US20110136683A1 - Systems and Methods for Expression-Based Discrimination of Distinct Clinical Disease States in Prostate Cancer - Google Patents
Systems and Methods for Expression-Based Discrimination of Distinct Clinical Disease States in Prostate Cancer Download PDFInfo
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- US20110136683A1 US20110136683A1 US12/994,408 US99440809A US2011136683A1 US 20110136683 A1 US20110136683 A1 US 20110136683A1 US 99440809 A US99440809 A US 99440809A US 2011136683 A1 US2011136683 A1 US 2011136683A1
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- 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
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- C12Q2600/112—Disease subtyping, staging or classification
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Definitions
- This invention relates to the field of diagnostics and in particular to systems and methods for classifying prostate cancer into distinct clinical disease states.
- Prostate cancer is the most common malignancy affecting U.S. men, with approximately 240,000 new cases diagnosed each year.
- the incidence of prostate cancer is increasing, in part due to increased surveillance efforts from the application of routine molecular testing such as prostate-specific antigen (PSA).
- PSA prostate-specific antigen
- prostate cancer is a slow-growing, organ-confined or localized malignancy that poses little risk of death.
- the most common treatments for prostate cancer in the U.S. are surgical procedures such as radical prostatectomy, where the entire prostate is removed from the patient. This procedure on its own is highly curative for most but not all men.
- RNA-based e.g., gene or non-coding RNA expression
- protein-based e.g., protein expression or modification
- FFPE-derived RNA is typically degraded and fragmented to between 100-300 bp in size and without poly-A tails making it of little use for traditional 3′-biased gene expression profiling, which requires larger microgram quantities of RNA with intact poly-A tails to prime cDNA synthesis.
- An object of the present invention is to provide systems and methods for expression-based discrimination of distinct clinical disease states in prostate cancer.
- a system for expression-based assessment of risk of prostate cancer recurrence after prostatectomy comprising one or more polynucleotides, each of said polynucleotides capable of specifically hybridizing to a RNA transcript of a gene selected from the group of genes set forth in Table 3 and/or 6.
- a nucleic acid array for expression-based assessment of prostate cancer recurrence risk comprising at least ten probes immobilized on a solid support, each of said probes being between about 15 and about 500 nucleotides in length, each of said probes being derived from a sequence corresponding to, or complementary to, a transcript of a gene selected from the group of genes set forth in Table 3 and/or 6, or a portion of said transcript.
- a method for expression-based assessment of prostate cancer recurrence comprising: (a) determining the expression level of one or more transcripts of one or more genes in a test sample obtained from said subject to provide an expression pattern profile, said one or more genes selected from the group of genes set forth in Table 3 and/or 6, and (c) comparing said expression pattern profile with a reference expression pattern profile.
- kits for characterizing the expression of one or more nucleic acid sequences depicted in SEQ ID NOs: 1-2114 comprising one or more nucleic acids selected from (a) a nucleic acid depicted in any of SEQ ID NOs: 1-2114; (b) an RNA form of any of the nucleic acids depicted in SEQ ID NOs: 1-2114; (c) a peptide nucleic acid form of any of the nucleic acids depicted in SEQ ID NOs: 1-2114; (d) a nucleic acid comprising at least 20 consecutive bases of any of (a-c); (e) a nucleic acid comprising at least 25 consecutive bases having at least 90% sequence identity to any of (a-c); or (f) a complement to any of (a-e); and optionally instructions for correlating the expression level of said one or more nucleic acid sequences with the disease state of prostate cancer tissue.
- an array of probe nucleic acids certified for use in expression-based assessment of prostate cancer recurrence risk comprising at least two different probe nucleic acids that specifically hybridize to corresponding different target nucleic acids depicted in one of SEQ ID NOs: 1-2114, an RNA form thereof, or a complement to either thereof.
- a device for classifying a biological sample from a prostate cancer as recurrent or non-recurrent comprising means for measuring the expression level of one or more transcripts of one or more genes selected from the group of genes set forth in Table 3 and/or 6; means for correlating the expression level with a classification of prostate cancer status; and means for outputting the prostate cancer status.
- a computer-readable medium comprising one or more digitally-encoded expression pattern profiles representative of the level of expression of one or more transcripts of one or more genes selected from the group of genes set forth in Table 3 and/or 6, each of said one or more expression pattern profiles being associated with a value wherein each of said values is correlated with the presence of recurrent or non-recurrent prostate cancer.
- FIG. 1 A) Principle components analysis (PCA) of 2,114 RNAs identified to be differentially expressed between tumors from patients with differing clinical outcome (see Table 2 for comparisons evaluated), PCA plot of 22 prostate cancer tumors shows tight clustering of samples by clinical outcome of patients (circles, NED; diamonds, PSA; squares, SYS).
- PCA Principle components analysis
- Table 4 Two-way hierarchical clustering dendrogram and expression matrix of 526 target sequences (Table 4) RNAs filtered using linear regression (p ⁇ 0.01) to identify RNAs that followed either SYS>PSA>NED or NED>PSA>SYS trend in differential expression.
- FIG. 2 Histograms showing distribution patient's tumor expression levels of a ‘metagene’ generated from a linear combination of the 526 RNAs for each clinical group. The histograms bin samples with similar metagene expression values and significantly separate three modes of patient metagene scores (ANOVA, p ⁇ 0.000001) corresponding to the three clinical status groups evaluated.
- FIG. 3 Scatter plots summarizing the mean ( ⁇ standard deviation) of metagene expression values for tumor samples from patients in the three clinical status groups (NED; PSA; SYS). Metagenes were generated from a linear combinations of 6 ( ⁇ ), 18 ( ⁇ ) or 20 ( ⁇ ) RNAs and demonstrate highly significant differential expression between clinical groups (ANOVA, p ⁇ 0.000001).
- FIG. 4 Box plots showing interquartile range and distribution of ‘POP’ scores for each clinical group using an 18-target sequence metagene (Table 7) to derive patient outcome predictor scores scaled and normalized on a data range of 0-100 points. T-tests were used to evaluate the statistical significance of differences in POP scores between NED and PSA (*) as well as between PSA and SYS (**) clinical groups (p ⁇ 7 ⁇ 10 ⁇ 7 and p ⁇ 1 ⁇ 10 ⁇ 6 , respectively).
- FIG. 5 Box plots showing interquartile range and distribution of ‘POP’ scores for each clinical group using a 10-target sequence metagene (Table 9) to derive patient outcome predictor scores scaled and normalized on a data range of 0-100 points. T-tests were used to evaluate the statistical significance of differences in POP scores between ‘recurrent’ (i.e., ‘SYS’) and non-recurrent (i.e., ‘PSA’ and ‘NED’) patient groups (**, p ⁇ 4 ⁇ 10 ⁇ 10 ).
- FIG. 6 Box plots showing interquartile range and distribution of ‘POP’ scores for each clinical group using a 41-target sequence metagene (Table 10) to derive patient outcome predictor scores scaled and normalized on a data range of 0-100 points. T-tests were used to evaluate the statistical significance of differences in POP scores between ‘recurrent’ (i.e., ‘SYS’) and non-recurrent (i.e., ‘PSA’ and ‘NED’) patient groups (**, p ⁇ 2 ⁇ 10 ⁇ 11 ).
- FIG. 7 Box plots showing interquartile range and distribution of ‘POP’ scores for each clinical group using a 148-target sequence metagene to derive patient outcome predictor scores scaled and normalized on a data range of 0-100 points. T-tests were used to evaluate the statistical significance of differences in POP scores between ‘recurrent’ (i.e., ‘SYS’) and non-recurrent (i.e., ‘PSA’ and ‘NED’) patient groups (**, p ⁇ 9 ⁇ 10 ⁇ 12 ).
- the present invention provides a system and method for assessing prostate cancer recurrence risk by distinguishing clinically distinct disease states in men with prostate cancer at the time of initial diagnosis or surgery.
- the system and methods are based on the identification of gene transcripts following a retrospective analysis of tumor samples that are differentially expressed in prostate cancer in a manner dependent on prostate cancer aggressiveness as indicated by long-term post-prostatectomy clinical outcome.
- These gene transcripts can be considered as a library which can be used as a resource for the identification of sets of specific target sequences (“prostate cancer prognostic sets”), which may represent the entire library of gene transcripts or a subset of the library and the detection of which is indicative of prostate cancer recurrence risk.
- the invention further provides for probes capable of detecting these target sequences and primers that are capable of amplifying the target sequences.
- the system and method for assessing prostate cancer recurrence risk are prognostic for a post surgery clinical outcome selected from no evidence of disease (‘NED’), biochemical relapse (two successive increases in prostate-specific antigen levels; (‘PSA’) and systemic prostate cancer systemic metastases (‘SYS’).
- NED no evidence of disease
- PSA prostate-specific antigen levels
- SYS systemic prostate cancer systemic metastases
- the target sequences comprised by the prostate cancer prognostic set are sequences based on or derived from the gene transcripts from the library, or a subset thereof. Such sequences are occasionally referred to herein as “probe selection regions” or “PSRs.”
- the target sequences comprised by the prostate classification set are sequences based on the gene transcripts from the library, or a subset thereof, and include both coding and non-coding sequences.
- the systems and methods provide for the molecular analysis of the expression levels of one or more of the target sequences as set forth in SEQ ID NOs: 1-2114 (Table 4). Increased relative expression of one or more target sequences in a ‘NED’ Group corresponding to the sequences as set forth in SEQ ID NOs: 1-913 is indicative of or predictive of a non-recurrent form of prostate cancer and can be correlated with an increased likelihood of a long-term NED prognosis or low risk of prostate cancer recurrence.
- Increased relative expression of one or more target sequences in a ‘SYS’ Group corresponding to the sequences as set forth in SEQ ID NOs: 914-2114 is indicative of or predictive of an aggressive form of prostate cancer and can be correlated with an increased likelihood of a long-term SYS prognosis or high risk of prostate cancer recurrence.
- intermediate relative levels of one or more target sequences in a ‘PSA’ Group corresponding to target sequences set forth in Table 7 is indicative of or predictive of biochemical recurrence. Subsets and combinations of these target sequences or probes complementary thereto may be used as described herein.
- polynucleotide refers to a polymer of greater than one nucleotide in length of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), hybrid RNA/DNA, modified RNA or DNA, or RNA or DNA mimetics, including peptide nucleic acids (PNAs).
- the polynucleotides may be single- or double-stranded.
- the term includes polynucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as polynucleotides having non-naturally-occurring portions which function similarly.
- backbone backbone linkages
- Such modified or substituted polynucleotides are well-known in the art and for the purposes of the present invention, are referred to as “analogues.”
- Complementary or “substantially complementary” refers to the ability to hybridize or base pair between nucleotides or nucleic acids, such as, for instance, between a sensor peptide nucleic acid or polynucleotide and a target polynucleotide.
- Complementary nucleotides are, generally, A and T (or A and U), or C and G.
- Two single-stranded polynucleotides or PNAs are said to be substantially complementary when the bases of one strand, optimally aligned and compared and with appropriate insertions or deletions, pair with at least about 80% of the bases of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%.
- substantial complementarity exists when a polynucleotide will hybridize under selective hybridization conditions to its complement.
- selective hybridization will occur when there is at least about 65% complementarity over a stretch of at least 14 to 25 bases, for example at least about 75%, or at least about 90% complementarity. See, M. Kanehisa Nucleic Acids Res. 12:203 (1984).
- Preferential binding or “preferential hybridization” refers to the increased propensity of one polynucleotide to bind to its complement in a sample as compared to a noncomplementary polymer in the sample.
- Hybridization conditions will typically include salt concentrations of less than about 1M, more usually less than about 500 mM, for example less than about 200 mM.
- the hybridization can be done in solutions containing little or no salt.
- Hybridization temperatures can be as low as 5° C., but are typically greater than 22° C., and more typically greater than about 30° C., for example in excess of about 37° C. Longer fragments may require higher hybridization temperatures for specific hybridization as is known in the art.
- hybridization conditions which may be controlled include buffer type and concentration, solution pH, presence and concentration of blocking reagents to decrease background binding such as repeat sequences or blocking protein solutions, detergent type(s) and concentrations, molecules such as polymers which increase the relative concentration of the polynucleotides, metal ion(s) and their concentration(s), chelator(s) and their concentrations, and other conditions known in the art.
- Multiplexing herein refers to an assay or other analytical method in which multiple analytes can be assayed simultaneously.
- a “target sequence” as used herein refers to a region of the genome against which one or more probes can be designed.
- a probe is any polynucleotide capable of selectively hybridizing to a target sequence or its complement, or to an RNA version of either.
- a probe may comprise ribonucleotides, deoxyribonucleotides, peptide nucleic acids, and combinations thereof.
- a probe may optionally comprise one or more labels.
- a probe may be used to amplify one or both strands of a target sequence or an RNA form thereof, acting as a sole primer in an amplification reaction or as a member of a set of primers.
- NED describes a clinically distinct disease state in which patients show no evidence of disease (‘NED’) at least 5 years after surgery
- PSA describes a clinically distinct disease state in which patients show biochemical relapse only (two successive increases in prostate-specific antigen levels but no other symptoms of disease with at least 5 years follow up after surgery; ‘PSA’) and ‘SYS’ describes a clinically distinct disease state in which patients develop biochemical relapse and present with systemic prostate cancer disease or metastases (‘SYS’) within five years after the initial treatment with radical prostatectomy.
- the term “about” refers to approximately a +/ ⁇ 10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
- the system of the present invention is based on the identification of a library of gene and RNA transcripts that are differentially expressed in prostate cancer in a manner dependent on prostate cancer aggressiveness as indicated by the post-prostatectomy clinical outcome of the patient.
- relative over expression of one or more of the gene transcripts in a prostate cancer sample compared to a reference sample or expression profile or signature there from may be prognostic of a clinically distinct disease outcome post-prostatectomy selected from no evidence of disease (‘NED’), biochemical relapse (‘PSA’) and prostate cancer disease systemic recurrence or metastases (‘SYS’).
- the reference sample can be, for example, from prostate cancer sample(s) of one or more references subject(s) with a known post-prostatectomy clinical outcomes.
- the reference expression profile or signature may optionally be normalized to one or more appropriate reference gene transcripts.
- expression of one or more of the gene transcripts in a prostate cancer sample may be compared to an expression profile or signature from normal prostate tissue.
- Expression profiles or signatures from prostate cancer samples may be normalized to one or more house keeping gene transcripts such that normalized over and/or under expression of one or more of the gene transcripts in a sample may be indicative of a clinically distinct disease state or prognosis.
- the Prostate Prognostic Library in accordance with the present invention comprises one or more gene or RNA transcripts whose relative and/or normalized expression is indicative of prostate cancer recurrence and which may be prognostic for post-prostatectomy clinical outcome of a patient.
- RNA transcripts that showed differential expression in prostate cancer samples from patients with clinically distinct disease outcomes after initial treatment with radical prostatectomy are shown in Table 3.
- the library comprises one or more of the gene transcripts of the genes listed in Table 3.
- the library comprises at least one transcript from at least one gene selected from those listed in Table 3. In one embodiment, the library comprises at least one transcript from each of at least 5 genes selected from those listed in Table 3. In another embodiment, the library comprises at least one transcript from each of at least 10 genes selected from those listed in Table 3. In a further embodiment, the library comprises at least one transcript from each of at least 15 genes selected from those listed in Table 1. In other embodiments, the library comprises at least one transcript from each of at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 and at least 65 genes selected from those listed in Table 3. In a further embodiment, the library comprises at least one transcript from all of the genes listed in Table 3. In a further embodiment, the library comprises at all transcripts from all of the genes listed in Table 3.
- the library comprises at least one transcript from at least one gene selected from the group consisting of [NM — 001004722]; [NM — 001005522]; [NM — 001013671]; [NM — 001033517]; [NM — 183049]; [NM — 212559]; 5′-3′ exoribonuclease 1; A kinase (PRKA) anchor protein (yotiao) 9; AarF domain containing kinase 4; Abhydrolase domain containing 3; Aconitase 1, soluble; Actinin, alpha 1; ADAM metallopeptidase domain 19 (meltrin beta); Adaptor-related protein complex 1, gamma 2 subunit; Adenosine deaminase, RNA-specific, B2 (RED2 homolog rat); Adenylate cyclase 3; ADP-ribosylation factor GTPase activating protein 3; ADP-rib
- Anthrax toxin receptor 1 Anthrax toxin receptor 1; Antizyme inhibitor 1; Arachidonate 12-lipoxygenase, 12R type; Arginine vasopressin receptor 1A; Arginine-glutamic acid dipeptide (RE) repeats; ARP3 actin-related protein 3 homolog (yeast); Arrestin 3, retinal (X-arrestin); Arrestin domain containing 1; Aryl hydrocarbon receptor interacting protein-like 1; Aryl hydrocarbon receptor nuclear translocator; Ataxin 1; ATM/ATR-Substrate Chk2-Interacting Zn2+-finger protein; ATPase, Class I, type 8B, member 1; ATPase, Na+/K+ transporting, alpha 1 polypeptide; ATP-binding cassette, sub-family F (GCN20), member 1; Autism susceptibility candidate 2; Baculoviral IAP repeat-containing 6 (apollon); Basonuclin 2; Brain-specific angiogenesis inhibitor 3; Bromodomain
- the library comprises at least one transcript from at least one gene selected from the group consisting of Replication factor C (activator 1) 3; Tripartite motif-containing 61; Citrate lyase beta like; Ankyrin repeat domain 15; UDP-glucose ceramide glucosyltransferase-like 2; Hypothetical protein FLJ12949; Chromosome 22 open reading frame 13; Phosphatidylinositol glycan anchor biosynthesis, class O; Solute carrier family 43, member 1; Rabaptin, RAB GTPase binding effector protein 1; Zinc finger protein 14 homolog; Hypothetical gene supported by AK128346; Adenylate cyclase 3; Phosphatidylinositol transfer protein, beta; Zinc finger protein 667; Gremlin 1, cysteine knot superfamily, homolog; Ankyrin 3, node of Ranvier (ankyrin G) and Maltase-glucoamylase (alpha-
- the library comprises at least one transcript from at least one gene selected from the group consisting of Replication factor C (activator 1) 3; Ankyrin repeat domain 15; Hypothetical protein FLJ12949; Solute carrier family 43, member 1; Thioredoxin-like 2; Polymerase (RNA) II (DNA directed) polypeptide L; Syntaxin 5; Leucine rich repeat containing 16; Calcium channel, voltage-dependent, beta 4 subunit; [NM — 001005522]; G protein-coupled receptor kinase interactor 2; Ankyrin 3, node of Ranvier (ankyrin G); Gremlin 1, cysteine knot superfamily, homolog; Zinc finger protein 667; Hypothetical gene supported by AK128346; Transmembrane 9 superfamily member 2; Potassium channel, subfamily K, member 1; Chromodomain helicase DNA binding protein 2; Microcephaly, primary autosomal recessive 1; Chromosome 21 open reading frame 34 and Dual specificity phosphatase
- the library comprises at least one transcript from at least one gene selected from the group consisting of Replication factor C (activator 1) 3; Tripartite motif-containing 61; Citrate lyase beta like; Ankyrin repeat domain 15; Ankyrin 3, node of Ranvier (ankyrin G) and Maltase-glucoamylase (alpha-glucosidase).
- activator 1 Replication factor C
- Tripartite motif-containing 61 Tripartite motif-containing 61
- Citrate lyase beta like Citrate lyase beta like
- Ankyrin repeat domain 15 Ankyrin 3, node of Ranvier (ankyrin G)
- Maltase-glucoamylase alpha-glucosidase
- the library comprises at least one transcript from at least one gene selected from the group consisting of Replication factor C (activator 1) 3; Ankyrin repeat domain 15; Hypothetical protein FLJ12949; Solute carrier family 43, member 1; Thioredoxin-like 2; Polymerase (RNA) II (DNA directed) polypeptide L; Syntaxin 5; Leucine rich repeat containing 16; Calcium channel, voltage-dependent, beta 4 subunit; [NM — 001005522]; G protein-coupled receptor kinase interactor 2; Ankyrin 3, node of Ranvier (ankyrin G); Gremlin 1, cysteine knot superfamily, homolog; Zinc finger protein 667; Hypothetical gene supported by AK128346; Transmembrane 9 superfamily member 2; Potassium channel, subfamily K, member 1; Chromodomain helicase DNA binding protein 2; Chromosome 9 open reading frame 94; Chromosome 21 open reading frame 34; and Dual specificity phosphatase 5.
- the library comprises at least one transcript from at least one gene selected from the group consisting of Citrate lyase beta like; Phosphodiesterase 4D, cAMP-specific; Ectodysplasin A receptor; DEP domain containing 6; Basonuclin 2; Chromosome 2 open reading frame 3; FLJ25476 protein; Staphylococcal nuclease and tudor domain containing 1; Hermansky-Pudlak syndrome 5 and Chromosome 12 open reading frame 30.
- the library comprises at least one transcript from at least one gene selected from the group consisting of Replication factor C (activator 1) 3; Tripartite motif-containing 61; Citrate lyase beta like; Adaptor-related protein complex 1, gamma 2 subunit; Kallikrein-related peptidase 2; Phosphodiesterase 4D, cAMP-specific; Cytochrome P450, family 4, subfamily F, polypeptide 11; Ectodysplasin A receptor
- Phospholipase C beta 1; KIAA1244; Paraoxonase 2; Arachidonate 12-lipoxygenase, 12R type; Cut-like 2; Chemokine (C-X-C motif) ligand 12; Rho guanine nucleotide exchange factor (GEF) 5; Olfactory receptor, family 2, subfamily A, member 4; Chromosome 19 open reading frame 42; Hypothetical gene supported by AK128346; Phosphoglucomutase 5; Hyaluronan binding protein 4; NECAP endocytosis associated 2 Myeloid/lymphoid or mixed-lineage leukemia; translocated to, 4; Signal transducer and activator of transcription 1; Chromosome 2 open reading frame 3; FLJ25476 protein; Staphylococcal nuclease and tudor domain containing 1; Transmembrane protein 18; Hermansky-Pudlak syndrome 5; Chromosome 12 open reading frame 30;
- the invention also contemplates that alternative libraries may be designed that include transcripts of one or more of the genes in Table 3, together with additional gene transcripts that have previously been shown to be associated with prostate cancer systemic progression.
- the publication and sequence databases can be mined using a variety of search strategies to identify appropriate additional candidates for inclusion in the library.
- search strategies for example, currently available scientific and medical publication databases such as Medline, Current Contents, OMIM (online Mendelian inheritance in man), various Biological and Chemical Abstracts, Journal indexes, and the like can be searched using term or key-word searches, or by author, title, or other relevant search parameters.
- databases are publicly available, and strategies and procedures for identifying publications and their contents, for example, genes, other nucleotide sequences, descriptions, indications, expression pattern, etc, are well known to those skilled in the art. Numerous databases are available through the internet for free or by subscription, see, for example, the National Center Biotechnology Information (NCBI), Infotrieve, Thomson ISI, and Science Magazine (published by the AAAS) websites. Additional or alternative publication or citation databases are also available that provide identical or similar types of information, any of which can be employed in the context of the invention. These databases can be searched for publications describing altered gene expression between recurrent and non-recurrent prostate cancer.
- Additional potential candidate genes may be identified by searching the above described databases for differentially expressed proteins and by identifying the nucleotide sequence encoding the differentially expressed proteins.
- a list of genes whose altered expression is between patients with recurrent disease and non-recurrent prostate cancer is presented in Table 6.
- a Prostate Prognostic Set comprises one or more target sequences identified within the gene transcripts in the prostate prognostic library, or a subset of these gene transcripts.
- the target sequences may be within the coding and/or non-coding regions of the gene transcripts.
- the set can comprise one or a plurality of target sequences from each gene transcript in the library, or subset thereof. The relative and/or normalized level of these target sequences in a sample is indicative of the level of expression of the particular gene transcript and thus of prostate cancer recurrence risk.
- the relative and/or normalized expression level of one or more of the target sequences may be indicative of an recurrent form of prostate cancer and therefore prognostic for prostate cancer systemic progression while the relative and/or normalized expression level of one or more other target sequences may be indicative of a non-recurrent form of prostate cancer and therefore prognostic for a NED clinical outcome.
- one embodiment of the present invention provides for a library or catalog of candidate target sequences derived from the transcripts (both coding and non-coding regions) of at least one gene suitable for classifying prostate cancer recurrence risk.
- the present invention provides for a library or catalog of candidate target sequences derived from the non-coding regions of transcripts of at least one gene suitable for classifying prostate cancer recurrence risk.
- the library or catalog of candidate target sequences comprises target sequences derived from the transcripts of one or more of the genes set forth in Table 3 and/or Table 6. The library or catalog in affect provides a resource list of transcripts from which target sequences appropriate for inclusion in a Prostate Cancer Prognostic set can be derived.
- an individual Prostate Cancer Prognostic set may comprise target sequences derived from the transcripts of one or more genes exhibiting a positive correlation with recurrent prostate cancer. In one embodiment, an individual Prostate Cancer Prognostic Set may comprise target sequences derived from the transcripts of one or more genes exhibiting a negative correlation with recurrent prostate cancer. In one embodiment, an individual Prostate Cancer Prognostic Set may comprise target sequences derived from the transcripts of two or more genes, wherein at least one gene has a transcript that exhibits a positive correlation with recurrent prostate cancer and at least one gene has a transcript that exhibits negative correlation with recurrent prostate cancer.
- the Prostate Cancer Prognostic Set comprises target sequences derived from the transcripts of at least one gene. In one embodiment, the Prostate Cancer Prognostic Set comprises target sequences derived from the transcripts of at least 5 genes. In another embodiment, the Prostate Cancer Prognostic set comprises target sequences derived from the transcripts of at least 10 genes. In a further embodiment, the Prostate Cancer Prognostic set comprises target sequences derived from the transcripts of at least 15 genes. In other embodiments, the Prostate Cancer Prognostic set comprises target sequences derived from the transcripts of at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 and at least 65 genes.
- target sequences can be identified by screening for target sequences that have been annotated to be associated with each specific gene locus from a number of annotation sources including GenBank, RefSeq, Ensembl, dbEST, GENSCAN, TWINSCAN, Exoniphy, Vega, microRNAs registry and others (see Affymetrix Exon Array design note).
- target sequences can be further evaluated for potential cross-hybridization against other putative transcribed sequences in the design (but not the entire genome) to identify only those target sequences that are predicted to uniquely hybridize to a single target.
- the set of target sequences that are predicted to uniquely hybridize to a single target can be further filtered using a variety of criteria including, for example, sequence length, for their mean expression levels across a wide selection of human tissues, as being representive of transcripts expressed either as novel alternative (i.e., non-consensus) exons, alternative retained introns, novel exons 5′ or 3′ of the gene's transcriptional start site or representing transcripts expressed in a manner antisense to the gene, amongst others.
- sequence length for their mean expression levels across a wide selection of human tissues, as being representive of transcripts expressed either as novel alternative (i.e., non-consensus) exons, alternative retained introns, novel exons 5′ or 3′ of the gene's transcriptional start site or representing transcripts expressed in a manner antisense to the gene, amongst others.
- the Prostate Classification Set comprises target sequences derived from 382,253 base pair 3′ of Replication factor C (activator 1) 3, 38 kDa; 58,123 base pair 3′ of Tripartite motif-containing 61; in intron #3 of Citrate lyase beta like; in intron #2 of Ankyrin repeat domain 15; in exon #1 of UDP-glucose ceramide glucosyltransferase-like 2; in exon of #19 of Hypothetical protein FLJ12949; in intron #4 of Chromosome 22 open reading frame 13; in exon #2 of phatidylinositol glycan anchor biosynthesis, class O; in exon #15 of Solute carrier family 43, member 1; in exon #1 of Rabaptin, RAB GTPase binding effector protein 1; in intron #38 of Maltase-glucoamylase (alpha-glucosidase); in intron #23 of Ankyrin 3, node of Ranvier (activator 1)
- the Prostate Classification Set comprises target sequences derived from 382,253 base pair 3′ of Replication factor C (activator 1) 3; in intron #2 of Ankyrin repeat domain 15; in exon #19 of Hypothetical protein FLJ12949; in exon #15 of Solute carrier family 43, member 1; 313,721 base pair 3′ of Thioredoxin-like 2; in exon #2 of Polymerase (RNA) II (DNA directed) polypeptide L, 7.6 kDa; in intron #10 of Syntaxin 5; 141,389 base pair 5′ of Leucine rich repeat containing 16; in intron #2 of Calcium channel, voltage-dependent, beta 4 subunit; 5,474 base pair 5′ of [NM — 001005522]; in intron #14 of G protein-coupled receptor kinase interactor 2; in intron #23 of Ankyrin 3, node of Ranvier (ankyrin G); 71,333 base pair 3′ of Gremlin 1, cysteine knot superfamily
- the Prostate Classification Set comprises target sequences derived from 382,253 base pair 3′ of Replication factor C (activator 1) 3, 38 kDa; 58,123 base pair 3′ of Tripartite motif-containing 61; in intron #3 of Citrate lyase beta like; in intron #2 of Ankyrin repeat domain 15; in intron #38 of Maltase-glucoamylase (alpha-glucosidase); and in intron #23 of Ankyrin 3, node of Ranvier (ankyrin G).
- the Prostate Classification Set comprises target sequences derived from 382,253 base pair 3′ of Replication factor C (activator 1) 3, 38 kDa; in intron #2 of Ankyrin repeat domain 15; in exon #19 of Hypothetical protein FLJ12949; in exon #15 of Solute carrier family 43, member 1; 313,721 base pair 3′ of Thioredoxin-like 2; in exon #2 of Polymerase (RNA) II (DNA directed) polypeptide L, 7.6 kDa; in intron #10 of Syntaxin 5; 141,389 base pair 5′ of Leucine rich repeat containing 16; in intron #2 of Calcium channel, voltage-dependent, beta 4 subunit; 5,474 base pair 5′ of [NM — 001005522]; in intron #14 of G protein-coupled receptor kinase interactor 2; in intron #2 of Ankyrin 3, node of Ranvier (ankyrin G); 71,333 base pair of 3′ Gremlin 1, cyste
- the Prostate Classification Set comprises target sequences derived from in intron #3 of Citrate lyase beta like; 210,560 base pair 5′ of Phosphodiesterase 4D; 189,722 base pair 5′ of Ectodysplasin A receptor; 3,510 base pair 3′ of DEP domain containing 6; in exon #6 of Basonuclin 2; in intron #1 of Chromosome 2 open reading frame 3; in intron #1 of FLJ25476 protein; in intron #10 of Staphylococcal nuclease and tudor domain containing 1; in exon #22 of Hermansky-Pudlak syndrome 5; and in exon #24 of Chromosome 12 open reading frame 30.
- the Prostate Classification Set comprises target sequences derived from 382,253 base pair 3′ of Replication factor C (activator 1) 3, 38 kDa; 58,123 base pair 3′ of Tripartite motif-containing 61; in intron #3 of Citrate lyase beta like; in intron #1 of Adaptor-related protein complex 1, gamma 2 subunit; in intron #2 of Kallikrein-related peptidase 2; 210,560 base pair 5′ of Phosphodiesterase 4D; 3,508 base pair 3′ of Cytochrome P450, family 4, subfamily F, polypeptide 11; 189,722 base pair 5′ of Ectodysplasin A receptor; in intron #2 of Phospholipase C, beta 1; in intron #10 of KIAA1244; in intron #2 of Paraoxonase 2; 11,235 base pair 3′ of Arachidonate 12-lipoxygenase, 12R type; in exon #22 of Cut-like 2;
- the potential set of target sequences can be filtered for their expression levels using the multi-tissue expression data made publicly available by Affymetrix at (http://www.affymetrix.com/stipport/technical/sample_data/exon_array_data.affx) such that probes with, for example, elevated expression across numerous tissues (non-specific) or no expression in prostate tissue can be excluded.
- each target sequence suitable for use in the Prostate Cancer Prognostic Set may be validated to confirm differential relative or normalized expression in recurrent prostate cancer or non-recurrent prostate cancer.
- Validation methods are known in the art and include hybridization techniques such as microarray analysis or Northern blotting using appropriate controls, and may include one or more additional steps, such as reverse transcription, transcription, PCR, RT-PCR and the like. The validation of the target sequences using these methods is well within the abilities of a worker skilled in the art.
- individual Prostate Cancer Prognostic Sets provide for at least a determination of a minimal expression signature, capable of distinguishing recurrent from non-recurrent forms of prostate cancer.
- Means for determining the appropriate number of target sequences necessary to obtain a minimal expression signature are known in the art and include the Nearest Shrunken Centroids (NSC) method.
- a standardized centroid is computed for each class. This is the average gene expression for each gene in each class divided by the within-class standard deviation for that gene.
- Nearest centroid classification takes the gene expression profile of a new sample, and compares it to each of these class centroids. The class whose centroid that it is closest to, in squared distance, is the predicted class for that new sample.
- Nearest shrunken centroid classification “shrinks” each of the class centroids toward the overall centroid for all classes by an amount called the threshold. This shrinkage consists of moving the centroid towards zero by threshold, setting it equal to zero if it hits zero.
- centroid of 3.2 would be shrunk to 1.2
- centroid of ⁇ 3.4 would be shrunk to ⁇ 1.4
- centroid of 1.2 would be shrunk to zero.
- the new sample is classified by the usual nearest centroid rule, but using the shrunken class centroids. This shrinkage can make the classifier more accurate by reducing the effect of noisy genes and provides an automatic gene selection.
- a gene is shrunk to zero for all classes, then it is eliminated from the prediction rule. Alternatively, it may be set to zero for all classes except one, and it can be learned that the high or low expression for that gene characterizes that class. The user decides on the value to use for threshold.
- PAM does K-fold cross-validation for a range of threshold values.
- the samples are divided up at random into K roughly equally sized parts.
- the classifier is built on the other K ⁇ 1 parts then tested on the remaining part. This is done for a range of threshold values, and the cross-validated misclassification error rate is reported for each threshold value.
- the user would choose the threshold value giving the minimum cross-validated misclassification error rate.
- minimal expression signatures can be established through the use of optimization algorithms such as the mean variance algorithm widely used in establishing stock portfolios.
- This method is described in detail in US patent publication number 20030194734.
- the method calls for the establishment of a set of inputs (stocks in financial applications, expression as measured by intensity here) that will optimize the return (e.g., signal that is generated) one receives for using it while minimizing the variability of the return.
- the method calls for the establishment of a set of inputs (e.g., expression as measured by intensity) that will optimize the signal while minimizing variability.
- Many commercial software programs are available to conduct such operations. “Wagner Associates Mean-Variance Optimization Application,” referred to as “Wagner Software” throughout this specification, is preferred.
- This software uses functions from the “Wagner Associates Mean-Variance Optimization Library” to determine an efficient frontier and optimal portfolios in the Markowitz sense is preferred. Use of this type of software requires that microarray data be transformed so that it can be treated as an input in the way stock return and risk measurements are used when the software is used for its intended financial analysis purposes.
- the process of selecting a minimal expression signature can also include the application of heuristic rules.
- such rules are formulated based on biology and an understanding of the technology used to produce clinical results. More preferably, they are applied to output from the optimization method.
- the mean variance method of portfolio selection can be applied to microarray data for a number of genes differentially expressed in subjects with cancer. Output from the method would be an optimized set of genes that could include some genes that are expressed in peripheral blood as well as in diseased tissue.
- heuristic rules can be applied that are not necessarily related to the biology in question. For example, one can apply a rule that only a prescribed percentage of the portfolio can be represented by a particular gene or group of genes.
- Commercially available software such as the Wagner Software readily accommodates these types of heuristics. This can be useful, for example, when factors other than accuracy and precision (e.g., anticipated licensing fees) have an impact on the desirability of including one or more genes.
- the Prostate Cancer Prognostic Set for obtaining a minimal expression signature comprises at least one, two, three, four, five, six, eight, 10, 15, 20, 25 or more of target sequences shown to have a positive correlation with non-recurrent prostate disease, for example those depicted in SEQ ID NOs:1-913 or a subset thereof.
- the Prostate Cancer Prognostic Set for obtaining a minimal expression signature comprises at least one, two, three, four, five, six, eight, 10, 15, 20, 25 or more of those target sequences shown to have a positive correlation with recurrent prostate cancer, for example those depicted in of SEQ ID NOs: 914-2114, or a subset thereof.
- the Prostate Cancer Prognostic Set for obtaining a minimal expression signature comprises at least one, two, three, four, five, six, eight, 10, 15, 20, 25 or more of target sequences shown to have a correlation with non-recurrent or recurrent prostate cancer, for example those depicted in SEQ ID NOs:1-2114 or a subset thereof.
- the Prostate Cancer Prognostic Set comprises target sequences for detecting expression products of SEQ IDs:1-2114. In some embodiments, the Prostate Cancer Prognostic Set comprises probes for detecting expression levels of sequences exhibiting positive and negative correlation with a disease status of interest are employed.
- a combination target sequences useful in these methods were found to include those encoding RNAs corresponding to SEQ ID NOs: 1-913 (found at increased expression in prostate cancer samples from NED patients) and/or corresponding to SEQ ID NOs: 914-2114 (found at increased expression levels in prostate cancer samples from SYS patients), where intermediate levels of certain target sequences (Table 7) are observed in prostate cancer samples from PSA patients with biochemical recurrence, where the RNA expression levels are indicative of a disease state or outcome. Subgroups of these target sequences, as well as individual target sequences, have been found useful in such methods.
- an RNA signature corresponding to SEQ ID NOs: 1, 4, 6, 9, 14-16, 18-21 915-917, 920, 922, 928, 929, 931, 935 and 936 (the 21-RNA′ signature) and/or SEQ ID NOs: 1-11, 914-920 (the ‘18-RNA’ signature) and/or SEQ ID NOs: 1-4, 914,915) (the ‘6-RNA’ signature) and/or SEQ ID NOs: 1, 4, 6, 9, 14-16, 18-21, 915-917, 920, 922, 928, 929, 931, 935 and 936 (the ‘20-RNA’ signature) and/or SEQ ID NOs 3, 36, 60, 63, 926, 971, 978, 999, 1014 and 1022 (the ‘10-RNA’ signature) and/or SEQ ID NOs 1-3, 32, 33, 36, 46, 60, 63, 66, 69, 88, 100, 241, 265, 334, 437, 920,
- Exemplary subsets and combinations of interest also include at least five, six, 10, 15, 18, 20, 23, 25, 27, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 750, 1000, 1200, 1400, 1600, 1800, 2000, or all 2114 target sequences in Table 4; at least five, six, 10, 15, 18, 20, 23, 25, 27, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, or all 526 target sequences in Table 7; SEQ ID NOs:1, 4, 915, 6, 916, 9, 917, 920, 922, 14, 15, 16, 928, 929, 18, 19, 931, 20, 21, 935, 936, or combinations thereof; SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 914, 915, 916, 917,
- Exemplary subsets of interest include those described herein, including in the examples.
- Exemplary combinations of interest include those utilizing one or more of the sequences listed in Tables 5, 7, 8, 9 or 10. Of particular interest are those combinations utilizing at least one sequence exhibiting positive correlation with the trait of interest, as well as those combinations utilizing at least one sequence exhibiting negative correlation with the trait of interest. Also of interest are those combinations utilizing at least two, at least three, at least four, at least five or at least six of those sequences exhibiting such a positive correlation, in combination with at least two, at least three, at least four, at least five, or at least six of those sequences exhibiting such a negative correlation. Exemplary combinations include those utilizing at least one, two, three, four, five or six of the target sequences depicted in Tables 5 and 6.
- increased relative expression of one or more of SEQ IDs:1-913, decreased relative expression of one or more of SEQ ID NOs:914-2114 or a combination of any thereof is indicative/predictive of the patient exhibiting no evidence of disease for at least seven years or more after surgery.
- increased relative expression of SEQ IDs:914-2114, decreased relative expression of one or more of SEQ ID NOs:1-913 or a combination of any thereof is indicative/predictive of the patient exhibiting systemic prostate cancer.
- Increased or decreased expression of target sequences represented in these sequence listings, or of the target sequences described in the examples, may be utilized in the methods of the invention.
- the Prostate Cancer Prognostic Set can optionally include one or more target sequences specifically derived from the transcripts of one or more housekeeping genes and/or one or more internal control target sequences and/or one or more negative control target sequences. In one embodiment, these target sequences can, for example, be used to normalize expression data.
- Housekeeping genes from which target sequences for inclusion in a Prostate Cancer Prognostic Set can be derived from are known in the art and include those genes in which are expressed at a constant level in normal and prostate cancer tissue.
- target sequences described herein may be used alone or in combination with each other or with other known or later identified disease markers.
- the system of the present invention provides for combinations of polynucleotide probes that are capable of detecting the target sequences of the Prostate Cancer Prognostic Sets.
- Individual polynucleotide probes comprise a nucleotide sequence derived from the nucleotide sequence of the target sequences or complementary sequences thereof.
- the nucleotide sequence of the polynucleotide probe is designed such that it corresponds to, or is complementary to the target sequences.
- the polynucleotide probe can specifically hybridize under either stringent or lowered stringency hybridization conditions to a region of the target sequences, to the complement thereof, or to a nucleic acid sequence (such as a cDNA) derived therefrom.
- polynucleotide probe sequences and determination of their uniqueness may be carried out in silico using techniques known in the art, for example, based on a BLASTN search of the polynucleotide sequence in question against gene sequence databases, such as the Human Genome Sequence, UniGene, dbEST or the non-redundant database at NCBI.
- the polynucleotide probe is complementary to a region of a target mRNA derived from a target sequence in the Prostate Cancer Prognostic Set.
- Computer programs can also be employed to select probe sequences that will not cross hybridize or will not hybridize non-specifically.
- nucleotide sequence of the polynucleotide probe need not be identical to its target sequence in order to specifically hybridize thereto.
- the polynucleotide probes of the present invention therefore, comprise a nucleotide sequence that is at least about 75% identical to a region of the target gene or mRNA.
- nucleotide sequence of the polynucleotide probe is at least about 90% identical a region of the target gene or mRNA.
- nucleotide sequence of the polynucleotide probe is at least about 95% identical to a region of the target gene or mRNA.
- nucleotide sequence of the polynucleotide probes of the present invention may exhibit variability by differing (e.g. by nucleotide substitution, including transition or transversion) at one, two, three, four or more nucleotides from the sequence of the target gene.
- the probes can be designed to have ⁇ 50% G content and/or between about 25% and about 70% G+C content.
- Strategies to optimize probe hybridization to the target nucleic acid sequence can also be included in the process of probe selection.
- Hybridization under particular pH, salt, and temperature conditions can be optimized by taking into account melting temperatures and by using empirical rules that correlate with desired hybridization behaviours.
- Computer models may be used for predicting the intensity and concentration-dependence of probe hybridization.
- a probe in order to represent a unique sequence in the human genome, a probe should be at least 15 nucleotides in length. Accordingly, the polynucleotide probes of the present invention range in length from about 15 nucleotides to the full length of the target sequence or target mRNA. In one embodiment of the invention, the polynucleotide probes are at least about 15 nucleotides in length. In another embodiment, the polynucleotide probes are at least about 20 nucleotides in length. In a further embodiment, the polynucleotide probes are at least about 25 nucleotides in length.
- the polynucleotide probes are between about 15 nucleotides and about 500 nucleotides in length. In other embodiments, the polynucleotide probes are between about 15 nucleotides and about 450 nucleotides, about 15 nucleotides and about 400 nucleotides, about 15 nucleotides and about 350 nucleotides, about 15 nucleotides and about 300 nucleotides in length.
- the polynucleotide probes of a Prostate Cancer Prognostic Set can comprise RNA, DNA, RNA or DNA mimetics, or combinations thereof, and can be single-stranded or double-stranded.
- the polynucleotide probes can be composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as polynucleotide probes having non-naturally-occurring portions which function similarly.
- Such modified or substituted polynucleotide probes may provide desirable properties such as, for example, enhanced affinity for a target gene and increased stability.
- the system of the present invention further provides for primers and primer pairs capable of amplifying target sequences defined by the Prostate Cancer Prognostic Set, or fragments or subsequences or complements thereof.
- the nucleotide sequences of the Prostate Cancer Prognostic set may be provided in computer-readable media for in silico applications and as a basis for the design of appropriate primers for amplification of one or more target sequences of the Prostate Cancer Prognostic Set.
- Primers based on the nucleotide sequences of target sequences can be designed for use in amplification of the target sequences.
- a pair of primers will be used.
- the exact composition of the primer sequences is not critical to the invention, but for most applications the primers will hybridize to specific sequences of the Prostate Cancer Prognostic Set under stringent conditions, particularly under conditions of high stringency, as known in the art.
- the pairs of primers are usually chosen so as to generate an amplification product of at least about 50 nucleotides, more usually at least about 100 nucleotides. Algorithms for the selection of primer sequences are generally known, and are available in commercial software packages.
- primers may be used in standard quantitative or qualitative PCR-based assays to assess transcript expression levels of RNAs defined by the Prostate Cancer Prognostic Set.
- these primers may be used in combination with probes, such as molecular beacons in amplifications using real-time PCR.
- the primers or primer pairs when used in an amplification reaction, specifically amplify at least a portion of a nucleic acid depicted in one of SEQ ID NOs: 1-2114 (or subgroups thereof as set forth herein), an RNA form thereof, or a complement to either thereof.
- a nucleoside is a base-sugar combination and a nucleotide is a nucleoside that further includes a phosphate group covalently linked to the sugar portion of the nucleoside.
- the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound, with the normal linkage or backbone of RNA and DNA being a 3′ to 5′ phosphodiester linkage.
- polynucleotide probes or primers useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages.
- oligonucleotides having modified backbones include both those that retain a phosphorus atom in the backbone and those that lack a phosphorus atom in the backbone.
- modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleotides.
- Exemplary polynucleotide probes or primers having modified oligonucleotide backbones include, for example, those with one or more modified internucleotide linkages that are phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′ amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl-phosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′.
- Exemplary modified oligonucleotide backbones that do not include a phosphorus atom are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
- Such backbones include morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulphone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulphamate backbones; methyleneimino and methylenehydrazino backbones; sulphonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.
- the present invention also contemplates oligonucleotide mimetics in which both the sugar and the internucleoside linkage of the nucleotide units are replaced with novel groups.
- the base units are maintained for hybridization with an appropriate nucleic acid target compound.
- An example of such an oligonucleotide mimetic which has been shown to have excellent hybridization properties, is a peptide nucleic acid (PNA) [Nielsen et al., Science, 254:1497-1500 (1991)].
- PNA peptide nucleic acid
- the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
- LNAs locked nucleic acids
- the nucleobases are retained and are bound directly or indirectly to aza-nitrogen atoms of the amide portion of the backbone.
- the present invention also contemplates polynucleotide probes or primers comprising “locked nucleic acids” (LNAs), which are novel conformationally restricted oligonucleotide analogues containing a methylene bridge that connects the 2′-O of ribose with the 4′-C (see, Singh et al., Chem. Commun., 1998, 4:455-456).
- LNA and LNA analogues display very high duplex thermal stabilities with complementary DNA and RNA, stability towards 3′-exonuclease degradation, and good solubility properties.
- LNAs form duplexes with complementary DNA or RNA or with complementary LNA, with high thermal affinities.
- the universality of LNA-mediated hybridization has been emphasized by the formation of exceedingly stable LNA:LNA duplexes (Koshkin et al., J. Am. Chem. Soc., 1998, 120:13252-13253).
- LNA:LNA hybridization was shown to be the most thermally stable nucleic acid type duplex system, and the RNA-mimicking character of LNA was established at the duplex level.
- Introduction of three LNA monomers (T or A) resulted in significantly increased melting points toward DNA complements.
- Modified polynucleotide probes or primers may also contain one or more substituted sugar moieties.
- oligonucleotides may comprise sugars with one of the following substituents at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
- Examples of such groups are: O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10.
- the oligonucleotides may comprise one of the following substituents at the 2′ position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
- 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE)
- 2′-dimethylaminooxyethoxy (O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2′-DMAOE)
- 2′-methoxy (2′-O—CH 3 )
- 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 )
- 2′-fluoro 2′-F
- polynucleotide probes or primers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
- Polynucleotide probes or primers may also include modifications or substitutions to the nucleobase.
- “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
- Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substitute
- nucleobases include those disclosed in U.S. Pat. No. 3,687,808; The Concise Encyclopedia Of Polymer Science And Engineering, (1990) pp 858-859, Kroschwitz, J. I., ed. John Wiley & Sons; Englisch et al., Angewandte Chemie, Int. Ed., 30:613 (1991); and Sanghvi, Y. S., (1993) Antisense Research and Applications , pp 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press. Certain of these nucleobases are particularly useful for increasing the binding affinity of the polynucleotide probes of the invention.
- 5-substituted pyrimidines include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. [Sanghvi, Y. S., (1993) Antisense Research and Applications , pp 276-278, Crooke, S. T. and Lebleu, B., ed., CRC Press, Boca Raton].
- nucleotide sequence of the entire length of the polynucleotide probe or primer does not need to be derived from the target sequence.
- the polynucleotide probe may comprise nucleotide sequences at the 5′ and/or 3′ termini that are not derived from the target sequences.
- Nucleotide sequences which are not derived from the nucleotide sequence of the target sequence may provide additional functionality to the polynucleotide probe. For example, they may provide a restriction enzyme recognition sequence or a “tag” that facilitates detection, isolation, purification or immobilisation onto a solid support.
- the additional nucleotides may provide a self-complementary sequence that allows the primer/probe to adopt a hairpin configuration.
- Such configurations are necessary for certain probes, for example, molecular beacon and Scorpion probes, which can be used in solution hybridization techniques.
- the polynucleotide probes or primers can incorporate moieties useful in detection, isolation, purification, or immobilisation, if desired.
- moieties are well-known in the art (see, for example, Ausubel et al., (1997 & updates) Current Protocols in Molecular Biology , Wiley & Sons, New York) and are chosen such that the ability of the probe to hybridize with its target sequence is not affected.
- Suitable moieties are detectable labels, such as radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, and fluorescent microparticles, as well as antigens, antibodies, haptens, avidin/streptavidin, biotin, haptens, enzyme cofactors/substrates, enzymes, and the like.
- a label can optionally be attached to or incorporated into a probe or primer polynucleotide to allow detection and/or quantitation of a target polynucleotide representing the target sequence of interest.
- the target polynucleotide may be the expressed target sequence RNA itself, a cDNA copy thereof, or an amplification product derived therefrom, and may be the positive or negative strand, so long as it can be specifically detected in the assay being used.
- an antibody may be labeled.
- labels used for detecting different targets may be distinguishable.
- the label can be attached directly (e.g., via covalent linkage) or indirectly, e.g., via a bridging molecule or series of molecules (e.g., a molecule or complex that can bind to an assay component, or via members of a binding pair that can be incorporated into assay components, e.g. biotin-avidin or streptavidin).
- a bridging molecule or series of molecules e.g., a molecule or complex that can bind to an assay component, or via members of a binding pair that can be incorporated into assay components, e.g. biotin-avidin or streptavidin.
- Many labels are commercially available in activated forms which can readily be used for such conjugation (for example through amine acylation), or labels may be attached through known or determinable conjugation schemes, many of which are known in the art.
- Labels useful in the invention described herein include any substance which can be detected when bound to or incorporated into the biomolecule of interest. Any effective detection method can be used, including optical, spectroscopic, electrical, piezoelectrical, magnetic, Raman scattering, surface plasmon resonance, colorimetric, calorimetric, etc.
- a label is typically selected from a chromophore, a lumiphore, a fluorophore, one member of a quenching system, a chromogen, a hapten, an antigen, a magnetic particle, a material exhibiting nonlinear optics, a semiconductor nanocrystal, a metal nanoparticle, an enzyme, an antibody or binding portion or equivalent thereof, an aptamer, and one member of a binding pair, and combinations thereof.
- Quenching schemes may be used, wherein a quencher and a fluorophore as members of a quenching pair may be used on a probe, such that a change in optical parameters occurs upon binding to the target introduce or quench the signal from the fluorophore.
- a molecular beacon Suitable quencher/fluorophore systems are known in the art.
- the label may be bound through a variety of intermediate linkages.
- a polynucleotide may comprise a biotin-binding species, and an optically detectable label may be conjugated to biotin and then bound to the labeled polynucleotide.
- a polynucleotide sensor may comprise an immunological species such as an antibody or fragment, and a secondary antibody containing an optically detectable label may be added.
- Chromophores useful in the methods described herein include any substance which can absorb energy and emit light.
- a plurality of different signaling chromophores can be used with detectably different emission spectra.
- the chromophore can be a lumophore or a fluorophore.
- Typical fluorophores include fluorescent dyes, semiconductor nanocrystals, lanthanide chelates, polynucleotide-specific dyes and green fluorescent protein.
- Coding schemes may optionally be used, comprising encoded particles and/or encoded tags associated with different polynucleotides of the invention.
- a variety of different coding schemes are known in the art, including fluorophores, including SCNCs, deposited metals, and RF tags.
- Polynucleotides from the described target sequences may be employed as probes for detecting target sequences expression, for ligation amplification schemes, or may be used as primers for amplification schemes of all or a portion of a target sequences.
- amplified either strand produced by amplification may be provided in purified and/or isolated form.
- polynucleotides of the invention include a nucleic acid depicted in (a) any one of SEQ ID NOs: 1-2114, or a subgroup thereof as set forth herein; (b) an RNA form of any one of the nucleic acids depicted in SEQ ID NOs: 1-2114, or a subgroup thereof as set forth herein; (c) a peptide nucleic acid form of any of the nucleic acids depicted in SEQ ID NOs: 1-2114, or a subgroup thereof as set forth herein; (d) a nucleic acid comprising at least 20 consecutive bases of any of (a-c); (e) a nucleic acid comprising at least 25 bases having at least 90% sequenced identity to any of (a-c); and (f) a complement to any of (a-e).
- Complements may take any polymeric form capable of base pairing to the species recited in (a)-(e), including nucleic acid such as RNA or DNA, or may be a neutral polymer such as a peptide nucleic acid.
- Polynucleotides of the invention can be selected from the subsets of the recited nucleic acids described herein, as well as their complements.
- polynucleotides of the invention comprise at least 20 consecutive bases as depicted in SEQ ID NOs:1-2114, or a complement thereto.
- the polynucleotides may comprise at least 21, 22, 23, 24, 25, 27, 30, 32, 35 or more consecutive bases as depicted in SEQ ID NOs:1-2114, as applicable.
- the nucleic acid in (a) can be selected from those in Table 3, and from SEQ ID NOs:1, 4, 915, 6, 916, 9, 917, 920, 922, 14, 15, 16, 928, 929, 18, 19, 931, 20, 21, 935, and 936; or from SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 914, 915, 916, 917, 918, 919, and 920; or from SEQ ID NOs: 1, 4, 6, 9, 14-16, 18-21, 915-917, 920, 922, 928, 929, 931, 935 and 936; or from SEQ ID NOs 3, 36, 60, 63, 926, 971, 978, 999, 1014 and 1022; or from SEQ ID NOs 1-3, 32, 33, 36, 46, 60, 63, 66, 69, 88, 100, 241, 265, 334, 437, 920, 925, 934, 945, 947, 954, 971,
- the polynucleotides may be provided in a variety of formats, including as solids, in solution, or in an array.
- the polynucleotides may optionally comprise one or more labels, which may be chemically and/or enzymatically incorporated into the polynucleotide.
- solutions comprising polynucleotide and a solvent are also provided.
- the solvent may be water or may be predominantly aqueous.
- the solution may comprise at least two, three, four, five, six, seven, eight, nine, ten, twelve, fifteen, seventeen, twenty or more different polynucleotides, including primers and primer pairs, of the invention. Additional substances may be included in the solution, alone or in combination, including one or more labels, additional solvents, buffers, biomolecules, polynucleotides, and one or more enzymes useful for performing methods described herein, including polymerases and ligases.
- the solution may further comprise a primer or primer pair capable of amplifying a polynucleotide of the invention present in the solution.
- one or more polynucleotides provided herein can be provided on a substrate.
- the substrate can comprise a wide range of material, either biological, nonbiological, organic, inorganic, or a combination of any of these.
- the substrate may be a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO 2 , SiN 4 , modified silicon, or any one of a wide variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, cross-linked polystyrene, polyacrylic, polylactic acid, polyglycolic acid, poly(lactide coglycolide), polyanhydrides, poly(methyl methacrylate), poly(ethylene-co-vinyl acetate), polysiloxanes, polymeric silica, latexes, dextran polymers, epoxies, polycarbonates,
- Substrates can be planar crystalline substrates such as silica based substrates (e.g. glass, quartz, or the like), or crystalline substrates used in, e.g., the semiconductor and microprocessor industries, such as silicon, gallium arsenide, indium doped GaN and the like, and includes semiconductor nanocrystals.
- silica based substrates e.g. glass, quartz, or the like
- crystalline substrates used in, e.g., the semiconductor and microprocessor industries such as silicon, gallium arsenide, indium doped GaN and the like, and includes semiconductor nanocrystals.
- the substrate can take the form of an array, a photodiode, an optoelectronic sensor such as an optoelectronic semiconductor chip or optoelectronic thin-film semiconductor, or a biochip.
- the location(s) of probe(s) on the substrate can be addressable; this can be done in highly dense formats, and the location(s) can be microaddressable or nanoaddressable.
- Silica aerogels can also be used as substrates, and can be prepared by methods known in the art. Aerogel substrates may be used as free standing substrates or as a surface coating for another substrate material.
- the substrate can take any form and typically is a plate, slide, bead, pellet, disk, particle, microparticle, nanoparticle, strand, precipitate, optionally porous gel, sheets, tube, sphere, container, capillary, pad, slice, film, chip, multiwell plate or dish, optical fiber, etc.
- the substrate can be any form that is rigid or semi-rigid.
- the substrate may contain raised or depressed regions on which an assay component is located.
- the surface of the substrate can be etched using known techniques to provide for desired surface features, for example trenches, v-grooves, mesa structures, or the like.
- Surfaces on the substrate can be composed of the same material as the substrate or can be made from a different material, and can be coupled to the substrate by chemical or physical means.
- Such coupled surfaces may be composed of any of a wide variety of materials, for example, polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, membranes, or any of the above-listed substrate materials.
- the surface can be optically transparent and can have surface Si—OH functionalities, such as those found on silica surfaces.
- the substrate and/or its optional surface can be chosen to provide appropriate characteristics for the synthetic and/or detection methods used.
- the substrate and/or surface can be transparent to allow the exposure of the substrate by light applied from multiple directions.
- the substrate and/or surface may be provided with reflective “mirror” structures to increase the recovery of light.
- the substrate and/or its surface is generally resistant to, or is treated to resist, the conditions to which it is to be exposed in use, and can be optionally treated to remove any resistant material after exposure to such conditions.
- the substrate or a region thereof may be encoded so that the identity of the sensor located in the substrate or region being queried may be determined Any suitable coding scheme can be used, for example optical codes, RFID tags, magnetic codes, physical codes, fluorescent codes, and combinations of codes.
- the polynucleotide probes or primers of the present invention can be prepared by conventional techniques well-known to those skilled in the art.
- the polynucleotide probes can be prepared using solid-phase synthesis using commercially available equipment.
- modified oligonucleotides can also be readily prepared by similar methods.
- the polynucleotide probes can also be synthesized directly on a solid support according to methods standard in the art. This method of synthesizing polynucleotides is particularly useful when the polynucleotide probes are part of a nucleic acid array.
- Polynucleotide probes or primers can be fabricated on or attached to the substrate by any suitable method, for example the methods described in U.S. Pat. No. 5,143,854, PCT Publ. No. WO 92/10092, U.S. patent application Ser. No. 07/624,120, filed Dec. 6, 1990 (now abandoned), Fodor et al., Science, 251: 767-777 (1991), and PCT Publ. No. WO 90/15070). Techniques for the synthesis of these arrays using mechanical synthesis strategies are described in, e.g., PCT Publication No. WO 93/09668 and U.S. Pat. No. 5,384,261.
- Still further techniques include bead based techniques such as those described in PCT Appl. No. PCT/US93/04145 and pin based methods such as those described in U.S. Pat. No. 5,288,514. Additional flow channel or spotting methods applicable to attachment of sensor polynucleotides to a substrate are described in U.S. patent application Ser. No. 07/980,523, filed Nov. 20, 1992, and U.S. Pat. No. 5,384,261.
- polynucleotide probes of the present invention can be prepared by enzymatic digestion of the naturally occurring target gene, or mRNA or cDNA derived therefrom, by methods known in the art.
- the present invention further provides methods for characterizing prostate cancer sample for recurrence risk.
- the methods use the Prostate Cancer Prognostic Sets, probes and primers described herein to provide expression signatures or profiles from a test sample derived from a subject having or suspected of having prostate cancer.
- such methods involve contacting a test sample with Prostate Cancer Prognostic probes (either in solution or immobilized) under conditions that permit hybridization of the probe(s) to any target nucleic acid(s) present in the test sample and then detecting any probe:target duplexes formed as an indication of the presence of the target nucleic acid in the sample.
- Expression patterns thus determined are then compared to one or more reference profiles or signatures.
- the expression pattern can be normalized.
- the methods use the Prostate Cancer Prognostic Sets, probes and primers described herein to provide expression signatures or profiles from a test sample derived from a subject to classify the prostate cancer as recurrent or non-recurrent.
- such methods involve the specific amplification of target sequences nucleic acid(s) present in the test sample using methods known in the art to generate an expression profile or signature which is then compared to a reference profile or signature.
- the invention further provides for prognosing patient outcome, predicting likelihood of recurrence after prostatectomy and/or for designating treatment modalities.
- the methods generate expression profiles or signatures detailing the expression of the 2114 target sequences having altered relative expression with different prostate cancer outcomes. In one embodiment, the methods generate expression profiles or signatures detailing the expression of the subsets of these target sequences having 526 or 18 target sequences as described in the examples.
- increased relative expression of one or more of SEQ IDs:1-913, decreased relative expression of one or more of SEQ ID NOs:914-2114 or a combination of any thereof is indicative of a non-recurrent form of prostate cancer and may be predictive a NED clinical outcome after surgery.
- increased relative expression of SEQ IDs:914-2114, decreased relative expression of one or more of SEQ ID NOs:1-913 or a combination of any thereof is indicative of a recurrent form of prostate cancer and may be predictive of a SYS clinical outcome after surgery.
- Increased or decreased expression of target sequences represented in these sequence listings, or of the target sequences described in the examples, may be utilized in the methods of the invention.
- intermediate levels of expression of one or more target sequences depicted in Table 7 indicate a probability of future biochemical recurrence.
- the methods detect combinations of expression levels of sequences exhibiting positive and negative correlation with a disease status. In one embodiment, the methods detect a minimal expression signature.
- any method of detecting and/or quantitating the expression of the encoded target sequences can in principle be used in the invention.
- Such methods can include Northern blotting, array or microarray hybridization, by enzymatic cleavage of specific structures (e.g., an Invader® assay, Third Wave Technologies, e.g. as described in U.S. Pat. Nos. 5,846,717, 6,090,543; 6,001,567; 5,985,557; and 5,994,069) and amplification methods, e.g. RT-PCR, including in a TaqMan® assay (PE Biosystems, Foster City, Calif., e.g. as described in U.S. Pat. Nos.
- nucleic acids may be amplified, labeled and subjected to microarray analysis.
- Single-molecule sequencing e.g., Illumina, Helicos, PacBio, ABI SOLID
- in situ hybridization bead-array technologies
- bead-array technologies e.g., Luminex xMAP, Illumina BeadChips
- branched DNA technology e.g., Panomics, Genisphere
- the expressed target sequences can be directly detected and/or quantitated, or may be copied and/or amplified to allow detection of amplified copies of the expressed target sequences or its complement.
- degraded and/or fragmented RNA can be usefully analyzed for expression levels of target sequences, for example RNA having an RNA integrity number of less than 8.
- quantitative RT-PCR assays are used to measure the expression level of target sequences depicted in SEQ IDs: 1-2114.
- a GeneChip or microarray can be used to measure the expression of one or more of the target sequences.
- Molecular assays measure the relative expression levels of the target sequences, which can be normalized to the expression levels of one or more control sequences, for example array control sequences and/or one or more housekeeping genes, for example GAPDH. Increased (or decreased) relative expression of the target sequences as described herein, including any of SEQ ID NOs:1-2114, may thus be used alone or in any combination with each other in the methods described herein. In addition, negative control probes may be included.
- Diagnostic samples for use with the systems and in the methods of the present invention comprise nucleic acids suitable for providing RNAs expression information.
- the biological sample from which the expressed RNA is obtained and analyzed for target sequence expression can be any material suspected of comprising prostate cancer tissue or cells.
- the diagnostic sample can be a biological sample used directly in a method of the invention.
- the diagnostic sample can be a sample prepared from a biological sample.
- the sample or portion of the sample comprising or suspected of comprising prostate cancer tissue or cells can be any source of biological material, including cells, tissue or fluid, including bodily fluids.
- the source of the sample include an aspirate, a needle biopsy, a cytology pellet, a bulk tissue preparation or a section thereof obtained for example by surgery or autopsy, lymph fluid, blood, plasma, serum, tumors, and organs.
- the samples may be archival samples, having a known and documented medical outcome, or may be samples from current patients whose ultimate medical outcome is not yet known.
- the sample may be dissected prior to molecular analysis.
- the sample may be prepared via macrodissection of a bulk tumor specimen or portion thereof, or may be treated via microdissection, for example via Laser Capture Microdissection (LCM).
- LCD Laser Capture Microdissection
- the sample may initially be provided in a variety of states, as fresh tissue, fresh frozen tissue, fine needle aspirates, and may be fixed or unfixed. Frequently, medical laboratories routinely prepare medical samples in a fixed state, which facilitates tissue storage.
- fixatives can be used to fix tissue to stabilize the morphology of cells, and may be used alone or in combination with other agents. Exemplary fixatives include crosslinking agents, alcohols, acetone, Bouin's solution, Zenker solution, Hely solution, osmic acid solution and Carnoy solution.
- Crosslinking fixatives can comprise any agent suitable for forming two or more covalent bonds, for example an aldehyde.
- Sources of aldehydes typically used for fixation include formaldehyde, paraformaldehyde, glutaraldehyde or formalin.
- the crosslinking agent comprises formaldehyde, which may be included in its native form or in the form of paraformaldehyde or formalin.
- One or more alcohols may be used to fix tissue, alone or in combination with other fixatives.
- exemplary alcohols used for fixation include methanol, ethanol and isopropanol.
- Formalin fixation is frequently used in medical laboratories.
- Formalin comprises both an alcohol, typically methanol, and formaldehyde, both of which can act to fix a biological sample.
- the biological sample may optionally be embedded in an embedding medium.
- embedding media used in histology including paraffin, Tissue-Tek® V.I.P.TM, Paramat, Paramat Extra, Paraplast, Paraplast X-tra, Paraplast Plus, Peel Away Paraffin Embedding Wax, Polyester Wax, Carbowax Polyethylene Glycol, PolyfinTM, Tissue Freezing Medium TFMTM, Cryo-GelTM, and OCT Compound (Electron Microscopy Sciences, Hatfield, Pa.).
- the embedding material may be removed via any suitable techniques, as known in the art.
- the embedding material may be removed by extraction with organic solvent(s), for example xylenes.
- Kits are commercially available for removing embedding media from tissues. Samples or sections thereof may be subjected to further processing steps as needed, for example serial hydration or dehydration steps.
- the sample is a fixed, wax-embedded biological sample.
- samples from medical laboratories are provided as fixed, wax-embedded samples, most commonly as formalin-fixed, paraffin embedded (FFPE) tissues.
- FFPE formalin-fixed, paraffin embedded
- the target polynucleotide that is ultimately assayed can be prepared synthetically (in the case of control sequences), but typically is purified from the biological source and subjected to one or more preparative steps.
- the RNA may be purified to remove or diminish one or more undesired components from the biological sample or to concentrate it. Conversely, where the RNA is too concentrated for the particular assay, it may be diluted.
- RNA can be extracted and purified from biological samples using any suitable technique.
- a number of techniques are known in the art, and several are commercially available (e.g., FormaPureTM nucleic acid extraction kit, Agencourt Biosciences, Beverly Mass., High Pure FFPE RNA Micro KitTM, Roche Applied Science, Indianapolis, Ind.).
- RNA can be extracted from frozen tissue sections using TRIzol (Invitrogen, Carlsbad, Calif.) and purified using RNeasy Protect kit (Qiagen, Valencia, Calif.). RNA can be further purified using DNAse I treatment (Ambion, Austin, Tex.) to eliminate any contaminating DNA.
- RNA concentrations can be made using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Rockland, Del.). RNA integrity can be evaluated by running electropherograms, and RNA integrity number (RIN, a correlative measure that indicates intactness of mRNA) can be determined using the RNA 6000 PicoAssay for the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, Calif.).
- Reverse transcription can be performed using the Omniscript kit (Qiagen, Valencia, Calif.), Superscript III kit (Invitrogen, Carlsbad, Calif.), for RT-PCR.
- Target-specific priming can be performed in order to increase the sensitivity of detection of target sequences and generate target-specific cDNA.
- TaqMan® RT-PCR can be performed using Applied Biosystems Prism (ABI) 7900 HT instruments in a 5 ⁇ l volume with target sequence-specific cDNA equivalent to 1 ng total RNA. Primers and probes concentrations for TaqMan analysis are added to amplify fluorescent amplicons using PCR cycling conditions such as 95° C. for 10 minutes for one cycle, 95° C. for 20 seconds, and 60° C. for 45 seconds for 40 cycles. A reference sample can be assayed to ensure reagent and process stability. Negative controls (i.e., no template) should be assayed to monitor any exogenous nucleic acid contamination.
- the nucleic acid portion of the sample comprising RNA that is or can be used to prepare the target polynucleotide(s) of interest can be subjected to one or more preparative reactions.
- These preparative reactions can include in vitro transcription (IVT), labeling, fragmentation, amplification and other reactions.
- mRNA can first be treated with reverse transcriptase and a primer to create cDNA prior to detection, quantitation and/or amplification; this can be done in vitro with purified mRNA or in situ, e.g., in cells or tissues affixed to a slide.
- amplification is meant any process of producing at least one copy of a nucleic acid, in this case an expressed RNA, and in many cases produces multiple copies.
- An amplification product can be RNA or DNA, and may include a complementary strand to the expressed target sequence.
- DNA amplification products can be produced initially through reverse translation and then optionally from further amplification reactions.
- the amplification product may include all or a portion of a target sequence, and may optionally be labeled.
- a variety of amplification methods are suitable for use, including polymerase-based methods and ligation-based methods.
- Exemplary amplification techniques include the polymerase chain reaction method (PCR), the ligase chain reaction (LCR), ribozyme-based methods, self sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), the use of Q Beta replicase, reverse transcription, nick translation, and the like.
- Asymmetric amplification reactions may be used to preferentially amplify one strand representing the target sequence that is used for detection as the target polynucleotide.
- the presence and/or amount of the amplification product itself may be used to determine the expression level of a given target sequence.
- the amplification product may be used to hybridize to an array or other substrate comprising sensor polynucleotides which are used to detect and/or quantitate target sequence expression.
- the first cycle of amplification in polymerase-based methods typically forms a primer extension product complementary to the template strand.
- the template is single-stranded RNA
- a polymerase with reverse transcriptase activity is used in the first amplification to reverse transcribe the RNA to DNA, and additional amplification cycles can be performed to copy the primer extension products.
- the primers for a PCR must, of course, be designed to hybridize to regions in their corresponding template that will produce an amplifiable segment; thus, each primer must hybridize so that its 3′ nucleotide is paired to a nucleotide in its complementary template strand that is located 3′ from the 3′ nucleotide of the primer used to replicate that complementary template strand in the PCR.
- the target polynucleotide can be amplified by contacting one or more strands of the target polynucleotide with a primer and a polymerase having suitable activity to extend the primer and copy the target polynucleotide to produce a full-length complementary polynucleotide or a smaller portion thereof.
- Any enzyme having a polymerase activity that can copy the target polynucleotide can be used, including DNA polymerases, RNA polymerases, reverse transcriptases, enzymes having more than one type of polymerase or enzyme activity.
- the enzyme can be thermolabile or thermostable. Mixtures of enzymes can also be used.
- Exemplary enzymes include: DNA polymerases such as DNA Polymerase I (“Pol I”), the Klenow fragment of Pol I, T4, T7, Sequenase® T7, Sequenase® Version 2.0 T7, Tub, Taq, Tth, Pfx, Pfu, Tsp, Tfl, Tli and Pyrococcus sp GB-D DNA polymerases; RNA polymerases such as E. coli , SP6, T3 and T7 RNA polymerases; and reverse transcriptases such as AMV, M-MuLV, MMLV, RNAse H ⁇ MMLV (SuperScript®), SuperScript® II, ThermoScript®, HIV-1, and RAV2 reverse transcriptases.
- DNA polymerases such as DNA Polymerase I (“Pol I”), the Klenow fragment of Pol I, T4, T7, Sequenase® T7, Sequenase® Version 2.0 T7, Tub, Taq, Tth, Pfx,
- Exemplary polymerases with multiple specificities include RAV2 and Tli (exo-) polymerases.
- Exemplary thermostable polymerases include Tub, Taq, Tth, Pfx, Pfu, Tsp, Tfl, Tli and Pyrococcus sp.
- GB-D DNA polymerases are commercially available.
- Suitable reaction conditions are chosen to permit amplification of the target polynucleotide, including pH, buffer, ionic strength, presence and concentration of one or more salts, presence and concentration of reactants and cofactors such as nucleotides and magnesium and/or other metal ions (e.g., manganese), optional cosolvents, temperature, thermal cycling profile for amplification schemes comprising a polymerase chain reaction, and may depend in part on the polymerase being used as well as the nature of the sample.
- Cosolvents include formamide (typically at from about 2 to about 10%), glycerol (typically at from about 5 to about 10%), and DMSO (typically at from about 0.9 to about 10%).
- Techniques may be used in the amplification scheme in order to minimize the production of false positives or artifacts produced during amplification. These include “touchdown” PCR, hot-start techniques, use of nested primers, or designing PCR primers so that they form stem-loop structures in the event of primer-dimer formation and thus are not amplified.
- Techniques to accelerate PCR can be used, for example centrifugal PCR, which allows for greater convection within the sample, and comprising infrared heating steps for rapid heating and cooling of the sample.
- One or more cycles of amplification can be performed.
- An excess of one primer can be used to produce an excess of one primer extension product during PCR; preferably, the primer extension product produced in excess is the amplification product to be detected.
- a plurality of different primers may be used to amplify different target polynucleotides or different regions of a particular target polynucleotide within the sample.
- An amplification reaction can be performed under conditions which allow an optionally labeled sensor polynucleotide to hybridize to the amplification product during at least part of an amplification cycle.
- an assay is performed in this manner, real-time detection of this hybridization event can take place by monitoring for light emission or fluorescence during amplification, as known in the art.
- amplification product is to be used for hybridization to an array or microarray
- suitable commercially available amplification products are available. These include amplification kits available from NuGEN, Inc. (San Carlos, Calif.), including the WT-OvationTM System, WT-OvationTM System v2, WT-OvationTM Pico System, WT-OvationTM FFPE Exon Module, WT-OvationTM FFPE Exon Module RiboAmp and RiboAmp Plus RNA Amplification Kits (MDS Analytical Technologies (formerly Arcturus) (Mountain View, Calif.), Genisphere, Inc.
- Amplified nucleic acids may be subjected to one or more purification reactions after amplification and labeling, for example using magnetic beads (e.g., RNAClean magnetic beads, Agencourt Biosciences).
- magnetic beads e.g., RNAClean magnetic beads, Agencourt Biosciences.
- RNA biomarkers can be analyzed using real-time quantitative multiplex RT-PCR platforms and other multiplexing technologies such as GenomeLab GeXP Genetic Analysis System (Beckman Coulter, Foster City, Calif.), SmartCycler® 9600 or GeneXpert(R) Systems (Cepheid, Sunnyvale, Calif.), ABI 7900 HT Fast Real Time PCR system (Applied Biosystems, Foster City, Calif.), LightCycler® 480 System (Roche Molecular Systems, Pleasanton, Calif.), xMAP 100 System (Luminex, Austin, Tex.) Solexa Genome Analysis System (Illumina, Hayward, Calif.), OpenArray Real Time qPCR (BioTrove, Woburn, Mass.) and BeadXpress System (Illumina, Hayward, Calif.).
- GenomeLab GeXP Genetic Analysis System Beckman Coulter, Foster City, Calif.
- SmartCycler® 9600 or GeneXpert(R) Systems Cepheid, Sunnyvale, Calif.
- an “array” is a spatially or logically organized collection of polynucleotide probes. Any array comprising sensor probes specific for two or more of SEQ ID NOs: 1-2114 or a product derived therefrom can be used. Desirably, an array will be specific for 5, 10, 15, 20, 25, 30, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 1000, 1200, 1400, 1600, 1800, 2000 or more of SEQ ID NOs: 1-2114. Expression of these sequences may be detected alone or in combination with other transcripts.
- an array which comprises a wide range of sensor probes for prostate-specific expression products, along with appropriate control sequences.
- An array of interest is the Human Exon 1.0 ST Array (HuEx 1.0 ST, Affymetrix, Inc., Santa Clara, Calif.).
- the polynucleotide probes are attached to a solid substrate and are ordered so that the location (on the substrate) and the identity of each are known.
- the polynucleotide probes can be attached to one of a variety of solid substrates capable of withstanding the reagents and conditions necessary for use of the array.
- Examples include, but are not limited to, polymers, such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, polypropylene and polystyrene; ceramic; silicon; silicon dioxide; modified silicon; (fused) silica, quartz or glass; functionalized glass; paper, such as filter paper; diazotized cellulose; nitrocellulose filter; nylon membrane; and polyacrylamide gel pad. Substrates that are transparent to light are useful for arrays that will be used in an assay that involves optical detection.
- array formats include membrane or filter arrays (for example, nitrocellulose, nylon arrays), plate arrays (for example, multiwell, such as a 24-, 96-, 256-, 384-, 864- or 1536-well, microtitre plate arrays), pin arrays, and bead arrays (for example, in a liquid “slurry”).
- Arrays on substrates such as glass or ceramic slides are often referred to as chip arrays or “chips.” Such arrays are well known in the art.
- the Prostate Cancer Prognosticarray is a chip.
- Array data can be managed and analyzed using techniques known in the art.
- the Genetrix suite of tools can be used for microarray analysis (Epicenter Software, Pasadena, Calif.).
- Probe set modeling and data pre-processing can be derived using the Robust Multi-Array (RMA) algorithm or variant GC-RMA, Probe Logarithmic Intensity Error (PLIER) algorithm or variant iterPLIER.
- RMA Robust Multi-Array
- PLIER Probe Logarithmic Intensity Error
- Variance or intensity filters can be applied to pre-process data using the RMA algorithm, for example by removing target sequences with a standard deviation of ⁇ 10 or a mean intensity of ⁇ 100 intensity units of a normalized data range, respectively.
- one or more pattern recognition methods can be used in analyzing the expression level of target sequences.
- the pattern recognition method can comprise a linear combination of expression levels, or a nonlinear combination of expression levels.
- expression measurements for RNA transcripts or combinations of RNA transcript levels are formulated into linear or non-linear models or algorithms (i.e., an ‘expression signature’) and converted into a likelihood score.
- This likelihood score indicates the probability that a biological sample is from a patient who will exhibit no evidence of disease, who will exhibit systemic cancer, or who will exhibit biochemical recurrence.
- the likelihood score can be used to distinguish these disease states.
- the models and/or algorithms can be provided in machine readable format, and may be used to correlate expression levels or an expression profile with a disease state, and/or to designate a treatment modality for a patient or class of patients.
- results of the expression level analysis can be used to correlate increased expression of RNAs corresponding to SEQ ID NOs: 1-2114, or subgroups thereof as described herein, with prostate cancer outcome, and to designate a treatment modality.
- Factors known in the art for diagnosing and/or suggesting, selecting, designating, recommending or otherwise determining a course of treatment for a patient or class of patients suspected of having prostate cancer can be employed in combination with measurements of the target sequence expression. These techniques include cytology, histology, ultrasound analysis, MRI results, CT scan results, and measurements of PSA levels.
- a certified test comprises a means for characterizing the expression levels of one or more of the target sequences of interest, and a certification from a government regulatory agency endorsing use of the test for classifying the prostate disease status of a biological sample.
- the certified test may comprise reagents for amplification reactions used to detect and/or quantitate expression of the target sequences to be characterized in the test.
- An array of probe nucleic acids can be used, with or without prior target amplification, for use in measuring target sequence expression.
- test is submitted to an agency having authority to certify the test for use in distinguishing prostate disease status and/or outcome.
- Results of detection of expression levels of the target sequences used in the test and correlation with disease status and/or outcome are submitted to the agency.
- a certification authorizing the diagnostic and/or prognostic use of the test is obtained.
- portfolios of expression levels comprising a plurality of normalized expression levels of the target sequences described herein, including SEQ ID NOs:1-2114. Such portfolios may be provided by performing the methods described herein to obtain expression levels from an individual patient or from a group of patients.
- the expression levels can be normalized by any method known in the art; exemplary normalization methods that can be used in various embodiments include Robust Multichip Average (RMA), probe logarithmic intensity error estimation (PLIER), non-linear fit (NLFIT) quantile-based and nonlinear normalization, and combinations thereof.
- Background correction can also be performed on the expression data; exemplary techniques useful for background correction include mode of intensities, normalized using median polish probe modeling and sketch-normalization.
- portfolios are established such that the combination of genes in the portfolio exhibit improved sensitivity and specificity relative to known methods.
- a small standard deviation in expression measurements correlates with greater specificity.
- Other measurements of variation such as correlation coefficients can also be used in this capacity.
- the invention also encompasses the above methods where the specificity is at least about 50% or at least about 60%.
- the invention also encompasses the above methods where the sensitivity is at least about 90%.
- the gene expression profiles of each of the target sequences comprising the portfolio can fixed in a medium such as a computer readable medium.
- a medium such as a computer readable medium.
- This can take a number of forms. For example, a table can be established into which the range of signals (e.g., intensity measurements) indicative of disease or outcome is input. Actual patient data can then be compared to the values in the table to determine the patient samples diagnosis or prognosis.
- patterns of the expression signals e.g., fluorescent intensity
- the expression profiles of the samples can be compared to a control portfolio. If the sample expression patterns are consistent with the expression pattern for a known disease or disease outcome, the expression patterns can be used to designate one or more treatment modalities. For patients with test scores consistent with systemic disease outcome after prostatectomy, additional treatment modalities such as adjuvant chemotherapy (e.g., docetaxel, mitoxantrone and prednisone), systemic radiation therapy (e.g., samarium or strontium) and/or anti-androgen therapy (e.g., surgical castration, finasteride, dutasteride) can be designated.
- adjuvant chemotherapy e.g., docetaxel, mitoxantrone and prednisone
- systemic radiation therapy e.g., samarium or strontium
- anti-androgen therapy e.g., surgical castration, finasteride, dutasteride
- Such patients would likely be treated immediately with anti-androgen therapy alone or in combination with radiation therapy in order to eliminate presumed micro-metastatic disease, which cannot be detected clinically but can be revealed by the target sequence expression signature. Such patients can also be more closely monitored for signs of disease progression.
- adjuvant therapy would not likely be recommended by their physicians in order to avoid treatment-related side effects such as metabolic syndrome (e.g., hypertension, diabetes and/or weight gain) or osteoporosis.
- Patients with samples consistent with NED could be designated for watchful waiting, or for no treatment.
- Patients with test scores that do not correlate with systemic disease but who have successive PSA increases could be designated for watchful waiting, increased monitoring, or lower dose or shorter duration anti-androgen therapy.
- Target sequences can be grouped so that information obtained about the set of target sequences in the group can be used to make or assist in making a clinically relevant judgment such as a diagnosis, prognosis, or treatment choice.
- a patient report comprising a representation of measured expression levels of a plurality of target sequences in a biological sample from the patient, wherein the representation comprises expression levels of target sequences corresponding to any one, two, three, four, five, six, eight, ten, twenty, thirty, fifty or more of the target sequences depicted in SEQ ID NOs: 1-2114, or of the subsets described herein, or of a combination thereof.
- the representation of the measured expression level(s) may take the form of a linear or nonlinear combination of expression levels of the target sequences of interest.
- the patient report may be provided in a machine (e.g., a computer) readable format and/or in a hard (paper) copy.
- the report can also include standard measurements of expression levels of said plurality of target sequences from one or more sets of patients with known disease status and/or outcome.
- the report can be used to inform the patient and/or treating physician of the expression levels of the expressed target sequences, the likely medical diagnosis and/or implications, and optionally may recommend a treatment modality for the patient.
- the articles can also include instructions for assessing the gene expression profiles in such media.
- the articles may comprise a readable storage form having computer instructions for comparing gene expression profiles of the portfolios of genes described above.
- the articles may also have gene expression profiles digitally recorded therein so that they may be compared with gene expression data from patient samples.
- the profiles can be recorded in different representational format. A graphical recordation is one such format. Clustering algorithms can assist in the visualization of such data.
- Kits for performing the desired method(s) comprise a container or housing for holding the components of the kit, one or more vessels containing one or more nucleic acid(s), and optionally one or more vessels containing one or more reagents.
- the reagents include those described in the composition of matter section above, and those reagents useful for performing the methods described, including amplification reagents, and may include one or more probes, primers or primer pairs, enzymes (including polymerases and ligases), intercalating dyes, labeled probes, and labels that can be incorporated into amplification products.
- the kit comprises primers or primer pairs specific for those subsets and combinations of target sequences described herein. At least two, three, four or five primers or pairs of primers suitable for selectively amplifying the same number of target sequence-specific polynucleotides can be provided in kit form. In some embodiments, the kit comprises from five to fifty primers or pairs of primers suitable for amplifying the same number of target sequence-representative polynucleotides of interest.
- the primers or primer pairs of the kit when used in an amplification reaction, specifically amplify at least a portion of a nucleic acid depicted in one of SEQ ID NOs: 1-2114 (or subgroups thereof as set forth herein), an RNA form thereof, or a complement to either thereof.
- the kit may include a plurality of such primers or primer pairs which can specifically amplify a corresponding plurality of different nucleic acids depicted in one of SEQ ID NOs: 1-2114 (or subgroups thereof as set forth herein), RNA forms thereof, or complements thereto.
- At least two, three, four or five primers or pairs of primers suitable for selectively amplifying the same number of target sequence-specific polynucleotides can be provided in kit form.
- the kit comprises from five to fifty primers or pairs of primers suitable for amplifying the same number of target sequence-representative polynucleotides of interest.
- the reagents may independently be in liquid or solid form.
- the reagents may be provided in mixtures.
- Control samples and/or nucleic acids may optionally be provided in the kit.
- Control samples may include tissue and/or nucleic acids obtained from or representative of prostate tumor samples from patients showing no evidence of disease, as well as tissue and/or nucleic acids obtained from or representative of prostate tumor samples from patients that develop systemic prostate cancer.
- the nucleic acids may be provided in an array format, and thus an array or microarray may be included in the kit.
- the kit optionally may be certified by a government agency for use in prognosing the disease outcome of prostate cancer patients and/or for designating a treatment modality.
- kit Instructions for using the kit to perform one or more methods of the invention can be provided with the container, and can be provided in any fixed medium.
- the instructions may be located inside or outside the container or housing, and/or may be printed on the interior or exterior of any surface thereof.
- a kit may be in multiplex form for concurrently detecting and/or quantitating one or more different target polynucleotides representing the expressed target sequences.
- the devices can comprise means for characterizing the expression level of a target sequence of the invention, for example components for performing one or more methods of nucleic acid extraction, amplification, and/or detection.
- Such components may include one or more of an amplification chamber (for example a thermal cycler), a plate reader, a spectrophotometer, capillary electrophoresis apparatus, a chip reader, and or robotic sample handling components. These components ultimately can obtain data that reflects the expression level of the target sequences used in the assay being employed.
- the devices may include an excitation and/or a detection means. Any instrument that provides a wavelength that can excite a species of interest and is shorter than the emission wavelength(s) to be detected can be used for excitation. Commercially available devices can provide suitable excitation wavelengths as well as suitable detection components.
- Exemplary excitation sources include a broadband UV light source such as a deuterium lamp with an appropriate filter, the output of a white light source such as a xenon lamp or a deuterium lamp after passing through a monochromator to extract out the desired wavelength(s), a continuous wave (cw) gas laser, a solid state diode laser, or any of the pulsed lasers.
- Emitted light can be detected through any suitable device or technique; many suitable approaches are known in the art.
- a fluorimeter or spectrophotometer may be used to detect whether the test sample emits light of a wavelength characteristic of a label used in an assay.
- the devices typically comprise a means for identifying a given sample, and of linking the results obtained to that sample.
- Such means can include manual labels, barcodes, and other indicators which can be linked to a sample vessel, and/or may optionally be included in the sample itself, for example where an encoded particle is added to the sample.
- the results may be linked to the sample, for example in a computer memory that contains a sample designation and a record of expression levels obtained from the sample. Linkage of the results to the sample can also include a linkage to a particular sample receptacle in the device, which is also linked to the sample identity.
- the devices also comprise a means for correlating the expression levels of the target sequences being studied with a prognosis of disease outcome.
- Such means may comprise one or more of a variety of correlative techniques, including lookup tables, algorithms, multivariate models, and linear or nonlinear combinations of expression models or algorithms.
- the expression levels may be converted to one or more likelihood scores, reflecting a likelihood that the patient providing the sample will exhibit a particular disease outcome.
- the models and/or algorithms can be provided in machine readable format, and can optionally further designate a treatment modality for a patient or class of patients.
- the device also comprises output means for outputting the disease status, prognosis and/or a treatment modality.
- output means can take any form which transmits the results to a patient and/or a healthcare provider, and may include a monitor, a printed format, or both.
- the device may use a computer system for performing one or more of the steps provided.
- FFPE Formalin-fixed paraffin embedded
- RNA Extraction RNA was extracted and purified from FFPE tissue sections using a modified protocol for the commercially available High Pure FFPE RNA Micro nucleic acid extraction kit (Roche Applied Sciences, Indianapolis, Ind.). RNA concentrations were calculated using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Rockland, Del.).
- RNA Amplification and GeneChip Hybridization Purified RNA was subjected to whole-transcriptome amplification using the WT-Ovation FFPE system including the WT-Ovation Exon and FL-Ovation Biotin V2 labeling modules, with the following modifications. Fifty (50) nanograms of RNA extracted from FFPE sections was used to generate amplified Ribo-SPIA product. For the WT-Ovation Exon sense-target strand conversion kit 4 ug of Ribo-SPIA product were used. All clean-up steps were performed with RNAClean magnetic beads (Agencourt Biosciences).
- Microarray Analysis All data management and analysis was conducted using the Genetrix suite of tools for microarray analysis (Epicenter Software, Pasadena, Calif.). Probe set modeling and data pre-processing were derived using the Robust Multi-Array (RMA) algorithm. The mode of intensity values was used for background correction and RMA-sketch was used for normalization and probe modeling used a median polish routine. A variance filter was applied to data pre-processed using the RMA algorithm, by removing target sequences with a mean intensity of ⁇ 10 intensity units of a normalized data range. Target sequences typically comprise four individual probes that interrogate the expression of RNA transcripts or portions thereof.
- RMA Robust Multi-Array
- RNAs Target sequence annotations and the sequences (RNAs) that they interrogate were downloaded from the Affymetrix website (www.netaffx.com).
- Supervised analysis of differentially expressed RNA transcripts was determined based on the fold change in the average expression (at least 2 fold change) and the associated t-test, with a p-value cut-off of p ⁇ 0.001 between different prostate cancer patient disease states. Linear regression was also used to screen differentially expressed transcripts that displayed an expression pattern of NED>PSA>SYS or SYS>PSA>NED and genes were selected with a p-value cut-off of p ⁇ 0.01 for two-way hierarchical clustering using Pearson's correlation distance metric with complete-linkage cluster distances.
- NED no evidence of disease
- PSA patients with rising PSA or biochemical recurrence
- PSA systemic or recurrent disease
- RNAs RNA levels for RefSeq, dbEST and predicted transcripts
- Table 3 displays the number of target sequences identified in two-way comparisons between different clinical states using the appropriate t-tests and a p-value cut-off of p ⁇ 0.001. At total of 2,114 target sequences (Table 3) were identified as differentially expressed in these comparisons and a principle components analysis demonstrates that these target sequences discriminate the distinct clinical states into three clusters ( FIG. 1A ).
- FIG. 1C depicts a two-way hierarchical clustering dendrogram and expression matrix of 148 target sequences and 22 tumor samples.
- FIG. 2 shows a histogram plot of the metagene expression values for the summarized 526 target sequences in each of the three clinical groups. This 526-metagene achieved maximal separation between clinical groups and low variance within each clinical group. Metagenes comprised of smaller subsets of 21, 18 and 6 target sequences were also generated ( FIG. 3 , Tables 7 and 8). The distinctions between clinical groups with respect to the metagene scores were preserved, although increased within-group variance was observed when using fewer target sequences ( FIG. 3 ).
- POP Patient outcome predictor
- NSC Nearest Shrunken Centroids
- T-tests were used to evaluate the statistical significance of differences in POP scores between ‘recurrent’ (i.e., ‘SYS’) and non-recurrent (i.e., ‘PSA’ and ‘NED’) patient groups (indicated in the figures) and show that increasing the number of target sequences in the metagene combination increases the significance level of the differences in POP scores.
- the data generated from such methods can be used to determine a prognosis for disease outcome, and/or to recommend or designate one or more treatment modalities for patients, to produce patient reports, and to prepare expression profiles.
- RNA transcripts identified from comparison tests described in Table 2. Sequence listings are annotated with the Affymetrix Human Exon 1.0 ST probe selection region ID, proximal annotated gene from RefSeq, and overlap with coding sequence (CDS). SEQ ID Gene No Affy.
- RNA transcripts used to plot hierarchical clustering and expression matrix (‘heat map’) in FIG. 1B.
- the 526 RNA transcripts represent a subset of the differentially expressed transcripts (Table 3) between patients in the three clinical status groups (i.e., ‘SYS’, ‘PSA’ and ‘NED’) disease using linear regression and a p-value cut-off of p ⁇ 0.01. Weighting factors were from the regression coeffecient values; positive and negative values indicated transcripts correlated to increased expression in ‘SYS’ and ‘NED’ disease, respectively with intermediate expression values in the ‘PSA’ disease group. Weighting factors were used to derive 526-metagene values in FIG. 2.
- RNA transcripts used to plot hierarchical clustering and expression matrix (‘heat map’) in FIG. 1C.
- the 148 RNA transcripts represent a subset of the most differentially expressed transcripts between patients with clinically significant ‘recurrent’ (i.e., ‘SYS’) and ‘non-recurrent’ (i.e., ‘PSA’ and ‘NED’) disease. Weighting factors were from the test statistic values; positive and negative values indicated transcripts correlated to increased expression in recurrent and non-recurrent disease, respectively. Weighting factors were used to derive 148-metagene values, which were converted by scaling and normalizing into ‘POP’ scores depicted in FIG. 7.
- CHEK1 Chromogranin A (parathyroid secretory protein 1) CHGA Chromatin accessibility complex 1 CHRAC1 Ceroid-lipofuscinosis, neuronal 5 CLN5 Clusterin CLU Calponin 1, basic, smooth muscle CNN1 Cannabinoid receptor 1 (brain) CNR1 Collagen, type XVIII, alpha 1 COL18A1 Collagen, type I, alpha 1 COL1A1 Collagen, type IV, alpha 3 (Goodpasture antigen) COL4A3 COMM domain containing 5 COMMD5 Catechol-O-methyltransferase COMT Coatomer protein complex, subunit beta 2 (beta prime) COPB2 COP9 constitutive photomorphogenic homolog subunit 5 COPS5 ( Arabidopsis ) Cytoplasmic polyadenylation element binding protein 3 CPEB3 Cysteine-rich secretory protein 3 CRISP3 V-crk sarcoma virus CT10 oncogene homolog (avian)
- MSH6 Microseminoprotein, beta- MSMB Macrophage scavenger receptor 1 MSR1 Macrophage stimulating 1 receptor (c-met-related tyrosine MST1R kinase) Metastasis associated 1 MTA1 5,10-methylenetetrahydrofolate reductase (NADPH) MTHFR Myotrophin MTPN 5-methyltetrahydrofolate-homocysteine methyltransferase MTR Metastasis suppressor 1 MTSS1 Mucin 1, cell surface associated MUC1 MAX dimerization protein 1 MXD1 MAX interactor 1 MXI1 V-myb myeloblastosis viral oncogene homolog (avian) MYB V-myb myeloblastosis viral oncogene homolog (avian)-like 2 MYBL2 Myosin
- PARD3 PAS domain containing serine/threonine kinase PASK Pre-B-cell leukemia homeobox 1 PBX1 Proliferating cell nuclear antigen PCNA PCTAIRE protein kinase 1 PCTK1 Platelet-derived growth factor alpha polypeptide PDGFA Platelet-derived growth factor receptor, alpha polypeptide PDGFRA Platelet-derived growth factor receptor, beta polypeptide PDGFRB Protein disulfide isomerase family A, member 5 PDIA5 PDZ and LIM domain 5 PDLIM5 Phosphatidylethanolamine-binding protein 4 PEBP4 Phosphatidylethanolamine N-methyltransferase PEMT Placental growth factor, vascular endothelial growth factor- PGF related protein Phosphoglycerate kinase 1 PGK1 Progesterone receptor PGR Phosphatase and actin regulator 2 PHACTR2 PHD finger protein 20-like 1 PHF20L1 P
- RNA Peroxisome proliferator-activated receptor delta PPARD Peroxisome proliferator-activated receptor gamma PPARG Protein phosphatase 2 (formerly 2A), regulatory subunit A, PPP2R1B beta isoform Papillary renal cell carcinoma (translocation-associated) PRCC Peroxisomal proliferator-activated receptor A interacting PRIC285 complex 285 Protein kinase, cAMP-dependent, regulatory, type I, alpha PRKAR1A (tissue specific extinguisher 1) Protease, serine, 8 PRSS8 Prostate stem cell antigen PSCA Proteasome (prosome, macropain) 26S subunit, non- PSMD1 ATPase, 1 Patched homolog 1 ( Drosophila ) PTCH1 Patched homo
- RAD21 RAD23 homolog A S. cerevisiae ) RAD23A RAD50 homolog ( S. cerevisiae ) RAD50 RAD54 homolog B ( S. cerevisiae ) RAD54B V-raf-1 murine leukemia viral oncogene homolog 1 RAF1 V-ral simian leukemia viral oncogene homolog B (ras RALB related; GTP binding protein) RAP1, GTP-GDP dissociation stimulator 1 RAP1GDS1 RAP2A, member of RAS oncogene family RAP2A Retinoic acid receptor, alpha RARA RAS p21 protein activator (GTPase activating protein) 1 RASA1 Retinoblastoma 1 (including osteosarcoma) RB1 Retinoblastoma binding protein 6 RBBP6 Retinoblastoma-like 2 (p130) RBL2 Retinol dehydrogenas
- SEC14L1 Sema domain immunoglobulin domain (Ig), short basic SEMA3F domain, secreted, (semaphorin) 3F Serpin peptidase inhibitor, clade B (ovalbumin), member 5 SERPINB5 Serpin peptidase inhibitor, clade I (neuroserpin), member 1 SERPINI1 Splicing factor 1 SF1 Secreted frizzled-related protein 4 SFRP4 SH3-domain binding protein 2 SH3BP2 SH3 domain containing ring finger 2 SH3RF2 Sonic hedgehog homolog ( Drosophila ) SHH Seven in absentia homolog 1 ( Drosophila ) SIAH1 V-ski sarcoma viral oncogene homolog (avian) SKI SKI-like oncogene SKIL Solute carrier family 14 (urea transporter), member 1 (Kidd SLC14A1 blood group) Solute carrier family 20 (phosphate transporter), member 1 SLC20A1 Solute carrier family 14 (urea
- TRMT12 Transient receptor potential cation channel, subfamily M, TRPM8 member 8
- Trichorhinophalangeal syndrome I TRPS1 Tuberous sclerosis 1 TSC1 Tuberous sclerosis 2 TSC2 Tetraspanin 13 TSPAN13 Tetraspanin 14 TSPAN14 Tissue specific transplantation antigen P35B TSTA3 Tetratricopeptide repeat domain 29 TTC29 Thymidylate synthetase TYMS TYRO3 protein tyrosine kinase TYRO3 Ubiquitin-conjugating enzyme E2, J2 (UBC6 homolog, UBE2J2 yeast) UBX domain containing 3 UBXD3 Vesicle-associated membrane protein 2 (synaptobrevin 2) VAMP2 Vav 1 guanine nucleotide exchange factor VAV1 Vav 2 guanine nucleotide exchange factor VAV2 Versican VCAN Vascular endothelial growth factor A VEGFA Vestigial like
- WEE1 WNT1 inducible signaling pathway protein 1 WISP1 Wingless-type MMTV integration site family, member 10B WNT10B Wingless-type MMTV integration site family member 2 WNT2 Wingless-type MMTV integration site family, member 2B WNT2B Wingless-type MMTV integration site family, member 5A WNT5A Wingless-type MMTV integration site family, member 8B WNT8B Werner syndrome WRN Wilms tumor 1 WT1 Xanthine dehydrogenase XDH Xeroderma pigmentosum, complementation group A XPA Xeroderma pigmentosum, complementation group C XPC X-ray repair complementing defective repair in Chinese XRCC1 hamster cells 1 X-ray repair complementing defective repair in Chinese XRCC4 hamster cells 4 X-ray repair complementing defective repair in Chinese XRCC5 hamster cells 5 (double-strand-break rejoining; Ku autoantigen, 80 kDa) X-ray
- the 6-RNA metagene is a subset of the sequences listed in Table 7, also depicted in FIG. 3.
- 18-RNA metagene scores were scaled and normalized to generate ‘POP’ scores depicted in FIG. 4.
- Weighting factors were from the linear regression coefficient values; positive and negative values indicated transcripts correlated to increased expression in ‘SYS’ and ‘NED’ disease, respectively with intermediate expression values in the ‘PSA’ disease group.
- RNA transcripts used to derive 10-RNA metagene values, which were converted by scaling and normalizing into ‘POP’ scores depicted in FIG. 5.
- RNA transcripts were identified using Nearest Shrunken Centroids algorithm with leave-1-out cross-validation to distinguish ‘recurren’ (i.e., ‘SYS’) and ‘non-recurrent’ (i.e., ‘PSA’ and ‘NED’) disease from Table 3 RNA transcripts. Weighting factors were from the test statistic values; positive and negative values indicated transcripts correlated to increased expression in ‘recurrent’ and ‘non-recurrent’ disease, respectively. Seq ID Weights 3 ⁇ 5.48 36 ⁇ 4.93 60 ⁇ 5.72 63 ⁇ 4.79 926 4.61 971 4.68 978 5.27 999 4.74 1014 4.86 1022 6.29
- RNA transcripts used to derive 41-RNA metagene values, which were converted by scaling and normalizing into ‘POP’ scores depicted in FIG. 6.
- RNA transcripts were identified using Nearest Shrunken Centroids algorithm with leave-1-out cross-validation to distinguish ‘recurrent’ (i.e., ‘SYS’) and ‘non-recurrent’ (i.e., ‘PSA’ and ‘NED’) disease from Table 3 RNA transcripts. Weighting factors were from the test statistic values; positive and negative values indicated transcripts correlated to increased expression in ‘recurrent’ and ‘non-recurrent’ disease, respectively.
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Also Published As
Publication number | Publication date |
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US10865452B2 (en) | 2020-12-15 |
EP2291553A1 (de) | 2011-03-09 |
EP2806054A1 (de) | 2014-11-26 |
CA2725978A1 (en) | 2009-12-03 |
AU2009253675A1 (en) | 2009-12-03 |
WO2009143603A1 (en) | 2009-12-03 |
EP2291553A4 (de) | 2011-12-14 |
US20170191133A1 (en) | 2017-07-06 |
AU2009253675A2 (en) | 2011-01-27 |
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