WO2013045464A1 - Biomarqueurs d'adnc dans du sang total pour l'évaluation du cancer colorectal - Google Patents

Biomarqueurs d'adnc dans du sang total pour l'évaluation du cancer colorectal Download PDF

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Publication number
WO2013045464A1
WO2013045464A1 PCT/EP2012/068905 EP2012068905W WO2013045464A1 WO 2013045464 A1 WO2013045464 A1 WO 2013045464A1 EP 2012068905 W EP2012068905 W EP 2012068905W WO 2013045464 A1 WO2013045464 A1 WO 2013045464A1
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colorectal cancer
colorectal
sample
polynucleotide
biomarker
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PCT/EP2012/068905
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English (en)
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Orsolya GALAMB
Beata Kanne
Friedemann Krause
Katalin LEISZTER
Ralf Mauritz
Fabian Model
Bela Molnar
Kinga TOTH
Heiko Walch
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Roche Diagnostics Gmbh
F. Hoffmann-La Roche Ag
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Publication of WO2013045464A1 publication Critical patent/WO2013045464A1/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
    • C12Q2600/158—Expression markers

Definitions

  • Biomarkers, methods and kits for the assessment of whole blood samples wherein such assessment targets any of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyps.
  • Biomarkers disclosed are polynucleotides originating from genetic loci selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and
  • Biomarkers have been identified for assessment and further analysis of colorectal cancer, colorectal adenoma, and colorectal polyps (the latter two are also referred to as precursors of colorectal cancer). Originally, the biomarkers have been identified using an oligonucleotide array-based screening of R A from biopsies and blood from patients with colorectal cancer or colorectal adenoma. The biomarkers have been established to assess changes in blood samples against normal blood.
  • the biomarkers have been developed to a real-time PCR format suitable for assessing in vitro different stages selected from colorectal cancer, colorectal adenoma, and colorectal polyp using a blood sample from a patient.
  • the biomarkers of the present disclosure may be used for providing a cost effective and rapid procedure for assessment, including risk assessment, early diagnosis, establishing prognosis, monitoring patient treatment, detecting relapse, and discovery of therapeutic intervention of colorectal cancer.
  • the present disclosure relates to a method for assessing an object selected from colorectal cancer, colorectal adenoma, and colorectal polyp, wherein a whole blood sample is assessed, and wherein the sample is derived from an individual; the assessment is based on measuring the level of a polynucleotide originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1 , CSNK1D, C19orf43, and LRR 3 in the sample.
  • the field of art of this disclosure concerns biomarkers for the assessment of colorectal cancer or a precursor thereof. These biomarkers are useful for risk assessment, early detection, establishing prognosis, evaluation of intervention, recurrence of neoplastic tissue, and discovery of therapeutic intervention, and methods of use thereof.
  • biomarkers are useful for risk assessment, early detection, establishing prognosis, evaluation of intervention, recurrence of neoplastic tissue, and discovery of therapeutic intervention, and methods of use thereof.
  • clinical procedures providing for the risk assessment and early detection of colorectal cancer have been long sought.
  • colorectal cancer is the second leading cause of cancer-related deaths in the Western world.
  • One picture that has clearly emerged through decades of research into colorectal cancer is that early detection is critical to enhanced survival rates.
  • the currently accepted methods for colorectal cancer screening include the fecal occult blood test (FOBT), x-ray using double contrast between barium enema and air (DCBE), sigmoidoscopy, and colonoscopy.
  • FOBT fecal occult blood test
  • DCBE x-ray using double contrast between barium enema and air
  • sigmoidoscopy is an invasive procedure that visually examines the lower third of the colon using a lighted, flexible endoscope
  • colonoscopy is a procedure that examines the entire colon. In both cases, biopsy samples can be taken during the procedure.
  • PreGen-PlusTM which is a non-invasive test that isolates and analyzes DNA extracted from a patient's stool sample for alterations associated with the presence of colorectal cancer.
  • ColonSentryTM is also a blood-based colon cancer detection test that measures the expression of seven genes as a basis for estimating the patient's current risk of having colorectal cancer.
  • Another assay is based on the detection of methylated septin-9 DNA (mSEPT9) aiming at early detection of colorectal cancer by analyzing a blood sample. The test is based on the observation of epigenetic change of the septin-9 gene in a number of colorectal tumors. Further methylation markers for detection and prognosis of colon cancers were developed by Oncomethylome.
  • Signature Diagnostics markets an assay called Detector C which is a blood-based screening test for the detection of colorectal cancer. The test uses oligonucleotide chip technology to evaluate the expression of 202 genes that are altered in response to tumor formation and growth.
  • a first aspect of the teachings herein is the use of a polynucleotide originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6,
  • MALAT1, CSNK1D, C19orf43, and LRR 3 as a biomarker for and/or in the assessment of one or more selected member(s) of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp.
  • a second aspect of the teachings herein is a panel of biomarkers for assessing any member of the group consisting of colorectal cancer, colorectal adenoma and colorectal polyp, the panel comprising at least two polynucleotides, each polynucleotide corresponding to a different genetic locus, wherein at least one, and particulatly at least two of the polynucleotides originate(s) from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and LRRN3.
  • a third aspect of the teachings herein is a method for measuring the expression levels of a polynucleotide from a biomarker, the biomarker being an indicator for the presence of any member of the group consisting of colorectal cancer, colorectal adenoma and colorectal polyp in a human individual, the method comprising: selecting a biomarker comprising a contiguous polynucleotide included in a member of the group consisting of SEQ ID NOs: 1, 14, 27, 40, 53, 66, 79, 92, 105, a fragment thereof or its complement, and a variant thereof or its complement; obtaining a biological sample from the human individual; isolating cellular RNA from the sample and reverse-transcribing isolated RNA to obtain cDNA; amplifying copies of cDNA for the biomarker originating from the sample; and quantifying the level of cDNA amplified from the sample, thereby measuring the expression level of a polynucleotide from a biomark
  • a fourth aspect of the teachings herein is a kit for the assessment of any member of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp, the kit comprising: at least one reagent that is used in analysis of polynucleotide expression level for a biomarker for a member selected from the group consisting of colorectal cancer, colorectal adenoma and colorectal polyp, wherein the biomarker comprises a polynucleotide selected from SEQ ID NOs: 1, 4, 8, 12, 14, 17, 21, 25, 27, 30, 34, 38, 40, 43, 47, 51, 53, 56, 60, 64, 66, 69, 73, 77, 79, 82, 86, 90, 92, 95, 99, 103, 105, 108, 112, a complement therof, a variant thereof, and a fragment thereof; and instructions for using the kit for analyzing the polynucleotide expression level.
  • a molecule also includes a plurality of molecules (i.e. one or more).
  • polynucleotide denotes a single-stranded DNA or RNA molecule, or its respective complement, wherein in an embodiment the polynucleotide is a polymer of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleoside monomers.
  • a particular embodiment is a polymer of 15 to 35 nucleoside monomers.
  • polynucleotide also encompasses homo- and heteroduplex double- stranded nucleic acid molecules of which each strand comprises 8 or more nucleoside monomers.
  • heteroduplices are encompassed of which one strand comprises one or more non-natural nucleoside analog(s).
  • the strand with the nucleoside analog(s) comprises 8 or more monomers.
  • non-natural nucleoside analog is a LNA (locked nucleic acid) monomer.
  • Other non-natural nucleoside analogs are known to the art and encompassed herein.
  • Particular embodiments are non-natural nucleoside analogs capable of increasing the melting point of a heteroduplex with RNA or DNA. Being directed to polynucleotides capable of forming hybrids, or being hybridized or in the process of hybridization, the term "duplex" denotes the double helix formed between a RNA or DNA molecule and its complement.
  • a "variant" of a polynucleotide differs from the polynucleotide by one or more mutations while preserving substantial sequence similarity as well as specificity in hybridization processes in the procedures disclosed herein.
  • a mutation can be a point mutation wherein at a given position a first nucleobase is substituted by a second nucleobase or an analog thereof. Further mutations are deletions or insertions.
  • a polynucleotide and its variant have a sequence identity of a value selected from 95%, 96%, 97%, 98%, 99%, and a value higher than 95% and below 100%.
  • a “fragment”, and particularly a “fragment of a polynucleotide” is understood to be an incomplete or isolated portion of the polynucleotide, comprising at least a single strand with an undisrupted (contiguous) nucleotide sequence of 8 or more positions, according to the entire polynucleotide.
  • the fragment thereof comprises an undisrupted nucleotide sequence of 17, 18, 19, 20, 25, 50, 75, 100, 200, 300, 500, 700, 1000 or more positions.
  • a polynucleotide or a nucleotide sequence "originating from” a genetic locus is understood to include a nucleic acid molecule (DNA or R A) which results from transcription of the genetic locus, i.e. a transcript, or from reverse-transcription of a transcript.
  • the term polynucleotide "originating from” a genetic locus encompasses a nucleic acid which is obtainable as a result of a sample preparation process in combination with one or more process(es) of treatment, that is treatment of the processed sample and/or treatment of the sample prior to applying the sample preparation process.
  • the nucleic acid obtainable as a result of the processes is double-stranded or single - stranded, and its sequence is comprised in or complementary to the sequence of any of the complements of the respective genetic locus.
  • genomic locus denotes a genomic region which is transcribed in a tumor cell and/or a non-tumor cell.
  • a transcribed region of a gene can comprise any of non-coding leader, non-coding trailer, intron and exon sequences.
  • the term “genetic locus” further includes any transcribed region of a pseudogene, any transcribed region with established or putative regulatory function, and any other transcribed region.
  • a transcribed region generally, is genomic DNA which is a template for a DNA-dependent RNA polymerase at least once or more times in the lifetime of a tumor cell and/or a non-tumor cell.
  • a “biomarker” is a biological molecule which indicates a particular disease state or another physiological state of an organism.
  • biomarker includes a substance whose detection indicates a particular disease state.
  • a biomarker may also indicate a change in gene or protein expression that correlates with the risk or progression of a disease, or with the susceptibility of the disease to a given treatment.
  • Biomarkers are characteristic biological properties that can be detected and measured in parts of the body like the blood or tissue. They may indicate either normal or diseased processes in the body. Biomarkers can be specific cells, molecules, or genes, gene products, enzymes, or hormones.
  • the term “biomarker” is encompassed in the broader term “marker” which denotes an object to indicate a position or a status or which serves as a standard of comparison or as an indication of what may be expected.
  • a panel of biomarkers is a selection of two or more biomarkers.
  • a “marker of cancer” and in particular a “marker of colorectal cancer” in the sense of the present disclosure is any marker that, either alone or in combination with one or more further biomarker(s), adds relevant information in the assessment of colorectal cancer.
  • a “marker of colorectal adenoma” and a “marker of colorectal polyp” adds relevant information in the assessment of the respective neoplastic potential precursor of colorectal cancer.
  • colorectal adenoma or colorectal polyp can be improved by combining the marker with another biomarker or with a diagnostic result, or by including the marker into a panel of biomarkers.
  • sample refers to a biological sample obtained for the purpose of evaluation in vitro.
  • the sample or patient sample is whole blood, e.g. peripheral blood; however, further samples are possible such as plasma, serum, stool and tissue. It is understood that any such evaluation is made in vitro, which implies that the sample material is physically separated from the patient's body in the process of such evaluation. The patient sample is discarded afterwards.
  • the patient sample is solely used for the in vitro diagnostic method as disclosed, and the material of the patient sample is not transferred back into the patient's body.
  • the expressions "assessment of an object or “assess” or “assessing” an object, wherein the object is selected from colorectal cancer, colorectal adenoma, and colorectal polyp, are used to indicate that the disclosed subject-matter will (alone or together with other markers or variables, e.g., the classification criteria set forth by the AJCC or the Dukes stage classification, see below) e.g., aid the physician to establish or confirm the absence or presence of colorectal cancer, colorectal adenoma, and colorectal polyp, or aid the physician in the prognosis, the detection of recurrence (follow-up of patients after surgery) and/or the monitoring of treatment, especially of chemotherapy.
  • markers or variables e.g., the classification criteria set forth by the AJCC or the Dukes stage classification, see below
  • assessing and “assessment” encompass the use of technical items and execution of procedural steps which yield the results on which the physician establishes or confirms the presence or absence of an object selected from colorectal cancer, colorectal adenoma, and colorectal polyp.
  • a "colorectal polyp” is understood as being a clump of cells on the inside (the lining) of the colon or the rectum.
  • a colorectal polyp may give rise to colorectal cancer.
  • Colorectal polyps are conventionally divided into two groups - non-neoplastic polyps and neoplastic polyps.
  • Neoplastic polyps are also known as adenomatous polyps or adenomas.
  • Non-neoplastic polyps include juvenile, hyperplastic, inflammatory, and lymphoid polyps.
  • Non-neoplastic polyps have not been considered precursors of cancer while the neoplastic polyps bear the risk of being precursors of colorectal cancer.
  • the gastroenterologist uses a colonoscopy to find and remove polyps and adenomas to prevent them from acquiring genetic changes that will lead to an invasive adenocarcinoma.
  • a particular desire is to have a biomarker indicating the presence of a polyp.
  • adenoma is a benign tumor of glandular origin which can grow from many organs including the colon and/or the rectum ("colorectal adenoma"). Although these growths are benign, over time they may progress to become malignant, at which point they are referred to as "adenocarcinomas". Most colorectal adenomas are polypoid. Large flat and depressed colorectal adenomas may be more likely to be severely dysplastic and give rise to malignancies. Colorectal adenomas are found commonly at colonoscopy. They are removed because of their tendency to become malignant and to lead to colorectal cancer. In medical practice it is desired to have alternative means to detect colon adenoma. A particular desire is to have a biomarker indicating the presence of colon adenoma.
  • a precursor stage poses the risk of developing further into colorectal cancer, i.e. a malignant cancer.
  • Adenocarcinoma is defined as a malignant tumor that grows on the glandular epithelial cells of an internal organ.
  • Cold- cancer is a cancer of the colon and/or the rectum, a malignant tumor arising from the inner wall of the large intestine. The majority of colorectal cancer is of the adenocarcinoma type.
  • Colorectal cancer staging is performed for diagnostic and research purposes, and to determine the best method of treatment. The systems for staging colorectal cancers depend on the extent of local invasion, the degree of lymph node involvement and whether there is distant metastasis.
  • TNM tumors/nodes/metastases
  • AJCC American Joint Committee on Cancer
  • AJCC AJCC Cancer Staging Manual
  • An alternative staging system for colorectal cancer is the Dukes classification. It identifies stages as
  • a particular goal of the disclosure is to provide a biomarker indicating the presence of colorectal cancer at an early stage.
  • a further goal of the disclosure is to provide a biomarker indicating the presence of a precursor of colorectal cancer, i.e. the presence of polyp or adenoma tissue in the colon or the rectum.
  • Biomarkers for cancer in general and colorectal cancer in particular have several potential uses in the management of patient care. Ideally, they would be used for risk assessment, for early diagnosis, for establishing prognosis, for monitoring treatment, and for detecting relapse. Additionally, such markers could play a valuable role in developing therapeutic interventions.
  • sampling methods used in conjunction with biomarker analysis are minimally invasive or non-invasive.
  • sampling methods are directed to sampling whole blood, plasma, serum, stool, swabs, and the like.
  • Non-invasive and minimally invasive methods increase patient compliance, and generally reduce cost.
  • biomarkers are specificity and sensitivity.
  • Specificity of the biomarker is a percentage of correctly analysed healthy subjects of all healthy subjects or the probability for the negative diagnosis of healthy subjects.
  • Stress ist the percentage of correctly analysed diseased patients of all diseased patients or the probability for a positive diagnosis of diseased patients.
  • biomarkers would have 100% clinical specificity and 100% clinical sensitivity.
  • no single biomarker has been identified that has an acceptably high degree of specificity and sensitivity required to be effective in for the broad range of needs in patient care management.
  • single serum biomarkers such as alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) have proven to provide value in some aspects of patient care management.
  • AFP alpha-fetoprotein
  • CEA carcinoembryonic antigen
  • elevated serum levels of CEA were first discovered in patients with adenocarcinoma of the colon. Elevated levels can be found in a variety of benign and malignant conditions other than colorectal cancer. Additionally, the production of CEA by early localized tumors of the colon is in the normal range. Therefore
  • CEA lacks both the sensitivity and specificity required to be of value for risk assessment or early diagnosis. Further, elevated levels of CEA correlate poorly with colon tumor differentiation and stage, rendering CEA as a biomarker for prognosis of colorectal cancer of limited value.
  • the two areas for which CEA has proven helpful clinically in managing patient care are in evaluating the effectiveness of treatment, and for detecting relapse. Illustrative of this, numerous studies have found that there is high correlation between elevated serum levels of CEA preceding clinical detection of recurrence of colorectal cancer. This has proven to be of value in managing the care of high-risk patents with second-look surgical procedures based on rising levels of CEA.
  • colorectal cancer it is highly desired to detect colorectal cancer at an early stage and/or to detect a precursor stage of colorectal cancer such as colorectal adenoma and colorectal polyp.
  • What is disclosed herein is based on discovery studies with mRNA isolated from biopsy and blood samples from patients using an oligonucleotide array-based screening approach. Out of a large number of candidate sequences, a panel of biomarkers has been established to assess changes in human tissue from surgical and biopsy samples and from blood samples against control tissue and blood, respectively. Clinical samples from patients were used to identify significant genomic alterations as correlates of the emergence and progression of disease. To this end, the Affymetrix® platform of oligonucleotide arrays and analysis means served in the analysis of tumor RNA expression. From these human subject studies, a plurality of biomarkers is disclosed herein.
  • the biomarkers can be of medical use when used as single markers. However, their technical advantage in the assessment of colorectal cancer, precursor and early stages of colorectal cancer is increased when a panel of these biomarkers is used. Further, what is disclosed are methods for measuring gene expression levels based on individual markers and on a panel of markers. Additionally, another aspect of what is disclosed are kits which provide the reagents and instructions for measuring gene and protein expression levels based on the panel. The panel, methods and kits are useful in the management of patient care for colorectal cancer. Additionally, the panel, methods and kits are believed useful as the basis for discovery of therapeutic interventions for colorectal cancer.
  • Measuring gene expression means to quantify the level at which a particular gene is expressed within a cell, tissue or organism. While the amount of a final gene product may be indicative for the level of expression of the respective gene, measurement of gene expression in line with the present disclosure is typically made by detecting and quantifying mRNA, and infer gene expression level. Thus, the level of a given mRNA in a given sample is used as a biomarker.
  • RT-qPCR reverse transcription quantitative polymerase chain reaction
  • qPCR quantitative PCR
  • the RT step first generates a DNA template from the mRNA by reverse transcription, which is called cDNA.
  • the cDNA serves as a template in the subsequent step of qPCR.
  • fluorescence of a detection probe changes as the DNA amplification process progresses.
  • Other qPCR embodiments make use of an intercalating dye which interacts with double-stranded DNA and which is capable of indicating newly formed DNA.
  • qPCR can produce an absolute measurement such as number of copies of mRNA present in the sample.
  • the qPCR method is very sensitive as detection of a single mRNA molecule is possible.
  • Arrays of different capture probes can be used to detect and/or quantify transcript levels for many genes at once (expression profiling).
  • Recent advances in microarray technology allow for the quantitation, even on a single array, of transcript levels for every known gene in several organism's genomes, including the human genome.
  • Table 1 gives a first overview of the marker genes and genetic loci; nucleotide sequences originating from these loci are used as biomarkers, according to the present disclosure.
  • a transcript of each marker gene or genetic locus alone advantageously serves as an indicator in the assessment of colorectal cancer or a precursor thereof.
  • a combination of two or more biomarkers as disclosed herein forms the basis for assessing colorectal cancer or a precursor thereof with enhanced specificity and sensitivity, and therefore provides enhanced management of patient care for colorectal cancer.
  • fragments and variants of the biomarkers described in Table 1 and in the sequence listings are also useful biomarkers, either alone or in a panel used for the analysis of colorectal cancer or a precursor thereof. What is meant by fragment is any incomplete or isolated portion of a polynucleotide in the sequence listing. It is recognized that almost daily, new discoveries are announced for gene variants, particularly for those genes under intense study, such as genes implicated in diseases like cancer. Therefore, the sequence listings given are exemplary of what is now reported for a gene, but it is recognized that for the purpose of an analytical methodology, variants of the gene, and fragments of the genetic loci are also included.
  • the disclosure incorporates any nucleotide sequence which is in the range of 80% to 100% identical to a nucleotide sequence disclosed herein or the complement thereof.
  • the identity is in the range of 90% to 100%, in yet another embodiment the identity is in the range of 95% and 100%.
  • Genomic locations are indicated by "Chromosome No.:start position-end position as defined by Ensembl release 63 - June 2011 ⁇ Wellcome Trust Sanger Institute
  • ASGR2 [synonyms: ASGP-R2
  • This receptor is a transmembrane protein that plays a critical role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N-acetylgalactosamine residues.
  • the asialoglycoprotem receptor may facilitate hepatic infection by multiple viruses including hepatitis B, and is also a target for liver-specific drug delivery.
  • the asialoglycoprotem receptor is a hetero-oligomeric protein composed of major and minor subunits, which are encoded by different genes.
  • the protein encoded by this gene is the less abundant minor subunit.
  • spliced transcript variants encoding multiple isoforms have been observed for this gene.
  • CLIPl [synonyms: CLIP
  • RESTIN I RSN gxHOMSA6696
  • CLIPl and its known effectors IQGAP1 and Cdc42 are present in the lamellar body- enriched fraction.
  • Lamellar bodies are tubulovesicular secretory organelles of epithelial cells related to lysosomes. Confocal microscopy analysis of skin cryosections has shown that CLIPl is expressed in differentiated keratinocytes and can be observed co-localized with Cdc42 and with the known lamellar body protein cathepsin D.
  • CLIPl also co-localizes with Rab7. It is hypothesized that CLIPl is involved together with Cdc42 and/or Rab7 in the intracellular trafficking of lamellar bodies.
  • ARHGAP18 [synonyms: FLJ25728
  • ARHGAP18 gene products belong to the human RhoGAP family with approximately 80 RhoGAP proteins known to be encoded in the human genome.
  • the RhoGAPs, GTPase-activating proteins have the ability to modulate Rho- mediated signaling pathways by controlling the balance between active and inactive Rho proteins.
  • Rho proteins belong to the Ras superfamily that is composed of over 50 members divided into 6 families, including Ras, Sar, Rho, Ran, Rab and Arf They participate in an array of physiological processes, such as cell migration, intercellular adhesion, cytokinesis, proliferation, differentiation and apoptosis.
  • Rho GTPases are important regulators of the actin cytoskeleton and consequently influence the shape and migration of cells. GTPases of the Rho family are strong regulators of signaling pathways that link growth factors and/or their receptors to adhesions and associated structures.
  • One signaling pathway mediated by Ras is initiated by the EGF receptor (EGFR), leading to cell proliferation. EGFR signaling can induce mitosis, proliferation, cell motility, differentiation and protein secretion.
  • EGFR EGF receptor
  • SLC16A6 [synonyms: MCT6
  • the monocarboxylate cotransporter (MCT) family comprises at least 14 members.
  • Members of the family catalyse proton-linked transport of metabolically important monocarboxylates such as lactate, pyruvate and ketone bodies. Lactic acid transport across the plasma membrane is fundamental for the metabolism of and pH regulation of all cells, removing lactic acid produced by glycolysis and allowing uptake by those cells utilizing it for gluconeogenesis or as a respiratory fuel.
  • MALATl [synonyms: HCN
  • CSNKID [synonyms: HCKID
  • C19orf43 [synonyms: MGC2803
  • LRRN3 [synonyms: FIGLER5
  • LRRN3 is expressed at very high levels in humans, about 2.3 times the average gene. It is most highly expressed in brain, heart, and testes tissues. It is also slightly expressed in kidney, muscle, pharynx, placental, and thymus tissue. The highest expression of the LRRN3 gene for the developmental stages is the fetal stage, but it is also expressed in the infant, juvenile, and adult stages, as can be seen in the EST profile.
  • biomarker for the assessment of any member of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp, the assessment including detection of any of these members, wherein the biomarker origins from a gene or a genetic locus selected from the group consisting of ASGR2, CLIPI, ARHGAP18, SLC16A6, MALATl, CSNKID, C19orf43, and LRRN3.
  • an embodiment is the use of (i) a partial or complete transcribed sequence or (ii) a fragment - of a gene or a genetic locus selected from the group consisting of ASGR2, CLIPI, ARHGAP18, SLC16A6, MALATl, CSNKID, C19orf43, and LRRN3, the use being that of a biomarker in the assessment of a member of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp.
  • Quantitative analysis of the respective markers revealed that in the diseased state (i.e. when a member of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp is present in the patient of whom a sample is assayed) the level of a marker is either higher or lower than in the non-diseased state.
  • Table 2 indicates the deviation for each maker locus.
  • ⁇ "diseased state” indicates the presence of neoplastic tissue, either colorectal cancer or a precursor thereof.
  • a particular embodiment is the use of any of the above markers in the assessment of an early stage of colorectal cancer, particularly a stage selected from Tis NO MO, Tl-4 NO MO, Dukes A, and Dukes B.
  • a further embodiment is a method for assessing in vitro an object selected from colorectal cancer, colorectal adenoma, and colorectal polyp, the method comprising measuring in a whole blood sample the concentration and/or activity of a biomarker, wherein the biomarker is a polynucleotide originating from a genetic locus selected from the group consisting of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALATl, CSNKID, C19orf43, and LRRN3, or a fragment of any of the aforementioned polynucleotides.
  • the biomarker is a transcript originating from a genetic locus selected from the group consisting of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALATl, CSNKID, C19orf43, and LRRN3, or a fragment of any of the aforementioned transcripts.
  • a panel of biomarkers with the specificity and sensitivity required for managing patient care for colorectal cancer.
  • entries ## 1-8 refer to the biomarkers.
  • a particular embodiment is a panel of biomarkers comprising two or more of the aforementioned polynucleotides.
  • biomarkers for colorectal cancer As previously mentioned, there is still a need for biomarkers for colorectal cancer as well as precursors thereof, the biomarkers having the specificity and sensitivity required to be effective for all aspects of patient care management. Therefore, the selection of an effective set of biomarkers is differentiating in providing the basis for effective determination of colorectal cancer.
  • expression levels of polynucleotides for the biomarkers indicated in SEQ ID NOs: 1, 14, 27, 40, 53, 66, 79, 92, and 105 and particularly SEQ ID NOs :4, 8, 12, 17, 21, 25, 30, 34, 38, 43, 47, 51, 56, 60, 64, 69, 73, 77, 82, 86, 90, 95, 99, 103, 108, and 112, are used in the determination of colorectal cancer or a precursor thereof.
  • Such analysis of polynucleotide expression levels is frequently referred to in the art as gene expression profiling.
  • gene expression profiling mRNA of a sample is first reverse-transcribed in a quantitative manner into a complementary DNA (cDNA).
  • the relative amount of cDNA derived from a given RNA species and resulting from the process of reverse transcription corresponds to the relative amount of the RNA species relative to all RNAs which are reverse-transcribed in the process.
  • the cDNA serves as template for qPCR using specific oligonucleotides as primers and a quantitative detection method for the DNA which is newly generated after each amplification round.
  • the deduced level of original target mRNA in the sample also in comparison to other samples, is a leading indicator of a biological state; as disclosed herein, the level of target mRNA is used as an indicator of colorectal cancer or a precursor thereof in comparison to healthy individuals or individuals with none of the above neoplastic growths.
  • Pairs of primers advantageously used in qPCR, as a subsequent step after the reverse transcription process and for sequences derived from each gene locus are listed in entries 1-8 of Tables 1 and 2. Since the combination of RT and qPCR amplifies copies of cDNA proportional to the original level of the corresponding mRNA in a sample, it has become a standard method that allows the analysis of even low levels of mRNA present in a biological sample. Compared to a normal/healthy/reference sample target sequences (polynucleotides) derived from certain genetic loci may either be up-regulated or down-regulated in any particular biological state, and hence mRNA levels shift accordingly.
  • Figures 1 and 2 show box plots and corresponding ROC diagrams reflecting results with regards to biomarkers for assessing precursors of colorectal cancer, i.e. adenoma and colorectal polyp tissue.
  • the basis of each assessmenrt was the level of RNA transcribed from the genetic loci as indicated.
  • Each assessment comprised assaying in vitro whole blood samples obtained from individuals.
  • each box contains the middle 50% of the respective data.
  • the middle line is the median, whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the box.
  • Data points in the box plots are indicative of the relative amount of target cDNA polynucleotide amplified (y-axis).
  • Figure 1 A LRRN3 (209841. s.at) norm. box plot
  • Figure 2 A MALAT1 (224568.x.a) norm. box plot
  • Figures 3 to 18 reflect the results of patients with colorectal cancer.
  • the labeling of the box-and-whisker plots indicate the healthy control ("normal"), TNM stages [Tl NO M0] and [T2 NO M0] grouped in “crc-1”, stages [T3 NO M0] and [T4 NO M0] grouped in “crc-2”, and stages [Tl-2 Nl M0], [T3-4
  • Figure 3 A ASGR2 (206130.s.at) norm. box plot
  • Figure 7 A ARHGAP18 (225173. at) norm. box plot
  • Figure 8 A ARHGAP18 (225173. at) raw box plot
  • Figure 11 A MALAT1 (224568.x.at) norm. box plot
  • Figure 13 A MALAT1 (223940.x.at) norm. box plot
  • Figure 15 A CSNK1D (1569263.at) norm. box plot
  • Figure 17 A C19orf43 (230213. at) norm. box plot
  • Figure 18 B C19orf43 (230213. at) raw ROC Methods and kits for determination of any of the polynucleotides shown, and expression profiling for a panel of molecular markers are also contemplated as part of the present disclosure.
  • the term "determination" encompasses qualitative and quantitative determination.
  • a method for gene expression profiling comprises measuring the level of one or more a polynucleotide(s) originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1 , CSNK1D, C19orf43, and LRR 3.
  • RNAs DNA sequences
  • RT reverse transcription
  • reagents such as one including a desoxynucleoside triphosphate mixture having all four desoxynucleoside triphosphates (e.g.
  • dATP, dGTP, dCTP, and dTTP are required for RT-qPCR. Additionally buffers, inhibitors and activators are also required for the RT-qPCR process.
  • fluorescent label is the intercalating dye SYBR Green, though numerous related chromophores capable of forming complexes with newly formed nucleic acids by way of intercalating exist, and are known in the art.
  • the hydrolysis probe principle is used. It is also known as the TaqMan® format. It relies on the 5 ' -3 ' exonuclease activity of Taq polymerase to cleave a dual-labeled probe (hydrolysis 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 hydrolysis (TaqMan®) probe significantly increases the specificity of the detection.
  • a hydrolysis probes consists of a fluorophore covalently attached to the 5 '-end of the oligonucleotide probe and a quencher at the 3 '-end.
  • fluorophores and quenchers are known to the art and commercially available.
  • the quencher molecule absorbs the fluorescence emitted by the fluorophore. As long as the fluorophore and the quencher remain in proximity, the quencher inhibit the fluorescence signal and therefore prevents its detection.
  • TaqMan® probes are designed such that they anneal within a DNA region amplified by a specific set of primers.
  • 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 to be detected.
  • the amount of fluorescence detected by way of spectroscopy in a real-time PCR thermal cycler is dependent on the fluorophore released, hence the amount of DNA template present in the PCR.
  • detection modes contemplated for the disclosed methods include, but are not limited to spectroscopic techniques, such as fluorescence and UV-Vis spectroscopy, scintillation counting, and mass spectroscopy.
  • labels for the purpose of detection and quantitation used in these methods include, but are not limited to chromophoric labels, scintillation labels, and mass labels.
  • the expression levels of polynucleotides and polypeptides measured using these methods may be normalized to a control established for the purpose of the targeted determination. These methods are believed useful in providing determinations as the basis of effective management of patient care for colorectal cancer. These methods may also be used in the discovery of therapeutic interventions for colorectal cancer.
  • kits having the reagents and procedures that facilitate the ready implementation of the methods, and provide consistency and quality control thereby.
  • kits for gene expression analysis in the assessment of colorectal cancer or a precursor thereof wherein the kit comprises the reagents and instructions necessary for the detection and quantitative analysis of a polynucleotide originating from any one of the genetic loci selected from the group consisting of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and LRR 3.
  • kit for gene expression profiling of a panel comprising ASGR2, CLIPl, ARHGAP18, CR2, SLC16A6, MALAT1, CSNK1D, C19orf43, CD200, and LRRN3.
  • the reagents may include primers, enzymes, and other reagents for the preparation, detection, and quantitation of cDNAs for the claimed panel of biomarkers, or individual biomarkers.
  • the method of creating cDNA from mRNA in a sample is referred to as the combination of reverse transcription and quantitative polymerase chain reaction (RT-qPCR).
  • RT-qPCR quantitative polymerase chain reaction
  • ID NOs: 2 and 3, 6 and 7, 10 and 11, 15 and 16, 19 and 20, 23 and 24, 28 and 29, 32 and 33, 36 and 37, 41 and 42, 45 and 46, 49 and 50, 54 and 55, 58 and 59, 62 and 63, 67 and 68, 71 and 72, 75 and 76, 80 and 81, 84 and 85, 88 and 89, 93 and 94, 97 and 98, 101 and 102, 106 and 107, and 110 and 111 are particularly suited for use in gene expression profiling using RT-qPCR based on the claimed panel. These primer pairs were specifically designed, selected, and tested accordingly.
  • reagents such as one including a desoxynucleoside triphosphate mixture having all four desoxynucleoside triphosphates (e.g. dATP, dGTP, dCTP, and dTTP), one having the reverse transcriptase enzyme, and one having a thermostable DNA polymerase are required for RT-qPCR.
  • buffers, inhibitors and activators used for the RT-qPCR process are suitable reagents for inclusion in the kit embodiment.
  • the reverse transcriptase and the thermostable DNA polymerase are provided together in a mixture, to enable one-step RT-qPCR.
  • the thermostable DNA polymerase aditionally comprises as a further enzymatic activity that of a reverse transcriptase.
  • fluorescence spectroscopy One method contemplated for detection of polynucleotides is fluorescence spectroscopy, and therefore fluorophores that are suited for fluorescence spectroscopy are desirable for labeling polynucleotides and may also be included in reagents of the kit embodiment.
  • labeled hydrolysis probes which can be used in the TaqMan® detection format may be included in the kits.
  • Such hydrolysis probes may comprise LNA nucleosides to facilitate provision of shorter hydrolysis probes.
  • kit embodiment for gene expression profiling teach the user the following steps: to obtain a biological sample; to isolate cellular RNA from the sample; to synthesize cDNA from the isolated RNA; to amplify copies of cDNA from the sample for the biomarker(s), and the panel for which the reagents are provided; and to quantify levels of cDNA amplified from the sample.
  • Various samples including whole blood from a variety of procedures may be used.
  • the instructions for obtaining a biological sample typically instruct the user to obtain a sample of blood (or another sample material as indicated elsewhere herein), optionally to stabilize the sample material, and to subject the sample to a nucleic acid preparation procedure.
  • the instructions may also include the step of comparing the cDNA levels quantified to a control.
  • consumable labware required for sample collection, preparation, and analysis may be provided with the kits.
  • a polynucleotide originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and LRRN3 as a biomarker for and/or in the assessment of neoplastic tissue selected from any of colorectal cancer and a precursor stage of colorectal cancer (a precursor stage being selected from any of colorectal adenoma and colorectal polyp), wherein in a specific embodiment the biomarker is the level of an RNA transcribed from the genetic locus, and in another specific embodiment the sample is whole blood.
  • the assessment comprises assaying in vitro (ex-vivo) a sample obtained from a patient (a human individual), particularly a blood sample.
  • assessment of one or more selected member(s) comprises assaying a polynucleotide selected from RNA, cDNA, and a complement thereof.
  • the assessment includes quantification of the polynucleotide.
  • the assessment includes comparing the presence and/or the level of the polynucleotide in the patient sample with a control.
  • colorectal polyp and/or colorectal adenoma are detected.
  • colorectal cancer is assessed at a stage selected from any of Tis NO MO, T 1-4 NO MO, Dukes A, and Dukes B.
  • colorectal cancer is assessed at a stage more advanced than any of T4 NO MO, and Dukes B.
  • the assessment is detection of (or the establishment of, or the confirmation of) the presence or absence of a member of the group selected from colorectal cancer, colorectal adenoma, and colorectal polyp.
  • any of the items 5 to 9 wherein, when compared to a sample from a healthy control, particularly a healthy control individual, the level of the polynucleotide is increased in the sample obtained from the human individual with colorectal neoplastic tissue, and the biomarker is selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, and C19orf43.
  • the colorectal neoplastic tissue is selected from any of colorectal cancer, colorectal adenoma, and colorectal polyp.
  • colorectal neoplastic tissue is selected from any of colorectal cancer and colorectal adenoma. 13. The use according to item 10, wherein the colorectal neoplastic tissue is colorectal cancer.
  • colorectal neoplastic tissue is selected from any of colorectal cancer, colorectal adenoma, and colorectal polyp.
  • biomarker is combined with one or more further biomarker(s).
  • the one or more further biomarker(s) is/are one or more polynucleotide(s) originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and LRRN3.
  • polynucleotide originating from the genetic locus ASGR2 is selected from SEQ ID NO: l, the complement of SEQ ID NO: l, a fragment thereof or its complement, and a variant thereof or its complement.
  • polynucleotide originating from the genetic locus CLIPl is selected from SEQ ID NO: 14, the complement of SEQ ID NO: 14, a fragment thereof or its complement, and a variant thereof or its complement. 1.
  • polynucleotide originating from the genetic locus ARHGAP18 is selected from SEQ ID NO:27, the complement of SEQ ID NO:27, a fragment thereof or its complement, and a variant thereof or its complement. 22.
  • polynucleotide originating from the genetic locus SLC16A6 is selected from SEQ ID NO:40, the complement of SEQ ID NO:40, a fragment thereof or its complement, and a variant thereof or its complement.
  • polynucleotide originating from the genetic locus MALATl is selected from SEQ ID NO:53, the complement of SEQ ID NO:53, a fragment thereof or its complement, and a variant thereof or its complement.
  • polynucleotide originating from the genetic locus MALATl is selected from SEQ ID NO: 66, the complement of SEQ ID NO:66, a fragment thereof or its complement, and a variant thereof or its complement.
  • polynucleotide originating from the genetic locus CSNKID is selected from SEQ ID NO:79, the complement of SEQ ID NO:79, a fragment thereof or its complement, and a variant thereof or its complement.
  • polynucleotide originating from the genetic locus C19orf43 is selected from SEQ ID NO:92, the complement of SEQ ID NO:92, a fragment thereof or its complement, and a variant thereof or its complement.
  • polynucleotide originating from the genetic locus LRRN3 is selected from SEQ ID NO: 105, the complement of SEQ ID NO: 105, a fragment thereof or its complement, and a variant thereof or its complement.
  • polynucleotides are a fragment of a polynucleotide originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and LRRN3, or a variant thereof.
  • at least one of the polynucleotides is a variant of a polynucleotide originating from a genetic locus selected from any of ASGR2, CLIPl, ARHGAP18, SLC16A6, MALAT1, CSNK1D, C19orf43, and LRRN3, or a fragment thereof.
  • a method for measuring the expression levels of a polynucleotide from a biomarker the biomarker being an indicator for the presence of any member of the group consisting of colorectal cancer, colorectal adenoma and colorectal polyp in a human individual, the method comprising: selecting a biomarker comprising a contiguous polynucleotide included in a member of the group consisting of SEQ ID NOs: 1, 14, 27, 40, 53, 66, 79, 92, 105, a fragment thereof or its complement, and a variant thereof or its complement; obtaining a biological sample from the human individual; in vitro (ex vivo) isolating cellular RNA from the sample and reverse-transcribing isolated RNA to obtain cDNA; amplifying copies of cDNA for the biomarker originating from the sample; and quantifying the level of cDNA amplified from the sample, thereby measuring the expression level of the polynucleotide from a biomarker,
  • the method includes the step of selecting a biomarker comprising at least one polynucleotide from SEQ ID NOs: 4, 8, 12, 17, 21, 25, 30, 34, 38, 43, 47, 51, 56, 60, 64, 69, 73, 77, 82, 86, 90, 95, 99, 103, 108, and 112.
  • step of amplifying copies of cDNA further comprises at least two pairs of primers chosen from the primer pairs of SEQ. ID NOs: 2 and 3, 6 and 7, 10 and 11, 15 and 16, 19 and 20, 23 and 24, 28 and 29, 32 and 33, 36 and 37, 41 and 42, 45 and 46, 49 and 50, 54 and 55, 58 and 59, 62 and 63, 67 and 68, 71 and 72, 75 and 76, 80 and 81, 84 and 85, 88 and 89, 93 and 94, 97 and 98, 101 and 102, 106 and 107, and 110 and 111.
  • step of reverse-transcribing isolated RNA to obtain cDNA comprises using enzymes and reagents for the preparation of cDNAs.
  • step of quantifying the level of cDNA further comprises labeling cDNA.
  • labeling cDNA includes at least one fluorophore capable of interacting with double stranded nucleic acid by way of intercalation.
  • labeling cDNA includes hybridization of a dual-labeled (fluorophore and quencher) hydrolysis probe to a the cDNA.
  • the management of patient care includes one or more of risk assessment, early diagnosis, establishing prognosis, monitoring patient treatment, and detecting relapse.
  • kits for the assessment of any member of the group consisting of colorectal cancer, colorectal adenoma, and colorectal polyp comprising: at least one reagent that is used in analysis of polynucleotide expression level for a biomarker for a member selected from the group consisting of colorectal cancer, colorectal adenoma and colorectal polyp, wherein the biomarker comprises a polynucleotide selected from SEQ ID NOs: 1, 4, 8, 12, 14, 17, 21, 25, 27, 30, 34, 38, 40, 43, 47, 51, 53, 56, 60, 64, 66, 69, 73, 77, 79, 82, 86, 90, 92, 95, 99, 103, 105, 108, 112, a complement therof, a variant thereof, and a fragment thereof; and instructions for using the kit for analyzing the polynucleotide expression level.
  • the kit according to item 55 comprising two or more reagents for use in analysis of polynucleotide expression levels for a plurality of biomarkers.
  • the plurality of biomarkers comprises two or more polynucleotides selected from 4, 8, 12, 17, 21, 25, 30, 34, 38, 43, 47, 51, 56, 60, 64, 69, 73, 77, 82, 86, 90, 95, 99, 103, 108, and 112.
  • the reagent comprises one or more pair(s) of primers chosen from the primer pairs of SEQ. ID NOs: 2 and 3, 6 and 7, 10 and 11, 15 and 16, 19 and 20, 23 and 24, 28 and 29, 32 and 33, 36 and 37, 41 and 42, 45 and 46, 49 and 50, 54 and 55, 58 and 59, 62 and 63, 67 and 68, 71 and 72, 75 and 76, 80 and 81, 84 and 85, 88 and 89, 93 and 94, 97 and 98, 101 and 102, 106 and 107, and 110 and 111.
  • the kit according to item 60 further comprising reagents for the preparation of cDNA.
  • 62 The kit according to any of items 55-59, comprising a reagent that is capable of detecting and quantifying polynucleotides.
  • 63 The kit according to item 62, wherein the reagent includes at least one chromophore, particularly a fluorophore capable of interacting with double- stranded nucleic acid by way of intercalation.
  • kit according to item 62 wherein the reagent includes at least one (dual- labeled) TaqMan® hydrolysis probe.
  • kit according to any of the items 55-64, further comprising consumable labware for at least one member of the group consisting of of sample collection, sample preparation, and sample analysis, wherein in a specific embodiment the consumable labware for sample preparation comprises materials and reagents for preparing RNA from whole blood.
  • colon biopsy specimen were taken before colonoscopy and put immediately in RNA later solution, then fresh frozen in less than 3 minutes.
  • RNA later solution RNA later solution
  • parellel 9 ml of peripheral blood samples were taken into Paxgene Blood RNA Tubes (Qiagen Inc, Germantown, US), before colonoscopy.
  • the biopsy specimen and the blood samples were stored at -80°C. In each case formalin fixed paraffin embedded specimen were collected und used for immunhistochemical analysis (FFPE).
  • FFPE immunhistochemical analysis
  • the samples with adenomas were either villous ones (including all villous adenomas >0 cm) or adenomas larger than 1 cm about with tubular phenotype only.
  • the diagnostic groups and the number of patients of each group are represented in Table 3 :
  • Biotinylated cRNA probes were synthesized from 5 ⁇ g total RNA and fragmented according to the Affymetrix description using One-cycle Target Labeling and Control Kit (Affymetrix) and Globin Reduction PNA oligomers (Applied Biosystems, Foster City, US) according to the "Globin
  • Globin reduction was done in order to reduce high amounts of globin transcripts in the cellular RNA purified from whole blood samples: during the reverse transcription step of the probe synthesis, globin-specific PNA oligonucleotides were added, which reduced the amount of cDNA generated from globin mRNA.
  • HGU133 Plus2.0 array (Affymetrix) at 45°C for 16 hours.
  • the slides were washed and stained using Fluidics Station 450 and antibody amplification staining method according to the manufacturer's instructions.
  • the fluorescent signals were detected by a GeneChip Scanner 3000.
  • Tabel 4 lists microarray target sequences for the capture of cDNAs of the genes / genomic loci ASGR2, CLIPl, ARHGAP18, CR2, SLC16A6, MALAT1, CSNK1D, C19orf43, CD200, and LRRN3, which proved to qualify as biomarkers for the assessment of colorectal cancer.
  • the Hydrolysis Probe or TaqMan® chemistry relies on the 5 -3' exonuclease activity of Taq polymerase, which degrades a hybridized non-extendible DNA probe during the extension step of the PCR.
  • This probe is designed to hybridize to a region within the amplicon and is duel labeled with a reporter dye and a quenching dye. The close proximity of the quencher suppresses the fluorescence of the reporter dye.
  • the exonuclease activity of Taq polymerase degrades the probe, the fluorescence of the reporter increases at a rate that is proportional to the amount of template present.
  • TaqMan® real-time PCR was used to measure the expression of 94 selected genes in 80 blood samples (35 samples which were also tested by microarrays and 45 independent samples) using an Applied Biosystems Micro Fluidic Card System. The measurements were performed using an ABI PRISM® 7900HT Sequence
  • RT-PCR assays were designed making use of LNA-based hydrolysis probes of the "Universal
  • UPL probe UPL; Roche Applied Science, Roche Diagnostics GmbH, Mannheim, Germany.
  • a UPL probe is a short hydrolysis probes. Such a probe is labeled at the 5' end with fluorescein (FAM) and at the 3' end with a dark quencher dye.
  • the length of a UPL probe is just 8-9 nucleotides and comprises a selected sequence.
  • Tm melting temperature
  • LNA Locked Nucleic Acids
  • LNA's are DNA nucleotide analogues with increased binding strengths compared to standard DNA nucleotides.
  • the basis for the design of primers and probes as listed in Table N were the sequences targeted by the capture probes of the Affymetrix hybridization microarrays of Example 2 and listed in Table 4.
  • ROC receiver-operating characteristics
  • the clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease or benign versus malignant disease.
  • the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds. On the y-axis is sensitivity, or the true-positive fraction [defined as
  • Each point on the ROC plot represents a sensitivity/ 1 -specificity pair corresponding to a particular decision threshold.
  • a test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity).
  • the theoretical plot for a test with no discrimination is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes.
  • Values range between 1.0 (perfect separation of the test values of the two groups) and 0.5 (no apparent distributional difference between the two groups of test values).
  • ROC plots are shown in the Figures.
  • box plots are shown. Each box contains the middle 50% of the respective data. The middle line is the median, whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the box.
  • RNAlater RNA stabilization reagent Qiagen
  • FFPE immunhistochemical analysis
  • Table 6 lists the groups of patients from which blood samples were obtained. Groups I and II together served as control. Table 6
  • SDHA succinate dehydrogenase complex, subunit A, flavoprotein housekeeping gene assay was performed in duplicate, as a control and for normalization. SDHA was selected as reference by comparing several standard housekeeping genes on normal blood and selecting the most stable gene closest to the median of all biomarker candidates.

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Abstract

La présente invention concerne des biomarqueurs, des méthodes et des kits pour l'évaluation d'échantillons de sang total, ladite évaluation ciblant n'importe quel élément faisant partie du groupe constitué par : cancer colorectal, adénome colorectal, et polype colorectal. Lesdits biomarqueurs sont des polynucléotides issus de loci génétiques choisis à partir de l'un quelconque des loci suivants : ASGR2, CLIPl, ARHGAP18, SLC16A6, MALATl, CSNKID, C19orf43, et LRRN3.
PCT/EP2012/068905 2011-09-26 2012-09-26 Biomarqueurs d'adnc dans du sang total pour l'évaluation du cancer colorectal WO2013045464A1 (fr)

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