMS3, and CMS4 for the sample.
  • CMS consensus molecular subtype
  • the stratification may be performed by the clinician, hospital, or health maintenance organization treating the subject, or may be performed by an entity remote or distinct from the treating clinician, hospital, or health maintenance organization treating the subject.
  • the CMS is CMS1, CMS2, CMS3, or CMS4. The present disclosure is not limited to a particular treatment course of action.
  • the treatment course of action is administration of an HSP90 inhibitor, a topoisomerase II inhibitor, 2-methoxyestradiol, disulfiram, atorvastatin, PF-03758309, rigosertib, tipifamib, YM155, indibulin, cytarabine, 8-chloro-adenosine, serdemetan, TH588, RAF265, KU-60019, BGB324, auranofin or SGC-CBP30 when the CMS is CMS1 or CMS4.
  • an HSP90 inhibitor a topoisomerase II inhibitor
  • 2-methoxyestradiol 2-methoxyestradiol
  • disulfiram atorvastatin
  • PF-03758309 rigosertib
  • tipifamib tipifamib
  • YM155 indibulin
  • cytarabine 8-chloro-adenosine
  • serdemetan TH588, RAF265,
  • the HSP90 inhibitor is, for example, luminespib, ganetespib, onalespib, SGX- 301, radicicol or a derivative thereof.
  • the radicicol derivative is OS- 47720.
  • the topoisomerase II inhibitor is valrubicin, daunorubicin, doxorubicin or idarubicin.
  • the treatment course of action is administration of luminespib, ganetespib, 2-methoxyestradiol, atorvastatin or disulfiram.
  • the biological sample is, for example, a tissue sample, a biopsy sample, a blood sample or a stool sample.
  • the colorectal cancer is stage I, II, III or IV.
  • identifying the CMS comprises determining the level of expression or presence of a mutation in one or more genes or proteins.
  • the CMS is determined by application of a CMS classifier algorithm. Suitable algorithms are described for example, in Guinney et al, Nature Medicine, 21: 1350; Eide et al., CMScaller: an R package for consensus molecular subtyping of colorectal cancer pre-clinical models, Scientific Reports 7: 16618 (2017) and Sveen et al. Colorectal cancer Consensus Molecular Subtypes translated to preclinical models uncover potentially targetable cancer-cell dependencies, (2017) Clin. Can. Res.
  • CMS subtypes are determined by analysis of a combination of genomic, mRNA, miRNA, and proteomic distinctions (e.g., expression, copy number, mutations, microsatellite stability, etc.) that distinguish the subtypes from each other.
  • the identification comprises the use of colorectal cancer informative reagent selected from, for example, a nucleic acid probe or probes that hybridizes to a respective gene product of the one or more genes, nucleic acid primers for the amplification and detection or sequencing of a respective gene product of the one or more genes, and an antigen binding protein that binds to a respective gene product of the one or more genes.
  • the gene product is an RNA transcript from the gene and the colorectal informative reagent is a nucleic acid probe or probes that hybridizes to the respective gene product of the one or more genes or nucleic acid primers for the amplification and detection or sequencing of the respective gene product of the one or more genes.
  • the gene product is a protein and the colorectal cancer informative reagent is an antibody recognizing the respective product of the one or more genes.
  • the method further comprises the step of administering the treatment course of action to the subject.
  • Additional embodiments provide a method for treating a subject diagnosed with colorectal cancer (CRC), comprising: a) identifying a consensus molecular subtype (CMS) of a colorectal cancer sample; b) determining a treatment course of action based on the CMS classification; and c) administering the treatment course of action to the subject.
  • CRC colorectal cancer
  • FIG. 1 Validation of molecular and clinicopathological characteristics of the CMS subtypes a) From a consecutive series of 409 patients with stage I-IV CRC, totally 323 (79%) tumors were confidently assigned to a CMS subtype (posterior probability larger than 0.5 from the random forest CMS classifier), while 46 tumors (11%) displayed mixed characteristics between two of the subtypes (posterior probability larger than 0.3 for both subtypes) and 40 tumors (10%) were indeterminate.
  • FIG. 3 CMS subtyping of CRC cell lines a) Confident CMS classification was obtained for 131 (87%) of 150 unique CRC cell lines using the cancer cell-specific CMS classifier, with similar distribution among the subtypes as for the consecutive patient series. In the cell line classification, the molecular and biological characteristics of the CMS subtypes were also recapitulated, as shown b) for MSI status, BRAF mutations, KRAS mutations and TP53 mutations, as well as c) by gene set expression enrichment analyses.
  • FIG. 4 Differential drug sensitivities between individual CMS subtypes
  • cell lines of two CMS subtypes were compared, as indicated, with respect to sensitivity to each of 460 drugs included in a high-throughput drug screen. Each dot represents one drug, and selected drugs are colored according to molecular targets, as indicated.
  • CMS1 cell lines were more sensitive to inhibitors of topoisomerases (orange), Hsp90-inhibitors (red; in particular luminespib, ganetespib and radicicol) and 2ME (green; combined angiogenesis- and tubulin-inhibitor).
  • CMS 1 cell lines were more sensitive to Hsp90-inhibitors and 2ME.
  • CMS2 cell lines were, in comparison with CMS3, more sensitive to EGFR-inhibitors, while the opposite was found for mitotic inhibitors.
  • CMS4 cell lines showed strong sensitivity to Hsp90-inhibitors, atorvastatin (blue; HMG-CoA reductase-inhibitor), 2ME and disulfiram (pink; inhibitor of acetaldehyde dehydrogenase). 2ME, 2-methoxyestradiol; DSS, drug sensitivity score
  • Hsp90-inhibitors red; luminespib, ganetespib and radicicol
  • 2ME green; combined angiogenesis- and tubulin-inhibitor
  • atorvastatin dark blue; HMG-CoA reductase-inhibit
  • a validation drug screen of five additional CMS1 and CMS4 cell lines confirmed strong sensitivity (red) to Hsp90-inhibitors, 2ME, atorvastatin and disulfiram in comparison both with two CMS3 cell lines included in the validation screen, and with the average sensitivity in CMS2 and CMS3 cell lines in the initial screen.
  • 2ME 2-methoxy estradiol
  • DSS drug sensitivity score
  • FIG. 6 Validation of strong relative ganetespib-sensitivity in CMS1 and CMS4 cell lines in published data Strong relative sensitivity to Hsp90-inhibition in CMS1 and CMS4 cell lines compared with CMS2 and CMS3 cell lines was validated in two published datasets; a) using the Hsp90-inhibitor ganetespib in He, S. et al. Invest. New Drugs 32, 577- 586 (2014); and b) using the Hsp90-inhibitor CCT018159 in the Genomics of Drug
  • FIG. 7 CMS and drug-sensitivity associations in extended panel of CRC cell lines. / ⁇ -values are for Kruskal-Wallis rank sum tests. Higher scores indicate higher drug sensitivity. Afatinib is included as an example of a drug with larger effect within CMS2.
  • FIG. 8 CMS-selective activity of luminespib in PDX models.
  • Tumor growth is plotted as the mean ⁇ standard error of tumor volume fold changes of all mice per treatment arm at the indicated time points.
  • sensitivity is defined as a statistical measure of performance of an assay (e.g., method, test), calculated by dividing the number of true positives by the sum of the true positives and the false negatives.
  • the term "specificity” is defined as a statistical measure of performance of an assay (e.g., method, test), calculated by dividing the number of true negatives by the sum of true negatives and false positives.
  • informative or “informativeness” refers to a quality of a marker or panel of markers, and specifically to the likelihood of finding a marker (or panel of markers) in a positive sample.
  • colonal cancer informative reagent refers to a reagent or reagents that are informative for identification of cancer gene markers described herein.
  • reagents are primers, probes or antibodies for detection of gene expression products (e.g., RNA transcripts or proteins) associated with CRC types CMSl-4.
  • the "consensus molecular subtype" or CMS refer to molecular subtypes of colorectal cancer (CRC) as defined in Guinney et al. (Nature Medicine, 21 : 1350; herein incorporated by reference in its entirety), and derivatives or adaptations of said subtypes comprising essentially the same molecular features, including the adapted CMS classifiers described herein (see, e.g., the CMS classifiers described in in Eide et al, CMScaller: an R package for consensus molecular subtyping of colorectal cancer pre-clinical models, Scientific Reports 7: 16618 (2017) and Sveen et al. Colorectal cancer Consensus Molecular Subtypes translated to preclinical models uncover potentially targetable cancer-cell dependencies, (2017) Clin. Can. Res. DOI:
  • the CMS is determined by application of the CMScaller algorithm, available on the world wide web at
  • CMS subtypes are a combination of genomic, mRNA, miRNA, and proteomic distinctions (e.g., expression, copy number, mutations, microsatellite stability, etc.) that distinguish the subtypes from each other.
  • the term “metastasis” is meant to refer to the process in which cancer cells originating in one organ or part of the body relocate to another part of the body and continue to replicate. Metastasized cells subsequently form tumors which may further metastasize. Metastasis thus refers to the spread of cancer from the part of the body where it originally occurs to other parts of the body.
  • the term “metastasized colorectal cancer cells” is meant to refer to colorectal cancer cells which have metastasized; colorectal cancer cells localized in a part of the body other than the colorectal.
  • an individual is suspected of being susceptible to metastasized colorectal cancer is meant to refer to an individual who is at an above-average risk of developing metastasized colorectal cancer.
  • individuals at a particular risk of developing colorectal cancer are those whose family medical history indicates above average incidence of colorectal cancer among family members and/or those who have already developed colorectal cancer and have been effectively treated who therefore face a risk of relapse and recurrence.
  • Other factors which may contribute to an above-average risk of developing metastasized colorectal cancer which would thereby lead to the classification of an individual as being suspected of being susceptible to metastasized colorectal cancer may be based upon an individual's specific genetic, medical and/or behavioral background and characteristics.
  • neoplasm refers to any new and abnormal growth of tissue.
  • a neoplasm can be a premalignant neoplasm or a malignant neoplasm.
  • neoplasm-specific marker refers to any biological material that can be used to indicate the presence of a neoplasm. Examples of biological materials include, without limitation, nucleic acids, polypeptides, carbohydrates, fatty acids, cellular components (e.g., cell membranes and mitochondria), and whole cells.
  • colonal neoplasm-specific marker refers to any biological material that can be used to indicate the presence of a colorectal neoplasm (e.g., a premalignant colorectal neoplasm, a malignant colorectal neoplasm, a metastatic colorectal neoplasm).
  • colorectal neoplasm-specific markers include, but are not limited to, the 13 gene signature described herein.
  • amplicon refers to a nucleic acid generated using primer pairs.
  • the amplicon is typically single-stranded DNA (e.g., the result of asymmetric amplification), however, it may be RNA or dsDNA.
  • amplifying or “amplification” in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide (e.g., a single polynucleotide molecule), where the amplification products or amplicons are generally detectable.
  • Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes.
  • the generation of multiple DNA copies from one or a few copies of a target or template DNA molecule during a polymerase chain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S. Patent No. 5,494,810; herein incorporated by reference in its entirety) are forms of amplification.
  • Additional types of amplification include, but are not limited to, allele-specific PCR (see, e.g., U.S. Patent No. 5,639,611; herein incorporated by reference in its entirety), assembly PCR (see, e.g., U.S. Patent No. 5,965,408; herein incorporated by reference in its entirety), helicase-dependent amplification (see, e.g., U.S. Patent No.
  • hot-start PCR see, e.g., U.S. Patent Nos. 5,773,258 and 5,338,671; each herein incorporated by reference in their entireties
  • intersequence-specfic PCR see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186; herein incorporated by reference in its entirety
  • ligation-mediated PCR see, e.g., Guilfoyle, R. et al, Nucleic Acids Research, 25: 1854-1858 (1997); U.S. Patent No.
  • methylation-specific PCR see, e.g., Herman, et al, (1996) PNAS 93(13) 9821-9826; herein incorporated by reference in its entirety
  • miniprimer PCR multiplex ligation-dependent probe amplification
  • multiplex PCR see, e.g., Chamberlain, et al, (1988) Nucleic Acids Research 16(23) 11141-11156; Ballabio, et al, (1990) Human Genetics 84(6) 571-573; Hayden, et al, (2008) BMC Genetics 9:80; each of which are herein incorporated by reference in their entireties
  • nested PCR see, overlap-extension PCR (see, e.g., Higuchi, et
  • the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
  • sequence “5'-A-G-T-3'” is complementary to the sequence “3'-T-C-A-5'.”
  • Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.
  • the term "primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced (e.g., in the presence of nucleotides and an inducing agent such as a biocatalyst (e.g. , a DNA polymerase or the like) and at a suitable temperature and pH).
  • the primer is typically single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is generally first treated to separate its strands before being used to prepare extension products.
  • the primer is an inducing agent
  • the primer is sufficiently long to prime the synthesis of extension products in the presence of the inducing agent.
  • the exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
  • the primer is a capture primer.
  • nucleic acid molecule refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA.
  • the term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4 acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil,
  • dihydrouracil inosine, N6-isopentenyladenine, 1-methyladenine, 1 -methylpseudo-uracil, 1- methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N- isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
  • nucleobase is synonymous with other terms in use in the art including “nucleotide,” “deoxynucleotide,” “nucleotide residue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” or deoxynucleotide triphosphate (dNTP).
  • oligonucleotide refers to a nucleic acid that includes at least two nucleic acid monomer units (e.g., nucleotides), typically more than three monomer units, and more typically greater than ten monomer units.
  • nucleic acid monomer units e.g., nucleotides
  • the exact size of an oligonucleotide generally depends on various factors, including the ultimate function or use of the oligonucleotide. To further illustrate, oligonucleotides are typically less than 200 residues long (e.g., between 15 and 100), however, as used herein, the term is also intended to encompass longer polynucleotide chains. Oligonucleotides are often referred to by their length.
  • oligonucleotide For example a 24 residue oligonucleotide is referred to as a "24-mer".
  • the nucleoside monomers are linked by phosphodiester bonds or analogs thereof, including phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like, including associated counterions, e.g., H + , NH 4 + , Na + , and the like, if such counterions are present.
  • oligonucleotides are typically single-stranded.
  • Oligonucleotides are optionally prepared by any suitable method, including, but not limited to, isolation of an existing or natural sequence, DNA replication or amplification, reverse transcription, cloning and restriction digestion of appropriate sequences, or direct chemical synthesis by a method such as the phosphotriester method of Narang et al. (1979) Meth Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetrahedron Lett. 22: 1859-1862; the triester method of Matteucci et al. (1981) J Am Chem Soc.
  • a "sequence" of a biopolymer refers to the order and identity of monomer units (e.g., nucleotides, etc.) in the biopolymer.
  • the sequence (e.g., base sequence) of a nucleic acid is typically read in the 5' to 3' direction.
  • the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.
  • the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • non-human animals refers to all non-human animals including, but are not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.
  • gene refers to a nucleic acid (e.g. , DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, RNA (e.g. , including but not limited to, mRNA, tRNA and rRNA) or precursor.
  • RNA e.g. , including but not limited to, mRNA, tRNA and rRNA
  • the polypeptide, RNA, or precursor can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g. , enzymatic activity, ligand binding, signal transduction, etc.) of the full-length or fragment are retained.
  • the term also encompasses the coding region of a structural gene and the including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA.
  • the sequences that are located 5' of the coding region and which are present on the mRNA are referred to as 5' untranslated sequences.
  • the sequences that are located 3' or downstream of the coding region and that are present on the mRNA are referred to as 3' untranslated sequences.
  • gene encompasses both cDNA and genomic forms of a gene.
  • a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences”.
  • Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript;
  • introns therefore are absent in the messenger RNA (mRNA) processed transcript.
  • mRNA messenger RNA
  • the mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
  • locus refers to a nucleic acid sequence on a chromosome or on a linkage map and includes the coding sequence as well as 5' and 3' sequences involved in regulation of the gene.
  • the present invention relates to compositions, systems, and methods for determining a treatment course of action.
  • the present invention relates to compositions, systems, and methods for utilizing gene expression profiles to determine drug sensitivity in colorectal cancer.
  • the present invention provides a for determining a treatment course of action in a subject diagnosed with colorectal cancer (CRC), comprising: a) identifying a consensus molecular subtype classification (CMS) for a colorectal cancer sample; and b) determining a treatment course of action based on the CMS classification.
  • CRC colorectal cancer
  • the CMS classification is determined by assaying a sample for the level of expression and/or presence or absence of a mutation in one or more genes.
  • the sample may be tissue (e.g. , a colorectal biopsy sample or other tissue sample), blood, stool or a fraction thereof (e.g. , plasma, serum, etc.).
  • tissue e.g. , a colorectal biopsy sample or other tissue sample
  • blood e.g. , blood, stool or a fraction thereof (e.g. , plasma, serum, etc.).
  • the patient sample is subjected to preliminary processing designed to isolate or enrich the sample for the pseudogenes or cells that contain the pseudogenes.
  • preliminary processing designed to isolate or enrich the sample for the pseudogenes or cells that contain the pseudogenes.
  • a variety of techniques known to those of ordinary skill in the art may be used for this purpose, including but not limited to: centrifugation; immunocapture; cell lysis; and, nucleic acid target capture (See, e.g. , EP Pat. No. 1 409 727, herein incorporated by reference in its entirety).
  • a marker includes, for example, nucleic acid(s) whose production or mutation or lack of production is characteristic of a colorectal neoplasm or a prognosis or treatment thereof.
  • the statistical analysis will vary. For example, where a particular combination of markers is highly specific for sensitivity of colorectal cancer to a particular treatment, the statistical significance of a positive result will be high.
  • markers may be used that show optimal function with different ethnic groups or sex, different geographic distributions, different stages of disease, different degrees of specificity or different degrees of sensitivity. Particular combinations may also be developed which are particularly sensitive to the effect of therapeutic regimens on disease progression. Subjects may be monitored after a therapy and/or course of action to determine the effectiveness of that specific therapy and/or course of action. Markers for other cancers, diseases, infections, and metabolic conditions are also contemplated for inclusion in a multiplex or panel format.
  • the methods are not limited to a particular type of mammal.
  • the mammal is a human.
  • the colorectal neoplasm is premalignant.
  • the colorectal neoplasm is malignant.
  • the colorectal neoplasm is colorectal cancer without regard to stage of the cancer (e.g., stage I, II, III, or IV).
  • the colorectal cancer is stage II.
  • nucleic acid techniques known to those of ordinary skill in the art, including but not limited to: nucleic acid sequencing; nucleic acid hybridization; and nucleic acid amplification. These techniques utilize colorectal informative reagents such as nucleic acid probes and primers that hybridize to or can be used to amplify gene products of the cancer marker genes so that the level of expression of the respective cancer marker gene can be determined.
  • nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing.
  • chain terminator Sanger
  • dye terminator sequencing Those of ordinary skill in the art will recognize that because RNA is less stable in the cell and more prone to nuclease attack experimentally RNA is usually reverse transcribed to DNA before sequencing.
  • Chain terminator sequencing uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. Extension is initiated at a specific site on the template DNA by using a short radioactive, or other labeled, oligonucleotide primer complementary to the template at that region.
  • the oligonucleotide primer is extended using a DNA polymerase, standard four deoxynucleotide bases, and a low concentration of one chain terminating nucleotide, most commonly a di-deoxynucleotide. This reaction is repeated in four separate tubes with each of the bases taking turns as the di-deoxynucleotide.
  • the DNA polymerase Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular di- deoxynucleotide is used.
  • the fragments are size-separated by electrophoresis in a slab polyacrylamide gel or a capillary tube filled with a viscous polymer. The sequence is determined by reading which lane produces a visualized mark from the labeled primer as you scan from the top of the gel to the bottom.
  • Dye terminator sequencing alternatively labels the terminators. Complete sequencing can be performed in a single reaction by labeling each of the di-deoxynucleotide chain- terminators with a separate fluorescent dye, which fluoresces at a different wavelength.
  • nucleic acid sequencing methods are contemplated for use in the methods of the present disclosure including, for example, chain terminator (Sanger) sequencing, dye terminator sequencing, and high-throughput sequencing methods. Many of these sequencing methods are well known in the art, See, e.g., Sanger et al, Proc. Natl. Acad. Sci. USA 74:5463-5467 (1997); Maxam et al, Proc. Natl. Acad. Sci. USA 74:560-564 (1977);
  • deep sequencing is utilized to provide an analysis of the sequence and frequency of RNA molecules in the samples.
  • Suitable deep sequencing techniques include, but are not limited to, next generation sequencing techniques such as single molecule real time sequencing (Pacific Biosciences), sequencing by synthesis
  • nucleic acid hybridization techniques include, but are not limited to, in situ hybridization (ISH), microarray, nuclease protection assay, and Southern or Northern blot.
  • ISH In situ hybridization
  • DNA ISH can be used to determine the structure of chromosomes.
  • RNA ISH is used to measure and localize mRNAs and other transcripts (e.g., pseudogenes) within tissue sections or whole mounts. Sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away.
  • ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.
  • gene expression is detected using fluorescence in situ hybridization (FISH).
  • FISH assays utilize bacterial artificial chromosomes (BACs). These have been used extensively in the human genome sequencing project (see Nature 409: 953-958 (2001)) and clones containing specific BACs are available through distributors that can be located through many sources, e.g., NCBI. Each BAC clone from the human genome has been given a reference name that unambiguously identifies it. These names can be used to find a corresponding GenBank sequence and to order copies of the clone from a distributor.
  • the present invention further provides a method of performing a FISH assay on human colorectal cells, human colorectal tissue or on the fluid surrounding the human colorectal cells or tissue.
  • Specific protocols are well known in the art and can be readily adapted for the present invention.
  • Guidance regarding methodology may be obtained from many references including: In situ Hybridization: Medical Applications (eds. G. R. Coulton and J. de Belleroche), Kluwer Academic Publishers, Boston (1992); In situ Hybridization: In Neurobiology; Advances in Methodology (eds. J. H. Eberwine, K. L. Valentino, and J. D. Barchas), Oxford University Press Inc., England (1994); In situ Hybridization: A Practical Approach (ed. D. G.
  • kits that are commercially available and that provide protocols for performing FISH assays (available from e.g. , Oncor, Inc., Gaithersburg, MD).
  • Patents providing guidance on methodology include U.S. 5,225,326; 5,545,524; 6,121,489 and 6,573,043. All of these references are hereby incorporated by reference in their entirety and may be used along with similar references in the art and with the information provided in the Examples section herein to establish procedural steps convenient for a particular laboratory.
  • the present invention utilizes nuclease protection assays.
  • Nuclease protection assays are useful for identification of one or more RNA molecules of known sequence even at low total concentration.
  • the extracted RNA is first mixed with antisense RNA or DNA probes that are complementary to the sequence or sequences of interest and the complementary strands are hybridized to form double-stranded RNA (or a DNA-RNA hybrid).
  • the mixture is then exposed to ribonucleases that specifically cleave only single-stranded RNA but have no activity against double-stranded RNA.
  • RNA regions are degraded to very short oligomers or to individual nucleotides; the surviving RNA fragments are those that were complementary to the added antisense strand and thus contained the sequence of interest.
  • Suitable nuclease protection assays include, but are not limited to those described in US 5,770,370; EP 2290101A3; US 20080076121; US 20110104693; each of which is incorporated herein by reference in its entirety.
  • the present invention utilizes the quantitative nuclease protection assay provided by HTG Molecular Diagnostics, Inc. (Tuscon, AZ).
  • DNA microarrays e.g. , cDNA microarrays and oligonucleotide microarrays
  • protein microarrays e.g., protein microarrays
  • tissue microarrays e.g., transfection or cell microarrays
  • chemical compound microarrays e.g., chemical compound microarrays
  • antibody microarrays e.g., antibody microarrays
  • a DNA microarray commonly known as gene chip, DNA chip, or biochip, is a collection of microscopic DNA spots attached to a solid surface (e.g. , glass, plastic or silicon chip) forming an array for the purpose of expression profiling or monitoring expression levels for thousands of genes simultaneously.
  • the affixed DNA segments are known as probes, thousands of which can be used in a single DNA microarray.
  • Microarrays can be used to identify disease genes or transcripts (e.g., genes described herein) by comparing gene expression in disease and normal cells.
  • Microarrays can be fabricated using a variety of technologies, including but not limiting: printing with fine-pointed pins onto glass slides; photolithography using pre-made masks; photolithography using dynamic micromirror devices; ink-jet printing; or, electrochemistry on microelectrode arrays.
  • Southern and Northern blotting is used to detect specific DNA or RNA sequences, respectively.
  • DNA or RNA extracted from a sample is fragmented, electrophoretically separated on a matrix gel, and transferred to a membrane filter.
  • the filter bound DNA or RNA is subject to hybridization with a labeled probe complementary to the sequence of interest. Hybridized probe bound to the filter is detected.
  • a variant of the procedure is the reverse Northern blot, in which the substrate nucleic acid that is affixed to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from a tissue and labeled.
  • Nucleic acids may be amplified prior to or simultaneous with detection.
  • Nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA).
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • TMA transcription-mediated amplification
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • PCR The polymerase chain reaction (U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159 and 4,965,188, each of which is herein incorporated by reference in its entirety), commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase copy numbers of a target nucleic acid sequence.
  • RT-PCR reverse transcriptase (RT) is used to make a complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA.
  • cDNA complementary DNA
  • TMA Transcription mediated amplification
  • a target nucleic acid sequence autocatalytically under conditions of substantially constant temperature, ionic strength, and pH in which multiple RNA copies of the target sequence autocatalytically generate additional copies.
  • TMA optionally incorporates the use of blocking moieties, terminating moieties, and other modifying moieties to improve TMA process sensitivity and accuracy.
  • the ligase chain reaction (Weiss, R., Science 254: 1292 (1991), herein incorporated by reference in its entirety), commonly referred to as LCR, uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid.
  • the DNA oligonucleotides are covalently linked by a DNA ligase in repeated cycles of thermal denaturation, hybridization and ligation to produce a detectable double-stranded ligated oligonucleotide product.
  • SDA uses cycles of annealing pairs of primer sequences to opposite strands of a target sequence, primer extension in the presence of a dNTPaS to produce a duplex hemiphosphorothioated primer extension product, endonuclease-mediated nicking of a hemimodified restriction endonuclease recognition site, and polymerase-mediated primer extension from the 3' end of the nick to displace an existing strand and produce a strand for the next round of primer annealing, nicking and strand displacement, resulting in geometric amplification of product.
  • thermophilic SDA uses thermophilic endonucleases and polymerases at higher temperatures in essentially the same method (EP Pat. No. 0 684 315).
  • amplification methods include, for example: nucleic acid sequence based amplification (U.S. Pat. No. 5,130,238, herein incorporated by reference in its entirety), commonly referred to as NASBA; one that uses an RNA replicase to amplify the probe molecule itself (Lizardi et al., BioTechnol. 6: 1197 (1988), herein incorporated by reference in its entirety), commonly referred to as replicase; a transcription based amplification method (Kwoh et al, Proc. Natl. Acad. Sci. USA 86:1173 (1989)); and, self-sustained sequence replication (Guatelli et al, Proc. Natl. Acad. Sci. USA 87: 1874 (1990), each of which is herein incorporated by reference in its entirety).
  • NASBA nucleic acid sequence based amplification
  • replicase a transcription based amplification method
  • self-sustained sequence replication (Guatelli et
  • Non-amplified or amplified nucleic acids can be detected by any conventional means.
  • the cancer marker genes described herein can be detected by hybridization with a detectably labeled probe and measurement of the resulting hybrids. Illustrative non- limiting examples of detection methods are described below.
  • One illustrative detection method provides for quantitative evaluation of the amplification process in real-time.
  • Evaluation of an amplification process in "real-time” involves determining the amount of amplicon in the reaction mixture either continuously or periodically during the amplification reaction, and using the determined values to calculate the amount of target sequence initially present in the sample.
  • a variety of methods for determining the amount of initial target sequence present in a sample based on real-time amplification are well known in the art. These include methods disclosed in U.S. Pat. Nos. 6,303,305 and 6,541,205, each of which is herein incorporated by reference in its entirety.
  • Another method for determining the quantity of target sequence initially present in a sample, but which is not based on a real-time amplification is disclosed in U.S. Pat. No. 5,710,029, herein incorporated by reference in its entirety.
  • Amplification products may be detected in real-time through the use of various self- hybridizing probes, most of which have a stem-loop structure.
  • Such self-hybridizing probes are labeled so that they emit differently detectable signals, depending on whether the probes are in a self-hybridized state or an altered state through hybridization to a target sequence.
  • “molecular torches” are a type of self-hybridizing probe that includes distinct regions of self-complementarity (referred to as “the target binding domain” and “the target closing domain") which are connected by a joining region (e.g. , non- nucleotide linker) and which hybridize to each other under predetermined hybridization assay conditions.
  • molecular torches contain single-stranded base regions in the target binding domain that are from 1 to about 20 bases in length and are accessible for hybridization to a target sequence present in an amplification reaction under strand displacement conditions.
  • hybridization of the two complementary regions, which may be fully or partially complementary, of the molecular torch is favored, except in the presence of the target sequence, which will bind to the single- stranded region present in the target binding domain and displace all or a portion of the target closing domain.
  • the target binding domain and the target closing domain of a molecular torch include a detectable label or a pair of interacting labels (e.g.
  • luminescent/quencher positioned so that a different signal is produced when the molecular torch is self-hybridized than when the molecular torch is hybridized to the target sequence, thereby permitting detection of probe:target duplexes in a test sample in the presence of unhybridized molecular torches.
  • Molecular torches and a variety of types of interacting label pairs are disclosed in U.S. Pat. No. 6,534,274, herein incorporated by reference in its entirety.
  • a TaqManTM detection system is utilized to detect and quantify expression of the cancer marker genes.
  • the TaqMan probe system relies on the 5 '-3' exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection. As in other real-time PCR methods, the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stages of the PCR; however, the TaqMan probe significantly increases the specificity of the detection.
  • TaqMan probes consist of a fluorophore covalently attached to the 5 '-end of the oligonucleotide probe and a quencher at the 3'-end.
  • fluorophores e.g. 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescein, acronym: TET
  • quenchers e.g. tetramethylrhodamine, acronym: TAMRA, or dihydrocyclopyrroloindole tripeptide minor groove binder, acronym: MGB
  • FRET Fluorescence Resonance Energy Transfer
  • TaqMan probes are designed such that they anneal within a DNA region amplified by a specific set of primers. As the Taq polymerase extends the primer and synthesizes the nascent strand (again, on a single-strand template, but in the direction opposite to that shown in the diagram, i.e. from 3' to 5' of the complementary strand), the 5' to 3' exonuclease activity of the polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the real-time PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR.
  • Molecular beacons include nucleic acid molecules having a target complementary sequence, an affinity pair (or nucleic acid arms) holding the probe in a closed conformation in the absence of a target sequence present in an amplification reaction, and a label pair that interacts when the probe is in a closed conformation. Hybridization of the target sequence and the target complementary sequence separates the members of the affinity pair, thereby shifting the probe to an open conformation. The shift to the open conformation is detectable due to reduced interaction of the label pair, which may be, for example, a fluorophore and a quencher (e.g., DABCYL and EDANS).
  • Molecular beacons are disclosed in U.S. Pat. Nos. 5,925,517 and 6,150,097, herein incorporated by reference in its entirety.
  • probe binding pairs having interacting labels such as those disclosed in U.S. Pat. No. 5,928,862 (herein incorporated by reference in its entirety) might be adapted for use in the present invention.
  • Probe systems used to detect single nucleotide polymorphisms (SNPs) might also be utilized in the present invention.
  • Additional detection systems include "molecular switches," as disclosed in U.S. Publ. No. 20050042638, herein incorporated by reference in its entirety.
  • Other probes, such as those comprising intercalating dyes and/or fluorochromes are also useful for detection of amplification products in the present invention. See, e.g. , U.S. Pat. No. 5,814,447 (herein incorporated by reference in its entirety).
  • Hybridization Protection Assay involves hybridizing a chemiluminescent oligonucleotide probe (e.g. , an acridinium ester- labeled (AE) probe) to the target sequence, selectively hydrolyzing the chemiluminescent label present on unhybridized probe, and measuring the chemiluminescence produced from the remaining probe in a luminometer.
  • a chemiluminescent oligonucleotide probe e.g. , an acridinium ester- labeled (AE) probe
  • AE acridinium ester- labeled
  • cancer marker genes described herein may be detected as proteins using a variety of protein techniques known to those of ordinary skill in the art, including but not limited to: protein sequencing; and, immunoassays.
  • Illustrative non-limiting examples of protein sequencing techniques include, but are not limited to, mass spectrometry and Edman degradation.
  • Mass spectrometry can, in principle, sequence any size protein but becomes computationally more difficult as size increases.
  • a protein is digested by an endoprotease, and the resulting solution is passed through a high pressure liquid chromatography column. At the end of this column, the solution is sprayed out of a narrow nozzle charged to a high positive potential into the mass spectrometer. The charge on the droplets causes them to fragment until only single ions remain. The peptides are then fragmented and the mass- charge ratios of the fragments measured.
  • the mass spectrum is analyzed by computer and often compared against a database of previously sequenced proteins in order to determine the sequences of the fragments. The process is then repeated with a different digestion enzyme, and the overlaps in sequences are used to construct a sequence for the protein.
  • the peptide to be sequenced is adsorbed onto a solid surface (e.g. , a glass fiber coated with polybrene).
  • a solid surface e.g. , a glass fiber coated with polybrene.
  • phenylisothiocyanate is added to the adsorbed peptide, together with a mildly basic buffer solution of 12% trimethylamine, and reacts with the amine group of the N-terminal amino acid.
  • the terminal amino acid derivative can then be selectively detached by the addition of anhydrous acid.
  • the derivative isomerizes to give a substituted
  • phenylthiohydantoin which can be washed off and identified by chromatography, and the cycle can be repeated.
  • the efficiency of each step is about 98%, which allows about 50 amino acids to be reliably determined.
  • immunoassays include, but are not limited to: immunoprecipitation; Western blot; ELISA; immunohistochemistry; immunocytochemistry; flow cytometry; and, immuno-PCR.
  • Polyclonal or monoclonal antibodies detectably labeled using various techniques known to those of ordinary skill in the art (e.g. , colorimetric, fluorescent, chemiluminescent or radioactive) are suitable for use in the immunoassays.
  • Immunoprecipitation is the technique of precipitating an antigen out of solution using an antibody specific to that antigen.
  • the process can be used to identify protein complexes present in cell extracts by targeting a protein believed to be in the complex.
  • the complexes are brought out of solution by insoluble antibody -binding proteins isolated initially from bacteria, such as Protein A and Protein G.
  • the antibodies can also be coupled to sepharose beads that can easily be isolated out of solution. After washing, the precipitate can be analyzed using mass spectrometry, Western blotting, or any number of other methods for identifying constituents in the complex.
  • a Western blot, or immunoblot is a method to detect protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate denatured proteins by mass. The proteins are then transferred out of the gel and onto a membrane, typically polyvinyldiflroride or nitrocellulose, where they are probed using antibodies specific to the protein of interest. As a result, researchers can examine the amount of protein in a given sample and compare levels between several groups.
  • An ELISA short for Enzyme-Linked Immunosorbent Assay, is a biochemical technique to detect the presence of an antibody or an antigen in a sample. It utilizes a minimum of two antibodies, one of which is specific to the antigen and the other of which is coupled to an enzyme.
  • the second antibody will cause a chromogenic or fluorogenic substrate to produce a signal.
  • Variations of ELISA include sandwich ELISA, competitive ELISA, and ELISPOT. Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations and also for detecting the presence of antigen.
  • Immunohistochemistry and immunocytochemistry refer to the process of localizing proteins in a tissue section or cell, respectively, via the principle of antigens in tissue or cells binding to their respective antibodies. Visualization is enabled by tagging the antibody with color producing or fluorescent tags.
  • color tags include, but are not limited to, horseradish peroxidase and alkaline phosphatase.
  • fluorophore tags include, but are not limited to, fluorescein isothiocyanate (FITC) or phycoerythrin (PE).
  • Flow cytometry is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an
  • a beam of light (e.g. , a laser) of a single frequency or color is directed onto a hydrodynamically focused stream of fluid.
  • a number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter (SSC) and one or more fluorescent detectors).
  • FSC Forward Scatter
  • SSC Segment Scatter
  • Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals in the particle may be excited into emitting light at a lower frequency than the light source.
  • FSC correlates with the cell volume and SSC correlates with the density or inner complexity of the particle (e.g. , shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness).
  • Immuno-polymerase chain reaction utilizes nucleic acid amplification techniques to increase signal generation in antibody-based immunoassays. Because no protein equivalence of PCR exists, that is, proteins cannot be replicated in the same manner that nucleic acid is replicated during PCR, the only way to increase detection sensitivity is by signal amplification.
  • the target proteins are bound to antibodies which are directly or indirectly conjugated to oligonucleotides. Unbound antibodies are washed away and the remaining bound antibodies have their oligonucleotides amplified. Protein detection occurs via detection of amplified oligonucleotides using standard nucleic acid detection methods, including real-time methods.
  • a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g. , the expression level a given marker or markers) into data of predictive value for a clinician.
  • the clinician can access the predictive data using any suitable means.
  • the present invention provides the further benefit that the clinician, who is not likely to be trained in genetics or molecular biology, need not understand the raw data.
  • the data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.
  • a sample e.g. , a biopsy or a serum or stool sample
  • a profiling service e.g., clinical lab at a medical facility, genomic profiling business, etc.
  • the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g.
  • the sample comprises previously determined biological information
  • the information may be directly sent to the profiling service by the subject (e.g. , an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems).
  • the profiling service Once received by the profiling service, the sample is processed and a profile is produced (i.e. , expression data), specific for the diagnostic or prognostic information desired for the subject.
  • the profile data is then prepared in a format suitable for interpretation by a treating clinician.
  • the prepared format may represent a diagnosis or risk assessment for the subject, along with recommendations for particular treatment options.
  • the data may be displayed to the clinician by any suitable method.
  • the profiling service generates a report that can be printed for the clinician (e.g. , at the point of care) or displayed to the clinician on a computer monitor.
  • the information is first analyzed at the point of care or at a regional facility.
  • the raw data is then sent to a central processing facility for further analysis and/or to convert the raw data to information useful for a clinician or patient.
  • the central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis.
  • the central processing facility can then control the fate of the data following treatment of the subject.
  • the central facility can provide data to the clinician, the subject, or researchers.
  • the subject is able to directly access the data using the electronic communication system.
  • the subject may chose further intervention or counseling based on the results.
  • the data is used for research use.
  • the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease or as a companion diagnostic to determine a treatment course of action.
  • compositions for use in the diagnostic methods described herein include, but are not limited to, kits comprising one or more colorectal cancer informative reagents as described above.
  • the kits comprise one or more colorectal cancer informative reagents for detecting altered gene expression in a sample from a subject having or suspected of having colorectal cancer of one or more two or more, five or more, 10 or more, 11 or more, 12 or more or 13.
  • the kits contain colorectal cancer informative reagents specific for a cancer gene marker, in addition to detection reagents and buffers.
  • the colorectal informative reagent is a probe(s) that specifically hybridizes to a respective gene product(s) of the one or more genes, a set(s) of primers that amplify a respective gene product(s) of the one or more genes, an antigen binding protein(s) that binds to a respective gene product(s) of the one or more genes, or a sequencing primer(s) that hybridizes to and allows sequencing of a respective gene product(s) of the one or more genes.
  • the probe and antibody compositions of the present invention may also be provided in the form of an array.
  • the kits contain all of the components necessary to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results.
  • kits include instructions for using the reagents contained in the kit for the detection and characterization of cancer in a sample from a subject.
  • the instructions further comprise the statement of intended use required by the U.S. Food and Drug Administration (FDA) in labeling in vitro diagnostic products.
  • FDA U.S. Food and Drug Administration
  • the FDA classifies in vitro diagnostics as medical devices and requires that they be approved through the 510(k) procedure.
  • Information required in an application under 510(k) includes: 1) The in vitro diagnostic product name, including the trade or proprietary name, the common or usual name, and the classification name of the device; 2) The intended use of the product; 3) The establishment registration number, if applicable, of the owner or operator submitting the 510(k) submission; the class in which the in vitro diagnostic product was placed under section 513 of the FD&C Act, if known, its appropriate panel, or, if the owner or operator determines that the device has not been classified under such section, a statement of that determination and the basis for the determination that the in vitro diagnostic product is not so classified; 4) Proposed labels, labeling and advertisements sufficient to describe the in vitro diagnostic product, its intended use, and directions for use.
  • photographs or engineering drawings should be supplied; 5) A statement indicating that the device is similar to and/or different from other in vitro diagnostic products of comparable type in commercial distribution in the U.S., accompanied by data to support the statement; 6) A 510(k) summary of the safety and effectiveness data upon which the substantial equivalence determination is based; or a statement that the 510(k) safety and effectiveness information supporting the FDA finding of substantial equivalence will be made available to any person within 30 days of a written request; 7) A statement that the submitter believes, to the best of their knowledge, that all data and information submitted in the premarket notification are truthful and accurate and that no material fact has been omitted; 8) Any additional information regarding the in vitro diagnostic product requested that is necessary for the FDA to make a substantial equivalency determination. Additional information is available at the Internet web page of the U.S. FDA. III. Methods of Use
  • the present invention provides colorectal cancer informative reagents and methods for determining a prognosis and/or treatment course of action for colorectal cancer in a subject.
  • the colorectal cancer can be stage I, II, III, or IV colorectal cancer.
  • the treatment course of action is administration of an HSP90 inhibitor, a topoisomerase II inhibitor, 2-methoxy estradiol, disulfiram, atorvastatin, PF- 03758309, rigosertib, tipifarnib, YM155, indibulin, cytarabine, 8-chloro-adenosine, serdemetan, TH588, RAF265, KU-60019, BGB324, auranofin or SGC-CBP30 when the CMS is CMS1 or CMS4.
  • the HSP90 inhibitor is, for example, luminespib, ganetespib, onalespib, SGX-301, radicicol or a derivative thereof.
  • the radicicol derivative is OS-47720.
  • the topoisomerase II inhibitor is valrubicin, daunorubicin, doxorubicin, or idarubicin.
  • the treatment course of action is administration of an alcohol dehydrogenase inhibitor or an Axl- inhibitor when the CMS is CMS4.
  • the alcohol dehydrogenase inhibitor is disulfiram and the Axl-inhibitor is BGB324.
  • the treatment course of action is administration of luminespib, ganetespib, 2-methoxyestradiol, atorvastatin or disulfiram.
  • treatments described herein are administered with one or more conventional treatments for CRC or in combination with surgical or radiation therapies. In some embodiments, treatments described herein are administered together with 5'- fluorouracil (5-FU).
  • 5-FU 5'- fluorouracil
  • the CMS classification is determined at one or more points during treatment (e.g., before, during, or after treatment with a particular agent).
  • Genes with subtype-specific expression were identified as genes with high relative expression in each CMS group in the TCGA training set. Differential expression analysis was done by comparing each subtype with the rest using the voom approach with quantile normalization in the R package limma, and genes with a log 2 fold-change > 1 and adjusted P- value ⁇ 0.1 in each subtype were retained. To enrich for genes likely to be informative in cell lines and PDX models, and to exclude genes with high expression in the tumor
  • NTP nearest template prediction
  • CMS4 (patient ID 43) and one of CMS2 (patient ID 1) were selected for drug treatment.
  • Experiments were conducted following the European Union's animal care directive (2010/63/EU) and were approved by the Ethical Committee of Animal Experimentation of VHIR (Vail d'Hebron Institute of Research)/VHIO (Vail d'Hebron Institute of Oncology; ID: 18/15 CEEA).
  • NOD-SCID NOD.CB17-Prfefc ⁇ /NcrCrl mice were purchased from Charles River Laboratories (Wilmington, MA, USA).
  • Luminespib 25 mg/kg in PBS, MCE, Monmouth Junction, NJ, USA was administered by intraperitoneal injection three times per week.
  • 5-FU 40mg/kg in PBS; Sigma- Aldrich, St. Louis, MO, USA
  • 5-FU 40mg/kg in PBS; Sigma- Aldrich, St. Louis, MO, USA
  • mice were euthanized and complete necropsies were performed.
  • Protein expression of HSP70 and Ki67 was analyzed in post-treatment tissue samples by immunohistochemistry.
  • Kaplan-Meier survival curves were compared with the log-rank test. Five-year relapse-free survival (RFS, considering relapse after complete resection or death from any cause as events) and overall survival (OS, considering death from any cause as events) were used as endpoints. Anti-tumor activity in PDX models was analyzed using a generalized linear mixed model of tumor volume fold changes, with random effects and treatment arm and time as covariates.
  • RF random forest
  • patients with CMS4 tumors had worse OS and RFS rates than patients with CMS 1-3 ( Figure 2).
  • a CMS classifier specific to cancer cells independent of the tumor microenvironment and optimized for CRC cell lines, was generated.
  • candidate genes with high relative expression in each subtype were identified in The Cancer Genome Atlas (TCGA) CRC gene expression data;
  • genes highly expressed in immune and stromal compartments were filtered out; and
  • NTP Nearest Template Prediction
  • the classifier was applied on a collection of 150 unique CRC cell lines (gene expression data was obtained from multiple publicly available datasets (13, 16, 24)and in- house expression analysis of 35 cell lines). Confident CMS classification was obtained for 131 (87%) of the cell lines, using a threshold for the false discovery rate (FDR) from NTP of 0.2. The distribution of cell lines among the subtypes was similar to the in-house patient series; 28 cell lines (19%) in CMS1; 51 (34%) in CMS2; 24 (16%) in CMS3; 28 (19%) in CMS4 ( Figure 3 a).
  • CMS2 additionally had up-regulation of HNF4A targets, while CMS3 was enriched for metabolic pathways.
  • CMS4 was specifically characterized by EMT activation, extracellular matrix organization and TGFp responses.
  • n 460 drugs.
  • DSS drug sensitivity scores
  • Hsp90-inhibitors luminespib, ganetespib and radicicol
  • 2ME 2ME
  • atorvastatin and indibulin another tubulin-inhibitor
  • Other drugs with notable selectivity for CMS 1 and CMS4 over CMS2 and CMS3 include the topoisomerase II inhibitors valrubicin, daunorubicin and doxorubicin, the survivin inhibitor YM155, the PAK inhibitor PF-03758309, the PLK inhibitor rigosertib, the farsenyltransferase inhibitor tipifarnib, and the anti-metabolite cytarabine. Additional drugs with differential sensitivity supporting CMS subtype-specific treatment of CRC are listed in tables 5-7.
  • CMS-associated drug sensitivities For independent biological validation of the CMS-associated drug sensitivities, five additional cell lines were identified as belonging to either the CMS l (LIM2405) or CMS4 (CAR1, HCA7, LIM2099 and OUMS23) subtypes based on publicly available gene expression data, and subsequently screened for drug sensitivities with the same experimental setup as in the initial discovery screen. In addition, two of the CMS3 cell lines from the initial screen were included as controls. Clear differential sensitivity in CMS l and CMS4 compared with CMS2 and CMS3 was validated for the three Hsp90-inhibitors (luminespib, ganetespib and radicicol), 2-ME, atorvastatin and disulfiram (Figure 5b). Notably, disulfiram had a biphasic dose-response curve.
  • HSP90 inhibition alleviates chemoresistance in CMS4 in vivo
  • CMS4 had a particularly poor response to fluoropyrimidines (P ⁇ 0.05 among MSS cell lines).
  • HSP90 inhibition may sensitize CRC cell lines to chemotherapy, and although monotherapy with HSP90 inhibitors has shown low efficacy in metastatic CRC (Cercek A, et al. Ganetespib, a novel Hsp90 inhibitor in patients with KRAS mutated and wild type, refractory metastatic colorectal cancer. Clin Colorectal Cancer 2014;13:207-12), response has been obtained by combination therapy with HSP90 inhibitors and capecitabine (5-FU pro-drug) in patients who have progressed on fluoropyrimidines (Bendell JC, et al.
  • transcriptomic CMS 1 and CMS4 groups of CRC by high-throughput drug screening, using a new and cancer cell-adapted CMS classifier.
  • HSP90 inhibition has previously been extensively investigated in cancer and has demonstrated anti-tumor activity in several solid tumor types, mainly as combination therapies (Wang H, et al. Effects of treatment with an Hsp90 inhibitor in tumors based on 15 phase II clinical trials. Mol Clin Oncol 2016;5:326- 34.).
  • low response rates are observed in unstratified patient populations.
  • single-agent treatment with ganetespib demonstrated good tolerance but low efficacy in chemotherapy -refractory metastatic disease, independent of KRAS mutation status (Cercek A, et al.
  • Ganetespib a novel Hsp90 inhibitor in patients with KRAS mutated and wild type, refractory metastatic colorectal cancer. Clin Colorectal Cancer 2014;13:207-12). Higher anti-tumor activity was seen in early clinical trials exploring combinations of HSP90 inhibitors with chemotherapies, including fluoropyrimidines (5-FU and capecitabine) (Bendell JC, et al. A phase I study of the Hsp90 inhibitor AUY922 plus capecitabine for the treatment of patients with advanced solid tumors. Cancer Invest
  • HSP90 inhibition downregulates thymidylate synthase and sensitizes colorectal cancer cell lines to the effect of 5FU-based chemotherapy.
  • Hsp90 inhibitors sensitise human colon cancer cells to topoisomerase I poisons by depletion of key anti-apoptotic and cell cycle checkpoint proteins. Biochem Pharmacol 2012;83:355-67).
  • Tumor localization (right, left, rectum, synchronous) 173, 122, 111, 3
  • Tumor differentiation grade (high, medium, low, unknown) 9, 336, 56, 8
  • MSI-status (MSI+, MSS, not scored) 72, 328, 9
  • KRAS mutant frequency, number of samples scored
  • MSI microsatellite instability
  • MSS microsatellite stable
  • CMS classes obtained from Guinney et al., Nat. Med. 21, 1350-1356 (2015); b CMS classes obtained using the RF algorithm in the CMSclassifier
  • CMS1/CMS4 cell lines CMS1/CMS4 cell lines.
  • DSS drug sensitivity score Table 7. Differential drug sensitivity between CMSl/4 and CMS2/3 cell lines - extended analysis

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Abstract

La présente invention concerne des compositions, des systèmes et des méthodes permettant de déterminer un plan d'action thérapeutique. En particulier, la présente invention concerne des compositions, des systèmes et des méthodes permettant d'utiliser des profils d'expression génique pour déterminer la sensibilité aux médicaments dans des cas de cancer colorectal.
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