WO2010135786A1 - Procédé permettant de diagnostiquer des néoplasmes et molécules destinées à être utilisées dans ce procédé - Google Patents

Procédé permettant de diagnostiquer des néoplasmes et molécules destinées à être utilisées dans ce procédé Download PDF

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WO2010135786A1
WO2010135786A1 PCT/AU2010/000660 AU2010000660W WO2010135786A1 WO 2010135786 A1 WO2010135786 A1 WO 2010135786A1 AU 2010000660 W AU2010000660 W AU 2010000660W WO 2010135786 A1 WO2010135786 A1 WO 2010135786A1
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sequence
seq
nucleic acid
length
similarity
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Susanne Pedersen
Aidan Mcevoy
Lawrence Charles Lapointe
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Clinical Genomics Pty. Ltd.
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Publication of WO2010135786A1 publication Critical patent/WO2010135786A1/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

  • the present invention relates generally to a nucleic acid molecule, the RNA and protein expression profiles of which are indicative of the onset, predisposition to the onset and/or progression of a large intestine neoplasm. More particularly, the present invention is directed to nucleic acid molecules, the expression profiles of which are indicative of the onset and/or progression of a colorectal neoplasm, such as an adenoma or an adenocarcinoma.
  • the expression profiles of the present invention are useful in a range of applications including, but not limited to, those relating to the diagnosis and/or monitoring of colorectal neoplasms, such as colorectal adenomas and adenocarcinomas.
  • the present invention provides novel polynucleotides and polypeptides translated therefrom. More particularly, the present invention is directed to SlOOAl 1 transcript variants, and derivatives and fragments thereof, and polypeptides translated therefrom.
  • Adenomas are benign tumours, or neoplasms, of epithelial origin which are derived from glandular tissue or exhibit clearly defined glandular structures. Some adenomas show recognisable tissue elements, such as fibrous tissue (fibroadenomas) and epithelial structure, while others, such as bronchial adenomas, produce active compounds that might give rise to clinical syndromes.
  • Adenomas may progress to become an invasive neoplasm and are then termed adenocarcinomas.
  • adenocarcinomas are defined as malignant epithelial tumours arising from glandular structures, which are constituent parts of many organs of the body.
  • the term adenocarcinoma is also applied to tumours showing a glandular growth pattern. These tumours may be sub-classified according to the substances that they produce, for example mucus secreting and serous adenocarcinomas, or to the microscopic arrangement of their cells into patterns, for example papillary and follicular adenocarcinomas.
  • These carcinomas may be solid or cystic (cystadenocarcinomas).
  • Each organ may produce tumours showing a variety of histological types, for example the ovary may produce both mucinous and cystadenocarcinoma.
  • Adenomas in different organs behave differently.
  • the overall chance of carcinoma being present within an adenoma i.e. a focus of cancer having developed within a benign lesion
  • this is related to size of an adenoma.
  • occurrence of a cancer within an adenoma is rare in adenomas of less than 1 centimetre.
  • Such a development is estimated at 40 to 50% in adenomas which are greater than 4 centimetres and show certain histopathological change such as villous change, or high grade dysplasia.
  • Adenomas with higher degrees of dysplasia have a higher incidence of carcinoma.
  • the predictors of the presence of cancer now or the future occurrence of cancer in the organ include size (especially greater than 9mm) degree of change from tubular to villous morphology, presence of high grade dysplasia and the morphological change described as "serrated adenoma".
  • the additional features of increasing age, familial occurrence of colorectal adenoma or cancer, male gender or multiplicity of adenomas predict a future increased risk for cancer in the organ - so-called risk factors for cancer. Except for the presence of adenomas and its size, none of these is objectively defined and all those other than number and size are subject to observer error and to confusion as to precise definition of the feature in question. Because such factors can be difficult to assess and define, their value as predictors of current or future risk for cancer is imprecise.
  • Colorectal adenomas represent a class of adenomas which are exhibiting an increasing incidence, particularly in more affluent countries.
  • the causes of adenoma, and of progression to adenocarcinoma, are still the subject of intensive research.
  • environmental factors such as diet
  • Colonic adenomas are localised areas of dysplastic epithelium which initially involve just one or several crypts and may not protrude from the surface, but with increased growth in size, usually resulting from an imbalance in proliferation and/or apoptosis, they may protrude.
  • Adenomas can be classified in several ways. One is by their gross appearance and the major descriptors include degrees of protrusion: flat sessile (i.e. protruding but without a distinct stalk) or pedunculated (i.e. having a stalk). Other gross descriptors include actual size in the largest dimension and actual number in the colon/rectum.
  • adenomas While small adenomas (less than say 5 or 10 millimetres) exhibit a smooth tan surface, pedunculated and especially larger adenomas tend to have a cobblestone or lobulated red- brown surface. Larger sessile adenomas may exhibit a more delicate villous surface.
  • Another set of descriptors include the histopathological classification; the prime descriptors of clinical value include degree of dysplasia (low or high), whether or not a focus of invasive cancer is present, degree of change from tubular gland formation to villous gland formation (hence classification is tubular, villous or tubulovillous), presence of admixed hyperplastic change and of so-called "serrated" adenomas and its subgroups.
  • Adenomas can be situated at any site in the colon and/or rectum although they tend to be more common in the rectum and distal colon. All of these descriptors, with the exception of number and size, are relatively subjective and subject to interobserver disagreement. - A -
  • adenomas are of value not just to ascertain the neoplastic status of any given adenomas when detected, but also to predict a person's future risk of developing colorectal adenomas or cancer.
  • Those features of an adenoma or number of adenomas in an individual that point to an increased future risk for cancer or recurrence of new adenomas include: size of the largest adenoma (especially 10mm or larger), degree of villous change (especially at least 25% such change and particularly 100% such change), high grade dysplasia, number (3 or more of any size or histological status) or presence of serrated adenoma features.
  • risk None except size or number is objective and all are relatively subjective and subject to interobserver disagreement. These predictors of risk for future neoplasia (hence "risk”) are vital in practice because they are used to determine the rate and need for and frequency of future colonoscopic surveillance. More accurate risk classification might thus reduce workload of colonoscopy, make it more cost-effective and reduce the risk of complications from unnecessary procedures.
  • Adenomas are generally asymptomatic, therefore rendering difficult their diagnosis and treatment at a stage prior to when they might develop invasive characteristics and so became cancer. It is technically impossible to predict the presence or absence of carcinoma based on the gross appearance of adenomas, although larger adenomas are more likely to show a region of malignant change than are smaller adenomas. Sessile adenomas exhibit a higher incidence of malignancy than pedunculated adenomas of the same size. Some adenomas result in blood loss which might be observed or detectable in the stools; while sometimes visible by eye, it is often, when it occurs, microscopic or "occult". Larger adenomas tend to bleed more than smaller adenomas.
  • the identification of molecular markers for adenomas would provide means for understanding the cause of adenomas and cancer, improving diagnosis of adenomas including development of useful screening tests, elucidating the histological stage of an adenoma, characterising a patient's future risk for colorectal neoplasia on the basis of the molecular state of an adenoma and facilitating treatment of adenomas.
  • SlOOAl 1 which to date has been thought to be characterised by the genomic sequence depicted in SEQ ID NO:1, in fact encodes 6 exons, one being entirely previously unknown and two having alternative transcription start sites, which transcribe to give rise to 3 alternatively spliced RNA transcript forms. It has also been determined that although all 3 transcripts show some level of increase in expression in the context of colorectal neoplasia development, two of the three forms (SlOOAl 1 -1 and SlOOAl 1-2) in fact show minimal expression in non-neoplastic tissue, with many non-neoplastic tissues not exhibiting detectable levels.
  • the findings of the present invention have therefore facilitated the development of a screening method to diagnose the onset, or predisposition thereto, of adenocarcinoma, adenoma and/or the monitoring of conditions characterised by the development of these types of neoplasms.
  • the term "derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of "a”, “and” and “the” include plural referents unless the context clearly dictates otherwise.
  • nucleotide sequence information prepared using the programme Patentln Version 3.4, presented herein after the bibliography.
  • Each nucleotide sequence is identified in the sequence listing by the numeric indicator ⁇ 210> followed by the sequence identifier (eg. ⁇ 210>l, ⁇ 210>2, etc).
  • the length, type of sequence (DNA, etc.) and source organism for each sequence is indicated by information provided in the numeric indicator fields ⁇ 21 1>, ⁇ 212> and ⁇ 213>, respectively.
  • Nucleotide sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (eg. SEQ ID NO: 1, SEQ ID NO: 2, etc).
  • sequence identifier referred to in the specification correlates to the information provided in numeric indicator field ⁇ 400> in the sequence listing, which is followed by the sequence identifier (eg. ⁇ 400>l, ⁇ 400>2, etc). That is SEQ ID NO: 1 as detailed in the specification correlates to the sequence indicated as ⁇ 400>l in the sequence listing.
  • One aspect of the present invention is directed to a method of screening for the onset or predisposition to the onset of a large intestine neoplasm in an individual, said method comprising measuring the level of expression of the SlOOAl 1-1, SlOOAl 1-2 and/or SlOOAl 1-3 splice variants in a biological sample from said individual wherein a higher level of expression of said splice variants relative to control levels is indicative of a neoplastic large intestine cell or a cell predisposed to the onset of a neoplastic state.
  • RNA transcripts which transcripts comprise an RNA sequence characterised by the sequence of one of:
  • SEQ ID NO:3 SEQ ID NO:3
  • SEQ ID NO:4 SEQ ID NO:4
  • SEQ ID NO:5 SEQ ID NO:5
  • RNA transcript in a biological sample from said individual wherein a higher level of said RNA transcript relative to control levels is indicative of a neoplastic large intestine cell or a cell predisposed to the onset of a neoplastic state.
  • the level of expression of which is assessed in accordance with the method of the present invention is one or more of the transcripts characterised by the sequence of one of:
  • SEQ ID NO:3 or a sequence having at least 90% similarity across the length of the sequence, or variant-of SEQ ID NO:3; or (ii) SEQ ID NO:4, or a sequence having at least 90% similarity across the length of the sequence, or variant of SEQ ID NO:4.
  • RNA transcript which transcript comprises one or more exon segments selected from:
  • an exon segment defined by SEQ ID NO:6 or a sequence having at least 90% similarity across the length of the sequence, or variant of SEQ ID NO:6;
  • an exon segment defined by SEQ ID NO: 7 or a sequence having at least 90% similarity across the length of the sequence, or variant of SEQ ID NO:7
  • an exon segment defined by SEQ ID NO: 8 or a sequence having at least 90% similarity across the length of the sequence, or variant of SEQ ID NO:8;
  • an exon segment defined by SEQ ID NO:9 or a sequence having at least 90% similarity across the length of the sequence, or variant of SEQ ID NO:9;
  • an exon segment defined by SEQ ID NO: 10 or a sequence having at least 90% similarity across the length of the sequence, or variant of SEQ ID NO: 10; and
  • an exon segment defined by SEQ ID NO: 1 1 or a sequence having at least 90% similarity across the length of the sequence, or variant of SEQ
  • RNA transcript or variant thereof relative to control levels is indicative of a neoplastic large intestine cell or a cell predisposed to the onset of a neoplastic state.
  • RNA transcript which transcript comprises one or more exon segments selected from:
  • RNA transcript or variant thereof relative to control levels is indicative of a neoplastic large intestine cell or a cell predisposed to the onset of a neoplastic state.
  • RNA transcript selected from:
  • RNA transcript which comprises each of the exon segments defined by SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO: 11, or a sequence having at least 90% similarity across the length of these sequences, or variants of SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:l l ;
  • RNA transcript which comprises each of the exon segments defined by SEQ ID NO:8 and SEQ ID NO: 10, or a sequence having at least 90% similarity across the length of these sequences, or variants of SEQ ID NO:8 and SEQ ID NO: 10;
  • RNA transcript which comprises each of the exon segments defined by SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO: 1 1, or a sequence having at least 90% similarity across the length of these sequences, or variants of SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:l l ;
  • RNA transcript or variant thereof relative to control levels is indicative of a neoplastic large intestine cell or a cell predisposed to the onset of a neoplastic state.
  • RNA transcript which transcript is selected from:
  • RNA transcript which comprises each of the exon segments defined by SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:1 1, or a sequence having at least 90% similarity across the length of these sequences, or variants of SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO: 1 1 ; and (ii) an RNA transcript which comprises each of the exon segments defined by SEQ ID NO:8 and SEQ ID NO: 10, or a sequence having at least 90% similarity across the length of these sequences, or variants of SEQ ID NO:8 and SEQ ID NO: 10;
  • RNA transcript or variant thereof relative to control levels is indicative of a neoplastic large intestine cell or a cell predisposed to the onset of a neoplastic state.
  • a related aspect of the present invention provides a molecular array, which array comprises a plurality of:
  • nucleic acid molecules comprising a nucleotide sequence corresponding to any one or more of the SlOOAl 1 sequences hereinbefore described or a sequence exhibiting at least 80% identity thereto or a functional derivative, fragment, variant or homologue of said nucleic acid molecule; or
  • nucleic acid molecules comprising a nucleotide sequence capable of hybridising to any one or more of the sequences of (i) under medium stringency conditions or a functional derivative, fragment, variant or homologue of said nucleic acid molecule; or
  • nucleic acid probes or oligonucleotides comprising a nucleotide sequence capable of hybridising to any one or more of the sequences of (i) under medium stringency conditions or a functional derivative, fragment, variant or homologue of said nucleic acid molecule; or
  • probes capable of binding to any one or more of the proteins encoded by the nucleic acid molecules of (i) or a derivative, fragment or, homologue thereof
  • Another aspect of the present invention provides a diagnostic kit for assaying biological samples comprising an agent for detecting one or more neoplastic marker reagents useful for facilitating the detection by the agent in the first compartment. Further means may also be included, for example, to receive a biological sample.
  • the agent may be any suitable detecting molecule.
  • Another aspect of the present invention is directed to an isolated nucleic acid molecule selected from the list consisting of:
  • nucleic acid molecule or molecule complementary thereto or, fragment or derivative thereof comprising one or more of the nucleotide sequences, as set forth in any one of SEQ ID NO:6-1 1 , or a nucleotide sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity over the length of the sequence, or a nucleotide sequence capable of hybridising to said nucleic acid molecule or complementary form thereof under low stringency conditions; and
  • nucleic acid molecule or derivative or fragment thereof comprising one or more of the nucleotide sequences substantially as set forth in any one of SEQ ID NO:6-1 1 or a fragment of said molecule.
  • Still another aspect of the present invention is directed to an isolated nucleic acid molecule selected from the list consisting of:
  • nucleic acid molecule or molecule complementary thereto or fragment or derivative thereof comprising a nucleotide sequence, as set forth in any one of SEQ ID NO:3 or 4, or a nucleotide sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity over the length of the sequence, or a nucleotide sequence capable of hybridising to said nucleic acid molecule or complementary form thereof under low stringency conditions; and (ii) An isolated nucleic acid molecule or derivative or fragment thereof comprising a nucleotide sequence substantially as set forth in any one of SEQ ID NO:3 or 4 or a fragment of said molecule.
  • Still another aspect of the present invention is directed to an isolated nucleic acid molecule selected from the list consisting of:
  • nucleic acid molecule or molecule complementary thereto or fragment or derivative thereof comprising a nucleotide sequence, as set forth in any one or more of SEQ ID NOs: 6, 7, 8, 9, 10 and 1 1, or a nucleotide sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity over the length of the sequence, or a nucleotide sequence capable of hybridising to said nucleic acid molecule or complementary form thereof under low stringency conditions; and
  • nucleic acid molecule or derivative or fragment thereof comprising a nucleotide sequence substantially as set forth in any one or more of SEQ ID NOs: 6, 7, 8, 9, 10 and 11 or a fragment of said molecule.
  • Yet another aspect of the present invention is directed to an isolated nucleic acid molecule selected from the list consisting of:
  • nucleic acid molecule or molecule complementary thereto or fragment or derivative thereof comprising a nucleotide sequence, as set forth in any one or more of SEQ ID NOs:6, 8 and 10, or a nucleotide sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity over the length of the sequence, or a nucleotide sequence capable of hybridising to said nucleic acid molecule or complementary form thereof under low stringency conditions; and (ii) An isolated nucleic acid molecule or derivative or fragment thereof comprising a nucleotide sequence substantially as set forth in any one or more of SEQ ID NOs:6, 8 and 10 or a fragment of said molecule.
  • the present invention is directed to a protein or derivative or fragment thereof encoded by the nucleotide sequence as set forth in any one or more of SEQ ID NOs:6-l 1 or the sequence complementary to a sequence capable of hybridising to SEQ ID NOs:6-l 1 under low stringency conditions or a protein molecule exhibiting at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to said protein across the length of the sequence.
  • the present invention is directed to a protein or derivative or fragment thereof encoded by the nucleotide sequence as set forth in any one of SEQ ID NOs:3 or 4 or the sequence complementary to a sequence capable of hybridising to SEQ ID NOs: 3 or 4 under low stringency conditions or a protein molecule exhibiting at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to said protein across the length of the sequence.
  • a further aspect of the invention provides an isolated cell which expresses an endogenous or recombinant protein or a functional fragment or derivative thereof of the present invention.
  • Yet another aspect of the present invention provides a method for identifying a S lOOAl 1 nucleic acid molecule as hereinbefore defined or a fragment or derivative thereof.
  • Still another aspect of the present invention is directed to antibodies to the subject protein or nucleic acid molecules including catalytic antibodies or derivatives, homologues, or mutants, of said antibodies.
  • kits comprising any one or more of the nucleic acid or protein molecules of the present invention.
  • Figure 1 is a depiction of the REFSEQ Sl 0OA 1 1 gene locus sequence (SEQ ID NO: 1) residing on human chromosome 1 on the minus strand, at Iq21 at map region 150271606 150276135 (Specification of chromosomal map positions refer to coordinates on human chromosome 16 using NCBI 36, March 2006, entry NC_000001.9).
  • Grey shading Exon sequences - See Table 3 for further information.
  • Underlining Alternative exon sequences - See Table 3 for further information.
  • 'Primer' Location of oligonucleotide primers used for PCR experiments - See Table 2 for further information.
  • Figure 2 is a depiction of the genomic DNA sequence comprising SEQ ID NO:1 and which includes the genome sequences for generation of the mature SlOOAl 1-1, SlOOAl 1-
  • Figure 3 is a schematic diagram of RNA transcript variants generated from SEQ ID NO: 1.
  • Figure 4 is a graphical representation of the detection of SEQ ID NO:1 expression in 71 colorectal tissue specimens.
  • the expression of SEQ ID NO:1 in a total of 71 colorectal specimens from 30 non-diseased controls ("normals"), 21 adenoma and 21 adenocarcinoma subjects were measured by hybridization to Affymetrix probeset IDs 200660_at.
  • Transcript expression levels were calculated by both Microarray Suite (MAS) 5.0 (Affymetrix) and the Robust Multichip Average (RMA) normalization techniques (Affymetrix. GeneChip expression data analysis fundamentals. Affymetrix, Santa Clara, CA USA, 2001 ; Hubbell et al.
  • Figure 5 is an image of the differential expression of SlOOAl 1 RNA variants in colorectal tissue specimens.
  • the expression of the 3 predicted RNA transcripts derived from the map region 150271606-150287008 of chromosome 1 was measured by end- point PCR using oligonucleotide primer sets specific for each SlOOAl 1 RNA transcript variant (Table 2).
  • Figure 6 is a graphical representation of the measurement of SEQ ID NO: 1 RNA concentration levels in colorectal tissue specimens.
  • Quantitative Real-Time PCR using forward and reverse oligonucleotide primers, 5'- ATCGAGTCCCT GATTGCTGT (Primer 6, Table 2) and 5'-CCATCACTGTTGGTGTCCAG (Primer 7, Table 2), was performed on RNA extracted from a total of 71 colorectal specimens from 30 non- diseased controls (left), 21 adenoma (middle) and 21 adenocarcinoma (right) subjects. Expression levels are relative to HPRTl using the Livak method (2- ⁇ CT).
  • Figure 7 is a schematic representation relating to the transcript activity from chromosome 1 map region 150286922-150287007, designated El (Table 4), which was measured by hybridisation to Affymetrix probeset ID 2435420 which is included on the commercially available Affymetrix Human Exon 1.0 ST Genechip.
  • Gene expression profiles were obtained by extracted RNA from colorectal tissue specimens from 5 non-diseased normals (left), 5 adenomas (middle) and 5 adenocarcinomas (right). A quality control analysis was performed to remove arrays not meeting essential quality control measures as defined by the manufacturer. Transcript expression levels were calculated using the Robust Multichip Average (RMA) normalization techniques (Affymetrix. GeneChip expression data analysis fundamentals. Affymetrix, Santa Clara, CA USA, 2001). DETAILED DESCRIPTION OF THE INVENTION
  • the present invention is predicated, in part, on the elucidation of the exon structure of SlOOAl 1, and its individual exon segments, and their involvement in terms of the assembly of SlOOAl 1 mRNA transcripts, including the formation of splice variants.
  • SlOOAl 1 splice variants which characterise large intestine cellular populations in terms of their neoplastic state. This finding has now facilitated the development of routine means of screening for the onset or predisposition to the onset of a large intestine neoplasm based on screening for upregulation of specific splice variants, relative to control expression levels.
  • SlOOAl 1 is modulated, in terms of differential changes to the level of expression of its splice variants, depending on whether the cell expressing that gene is neoplastic or not.
  • expression product or "expression of a gene” is a reference to either a transcription product (such as primary RNA or mRNA) or a translation product such as protein.
  • This gene and its expression products, whether they be RNA transcripts or encoded proteins, are collectively referred to as the "neoplastic marker".
  • one aspect of the present invention is directed to a method of screening for the onset or predisposition to the onset of a large intestine neoplasm in an individual, said method comprising measuring the level of expression of the SlOOAl 1-1, SlOOAl 1-2 and/or SlOOAl 1-3 splice variants in a biological sample from said individual wherein a higher level of expression of said splice variants relative to control levels is indicative of a neoplastic large intestine cell or a cell predisposed to the onset of a neoplastic state.
  • neoplasm should be understood as a reference to a lesion, tumour or other encapsulated or unencapsulated mass or other form of growth which comprises neoplastic cells.
  • a “neoplastic cell” should be understood as a reference to a cell exhibiting abnormal growth.
  • growth should be understood in its broadest sense and includes reference to proliferation.
  • an example of abnormal cell growth is the uncontrolled proliferation of a cell.
  • Another example is failed apoptosis in a cell, thus prolonging its usual life span.
  • the neoplastic cell may be a benign cell or a malignant cell.
  • the subject neoplasm is an adenoma or an adenocarcinoma.
  • an adenoma is generally a benign tumour of epithelial origin which is either derived from epithelial tissue or exhibits clearly defined epithelial structures. These structures may take on a glandular appearance. It can comprise a malignant cell population within the adenoma, such as occurs with the progression of a benign adenoma to a malignant adenocarcinoma.
  • said neoplastic cell is an adenoma or adenocarcinoma and even more preferably a colorectal adenoma or adenocarcinoma.
  • SlOOAH and its transcribed and translated expression products should be understood as a reference to all forms of this gene and to fragments thereof.
  • genes are known to exhibit allelic or polymorphic variation between individuals.
  • SlOOAl 1 should be understood to extend to such variants which, in terms of the present diagnostic applications, achieve the same outcome despite the fact that minor genetic variations between the actual nucleic acid sequences may exist between individuals.
  • splice variants should be understood to extend to alternative transcriptional forms of SlOOAl 1 which exhibit variation to exon expression and arrangement, such as in terms of multiple exon combinations or alternate 5'- or 3'- ends.
  • RNA eg mRNA, primary RNA transcript, miRNA, etc
  • cDNA e.g. cDNA
  • peptide isoforms which arise from alternative splicing or any other mutation, polymorphic or allelic variation. It should also be understood to include reference to any subunit polypeptides such as precursor forms which may be generated, whether existing as a monomer, multimer, fusion protein or other complex.
  • the S 100 A 1 1 genomic sequence comprises SEQ ID NO:2.
  • the SEQ ID NO:2 nucleic acid molecule has been determined to generate 6 exon segments, as follows:
  • RNA transcripts which each comprise one of the sequences depicted in SEQ ID NOs:3-5, Table 1 and are schematically depicted in Figure 3.
  • the protein expression products translated therefrom are depicted in SEQ ID NOs: 19-21, respectively.
  • sequences which are depicted in SEQ ID NOs:3-5 take the form of DNA since they have been assembled using SEQ ID NO:2.
  • the RNA transcripts which are generated either in vivo or in vitro would be characterised by comprising a corresponding sequence, albeit in RNA form.
  • screening for the "level of expression" of SlOOAl 1 splice variants may be achieved in a variety of ways including screening for any of the forms of RNA transcribed from SlOOAl 1, cDNA generated therefrom or a protein expression product. Changes to the levels of any of these products is indicative of changes to the expression of the subject gene. Still further, the molecule which is identified and measured may be a whole molecule or a fragment thereof.
  • fragmented SlOOAl 1 molecules are useful in the context of the method of the present invention.
  • RNA transcripts which transcripts comprise an RNA sequence characterised by the sequence of one of:
  • SEQ ID NO:3 SEQ ID NO:3
  • SEQ ID NO:4 SEQ ID NO:4
  • SEQ ID NO:5 SEQ ID NO:5
  • RNA transcript in a biological sample from said individual wherein a higher level of said RNA transcript relative to control levels is indicative of a neoplastic large intestine cell or a cell predisposed to the onset of a neoplastic state.
  • said RNA sequence is not SEQ ID NO:5.
  • said level of expression is measured by screening for the level of a polypeptide characterised by the sequence of one of: (i) SEQ ID NO: 19, or a sequence having at least 90% similarity across the length of the sequence; (ii) SEQ ID NO:20, or a sequence having at least 90% similarity across the length of the sequence; or (iii) SEQ ID NO:21, or a sequence having at least 90% similarity across the length of the sequence.
  • said polypeptide sequence is not SEQ ID NO:21.
  • RNA transcript being "characterised by" the sequence of any one of SEQ ID NOs:3-5 should be understood to mean that the subject RNA transcript comprises a corresponding RNA form of the DNA sequence information which is depicted in SEQ ID NOs:3-5. That is, each of the DNA nucleotides depicted in these sequences should be replaced with the corresponding RNA version of that nucleotide.
  • RNA transcript the level of expression of which is assessed in accordance with the method of the present invention, is one or more of the transcripts characterised by the sequence of one of:
  • SEQ ID NO:3 or a sequence having at least 90% similarity across the length of the sequence, or variant of SEQ ID NO:3; or (ii) SEQ ID NO:4, or a sequence having at least 90% similarity across the length of the sequence, or variant of SEQ ID NO:4.
  • RNA transcript is not characterised by the sequence of SEQ ID NO:5.
  • SlOOAl 1 has been determined to comprise 6 alternatively spliced exon segments which give rise to at least 3 RNA transcripts. Accordingly, screening for the expression of one or more of the exon segments themselves is indicative of the neoplastic state of the individual in issue. It has still further been determined that the identification of certain combinations of these exons is particularly useful in this regard. To this end, it should be appreciated that the specific exon combinations which are discussed may, in some RNA transcripts, have been spliced such that they are joined. In other transcripts, the subject exons may not be joined to one another but may be positioned, relative to one another, either proximally or distally along the transcript.
  • RNA transcript which transcript comprises one or more exon segments selected from:
  • RNA transcript which transcript comprises one or more exon segments selected from:
  • RNA transcript or variant thereof relative to control levels is indicative of a neoplastic large intestine cell or a cell predisposed to the onset of a neoplastic state.
  • RNA transcript selected from:
  • RNA transcript which comprises each of the exon segments defined by SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO: 1 1, or a sequence having at least 90% similarity across the length of these sequences, or variants of SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:l l ;
  • RNA transcript which comprises each of the exon segments defined by SEQ ID NO:8 and SEQ ID NO: 10, or a sequence having at least 90% similarity across the length of these sequences, or variants of SEQ ID NO:8 and SEQ ID NO: 10;
  • RNA transcript which comprises each of the exon segments defined by SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO: 11, or a sequence having at least 90% similarity across the length of these sequences, or variants of SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO: l l ; in a biological sample from said individual wherein a higher level of said RNA transcript or variant thereof relative to control levels is indicative of a neoplastic large intestine cell or a cell predisposed to the onset of a neoplastic state.
  • RNA transcript which transcript is selected from:
  • RNA transcript which comprises each of the exon segments defined by SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO: 11, or a sequence having at least 90% similarity across the length of these sequences, or variants of SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO: 1 1 ; or
  • RNA transcript which comprises each of the exon segments defined by SEQ ID NO:8 and SEQ ID NO: 10, or a sequence having at least 90% similarity across the length of these sequences, or variants of SEQ ID NO:8 and SEQ ID NO: 10;
  • RNA transcript or variant thereof relative to control levels is indicative of a neoplastic large intestine cell or a cell predisposed to the onset of a neoplastic state.
  • RNA transcript which comprises each of the exon segments defined by SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO: 1 1, or a sequence having at least 90% similarity across the length of these sequences, or variants of SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO: 1 1, in a biological sample from said individual wherein a higher level of said RNA transcript or variant thereof relative to control levels is indicative of a neoplastic large intestine cell or a cell predisposed to the onset of a neoplastic state.
  • said RNA transcript is not an RNA transcript which comprises each of the exon segments defined by SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11, or a sequence having at least 90% similarity across the length of these sequences, or variants of SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO: 11.
  • exon segments of said transcripts are spliced such that they are joined.
  • sequence similarity also referred to as “identity”
  • terms used to describe sequence relationships between two or more polynucleotides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units in length. Because two polynucleotides may each comprise (1) a sequence (i.e.
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence.
  • the comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • Altschul et al. Nucl. Acids Res. 25: 3389, 1997.
  • a detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, Chapter 15, 1994-1998).
  • a range of other algorithms may be used to compare the nucleotide and amino acid sequences such as but not limited to PILEUP, CLUSTALW, SEQUENCHER or VectorNTI.
  • sequence similarity and “sequence identity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide- by-nucvolide basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence identity will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
  • nucleic acid sequence identities may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art.
  • the extent of sequence identity may be determined using any computer program and associated parameters, including those described herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with the default parameters.
  • sequence comparison algorithm is a BLAST version algorithm.
  • Exemplary algorithms and programs include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl Acad. Sci. USA 85(8):2444-2448, 1988; Altschul et al.J. MoI. Biol.
  • BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practice the invention. They are described, e.g., in ; Altschul et al (1990), supra. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al. (1990) supra). These initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • HSPs high scoring sequence pairs
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873).
  • One measure of similarity provided by BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance.
  • the subject sequences are defined as exhibiting at least 90% similarity.
  • said percentage similarity is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %or 99%.
  • the "individual" who is the subject of testing may be any human or non-human mammal.
  • non-human mammals includes primates, livestock animals (e.g. horses, cattle, sheep, pigs, donkeys), laboratory test animals (e.g. mice, rats, rabbits, guinea pigs), companion animals (e.g. dogs, cats) and captive wild animals (e.g. deer, foxes).
  • livestock animals e.g. horses, cattle, sheep, pigs, donkeys
  • laboratory test animals e.g. mice, rats, rabbits, guinea pigs
  • companion animals e.g. dogs, cats
  • captive wild animals e.g. deer, foxes
  • control level is the "normal level”, which is the level of marker expressed by a corresponding large intestine cell or cellular population which is not neoplastic.
  • the normal (or "non-neoplastic" level may be determined using tissues derived from the same individual who is the subject of testing. However, it would be appreciated that this may be quite invasive for the individual concerned and it is therefore likely to be more convenient to analyse the test results relative to a standard result which reflects individual or collective results obtained from individuals other than the patient in issue. This latter form of analysis is in fact the preferred method of analysis since it enables the design of kits which require the collection and analysis of a single biological sample, being a test sample of interest.
  • the standard results which provide the normal level may be calculated by any suitable means which would be well known to the person of skill in the art.
  • a population of normal tissues can be assessed in terms of the level of the neoplastic marker of the present invention, thereby providing a standard value or range of values against which all future test samples are analysed.
  • the normal level may be determined from the subjects of a specific cohort and for use with respect to test samples derived from that cohort. Accordingly, there may be determined a number of standard values or ranges which correspond to cohorts which differ in respect of characteristics such as age, gender, ethnicity or health status.
  • Said "normal level” may be a discrete level or a range of levels. An increase in the expression level of the subject genes relative to normal levels is indicative of the tissue being neoplastic.
  • control level is a non-neoplastic level.
  • said large intestine tissue is preferably colorectal tissue.
  • said neoplasm is a colorectal adenoma or adenocarcinoma.
  • SlOOAl 1 specifically, SlOOAl 1-1 and SlOOAl 1-2
  • SlOOAl 1-1 and SlOOAl 1-2 markers are not only expressed at levels higher than normal levels, their expression pattern is uniquely characterised by the fact that in a proportion of normal tissues, expression levels above that of background levels are not detectable in non-neoplastic tissue.
  • background levels should be understood as the levels which are detectable in a negative control sample. That is, a sample in which the subject SlOOAl 1 molecule is not expressed.
  • said background level is that of the chosen testing methodoloty. This contrasts to SlOOAl 1-3 which is detectable in all non-neoplastic tissue, albeit at lower levels. This determination has therefore enabled the development of screening systems which are more sensitive.
  • control level is the background level.
  • the detection method of the present invention can be performed on any suitable biological sample.
  • a biological sample should be understood as a reference to any sample of biological material derived from an animal such as, but not limited to, cellular material, biological fluids (eg. blood), faeces, tissue biopsy specimens, surgical specimens or fluid which has been introduced into the body of an animal and subsequently removed (such as, for example, the solution retrieved from an enema wash).
  • the biological sample which is tested according to the method of the present invention may be tested directly or may require some form of treatment prior to testing. For example, a biopsy or surgical sample may require homogenisation prior to testing or it may require sectioning for in situ testing of the qualitative expression levels of individual genes.
  • a cell sample may require permeabilisation prior to testing. Further, to the extent that the biological sample is not in liquid form, (if such form is required for testing) it may require the addition of a reagent, such as a buffer, to mobilise the sample.
  • a reagent such as a buffer
  • the biological sample may be directly tested or else all or some of the nucleic acid or protein material present in the biological sample may be isolated prior to testing.
  • the sample may be partially purified or otherwise enriched prior to analysis. For example, to the extent that a biological sample comprises a very diverse cell population, it may be desirable to enrich for a sub-population of particular interest.
  • the target cell population or molecules derived therefrom pretreated prior to testing, for example, inactivation of live virus or being run on a gel. It should also be understood that the biological sample may be freshly harvested or it may have been stored (for example by freezing) prior to testing or otherwise treated prior to testing (such as by undergoing culturing).
  • said sample is a faecal (stool) sample, enema wash, surgical resection, tissue biopsy or blood sample.
  • the present invention is designed to screen for a neoplastic cell or cellular population, which is located in the large intestine.
  • cell or cellular population should be understood as a reference to an individual cell or a group of cells.
  • Said group of cells may be a diffuse population of cells, a cell suspension, an encapsulated population of cells or a population of cells which take the form of tissue.
  • RNA transcripts eg primary RNA or mRNA
  • RNA any form of RNA, such as primary RNA or mRNA.
  • the present invention also extends to detection methodology which is directed to screening for modulated levels or patterns of the neoplastic marker protein products as an indicator of the neoplastic state of a cell or cellular population.
  • detection methodology which is directed to screening for modulated levels or patterns of the neoplastic marker protein products as an indicator of the neoplastic state of a cell or cellular population.
  • one method is to screen for RNA transcripts and/or the corresponding protein product
  • the present invention is not limited in this regard and extends to screening for any other form of neoplastic marker expression product such as, for example, a primary RNA transcript. It is well within the skill of the person of skill in the art to determine the most appropriate screening target for any given situation.
  • nucleic acid molecule should be understood as a reference to both deoxyribonucleic acid molecules and ribonucleic acid molecules and fragments thereof.
  • the present invention therefore extends to both directly screening for RNA levels in a biological sample or screening for the complementary cDNA which has been reverse- transcribed from an RNA population of interest. It is well within the skill of the person of skill in the art to design methodology directed to screening for either DNA or RNA. As detailed above, the method of the present invention also extends to screening for the protein product translated from the subject RNA.
  • RNA transcripts In terms of screening for the upregulation of SlOOAl 1 it would also be well known to the person of skill in the art that changes which are detectable at the DNA level are indicative of changes to gene expression activity and therefore changes to expression product levels. Such changes include but are not limited to, changes to DNA methylation and chromatin proteins associated with the gene. Accordingly, reference herein to "screening the level of expression" of RNA transcripts and comparison of these transcript “levels of expression” to control transcript “levels of expression” should be understood to include assessing DNA factors which are related to transcription, such as gene/DNA methylation patterns or association with specific chromosomal proteins. That is, changes to levels of expression of RNA transcripts can be detected at the DNA level, RNA level or protein level.
  • protein should be understood to encompass peptides, polypeptides and proteins (including protein fragments).
  • the protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • Reference herein to a "protein” includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • the proteins encoded by SlOOAl 1 may be in multimeric form meaning that two or more molecules are associated together. Where the same protein molecules are associated together, the complex is a homomultimer.
  • An example of a homomultimer is a homodimer.
  • the complex is a heteromultimer such as a heterodimer.
  • fragment should be understood as a reference to a portion of the subject nucleic acid molecule or protein. As detailed hereinbefore, this is particularly relevant with respect to screening for modulated RNA levels in stool samples since the subject RNA is likely to have been degraded or otherwise fragmented due to the environment of the gut. One may therefore actually be detecting fragments of the subject RNA molecule, which fragments are identified by virtue of the use of a suitably specific probe.
  • references to the "onset" of a neoplasm should be understood as a reference to one or more cells of that individual exhibiting dysplasia.
  • the adenoma or adenocarcinoma may be well developed in that a mass of dysplastic cells has developed.
  • the adenoma or adenocarcinoma may be at a very early stage in that only relatively few abnormal cell divisions have occurred at the time of diagnosis.
  • the present invention also extends to the assessment of an individual's predisposition to the development of a neoplasm, such as an adenoma or adenocarcinoma.
  • changed levels of the neoplastic marker may be indicative of that individual's predisposition to developing a neoplasia, such as the future development of an adenoma or adenocarcinoma or another adenoma or adenocarcinoma.
  • the detection of converse changes in the levels of said marker may be desired under certain circumstances, for example, to monitor the effectiveness of therapeutic or prophylactic treatment directed to modulating a neoplastic condition, such as adenoma or adenocarcinoma development.
  • a neoplastic condition such as adenoma or adenocarcinoma development.
  • screening for a decrease in the levels of this marker subsequently to the onset of a therapeutic regime may be utilised to indicate reversal or other form of improvement of the subject individual's condition.
  • the method of the present invention is therefore useful as a one off test or as an on-going monitor of those individuals thought to be at risk of neoplasia development or as a monitor of the effectiveness of therapeutic or prophylactic treatment regimes directed to inhibiting or otherwise slowing neoplasia development.
  • mapping the modulation of SlOOAl 1 expression levels in any one or more classes of biological samples is a valuable indicator of the status of an individual or the effectiveness of a therapeutic or prophylactic regime which is currently in use.
  • the method of the present invention should be understood to extend to monitoring for increases or decreases in S lOOAl 1 expression levels in an individual relative to their normal level (as hereinbefore defined), or relative to one or more earlier marker expression levels determined from a biological sample of said individual.
  • Means of testing for t ⁇ e subject expressed neoplasm marker in a biological sample can be achieved by any suitable method, which would be well known to the person of skill in the art, such as but not limited to:
  • Imaging may be used following administration of imaging probes or reagents capable of disclosing altered expression of the marker in the intestinal tissues.
  • Molecular imaging (Moore et al, BBA, 1402:239-249, 1988; Weissleder et al, Nature Medicine 6:351-355, 2000) is the in vivo imaging of molecular expression that correlates with the macro-features currently visualized using "classical" diagnostic imaging techniques such as X-Ray, computed tomography (CT), MRI, Positron Emission Tomography (PET) or endoscopy.
  • CT computed tomography
  • PET Positron Emission Tomography
  • RNA expression in the cells by Fluorescent In Situ Hybridization (FISH), or in extracts from the cells by technologies such as Quantitative Reverse Transcriptase Polymerase Chain Reaction (QRTPCR) or Flow cytometric qualification of competitive RT-PCR products (Wedemeyer et al. , Clinical Chemistry 48:9 1398-1405, 2002).
  • FISH Fluorescent In Situ Hybridization
  • QRTPCR Quantitative Reverse Transcriptase Polymerase Chain Reaction
  • Flow cytometric qualification of competitive RT-PCR products Wedemeyer et al. , Clinical Chemistry 48:9 1398-1405, 2002.
  • a "microarray” is a linear or multi-dimensional array of preferably discrete regions, each having a defined area, formed on the surface of a solid support. The density of the discrete regions on a microarray is determined by the total numbers of target polynucleotides to be detected on the surface of a single solid phase support.
  • a DNA microarray is an array of oligonucleotide probes placed onto a chip or other surfaces used to detect complementary oligonucleotides from a complex nucleic acid mixture. Since the position of each particular group of probes in the array is known, the identities of the target polynucleotides can be determined based on their binding to a particular position in the microarray.
  • arrays are used in the analysis of differential gene expression, where the profile of expression of genes in different cells or tissues, often a tissue of interest and a control tissue, is compared and any differences in gene expression among the respective tissues are identified. Such information is useful for the identification of the types of genes expressed in a particular tissue type and diagnosis of conditions based on the expression profile.
  • RNA from the sample of interest is subjected to reverse transcription to obtain labelled cDNA. See U.S. Pat. No. 6,410,229 (Lockhart et al.)
  • the cDNA is then hybridized to oligonucleotides or cDNAs of known sequence arrayed on a chip or other surface in a known order.
  • the RNA is isolated from a biological sample and hybridised to a chip on which are anchored cDNA probes. The location of the oligonucleotide to which the labelled cDNA hybridizes provides sequence information on the cDNA, while the amount of labelled hybridized RNA or cDNA provides an estimate of the relative representation of the RNA or cDNA of interest.
  • nucleic acid probes corresponding to the subject nucleic acids are made.
  • the nucleic acid probes attached to the microarray are designed to be substantially complementary to the nucleic acids of the biological sample such that specific hybridization of the target sequence and the probes of the present invention occurs.
  • This complementarity need not be perfect, in that there may be any number of base pair mismatches that will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention. It is expected that the overall homology of the genes at the nucleotide level probably will be about 40% or greater, probably about 60% or greater, and even more probably about 80% or greater; and in addition that there will be corresponding contiguous sequences of about 8-12 nucleotides or longer. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. Thus, by “substantially complementary” herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions, particularly high stringency conditions.
  • a nucleic acid probe is generally single stranded but can be partly single and partly double stranded.
  • the strandedness of the probe is dictated by the structure, composition, and properties of the target sequence.
  • the oligonucleotide probes range from about 6, 8, 10, 12, 15, 20, 30 to about 100 bases long, with from about 10 to about 80 bases being preferred, and from about 15 to about 40 bases being particularly preferred. That is, generally entire genes are rarely used as probes. In some embodiments, much longer nucleic acids can be used, up to hundreds of bases.
  • the probes are sufficiently specific to hybridize to a complementary template sequence under conditions known by those of skill in the art.
  • the number of mismatches between the probe's sequences and their complementary template (target) sequences to which they hybridize during hybridization generally do not exceed 15%, usually do not exceed 10% and preferably do not exceed 5%, as-determined by BLAST (default settings).
  • Oligonucleotide probes can include the naturally-occurring heterocyclic bases normally found in nucleic acids (uracil, cytosine, thymine, adenine and guanine), as well as modified bases and base analogues. Any modified base or base analogue compatible with hybridization of the probe to a target sequence is useful in the practice of the invention.
  • the sugar or glycoside portion of the probe can comprise deoxyribose, ribose, and/or modified forms of these sugars, such as, for example, 2'- O-alkyl ribose.
  • the sugar moiety is 2'-deoxyribose; however, any sugar moiety that is compatible with the ability of the probe to hybridize to a target sequence can be used.
  • nucleoside units of the probe are linked by a phosphodiester backbone, as is well known in the art.
  • internucleotide linkages can include any linkage known to one of skill in the art that is compatible with specific hybridization of the probe including, but not limited to phosphorothioate, methylphosphonate, sulfamate (e.g., U.S. Pat. No. 5,470,967) and polyamide (i.e., peptide nucleic acids).
  • Peptide nucleic acids are described in Nielsen et al. (1991) Science 254: 1497-1500, U.S. Pat. No. 5,714,331, and Nielsen (1999) Curr. Opin. Biotechnol. 10:71-75.
  • the probe can be a chimeric molecule; i.e., can comprise more than one type of base or sugar subunit, and/or the linkages can be of more than one type within the same primer.
  • the probe can comprise a moiety to facilitate hybridization to its target sequence, as are known in the art, for example, intercalators and/or minor groove binders. Variations of the bases, sugars, and internucleoside backbone, as well as the presence of any pendant group on the probe, will be compatible with the ability of the probe to bind, in a sequence- specific fashion, with its target sequence. A large number of structural modifications, are possible within these bounds.
  • the probes according to the present invention may have structural characteristics such that they allow the signal amplification, such structural characteristics being, for example, branched DNA probes as those described by Urdea et al. ⁇ Nucleic Acids Symp. Ser., 24: 197-200 (1991)) or in the European Patent No. EP-0225,807.
  • synthetic methods for preparing the various heterocyclic bases, sugars, nucleosides and nucleotides that form the probe, and preparation of oligonucleotides of specific predetermined sequence are well-developed and known in the art.
  • a preferred method for oligonucleotide synthesis incorporates the teaching of U.S. Pat. No. 5,419,966.
  • Multiple probes may be designed for a particular target nucleic acid to account for polymorphism and/or secondary structure in the target nucleic acid, redundancy of data and the like.
  • more than one probe per sequence either overlapping probes or probes to different sections of a single target gene are used. That is, two, three, four or more probes, are used to build in a redundancy for a particular target.
  • the probes can be overlapping (i.e. have some sequence in common), or are specific for distinct sequences of a gene.
  • each probe or probe group corresponding to a particular target polynucleotide is situated in a discrete area of the microarray.
  • Probes may be in solution, such as in wells or on the surface of a micro-array, or attached to a solid support.
  • solid support materials that can be used include a plastic, a ceramic, a metal, a resin, a gel and a membrane.
  • Useful types of solid supports include plates, beads, magnetic material, microbeads, hybridization chips, membranes, crystals, ceramics and self-assembling monolayers.
  • One example comprises a two-dimensional or three-dimensional matrix, such as a gel or hybridization chip with multiple probe binding sites (Pevzner et al. , J. Biomol. Struc. & Dyn. 9:399-410, 1991 ; Maskos and Southern, Nuc. Acids Res. 20: 1679-84, 1992).
  • Hybridization chips can be used to construct very large probe arrays that are subsequently hybridized with a target nucleic acid. Analysis of the hybridization pattern of the chip can assist in the identification of the target nucleotide sequence. Patterns can be manually or computer analyzed, but it is clear that positional sequencing by hybridization lends itself to computer analysis and automation.
  • one may use an Affymetrix chip on a solid phase structural support in combination with a fluorescent bead based approach.
  • one may utilise a cDNA microarray.
  • the oligonucleotides described by Lockkart et al. i.e. Affymetrix synthesis probes in situ on the solid phase
  • nucleic acids can be attached or immobilized to a solid support in a wide variety of ways.
  • immobilized herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal.
  • the binding can be covalent or non-covalent.
  • non-covalent binding and grammatical equivalents herein is meant one or more of either electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as streptavidin, to the support and the non-covalent binding of the biotinylated probe to the streptavidin.
  • covalent binding and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.
  • Nucleic acid probes may be attached to the solid support by covalent binding such as by conjugation with a coupling agent or by covalent or non-covalent binding such as electrostatic interactions, hydrogen bonds or antibody-antigen coupling, or by combinations thereof.
  • Typical coupling agents include biotin/avidin, biotin/streptavidin, Staphylococcus aureus protein A/IgG antibody F c fragment, and streptavidin/protein A chimeras (T. Sano and C. R. Cantor, Bio/Technology 9: 1378- 81 (1991)), or derivatives or combinations of these agents.
  • Nucleic acids may be attached to the solid support by a photocleavable bond, an electrostatic bond, a disulfide bond, a peptide bond, a diester bond or a combination of these sorts of bonds.
  • the array may also be attached to the solid support by a selectively releasable bond such as 4,4'-dimethoxytrityl or its derivative.
  • Derivatives which have been found to be useful include 3 or 4 [bis-(4-methoxyphenyl)]-methyl- benzoic acid, N-succinimidyl-3 or 4 [bis-(4-methoxyphenyl)]-methyl-benzoic acid, N-succinimidyl-3 or 4 [bis-(4-methoxyphenyl)]-hydroxymethyl-benzoic acid, N- succinimidyl-3 or 4 [bis-(4-methoxyphenyl)]-chloromethyl-benzoic acid, and salts of these acids.
  • the probes are attached to the microarray in a wide variety of ways, as will be appreciated by those in the art.
  • the nucleic acids can either be synthesized first, with subsequent attachment to the microarray, or can be directly synthesized on the microarray.
  • the microarray comprises a suitable solid substrate.
  • substrate or “solid support” or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method.
  • the solid phase support of the present invention can be of any solid materials and structures suitable for supporting nucleotide hybridization and synthesis.
  • the solid phase support comprises at least one substantially rigid surface on which the oligonucleotide primers can be immobilized and the reverse transcriptase reaction performed.
  • the substrates with which the polynucleotide microarray elements are stably associated and may be fabricated from a variety of materials, including plastics, ceramics, metals, acrylamide, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, Teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids.
  • plastics plastics, ceramics, metals, acrylamide, cellulose, nitrocellulose, glass, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, Teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides
  • Substrates may be two-dimensional or three-dimensional in form, such as gels, membranes, thin films, glasses, plates, cylinders, beads, magnetic beads, optical fibers, woven fibers, etc.
  • a preferred form of array is a three-dimensional array.
  • a preferred three-dimensional array is a collection of tagged beads. Each tagged bead has different oligonucleotide primers attached to it. Tags are detectable by signalling means such as color (Luminex, Illumina) and electromagnetic field (Pharmaseq) and signals on tagged beads can even be remotely detected (e.g., using optical fibers).
  • the size of the solid support can be any of the standard microarray sizes, useful for DNA microarray technology, and the size may be tailored to fit the particular machine being used to conduct a reaction of the invention.
  • the substrates allow optical detection and do not appreciably fluoresce.
  • the surface of the microarray and the probe may be derivatized with chemical functional groups for subsequent attachment of the two.
  • the microarray is derivatized with a chemical functional group including, but not limited to, amino groups, carboxy groups, oxo groups and thiol groups, with amino groups being particularly preferred.
  • the probes can be attached using functional groups on the probes.
  • nucleic acids containing amino groups can be attached to surfaces comprising amino groups, for example using linkers as are known in the art; for example, homo-or hetero- bifunctional linkers as are well known.
  • additional linkers such as alkyl groups (including substituted and heteroalkyl groups) may be used.
  • the oligonucleotides are synthesized as is known in the art, and then attached to the surface of the solid support.
  • either the 5' or 3' terminus may be attached to the solid support, or attachment may be via an internal nucleoside.
  • the immobilization to the solid support may be very strong, yet non-covalent.
  • biotinylated oligonucleotides can be made, which bind to surfaces covalently coated with streptavidin, resulting in attachment.
  • the arrays may be produced according to any convenient methodology, such as preforming the polynucleotide microarray elements and then stably associating them with the surface.
  • the oligonucleotides may be synthesized on the surface, as is known in the art.
  • a number of different array configurations and methods for their production are known to those of skill in the art and disclosed in WO 95/251 16 and WO 95/35505 (photolithographic techniques), U.S. Pat. No. 5,445,934 (in situ synthesis by photolithography), U.S. Pat. No. 5,384,261 (in situ synthesis by mechanically directed flow paths); and U.S. Pat. No.
  • gene expression can also be quantified using liquid-phase assays.
  • PCR kinetic polymerase chain reaction
  • Kinetic PCR allows for the simultaneous amplification and quantification of specific nucleic acid sequences.
  • the specificity is derived from synthetic oligonucleotide primers designed to preferentially adhere to single-stranded nucleic acid sequences bracketing the target site. This pair of oligonucleotide primers form specific, non-covalently bound complexes on each strand of the target sequence. These complexes facilitate in vitro transcription of double-stranded DNA in opposite orientations.
  • Temperature cycling of the reaction mixture creates a continuous cycle of primer binding, transcription, and re-melting of the nucleic acid to individual strands. The result is an exponential increase of the target dsDNA product.
  • This product can be quantified in real time either through the use of an intercalating dye or a sequence specific probe.
  • SYBR(r) Green 1 is an example of an intercalating dye, that preferentially binds to dsDNA resulting in a concomitant increase in the fluorescent signal.
  • Sequence specific probes such as used with TaqMan technology, consist of a fluorochrome and a quenching molecule covalently bound to opposite ends of an oligonucleotide.
  • the probe is designed to selectively bind the target DNA sequence between the two oligonucleotide primers.
  • the fluorochrome is cleaved from the probe by the exonuclease activity of the polymerase resulting in signal dequenching.
  • the probe signalling method can be more specific than the intercalating dye method, but in each case, signal strength is proportional to the dsDNA product produced.
  • Each type of quantification method can be used in multi- well liquid phase arrays with each well representing oligonucleotide primers and/or probes specific to nucleic acid sequences of interest.
  • an array of probe/primer reactions can simultaneously quantify the expression of multiple gene products of interest. See Germer et al, Genome Res. 10:258-266 (2000); Heid et al, Genome Res. 6:986-994 (1996).
  • Testing for proteinaceous neoplastic marker expression product in a biological sample can be performed by any one of a number of suitable methods which are well known to those skilled in the art. Examples of suitable methods include, but are not limited to, antibody based screening of tissue sections, biopsy specimens or bodily fluid samples.
  • the presence of the marker protein may be determined in a number of ways such as by Western blotting, ELISA or flow cytometry procedures. These, of course, include both single-site and two-site or “sandwich” assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target. Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule.
  • a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent.
  • a first antibody having specificity for the marker or antigenic parts thereof is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking, covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample.
  • an aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes) and under suitable conditions (e.g. 25 0 C) to allow binding of any subunit present in the antibody.
  • the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the antigen.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the antigen.
  • An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody.
  • a second labelled antibody specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above.
  • the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample.
  • Reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fluorecein and rhodamine
  • fluorecein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome- labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome- labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope.
  • the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.
  • Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • methylation of DNA at CpG sites and modifications such as deacetylation of histone H3 on lysine 9, and methylation on lysine 9 or 27 are associated with inactive chromatin, while the converse state of a lack of DNA methylation, acetylation of lysine 9 of histone H3 is associated with open chromatin and active gene expression.
  • this epigenetic regulation of gene expression is frequently found to be disrupted (Esteller & Herman, 2000; Jones & Baylin, 2002).
  • Genes such as tumour suppressor or metastasis suppressor genes are often found to be silenced by DNA methylation, while other genes may be hypomethylated and inappropriately expressed.
  • this in some instances is characterised by a loss of methylation of the promoter or regulatory region of the gene.
  • Epigenetic alterations and chromatin changes in cancer are also evident in the altered association of modified histones with specific genes(Esteller, 2007); for example activated genes are often found associated with histone H3 that is acetylated on lysine 9 and methylated on lysine 4.
  • activated genes are often found associated with histone H3 that is acetylated on lysine 9 and methylated on lysine 4.
  • the use of antibodies targeted to altered histones allows for the isolation of DNA associated with particular chromatin states and has potential use in cancer diagnosis.
  • gene expression levels can be measured by a variety of methods known in the art.
  • gene transcription or translation products can be measured.
  • Gene transcription products, i.e., RNA can be measured, for example, by hybridization assays, run-off assays., Northern blots, or other methods known in the art.
  • Hybridization assays generally involve the use of oligonucleotide probes that hybridize to the single-stranded RNA transcription products.
  • the oligonucleotide probes are complementary to the transcribed RNA expression product.
  • a sequence- specific probe can be directed to hybridize to RNA or cDNA.
  • a "nucleic acid probe”, as used herein, can be a DNA probe or an RNA probe that hybridizes to a complementary sequence.
  • One of skill in the art would know how to design such a probe such that sequence specific hybridization will occur.
  • One of skill in the art will further know how to quantify the amount of sequence specific hybridization as a measure of the amount of gene expression for the gene was transcribed to produce the specific RNA.
  • hybridization sample is maintained under conditions that are sufficient to allow specific hybridization of the nucleic acid probe to a specific gene expression product.
  • Specific hybridization indicates near exact hybridization (e.g., with few if any mismatches).
  • Specific hybridization can be performed under high stringency conditions or moderate stringency conditions.
  • the hybridization conditions for specific hybridization are high stringency. For example, certain high stringency conditions can be used to distinguish perfectly complementary nucleic acids from those of less complementarity.
  • hybridization conditions By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions that will allow a given sequence to hybridize (e.g., selectively) with the most complementary sequences in the sample can be determined.
  • washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids. Generally, starting from the lowest temperature at which only homologous hybridization occurs, each 0 C. by which the final wash temperature is reduced (holding SSC concentration constant) allows an increase by 1% in the maximum mismatch percentage among the sequences that hybridize. Generally, doubling the concentration of SSC results in an increase in T m of about 17 0 C.
  • the wash temperature can be determined empirically for high, moderate or low stringency, depending on the level of mismatch sought.
  • a low stringency wash can comprise washing in a solution containing 0.2.times.SSC/0.1% SDS for 10 minutes at room temperature
  • a moderate stringency wash can comprise washing in a pre-warmed solution (42 0 C) solution containing 0.2.times.SSC/0.1% SDS for 15 minutes at 42 0 C
  • a high stringency wash can comprise washing in pre-warmed (68 0 C.) solution containing 0.1. times. SSC/0.1% SDS for 15 minutes at 68 0 C.
  • washes can be performed repeatedly or sequentially to obtain a desired result as known in the art.
  • Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of complementarity between the target nucleic acid molecule and the primer or probe used (e.g., the sequence to be hybridized).
  • a related aspect of the present invention provides a molecular array, which array comprises a plurality of:
  • nucleic acid molecules comprising a nucleotide sequence corresponding to any one or more of the SlOOAl 1 sequences hereinbefore described or a sequence exhibiting at least 80% identity thereto or a functional derivative, fragment, variant or homologue of said nucleic acid molecule; or
  • nucleic acid molecules comprising a nucleotide sequence capable of hybridising to any one or more of the sequences of (i) under medium stringency conditions or a functional derivative, fragment, variant or homologue of said nucleic acid molecule; or
  • nucleic acid probes or oligonucleotides comprising a nucleotide sequence capable of hybridising to any one or more of the sequences of (i) under medium stringency conditions or a functional derivative, fragment, variant or homologue of said nucleic acid molecule; or
  • probes capable of binding to any one or more of the proteins encoded by the nucleic acid molecules of (i) or a derivative, fragment or, homologue thereof
  • said percent identity is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • Low stringency includes and encompasses from at least about 1% v/v to at least " about 15% v/v formamide and from at least about IM to at least about 2M salt for hybridisation, and at least about IM to at least about 2M salt for washing conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.0 IM to at least about 0.15M salt for hybridisation, and at least about 0.0 IM to at least about 0.15M salt for washing conditions.
  • the T m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatched based pairs (Bonner et al (1973) J. MoI. Biol. 81 : 123).
  • the subject probes are designed to bind to the nucleic acid or protein to which they are directed with a level of specificity which minimises the incidence of non-specific reactivity.
  • a level of specificity which minimises the incidence of non-specific reactivity.
  • probes which are used to detect the subject proteins may take any suitable form including antibodies and aptamers.
  • a library or array of nucleic acid or protein probes provides rich and highly valuable information. Further, two or more arrays or profiles (information obtained from use of an array) of such sequences are useful tools for comparing a test set of results with a reference, such as another sample or stored calibrator. In using an array, individual probes typically are immobilized at separate locations and allowed to react for binding reactions. Oligonucleotide primers associated with assembled sets of markers are useful for either preparing libraries of sequences or directly detecting markers from other biological samples.
  • a library (or array, when referring to physically separated nucleic acids corresponding to at least some sequences in a library) of SlOOAl 1 marker or exons thereof exhibits highly desirable properties. These properties are associated with specific conditions, and may be characterized as regulatory profiles.
  • a profile as termed here refers to a set of members that provides diagnostic information of the tissue from which the markers were originally derived. A profile in many instances comprises a series of spots on an array made from deposited sequences.
  • a characteristic patient profile is generally prepared by use of an array.
  • An array profile may be compared with one or more other array profiles or other reference profiles.
  • the comparative results can provide rich information pertaining to disease states, developmental state, receptiveness to therapy and other information about the patient.
  • Another aspect of the present invention provides a diagnostic kit for assaying biological samples comprising an agent for detecting one or more neoplastic marker reagents useful for facilitating the detection by the agent in the first compartment. Further means may also be included, for example, to receive a biological sample.
  • the agent may be any suitable detecting molecule.
  • Another aspect of the present invention is directed to an isolated nucleic acid molecule selected from the list consisting of:
  • nucleic acid molecule or molecule complementary thereto or, fragment or derivative thereof comprising one or more of the nucleotide sequences, as set forth in any one of SEQ ID NO: 6-1 1, or a nucleotide sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity over the length of the sequence, or a nucleotide sequence capable of hybridising to said nucleic acid molecule or complementary form thereof under low stringency conditions; and
  • nucleic acid molecule or derivative or fragment thereof comprising one or more of the nucleotide sequences substantially as set forth in any one of SEQ ID NO:6-11 or a fragment of said molecule.
  • the SlOOAl 1 genomic sequence comprises SEQ ID NO:2.
  • the SEQ ID NO:2 nucleic acid molecule has been determined to encode 6 exon segments, as follows:
  • said sequence excludes the full length genomic DNA form of SlOOAl 1, this being SEQ ID NO:2 and the splice variant of SEQ ID NO:5.
  • RNA such as mRNA
  • cDNA corresponding to the coding regions (i.e. exons) optionally comprising 5'- or 3 '-untranslated sequences of the gene
  • RNA such as mRNA
  • cDNA corresponding to the coding region either with or without sequences associated with precursor forms of the protein, such as signal sequences, and optionally 5'- or 3'- untranslated sequences; and/or
  • genomic DNA fragments comprising one or more of the exons identified herein.
  • said nucleic acid molecules correspond to El, E2b and E3b.
  • transcripts of SlOOAl 1 which are generated from SEQ ID NO:2 reflect the splicing together of the transcribed exons and exon segments detailed earlier. Without limiting the present invention to any one theory or mode of action, these exons and exon segments have been determined to transcribe to 6 variant transcript forms as depicted in Figure 3.
  • nucleic acid molecule selected from the list consisting of:
  • nucleic acid molecule or molecule complementary thereto or fragment or derivative thereof comprising a nucleotide sequence, as set forth in any one of SEQ ID NO:3 or 4, or a nucleotide sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity over the length of the sequence, or a nucleotide sequence capable of hybridising to said nucleic acid molecule or complementary form thereof under low stringency conditions; and
  • nucleic acid molecule or derivative or fragment thereof comprising a nucleotide sequence substantially as set forth in any one of SEQ ID NO:3 or 4 or a fragment of said molecule.
  • Still another aspect of the present invention is directed to an isolated nucleic acid molecule selected from the list consisting of: (i) An isolated nucleic acid molecule or molecule complementary thereto or fragment or derivative thereof comprising a nucleotide sequence, as set forth in any one or more of SEQ ID NOs: 6, 7, 8, 9, 10 and 11, or a nucleotide sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity over the length of the sequence, or a nucleotide sequence capable of hybridising to said nucleic acid molecule or complementary form thereof under low stringency conditions; and
  • nucleic acid molecule or derivative or fragment thereof comprising a nucleotide sequence substantially as set forth in any one or more of SEQ ID NOs: 6, 7, 8, 9, 10 and 11 or a fragment of said molecule.
  • Yet another aspect of the present invention is directed to an isolated nucleic acid molecule selected from the list consisting of:
  • nucleic acid molecule or molecule complementary thereto or fragment or derivative thereof comprising a nucleotide sequence, as set forth in any one or more of SEQ ID NOs:6, 8 and 10, or a nucleotide sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity over the length of the sequence, or a nucleotide sequence capable of hybridising to said nucleic acid molecule or complementary form thereof under low stringency conditions; and
  • nucleic acid molecule or derivative or fragment thereof comprising a nucleotide sequence substantially as set forth in any one or more of SEQ ID NOs:6, 8 and 10 or a fragment of said molecule.
  • said nucleotide sequences are not SEQ ID NOs: 7, 9 or 1 1.
  • references to "gene” herein should be understood as a reference to any genomic locus or set of loci which give rise to mRNA transcripts from one or more promoters, including transcripts formed by the splicing of two or more exons as hereinbefore described. It would be appreciated that not all mRNA transcripts are necessarily translated to a protein expression product.
  • Yet another aspect of the present invention is directed to an isolated protein which is translated from any one of the exons, sequences or mRNA transcripts herein disclosed.
  • protein should be understood to encompass peptides, polypeptides and proteins. It should also be understood that these terms are used interchangeably herein.
  • the protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated with the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • Reference hereinafter to a "protein” includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • the protein of the present invention is preferably in isolated form.
  • isolated is meant a protein having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10% subject protein, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%, even still more preferably at least about 60-70%, yet even still more preferably 80-90% or greater of subject protein relative to other components as determined by molecular weight, amino acid sequence or other convenient means.
  • the protein of the present invention may also be considered, in a preferred embodiment, to be biologically pure.
  • the term "isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally occurring polynucleotide or protein present in a living plant is not isolated, but the same polynucleotide or protein, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or protein could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • an isolated material or composition can also be a "purified" composition, i.e., it does not require absolute purity; rather, it is intended as a relative definition.
  • Individual nucleic acids obtained from a library can be conventionally purified to electrophoretic homogeneity.
  • the invention provides nucleic acids which have been purified from genomic DNA or from other sequences in a library or other environment by at least one, two, three, four, five or more orders of magnitude.
  • Proteins of the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo. The proteins of the invention can be made and isolated using any method known in the art. Proteins of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers et al. (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn et al. (1980) Nucleic Acids Res. Symp. Ser.
  • peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge et al. (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automated synthesis may be achieved, e.g., using the ABI 43 IA Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
  • Proteins of the invention can also be synthesised and expressed as fusion proteins with one or more additional domains linked thereto, for example to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody- expressing B cells, and the like.
  • Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA).
  • an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams et al (1995) Biochemistry 34:1787-1797; Dobeli et al (1998) Protein Expr. Purif. 12:404-14).
  • histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying a region from the remainder of the fusion protein.
  • Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see e.g., Kroll et al. (1993) DNA Cell Biol, 12:441-53.
  • a chemically systhesised protein according to the present invention is conveniently synthesised based on molecules isolated from Bahia grass pollen. Isolation of these molecules may be accomplished by any suitable means such as by chromatographic separation, for example using CM-cellulose ion exchange chromatography followed by Sephadex (e. g. G-50 column) filtration. Many other techniques are available including HPLC, PAGE amongst others.
  • the subject protein may be synthesised by solid phase synthesis using F-moc chemistry as described by Carpino et al. (1991).
  • Polypeptides and fragments thereof may also be synthesised by alternative chemistries including, but not limited to, t-Boc chemistry as described in Stewart et al (1985) or by classical methods of liquid phase peptide synthesis.
  • Still another aspect of the present invention is directed to a protein or derivative or fragment thereof encoded by a nucleic acid molecule as hereinbefore defined.
  • the present invention is directed to a protein or derivative or fragment thereof encoded by the nucleotide sequence as set forth in any one or more of SEQ ID NOs:6-l 1 or the sequence complementary to a sequence capable of hybridising to SEQ ID NOs: 6-11 under low stringency conditions or a protein molecule exhibiting at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to said protein across the length of the sequence.
  • said nucleotide sequences are SEQ ID NOs:6, 8 and 10.
  • the present invention is directed to a protein or derivative or fragment thereof encoded by the nucleotide sequence as set forth in any one of SEQ ID NOs:3 or 4 or the sequence complementary to a sequence capable of hybridising to SEQ ID NOs: 3 or 4 under low stringency conditions or a protein molecule exhibiting at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to said protein across the length of the sequence.
  • said sequences exclude SEQ ID Nos:2 and 5.
  • the present invention is directed to a protein or derivative or fragment thereof characterised by the sequence as set forth in any one of SEQ ID NOs: 19 or 20 or a protein molecule exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to said protein across the length of the sequence.
  • nucleic acid or “nucleic acid sequence” as used herein refer to an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin which may be single- stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs).
  • DNA or RNA e.g., mRNA, rRNA, tRNA
  • PNA peptide nucleic acid
  • DNA-like or RNA-like material natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., iRNPs
  • the term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides.
  • the term also encompasses nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup et al. (1997) Biochemistry 36:8692-8698; Mull et al. (1996) Antisense Nucleic Acid Drug Dev 6: 153-156.
  • the present invention extends to antisense nucleic acid molecules siRNA and miRNA which are directed to the nucleic acid molecules hereinbefore defined.
  • the present invention should also be understood to extend to probes and primers directed to the nucleic acid molecules hereinbefore defined.
  • antisense nucleic acid molecules and probes and primers would be a matter of routine procedure to the person of skill in the art in light of the detailed teachings provided herein.
  • Said antisense molecules, probes and primers are preferably specific for their target molecule although it would be appreciated that the same cross-reactivity may occur depending on the sequence and length of the antisense molecule, probe or primer. Whether or not a level of cross-reactivity/promiscuity is acceptable is a judgement to be made by the skilled person and will depend on the particularities of the situation. In general, increased specificity can be effected by increasing the length of the probe or primer.
  • said probe or primer comprises a sequence of nucleotides of at least 10, 20, 30, 40 or 50 nucleotides, although the use of larger molecules are also contemplated, derived from or directed to the nucleotide sequences hereinbefore defined.
  • This sequence may be labelled with a reporter molecule capable of giving an identifiable signal.
  • the nucleic acid molecule of the present invention is preferably in isolated form or ligated to a vector, such as an expression vector.
  • isolated is meant a nucleic acid molecule having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10% subject nucleic acid molecule, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%, even still more preferably at least about 60-70%, yet even still more preferably 80-90% or greater of subject nucleic acid molecule relative to other components as determined by molecular weight, sequence or other convenient means.
  • the nucleic acid molecule of the present invention may also be considered, in a preferred embodiment, to be biologically pure.
  • the nucleic acids of the invention can be made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of mRNA or genomic DNA by PCR, and the like.
  • RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed or generated recombinantly. Recombinant proteins generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
  • these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661 ; Belousov et al. (1997) Nucleic Acids Res. 25:3440-3444; Frenkel et al. (1995) Free Radical Biology & Medicine 19(3):373-80; Blommers et al. (1994) Biochemistry 33:7886-96; Narang et al. (1979) Meth. Enzymol. 68:90; Brown et al. (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Patent No. 4,458,066.
  • nucleic acids such as, e.g., subcloning, labelling probes (e.g., random-primer labelling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), VOIS. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed.
  • Nucleic acids, vectors, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g.
  • the nucleic acids of the invention can be operably linked to a promoter.
  • a promoter can be one motif or an array of nucleic acid control sequences which direct transcription of a nucleic acid.
  • a promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
  • a "constitutive" promoter is a promoter which is active under most environmental and developmental conditions.
  • An “inducible” promoter is a promoter which is under environmental or developmental regulation.
  • tissue specific promoter is active in certain tissue types of an organism, but not in other tissue types from the same organism.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • the invention provides expression vectors and cloning vehicles comprising nucleic acids of the invention, e.g., sequences encoding the proteins of the invention.
  • Expression vectors and cloning vehicles of the invention can comprise viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), Pl -based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, Aspergillus and yeast).
  • Vectors of the invention can include chromosomal, non-chromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the aft, and are commercially available.
  • nucleic acids of the invention can be cloned, if desired, into any of a variety of vectors using routine molecular biological methods. Methods for cloning in vitro amplified nucleic acids are described, e.g., U.S. Pat. No. 5,426,039. To facilitate cloning of amplified sequences, restriction enzyme sites can be "built into” a PCR primer pair.
  • the invention provides libraries of expression vectors encoding polypeptides and peptides of the invention. These nucleic acids may be introduced into a genome or into the cytoplasm or a nucleus of a cell and expressed by a variety of conventional techniques, well described in the scientific and patent literature. See, e.g., Roberts et al. (1987) Nature 328:731; Schneider (1995) Protein Expr. Purif. 6435: 10; Sambrook, Tijssen or Ausubel.
  • the vectors can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic or recombinant methods.
  • the nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses which are stably or transiently expressed in cells (e.g., episomal expression systems).
  • Selection markers can be incorporated into expression cassettes and vectors to confer a selectable phenotype on transformed cells and sequences.
  • selection markers can code for episomal maintenance and replication such that integration into the host genome is not required.
  • the nucleic acid molecule may be ligated to an expression vector capable of expression in a prokaryotic cell (eg. E. col ⁇ ) or a eukaryotic cell (eg. yeast cells, fungal cells, insect cells, mammalian cells or plant cells).
  • the nucleic acid molecule may be ligated or fused or otherwise associated with a nucleic acid molecule encoding another entity such as, for example, a signal peptide. It may also comprise additional nucleotide sequence information fused, linked or otherwise associated with it either at the 3' or 5' terminal portions or at both the 3' and 5' terminal portions.
  • the nucleic acid molecule may also be part of a vector, such as an expression vector.
  • nucleic acids may be useful for recombinant production of protein by insertion into an appropriate vector and transfection into a suitable cell line.
  • expression vectors and host cell lines also form an aspect of the invention.
  • host cells transformed with a nucleic acid of the invention are cultured in a medium suitable for the particular cells concerned. Proteins can then be purified from cell culture medium, the host cells or both using techniques well known in the art such as ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis or immunopurification with antibodies specific for the peptide.
  • Nucleic acids may be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells such as Chinese hamster ovary cells (CHO). Suitable expression vectors, promoters, enhancers and other expression control elements are referred to in Sambruck et al (Cold Spring Harbour Laboratory Press, 1989). Other suitable expression vectors, promoters, enhancers and other expression elements are well known to those skilled in the art. Examples of suitable expression vectors in yeast include Yep Sec 1 (Baldari et al, 1987, Embo.
  • baculovirus vectors are freely available as are baculovirus and mammalian expression systems.
  • a baculovirus system is commercially available (ParMingen, San Diego, CA) for expression in insect cells while the pMsg vector is commercially available (Pharmacia, Piscataway, NJ) for expression in mammalian cells.
  • Host cells can be transformed to express the nucleic acids of the invention using conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection or electroporation. Suitable methods for transforming the host cells may be found in (Sambruck et al., 1989), and other laboratory texts.
  • the nucleic acid sequence of the invention may also be chemically synthesised using standard techniques.
  • expression cassette refers to a nucleotide sequence which is capable of effecting expression of a structural gene (i.e., a protein coding sequence, such as a polypeptide of the invention) in a host compatible with such sequences.
  • Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, e.g., enhancers. "Operably linked” as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence.
  • expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like.
  • a "vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid.
  • the vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.).
  • Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated.
  • Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Patent No. 5,217,879), and includes both the expression and non-expression plasmids.
  • RNA autonomous self-replicating circular or linear DNA or RNA
  • plasmids viruses, and the like, see, e.g., U.S. Patent No. 5,217,879
  • plasmids plasmids, viruses, and the like, see, e.g., U.S. Patent No. 5,217,879
  • a recombinant microorganism or cell culture is described as hosting an "expression vector” this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s).
  • the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is
  • the nucleic acid molecule may be placed operably under the control of a promoter sequence such as those discussed earlier. Suitable cells and virus particles for this purpose are also discussed earlier. Promoter sequences and culture conditions for cells or virus particles which produce high levels of expression will be well-known to those skilled in the relevant art.
  • the present invention also provides a transformed cell comprising a nucleic acid sequence of the invention, e.g., a sequence encoding the protein of the invention, or a vector of the invention.
  • the host cell may be any of the host cells familiar to those skilled in the art, including prokaryotic cells, such as bacterial cells, eukaryotic cells, such as fungal cells, yeast cells, mammalian cells, insect cells, or plant cells.
  • Exemplary bacterial cells include E. coli, Streptomyces, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • Exemplary insect cells include Drosophila S2 and Spodoptera Sf9.
  • Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line. The selection of an appropriate host is within the abilities of those skilled in the art.
  • the vector may be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti- mediated gene transfer. Particular methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, or electroporation.
  • Engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the invention. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter may be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells may be cultured for an additional period to allow them to produce the desired protein or fragment thereof.
  • appropriate means e.g., temperature shift or chemical induction
  • a further aspect of the invention provides an isolated cell which expresses an endogenous or recombinant protein or a functional fragment or derivative thereof of the present invention.
  • similarity and identity as used herein include exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, “similarity” and include “identity” differences between sequences which may encode different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide sequence comparisons are made at the level of identity rather than similarity.
  • sequence relationships between two or more polynucleotides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units in length. Because two polynucleotides may each comprise (1) a sequence (i.e.
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence.
  • the comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • sequence similarity refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide- by-nucleotide basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g.
  • sequence identity will be understood to mean the "match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
  • sequence comparison algorithms may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art.
  • the extent of sequence identity may be determined using any computer program and associated parameters, including those described herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with the default parameters.
  • sequence comparison algorithm is a BLAST version algorithm.
  • Exemplary algorithms and programs include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. ScL USA 85(8):2444-2448, 1988; Altschul et al., J. MoI. Biol. 215(3):403-410, 1990; Thompson et aU Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al, Methods Enzymol. 266:383- 402, 1996; Altschul et al, Nature Genetics 3:266-272, 1993).
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705.
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705.
  • BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practice the invention. They are described, e.g., in ; Altschul et al. (1990), supra. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al. (1990) supra). These initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • HSPs high scoring sequence pairs
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873).
  • One measure of similarity provided by BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a references sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST").
  • BLAST Basic Local Alignment Search Tool
  • five specific BLAST programs can be used to perform the following task: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and, (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • the BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.
  • High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art.
  • the scoring matrix used is the BLOSUM62 matrix (Gonnet et al, Science 256: 1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61, 1993).
  • the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).
  • the NCBI BLAST 2.2.2 programs is used, default options to blast. There are about 38 setting options in the BLAST 2.2.2 program.
  • all default values are used except for the default filtering setting (i.e., all parameters set to default except filtering which is set to OFF); in its place a "-F F" setting is used, which disables filtering.
  • Use of default filtering often results in Karlin-Altschul violations due to short length of sequence.
  • sequences or subsequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection.
  • sequence comparison one sequence can act as a reference sequence to which test sequences are compared.
  • sequence comparison algorithm test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a "comparison window" includes reference to a segment of any one of the numbers of contiguous residues. For example, in alternative aspects of the invention, contiguous residues ranging anywhere from 20 to the full length of an exemplary polypeptide or nucleic acid sequence of the invention are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. If the reference sequence has the requisite sequence identity to an exemplary polypeptide or nucleic acid sequence of the invention, that sequence is within the scope of the invention.
  • nucleic acid sequences of the invention can be substantially identical over the entire length of a polypeptide coding region.
  • the sequence of the invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. Accordingly, the invention provides computers, computer systems, computer readable mediums, computer programs products and the like recorded or stored thereon the nucleic acid and polypeptide sequences of the invention.
  • the words "recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid and/or polypeptide sequences of the invention.
  • Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media.
  • the computer readable media may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.
  • the invention provides isolated or recombinant nucleic acids that hybridize under low stringency conditions to an exemplary sequence of the invention.
  • the stringent conditions are highly stringent conditions or medium stringent conditions, as known in the art and as described herein. These methods may be used to isolate nucleic acids of the invention.
  • Hybridization refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing. Hybridization reactions can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations. Stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. For example, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature, altering the time of hybridization, as described in detail, below. In alternative aspects, nucleic acids of the invention are defined by their ability to hybridize under various stringency conditions (e.g., high, medium, and low), as set forth herein.
  • stringency conditions e.g., high, medium, and low
  • Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions.
  • low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions.
  • washing is carried out T m - 69.3 + 0.41 (G+C)% (Marmur and Doty, J. MoI.
  • T m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Eur. J. Biochem. 46: 83, 1974).
  • Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1 % w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.
  • nucleic acids of the invention are defined by their ability to hybridize under high stringency, these conditions comprise about 50% formamide at about 37°C to 42°C.
  • nucleic acids of the invention are defined by their ability to hybridize under reduced stringency comprising conditions in about 35% to 25% formamide at about 30 0 C to 35°C.
  • nucleic acids of the invention are defined by their ability to hybridize under high stringency comprising conditions at 42 0 C in 50% formamide, 5X SSPE, 0.3% SDS, and a repetitive sequence blocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/ml sheared and denatured salmon sperm DNA).
  • nucleic acids of the invention are defined by their ability to hybridize under reduced stringency conditions comprising 35% formamide at a reduced temperature of 35 0 C.
  • the filter may be washed with 6X SSC, 0.5% SDS at 5O 0 C. These conditions are considered to be “moderate” conditions above 25% formamide and “low” conditions below 25% formamide.
  • 6X SSC 0.5% SDS at 5O 0 C.
  • Moderate Hybridization
  • low stringency Hybridization
  • the temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly.
  • Nucleic acids of the invention are also defined by their ability to hybridize under high, medium, and low stringency conditions as set forth in Ausubel and Sambrook. Variations on the above ranges and conditions are well known in the art.
  • the above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence.
  • less stringent conditions may be used.
  • the hybridization temperature may be decreased in increments of 5°C from 68°C to 42°C in a hybridization buffer having a Na+ concentration of approximately IM.
  • the filter may be washed with 2X SSC, 0.5% SDS at the temperature of hybridization.
  • These conditions are considered to be “moderate” conditions above 50 0 C and "low” conditions below 50°C.
  • a specific example of “moderate” hybridization conditions is when the above hybridization is conducted at 55 0 C.
  • a specific example of "low stringency" hybridization conditions is when the above hybridization is conducted at 45°C.
  • the hybridization may be carried out in buffers, such as 6X SSC, containing formamide at a temperature of 42°C.
  • concentration of formamide in the hybridization buffer may be reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe.
  • the filter may be washed with 6X SSC, 0.5% SDS at 50 0 C.
  • 6X SSC 0.5% SDS at 50 0 C.
  • wash conditions used to identify nucleic acids within the scope of the invention include, e.g.: a salt concentration of about 0.02 molar at pH 7 and a temperature of at least about 50°C or about 55°C to about 60°C; or, a salt concentration of about 0.15 M NaCl at 72°C for about 15 minutes; or, a salt concentration of about 0.2X SSC at a temperature of at least about 50°C or about 55°C to about 6O 0 C for about 15 to about 20 minutes; or, the hybridization complex is washed twice with a solution with a salt concentration of about 2X SSC containing 0.1% SDS at room temperature for 15 minutes and then washed twice by 0.1 X SSC containing 0.1% SDS at 68 0 C for 15 minutes; or, equivalent conditions. See Sambrook, Tijssen and Ausubel
  • derivative includes portions, fragments and parts of the nucleic acid molecule or a translation product thereof.
  • a derivative may also comprise a single or multiple nucleotide or amino acid substitution, deletion and/or addition but which substitution, deletion and/or addition does not alter the functional activity of said protein.
  • reference to a "functional" protein or nucleic acid should be understood as a reference to a protein or nucleic acid which exhibits the same functional activity as the molecule of which it is a derivative.
  • a derivative of the nucleic acid molecule of the present invention also includes nucleic acid molecules capable of hybridising to the nucleotide sequence set forth hereinbefore under at least low stringency conditions.
  • the derivatives of the nucleic acid molecule of the present invention defined herein also includes oligonucleotides, PCR primers, probes, antisense molecules, molecules suitable for use in co- suppression and fusion nucleic acid molecules. These molecules are particularly useful in relation to the diagnostic utility of SlOOAl 1, such as in the context of enabling the generation of microarrays or otherwise facilitating the probing or amplification of a nucleic acid population to identify a SlOOAl 1 transcript or fragment thereof. Reference to “fragments” should therefore be understood to include reference to parts and portions, from natural, synthetic or recombinant sources.
  • said derivatives or fragments are functional derivatives or fragments.
  • Yet another aspect of the present invention provides a method for identifying a SlOOAl 1 nucleic acid molecule as hereinbefore defined or a fragment or derivative thereof.
  • said method comprises contacting genomic DNA, mRNA or cDNA with a hybridisation effective amount of a SlOOAl 1 probe and then detecting said hybridisation.
  • An alternative embodiment of the present invention provides a method for identifying a SlOOAl 1 genetic sequence or a fragment or derivative thereof comprising contacting two non-complementary nucleic acid "primer molecules" of at least 12 nucleotides in length derived from the nucleotide sequence hereinbefore described with a nucleic acid template molecule comprising nucleotide sequences related to the primer molecule sequences and amplifying specific nucleic acid molecule copies of the template molecule in an amplification reaction.
  • the nucleic acid primer molecule may consist of a combination of any of the nucleotides adenine, cytosine, guanine, thymidine, or inosine, or functional analogues or derivatives thereof, capable of being incorporated into a polynucleotide molecule provided that it is capable of hybridising under at least low stringency conditions to the nucleic acid molecule set forth in the sequences hereinbefore defined.
  • the nucleic acid primer molecules may further be each contained in an aqueous pool comprising other nucleic acid primer molecules. More preferably, the nucleic acid primer molecule is in a substantially pure form. Still another aspect of the present invention is directed to antibodies to the subject protein or nucleic acid molecules including catalytic antibodies or derivatives, homologues, or mutants, of said antibodies. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies or may be specifically raised. In the case of the latter, the molecule to which the antibody is to be raised may first need to be associated with a carrier molecule.
  • Both polyclonal and monoclonal antibodies are obtainable by immunization with the molecules of the present invention.
  • the methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of the antigen of interest, or antigenic part thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques.
  • antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
  • the use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology VoI II, ed. by Schwartz, 1981 ; Kohler and Milstein, Nature 256: 495-499, 1975; European Journal of Immunology 6: 51 1-519, 1976).
  • the antibody of the present invention specifically binds the molecule to which it was raised.
  • “specifically binds” is meant high avidity and/or high affinity binding of an antibody to a specific antigen.
  • Antibody binding to its epitope on this specific antigen is stronger than binding of the same antibody to any other epitope, particularly those that may be present in molecules in association with, or in the same sample, as the specific antigen of interest.
  • Antibodies that bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level. Such weak binding, or background binding, is readily discernible from the specific antibody binding to the polypeptide of interest, e.g. by use of appropriate controls.
  • An antibody as hereinbefore defined is also useful in purifying or screening for the molecules of the present invention.
  • Methods for the affinity purification of proteins using antibodies are well-known to those skilled in the art.
  • kits comprising any one or more of the nucleic acid protein or antibody molecules of the present invention together with a detection means.
  • Probeset 200660_at contains oligonucleotides complementary to E2, E3 and E4 (see Figure 3 for further details).
  • the clinical specimens included 68 colorectal tissues: 19 adenocarcinoma tissues, 19 adenoma tissues and 30 non-diseased controls, Figure 4.
  • Measuring nucleotide transcripts derived from the human Reference Sequence gene SlOOAIl has diagnostic utility for identification of colorectal neoplasia.
  • Oligonucleotide sequence primer sets were generated to each of the predicted 3 RNA variants (Table 1) from the map region 150271608-150292007 on the minus strand of human chromosome 1 ( Figure 2) and end-point PCR using these primer sets was performed to measure the existence of the three [3] RNA transcript variants in a total of 18 colorectal tissue specimens from 6 non-disease, 6 adenoma and 6 adenocarcinoma subjects.
  • the differential expression of the 3 predicted RNA transcripts is shown in Figure 5. Differential expression as measured by end-point PCR was observed for 2 of the 3 RNA variants (Table 1) e.g. SEQUENCE "SlOOAl 1_1" and SEQUENCE “SlOOAl 1_2" where in particular SEQUENCE "SlOOAl 1_1" demonstrated strong phenotype discrimination.
  • Quantitative real-time PCR using the oligonucleotide sequences, 5'- ATCGAGTCCCT GATTGCTGT (Primer 6, Table 2) and 5'-CCATCACTGTTGGTGTCCAG (Primer 7, Table 2), was performed to measure the RNA concentration level of SEQ ID NO:1 from map region 150272713 to 150271861 in a total of 71 colorectal tissue specimens: 30 non- diseased controls, 21 adenoma tissues and 20 adenocarcinoma tissues, Figure 6.
  • SEQ ID NO:1 which contains map region 150272713 to 150271861, has diagnostic utility as a means of detecting colorectal neoplasia.
  • the expression level from the minus strand of map region 150286922-150287007 on chromosome 1 was measured by determining the hybridization of RNA extracted from clinical specimens to Affymetrix Probe Set ID 2435420, which belongs to the Transcript Cluster ID 2435410 on the Affymetrix GeneChip "Human Exon 1.0 ST" array.
  • the probeset 2435420 anneals to map region 150286944-150286968 on chromosome 1, which is part of SEQUENCE “El” (see Figure 3 and Figure 1 for further details).
  • the clinical specimens included 15 colorectal tissues: 5 adenocarcinoma tissues, 5 adenoma tissues and 5 non-diseased controls, Figure 7.

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Abstract

La présente invention a globalement pour objet une molécule d'acide nucléique, dont les profils d'expression de l'ARN et des protéines sont indicatifs de la survenue, de la prédisposition à la survenue et/ou de la progression d'un gros néoplasme intestinal. Plus particulièrement, la présente invention concerne des variantes de transcription de S100A11, dont les profils d'expression sont indicatifs de la survenue et/ou de la progression d'un néoplasme colorectal, tel qu'un adénome ou un adénocarcinome. Les profils d'expression selon la présente invention sont utiles dans une gamme d'applications comprenant, sans y être limitées, celles concernant le diagnostic et/ou la surveillance des néoplasmes colorectaux, tels que les adénomes et les adénocarcinomes colorectaux.
PCT/AU2010/000660 2009-05-29 2010-05-27 Procédé permettant de diagnostiquer des néoplasmes et molécules destinées à être utilisées dans ce procédé WO2010135786A1 (fr)

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US11254985B2 (en) 2012-05-11 2022-02-22 Clinical Genomics Pty. Ltd. Diagnostic gene marker panel for colorectal cancer

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