The present invention relates to prostate cancer. More specifically, the present invention relates to a method to detect prostate cancer in a patient sample by detecting the RNA encoded by the gene PCA3. More particularly the present invention relates to a method for determining a predisposition, or presence of prostate cancer in a patient comprising: (a) contacting a biological sample of a patient with at least one oligonucleotide that hybridizes to a PCA3 polynucleotide; (b) detecting in the biological sample an amount of PCA3 and second prostate specific polynucleotides; and (c) comparing the amount of PCA3 polynucleotide that hybridizes to the oligonucleotide to a predetermined cut off value, and therefrom determining the presence or absence of prostate cancer in the biological sample. The present invention further relates to diagnostic kits for the detection of prostate cancer or the risk of developing same in a patient comprising: (a) at least one container means having disposed therein at least one oligonucleotide probe or primer that hybridizes to one a PCA3 nucleic acid or complement thereof; (b) at least one oligonucleotide probe or primer that hybridizes with a second prostate specific nucleic acid or complement thereof; and (c) reagents enabling a detection of PCA3 and of the second prostate specific nucleic acid when PCA3 or second prostate-specific nucleic acid sequence is present.
DescriptionThe present invention relates to prostate cancer. More specifically, the present invention relates to a method to detect prostate cancer in a patient sample by detecting an RNA encoded by the prostate cancer antigen PCA3 gene.
Over the last decade, cancer of the prostate has become the most commonly diagnosed malignancy among men and the second leading cause of male cancer deaths in the western population, following lung cancer.
Early detection and treatment of prostate cancer before it has spread from the prostate gland, reduces the mortality of the disease. This is particularly true for younger men who are at greater risk of dying from this pernicious but slowly growing malignancy. This realization has prompted increasing efforts for early diagnosis and treatment. Indeed, the American Cancer Society and the American Urological Association recommend that male population at large undergo annual screening for prostate cancer beginning at age 50. The recommended age for screening is lowered to 40 for men giving a family history of prostate cancer or other risk factors.
With this increasing focus on prostate cancer screening, more men than ever before are being routinely tested for prostate cancer. Not surprisingly, this practice has increased early detection of onset of the disease, as reflected by an apparent increase in the incidence of prostate cancer and decrease in the apparent average age of diagnosis. The clinical hope is that earlier detection of prostate cancer before it metastasizes will reduce the overall mortality rate. Healthcare payers look for early screening and detection to translate into a reduction in the healthcare burden, as early treatment can be less radical, more successful and therefore provided at a lower cost per treated patient. The key to accomplishing this goal remains providing better differential diagnostic tools.
Screening for prostate cancer now involves both palpation of the prostate by digital rectal examination (DRE) and assay of plasma levels of prostate specific antigen (PSA/hK3/hKLK3). PSA is a serine protease produced by the prostatic epithelium that is normally secreted in the seminal fluid to liquefy it. Disruption of the anatomic integrity of the prostate gland can compromise the cellular barriers that normally restrict PSA to within the duct system of the prostate, allowing it to disperse into blood or urine. A number of conditions can result in leakage of PSA into the blood. They include inflammation of the prostate, urinary retention, prostatic infection, benign prostatic hyperplasia (BPH), and prostate cancer. Physical manipulation of the prostate can also increase serum PSA levels, but a mild stimulus, such as digital rectal examination (DRE), does not normally increase serum PSA. It is therefore not surprising that screening of serum PSA as an indicator of prostate cancer is not absolutely predictive.
Despite the fact that measure of blood PSA levels can be the result from a variety of different causes, it is nonetheless the basis for primary screening for prostate cancer. Measurement of total PSA (tPSA) as a diagnostic assay to predict prostate cancer has been in use since 1991. Levels of 4 ng/ml or greater in blood serum are considered abnormal and predictive of prostate cancer. However, the sensitivity of such elevated tPSA levels is only 79%; thus leaving 21% of patients with prostate cancer undetected. The specificity for all tPSA values of 4 ng/ml or greater is very poor. In addition, estimates of specificity for tPSA levels >4.0 ng/ml are reported to be in the range of 20% to 59%, averaging around 33%. The vast majority of false positives are ultimately shown to be benign prostatic hyperplasia (BPH). The specificity is lowest for modestly elevated tPSA, in the low so-called gray zone of 4 to 10 ng/ml. This low level of specificity results in additional more invasive and costly diagnostic procedures, such as transrectal ultrasounds and prostate biopsies. Such tests when unnecessary are also very traumatic for the patient. The psychological impact of being diagnosed as positive until proven as a false positive should not be understated either.
Because of the shortcomings of tPSA, research has been focused on attempting to develop PSA derivatives to increase the sensitivity and specificity of this general diagnostic approach.
One modification is free PSA (fPSA), which was FDA approved in 1998. PSA in serum can be found either in an unbound form or complexed with circulating protease inhibitors, most commonly with alpha-1-antitrypsin (ACT). Clinicians have shown that the proportion of PSA bound to ACT was significantly higher in men with prostate cancer than in unaffected men or those with BPH. As a guideline, if 25% orless of total PSA is free, this is an indicator of possible prostate cancer. The fPSA assay was approved for use in men with tPSA's for 4 to 10 ng/ml. Thus, the fPSA assay was positioned to improve the specificity over that of tPSA alone. However, the predictivity of the fPSA test is not as good in people with really low or really high tPSA levels. Very low tPSA, regardless of measured fPSA, is predictive of not having cancer, while the converse is true with very high tPSA levels. The diagnostic usefulness of fPSA is relatively limited as it can be associated with either BPH or prostate cancer. The use of fPSA in combination with tPSA has been shown to reduce the number of unnecessary biopsies by about 20%.
Clearly, prostate biopsy is the gold standard for confirming prostate cancer. However, even a biopsy is not always 100% certain. The standard is the sextant biopsy where tissue sample collection is guided by transrectal ultrasound. Often six samples are not enough to detect the cancer and either a second biopsy procedure or more than six samples are required.
Despite the improvements in prostate cancer screening over the last ten years, there remains a large unmet need in diagnostic sensitivity and specificity, even when these tools are used in combination. Coupling this need with the large incidence of prostate cancer and the importance for early, accurate detection, the potential usefulness for a true differential diagnostic tool is very significant.
A new prostate cancer marker, PCA3, was discovered a few years ago by differential display analysis intended to highlight genes associated with prostate cancer development. PCA3 is located on chromosome 9 and composed of four exons. It encodes at least four different transcripts which are generated by alternative splicing and polyadenylation. By RT-PCR analysis, PCA3 expression was found to be limited to the prostate and absent in all other tissues tested, including testis, ovary, breast and bladder. Northern blot analysis showed that PCA3 is highly expressed in the vast majority of prostate cancers examined (47 out of 50) whereas no or very low expression is detected in BPH or normal prostate cells from the same patients [Cancer Res 1999 December 1;59(23):5975-9]. Moreover, a recent study comparing the clinical performance of RNA telomerase RT and RNA PCA3 detection in the case of prostate cancer showed that the PCA3 gene can be considered as a better marker (Cancer Res 2002 May 1;62(9):2695-8).
The PCA3 gene is composed of 4 exons (e1-e4) and 3 introns (i1-i3). While PCA3 appears to be recognized as the best prostate-cancer marker ever identified, this specificity has been contested in the literature. For example, Gandini et al. 2003, claim that the prostate-specific expression of PCA3 is restricted to that of exon 4 of the PCA3 gene. However, the applicants have shown in a recent patent application that this is not the case (Patent application CA 2,432,365).
In view of the fact that advanced prostate cancer remains a life threatening disease reaching a very significant proportion of the male population, there remains a need to provide the most specific, selective, and rapid prostate cancer detection methods and kits.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
The present invention relates to diagnostic methods and kits to detect prostate cancer, which are more specific and selective than the methods and kits of the prior art.
The present invention relates to a method to detect prostate cancer in a patient and especially from a urine sample thereof by detecting the RNA encoded by the PCA3 gene.
The invention further relates to a method of diagnosing the presence or predisposition to develop prostate cancer in a patient. Also disclosed is a method for monitoring the progression of prostate cancer in a patient.
In one particular embodiment, the present invention relates to a method to detect prostate cancer in urine samples by detecting the presence of RNA encoded by the PCA3 gene. In one embodiment, RNA encoded by the PCA3 gene is detected using an amplification method, which simultaneously amplifies a second prostate-specific nucleic acid sequence also contained in the sample.
In one further particular embodiment of the present invention, the amplified second prostate specific marker is selected from the group consisting of PSA, human kallikrein 2 (hK2/KLK2), prostate specific membrane antigen (PSMA), transglutaminase 4, acid phosphatase or PCGEM1 RNA.
In another embodiment of the present invention, the RNA is detected using an RNA amplification method. In a further embodiment, the RNA amplification method is coupled to real-time detection of the amplified products using fluorescence specific probes. In yet a further embodiment, the amplification method is PCR. In an additional embodiment the PCR is real-time PCR or a related method enabling detection in real-time of the amplified products.
In a related embodiment RNA encoded by the PCA3 gene is detected in a nucleic acid extract by an in vitro RNA amplification method named Nucleic Acid Based Amplification (NASBA). Of course other RNA amplification methods are known and the instant methods and kits are therefore not limited to NASBA. Non-limiting examples of such RNA amplification methods include transcriptase mediated amplification (TMA), strand displacement amplification (SDA) and ligase chain reaction (LCR).
In a further embodiment, the amplified products are detected in a homogenous phase using a fluorescent probe. In one embodiment, the Beacon approach is used. In another embodiment, the product is detected on solid phase using fluorescent or colorimetric method. It should thus be understood that numerous fluorescent, calorimetric or enzymatic methods can be used in accordance with the present invention to detect and/or quantify RNAs. Other types of labelled probes and primers or other types of detection methods may also be used in the present invention (e.g., hybridization assays such as Northern blots, dot blots or slot blots and radiolabelled probes and primers).
In one embodiment, the RNA encoded by the PCA3 gene is obtained from a cell contained in a voided urine sample from the patient.
In another embodiment, the urine sample is obtained after an attentive digital rectal examination (DRE). Of course, it should be understood that the present methods and kits could also be used on a urine sample obtained without DRE, or on other types of samples such as sperm or mixed urine and sperm (e.g., first urine sample following ejaculation), provided that the amplification method and/or detection method is sensitive enough to detect the targeted markers (PCA3 and second marker). Experiments showed that the methods and kits of the present invention can also be performed with these types of samples. Other samples that can be used include blood or serum.
In one embodiment, the cells collected from the urine sample are harvested and a total nucleic acid extraction is carried out. In one particular embodiment, total nucleic acid extraction is carried out using a solid phase band method on silica beads as described by BOOM et al., (1990, J. Clin. Microbiol. 28: 495-503). In another embodiment, the nucleic acids are purified using another target capture method (see below). Of course, it should be understood that numerous nucleic acid extraction and purification methods exist and thus, that other methods could be used in accordance with the present invention. Non-limiting examples include a phenol/chloroform extraction method and target capture purification method (see below). Other such methods are described in herein referenced textbooks. It should also be recognized that numerous means to stabilize or protect the prostate cells contained in the urine sample or other sample, as well as to stabilize or protect the RNA present in these cells are well known in the art.
In another embodiment, the methods of the present invention are carried out using a crude, unpurified, or semi-purified sample.
In one particular embodiment, the present invention also relates to a prostate cancer diagnostic kit for detecting the presence of PCA3 nucleic acid in a sample. Such kit generally comprises a first container means having disposed therein at least one oligonucleotide probe and/or primer that hybridizes to a PCA3 nucleic acid (e.g. PCA3 RNA) and a second container means containing at least one other oligonucleotide primer and/or probe that hybridizes to the above-mentioned second prostate-specific sequence. In another embodiment, a third container means contains a probe which specifically hybridizes to the PCA3 amplification product. In a preferred embodiment, the kit further includes other containers comprising additional components such as a additional oligonucleotide or primer and/or one or more of the following: buffers, reagents to be used in the assay (e.g. wash reagents, polymerases, internal controls (IC) or else) and reagents capable of detecting the presence of bound nucleic acid probe(s)/primer(s). Of course numerous embodiments of the kits of the present invention are possible. For example, the different container means can be divided in amplifying reagents and detection reagents. In one such an embodiment, a first container means contains amplification or hybridization reagents specific for the target nucleic acids of the present invention (e.g., PCA 3, second prostate specific and internal control nucleic acids) and the second container means contains detection reagents. Alternatively, the detection reagents and amplification reagents can be contained in the same container mean.
The present invention in addition relates to a prostate cancer diagnostic kit for detecting the presence of PCA3 nucleic acid in a sample. Such kit generally comprises a first container means having disposed therein at least one oligonucleotide probe and/or primer that hybridizes to a PCA3 mRNA and a second container means containing at least one other oligonucleotide primer and/or probe that hybridizes to the mRNA of the second prostate-specific sequence. In another embodiment, a third container means contains a probe which specifically hybridizes to the PCA3 amplification product. In a yet another embodiment a fourth container means contains a probe which specifically hybridizes to the second prostate specific mRNA. In a preferred embodiment, the kit further includes other containers comprising additional components such as a additional oligonucleotide or primer (e.g., for internal control) and/or one or more of the following: buffers, reagents to be used in the assay (e.g. wash reagents, polymerases, internal control nucleic acid or cells or else) and reagents capable of detecting the presence of bound nucleic acid probe(s)/primer(s). Of course the separation or assembly of reagents in same or different container means is dictated by the types of extraction, amplification or hybridization methods, and detection methods used as well as other parameters including stability, need for preservation etc.
Multiple methods and kits are encompassed by the present invention. For example, the detection and or amplification of the PCA3 nucleic acid sequence does not need to be identical to that of the second prostate specific polynucleotide or other targeted sequences. Thus for example a method or kit which would be RNA based for PCA3 could be DNA based for the second prostate marker or for other targeted sequences.
It should be understood by a person of ordinary skill that numerous statistical methods can be used in the context of the present invention to determine if the test is positive or negative. The decisional tree used is only one non-limiting example of such a statistical method.
Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Commonly understood definitions of molecular biology terms can be found for example in Dictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton et al., 1994, John Wiley & Sons, New York, N.Y.) or The Harper Collins Dictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York, N.Y.), Rieger et al., Glossary of genetics: Classical and molecular, 5th edition, Springer-Verlag, N.Y., 1991; Alberts et al., Molecular Biology of the Cell, 4th edition, Garland science, New-York, 2002; and, Lewin, Genes VII, Oxford University Press, New-York, 2000. Generally, the procedures of molecular biology methods and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as for example Sambrook et al. (2000, Molecular CloningâA Laboratory Manual, Third Edition, Cold Spring Harbor Laboratories); and Ausubel et al. (1994, Current Protocols in Molecular Biology, John Wiley & Sons, New-York).
In the present description, a number of terms are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.
Nucleotide sequences are presented herein by single strand, in the 5â² to 3â² direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission.
The present description refers to a number of routinely used recombinant DNA (rDNA) technology terms. Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency.
As used herein, ânucleic acid moleculeâ or âpolynucleotidesâ, refers to a polymer of nucleotides. Non-limiting examples thereof include DNA (e.g. genomic DNA, cDNA), RNA molecules (e.g. mRNA) and chimeras thereof. The nucleic acid molecule can be obtained by cloning techniques or synthesized. DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense]). Conventional ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) are included in the term ânucleic acidâ and polynucleotides as are analogs thereof. A nucleic acid backbone may comprise a variety of linkages known in the art, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (referred to as âpeptide nucleic acidsâ (PNA); Hydig-Hielsen et al., PCT Int'l Pub. No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages or combinations thereof. Sugar moieties of the nucleic acid may be ribose or deoxyribose, or similar compounds having known substitutions, e.g., 2â² methoxy substitutions (containing a 2â²-O-methylribofuranosyl moiety; see PCT No. WO 98/02582) and/or 2â² halide substitutions. Nitrogenous bases may be conventional bases (A, G, C, T, U), known analogs thereof (e.g., inosine or others; see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992), or known derivatives of purine or pyrimidine bases (see, Cook, PCT Int'l Pub. No. WO 93/13121) or âabasicâ residues in which the backbone includes no nitrogenous base for one or more residues (Arnold et al., U.S. Pat. No. 5,585,481). A nucleic acid may comprise only conventional sugars, bases and linkages, as found in RNA and DNA, or may include both conventional components and substitutions (e.g., conventional bases linked via a methoxy backbone, or a nucleic acid including conventional bases and one or more base analogs). The terminology âPCA3 nucleic acidâ or âPCA3 polynucleotidesâ refers to a native PCA3 nucleic acid sequence. In one embodiment, the PCA3 nucleic acid has the sequence has set forth in SEQ ID NOs 9,10 and 13. In another embodiment, the PCA3 nucleic acid encodes a PCA3 protein. In a further embodiment, the PCA3 nucleic acid is a non-coding nucleic acid sequence. In yet a further embodiment, the PCA3 sequence which is targeted by the PCA3 sequences encompassed by the present invention, is a natural PCA3 sequence found in a patient sample.
The term ârecombinant DNAâ as known in the art refers to a DNA molecule resulting from the joining of DNA segments. This is often referred to as genetic engineering. The same is true for ârecombinant nucleic acidâ.
The term âDNA segmentâ is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides. This sequence when read in accordance with the genetic code (e.g., an open reading frame or ORF), can encode a linear stretch or sequence of amino acids which can be referred to as a polypeptide, protein, protein fragment and the like.
The terminology âamplification pairâ or âprimer pairâ refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes.
âAmplificationâ refers to any known in vitro procedure for obtaining multiple copies (âampliconsâ) of a target nucleic acid sequence or its complement or fragments thereof. In vitro amplification refers to production of an amplified nucleic acid that may contain less than the complete target region sequence or its complement. Known in vitro amplification methods include, e.g., transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification and strand-displacement amplification (SDA). Replicase-mediated amplification uses self-replicating RNA molecules, and a replicase such as Qβ-replicase (e.g., Kramer et al., U.S. Pat. No. 4,786,600). PCR amplification is well known and uses DNA polymerase, primers and thermal cycling to synthesize multiple copies of the two complementary strands of DNA or cDNA (e.g., Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation (e.g., EP Pat. App. Pub. No.0 320 308). SDA is a method in which a primer contains a recognition site for a restriction endonuclease that permits the endonuclease to nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps (e.g., Walker et al., U.S. Pat. No. 5,422,252). Another known strand-displacement amplification method does not require endonuclease nicking (Dattagupta et al., U.S. Pat. No. 6,087,133). Transcription-mediated amplification is used in the present invention. Those skilled in the art will understand that the oligonucleotide primer sequences of the present invention may be readily used in any in vitro amplification method based on primer extension by a polymerase. (see generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25 and (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 2000, Molecular CloningâA Laboratory Manual, Third Edition, CSH Laboratories). As commonly known in the art, the oligos are designed to bind to a complementary sequence under selected conditions.
As used herein, the term âphysiologically relevantâ is meant to describe interactions that can modulate a function which is physiologically relevant. In the present invention, encompassed for example the transcription of a gene in its natural setting. Of course a binding of a protein to PCA3 may also be considered as a physiologically relevant function if this binding occur in a natural setting.
The term âDNAâ molecule or sequence (as well as sometimes the term âoligonucleotideâ) refers to a molecule comprised generally of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C). In âRNAâ, T is replaced by uracil (U). As used herein, particular DNA or RNA sequences may be described according to the normal convention of giving only the sequence in the 5â² to 3â² direction.
Agarose Gel Electrophoresis. The most commonly used technique (though not the only one) for fractionating double stranded DNA is agarose gel electrophoresis. The principle of this method is that DNA molecules migrate through the gel as though it were a sieve that retards the movement of the largest molecules to the greatest extent and the movement of the smallest molecules to the least extent. Note that the smaller the DNA fragment, the greater the mobility under electrophoresis in the agarose gel.
The DNA fragments fractionated by agarose gel electrophoresis can be visualized directly by a staining procedure if the numberof fragments included in the pattern is small. In ordertovisualize a small subset of these fragments, a methodology referred to as a hybridization procedure (e.g., Southern hybridization) can be applied.
âNucleic acid hybridizationâ refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al., 2000, supra and Ausubel et al., 1994, supra) and are commonly known in the art. In the case of a hybridization to a nitrocellulose filter (or other such support like nylon), as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 65° C. with a labeled probe in a solution containing high salt (6ÃSSC or 5ÃSSPE), 5à Denhardt's solution, 0.5% SDS, and 100 μg/ml denatured carrier DNA (e.g. salmon sperm DNA). The non-specifically binding probe can then be washed off the filter by several washes in 0.2ÃSSC/0.1% SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42° C. (moderate stringency) or 65° C. (high stringency). The salt and SDS concentration of the washing solutions may also be adjusted to accommodate for the desired stringency. The selected temperature and salt concentration is based on the melting temperature (Tm) of the DNA hybrid. Of course, RNA-DNA hybrids can also be formed and detected. In such cases, the conditions of hybridization and washing can be adapted according to well known methods by the person of ordinary skill. Stringent conditions will be preferably used (Sambrook et al., 2000, supra). Other protocols or commercially available hybridization kits (e.g., ExpressHyb⢠from BD Biosciences Clonetech) using different annealing and washing solutions can also be used as well known in the art.
A âprobeâ is meant to include a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid or its complement, under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection may either be direct (i.e, resulting from a probe hybridizing directly to the target or amplified sequence) or indirect (i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target or amplified sequence). A probe's âtargetâ generally refers to a sequence within an amplified nucleic acid sequence (i.e, a subset of the amplified sequence) that hybridizes specifically to at least a portion of the probe sequence by standard hydrogen bonding or âbase pairing.â Sequences that are âsufficiently complementaryâ allow stable hybridization of a probe sequence to a target sequence, even if the two sequences are not completely complementary. A probe may be labeled or unlabeled.
By âsufficiently complementaryâ is meant a contiguous nucleic acid base sequence that is capable of hybridizing to another sequence by hydrogen bonding between a series of complementary bases. Complementary base sequences may be complementary at each position in sequence by using standard base pairing (e.g., G:C, A:T or A:U pairing) or may contain one or more residues (including abasic residues) that are not complementary by using standard base pairing, but which allow the entire sequence to specifically hybridize with another base sequence in appropriate hybridization conditions. Contiguous bases of an oligomer are preferably at least about 80% (81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%), more preferably at least about 90% complementary to the sequence to which the oligomer specifically hybridizes. Appropriate hybridization conditions are well known to those skilled in the art, can be predicted readily based on sequence composition and conditions, or can be determined empirically by using routine testing (see Sambrook et al., Molecular Cloning, A Laboratoiy Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly at §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).
Nucleic acid sequences may be detected by using hybridization with a complementary sequence (e.g., oligonucleotide probes) (see U.S. Pat. No. 5,503,980 (Cantor), U.S. Pat. No. 5,202,231 (Drmanac et al.), U.S. Pat. No. 5,149,625 (Church et al.), U.S. Pat. No. 5,112,736 (Caldwell et al.), U.S. Pat. No. 5,068,176 (Vijg et al.), and U.S. Pat. No. 5,002,867 (Macevicz)). Hybridization detection methods may use an array of probes (e.g., on a DNA chip) to provide sequence information about the target nucleic acid which selectively hybridizes to an exactly complementary probe sequence in a set of four related probe sequences that differ one nucleotide (see U.S. Pat. Nos. 5,837,832 and 5,861,242 (Chee et al.)).
A detection step may use any of a variety of known methods to detect the presence of nucleic acid by hybridization to a probe oligonucleotide. One specific example of a detection step uses a homogeneous detection method such as described in detail previously in Arnold et al. Clinical Chemistry 35:1588-1594 (1989), and U.S. Pat. No. 5,658,737 (Nelson et al.), and U.S. Pat. Nos. 5,118,801 and 5,312,728 (Lizardi et al.).
The types of detection methods in which probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds (e.g protein detection by far western technology: Guichet et al., 1997, Nature 385(6616): 548-552; and Schwartz et al., 2001, EMBO 20(3): 510-519). Other detection methods include kits containing reagents of the present invention on a dipstick setup and the like. Of course, it might be preferable to use a detection method which is amenable to automation. A non-limiting example thereof includes a chip or other support comprising one or more (e.g. an array) of different probes.
A âlabelâ refers to a molecular moiety or compound that can be detected or can lead to a detectable signal. A label is joined, directly or indirectly, to a nucleic acid probe or the nucleic acid to be detected (e.g., an amplified sequence). Direct labeling can occur through bonds or interactions that link the label to the nucleic acid (e.g., covalent bonds or non-covalent interactions), whereas indirect labeling can occur through use a âlinkerâ or bridging moiety, such as additional oligonucleotide(s), which is either directly or indirectly labeled. Bridging moieties may amplify a detectable signal. Labels can include any detectable moiety (e.g., a radionuclide, ligand such as biotin or avidin, enzyme or enzyme substrate, reactive group, chromophore such as a dye or colored particle, luminescent compound including a bioluminescent, phosphorescent or chemiluminescent compound, and fluorescent compound). Preferably, the label on a labeled probe is detectable in a homogeneous assay system, i.e., in a mixture, the bound label exhibits a detectable change compared to an unbound label.
Other methods of labeling nucleic acids are known whereby a label is attached to a nucleic acid strand as it is fragmented, which is useful for labeling nucleic acids to be detected by hybridization to an array of immobilized DNA probes (e.g., see PCT No. PCT/IB99/02073).
A âhomogeneous detectable labelâ refers to a label whose presence can be detected in a homogeneous fashion based upon whether the labeled probe is hybridized to a target sequence. A homogeneous detectable label can be detected without physically removing hybridized from unhybridized forms of the labeled probe. Homogeneous detectable labels and methods of detecting them have been described in detail elsewhere (e.g., see U.S. Pat. Nos. 5,283,174, 5,656,207 and 5,658,737).
As used herein, âoligonucleotidesâ or âoligosâ define a molecule having two or more nucleotides (ribo or deoxyribonucleotides). The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill. An oligonucleotide can be synthesized chemically or derived by cloning according to well known methods. While they are usually in a single-stranded form, they can be in a double-stranded form and even contain a âregulatory regionâ. They can contain natural rare or synthetic nucleotides. They can be designed to enhance a chosen criteria like stability for example.
As used herein, a âprimerâ defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an initiation point for nucleic acid synthesis under suitable conditions. Primers can be, for example, designed to be specific for certain alleles so as to be used in an allele-specific amplification system. For example, a primer can be designed so as to be complementary to a short PCA3 RNA which is associated with a malignant state of the prostate, whereas a long PCA3 RNA is associated with a non-malignant state (benign) thereof (PCT/CA00/01154 published under No. WO 01/23550). The primer's 5â² region may be non-complementary to the target nucleic acid sequence and include additional bases, such as a promoter sequence (which is referred to as a âpromoter primerâ). Those skilled in the art will appreciate that any oligomer that can function as a primer can be modified to include a 5â² promoter sequence, and thus function as a promoter primer. Similarly, any promoter primer can serve as a primer, independent of its functional promoter sequence. Of course the design of a primer from a known nucleic acid sequence is well known in the art. As for the oligos, it can comprise a number of types of different nucleotides.
Transcription-associated amplification. Amplifying a target nucleic acid sequence by using at least two primers can be accomplished using a variety of known nucleic acid amplification methods, but preferably uses a transcription-associated amplification reaction that is substantially isothermal. By using such an in vitro amplification method, many strands of nucleic acid are produced from a single copy of target nucleic acid, thus permitting detection of the target in the sample by specifically binding the amplified sequences to one or more detection probes. Transcription-associated amplification methods have been described in detail elsewhere (e.g., U.S. Pat. Nos. 5,399,491 and 5,554,516). Briefly, transcription-associated amplification uses two types of primers (one being a promoter primer because it contains a promoter sequence for an RNA polymerase), two enzyme activities (a reverse transcriptase (RT) and an RNA polymerase), substrates (deoxyribonucleoside triphosphates, ribonucleoside triphosphates) and appropriate salts and buffers in solution to produce multiple RNA transcripts from a nucleic acid template. Initially, a promoter primer hybridizes specifically to a target sequence (e.g., RNA) and reverse transcriptase creates a first complementary DNA strand (cDNA) by extension from the 3â² end of the promoter primer. The cDNA is made available for hybridization with the second primer by any of a variety of methods, such as, by denaturing the target-cDNA duplex or using RNase H activity supplied by the RT that degrades RNA in a DNA:RNA duplex. A second primer binds to the cDNA and a new strand of DNA is synthesized from the end of the second primer using the RT activity to create a double-stranded DNA (dsDNA) having a functional promoter sequence at one end. An RNA polymerase binds to the dsDNA promoter sequence and transcription produces multiple transcripts (âampliconsâ). Amplicons are used in subsequent steps or cycles of the transcription-associated amplification process by serving as a new template for replication, thus generating many copies of amplified nucleic acid (i.e., about 100 to 3,000 copies of RNA are synthesized from each template).
NASBA. Nucleic Acid Sequence Based Amplification (NASBA) can be carried out in accordance with known techniques (Malek et al. Methods Mol Biol, 28:253-260). In an embodiment, the NASBA amplification starts with the annealing of an antisense primer P1 (containing the T7 RNA polymerase promoter) to the mRNA target. Reverse transcriptase (RTase) then synthesizes a complementary DNA strand. The double stranded DNA/RNA hybrid is recognized by RNase H that digests the RNA strand, leaving a single-stranded DNA molecule to which the sense primer P2 can bind. P2 serves as an anchor to the RTase that synthesizes a second DNA strand. The resulting double-stranded DNA has a functional T7 RNA polymerase promoter recognized by the respective enzyme. The NASBA reaction can then enter in the phase of cyclic amplification comprising six steps: (1) Synthesis of short antisense single-stranded RNA molecules (101 to 103 copies per DNA template) by the T7 RNA polymerase; (2) annealing of primer P2 to these RNA molecules; (3) synthesis of a complementary DNA strand by RTase; (4) digestion of the RNA strand in the DNA/RNA hybrid; (5) annealing of primer P1 to the single-stranded DNA; and (6) generation of double stranded DNA molecules by RTase. Because the NASBA reaction is isothermal (41° C.), specific amplification of ssRNA is possible if denaturation of dsDNA is prevented in the sample preparation procedure. It is thus possible to pick up RNA in a dsDNA background without getting false positive results caused by genomic dsDNA.
Polymerase chain reaction (PCR). Polymerase chain reaction can be carried out in accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188 (the disclosures of all three U.S. Patent are incorporated herein by reference). In general, PCR involves, a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected. An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith. The extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers. Following a sufficient number of rounds of synthesis of extension products, the sample is analyzed to assess whether the sequence or sequences to be detected are present. Detection of the amplified sequence may be carried out by visualization following EtBr staining of the DNA following gel electrophoresis, or using a detectable label in accordance with known techniques, and the like. For a review on PCR techniques (see PCR Protocols, A Guide to Methods and Amplifications, Michael et al. Eds, Acad. Press, 1990).
Ligase chain reaction (LCR) can be carried out in accordance with known techniques (Weiss, 1991, Science 254:1292). Adaptation of the protocol to meet the desired needs can be carried out by a person of ordinary skill. Strand displacement amplification (SDA) is also carried out in accordance with known techniques or adaptations thereof to meet the particular needs (Walker et al., 1992, Proc. Natl. Acad. Sci. USA 89:392-396; and ibid., 1992, Nucleic Acids Res. 20:1691-1696).
Target capture. In one embodiment, target capture is included in the method to increase the concentration or purity of the target nucleic acid before in vitro amplification. Preferably, target capture involves a relatively simple method of hybridizing and isolating the target nucleic acid, as described in detail elsewhere (e.g., see U.S. Pat. Nos. 6,110,678, 6,280,952, and 6,534,273). Generally speaking, target capture can be divided in two family, sequence specific and non sequence specific. In the non-specific method, a reagent (e.g., silica beads) is used to capture non specifically nucleic acids. In the sequence specific method an oligonucleotide attached to a solid support is contacted with a mixture containing the target nucleic acid under appropriate hybridization conditions to allow the target nucleic acid to be attached to the solid support to allow purification of the target from other sample components. Target capture may result from direct hybridization between the target nucleic acid and an oligonucleotide attached to the solid support, but preferably results from indirect hybridization with an oligonucleotide that forms a hybridization complex that links the target nucleic acid to the oligonucleotide on the solid support. The solid support is preferably a particle that can be separated from the solution, more preferably a paramagnetic particle that can be retrieved by applying a magnetic field to the vessel. After separation, the target nucleic acid linked to the solid support is washed and amplified when the target sequence is contacted with appropriate primers, substrates and enzymes in an in vitro amplification reaction.
Generally, capture oligomer sequences include a sequence that specifically binds to the target sequence, when the capture method is indeed specific, and a âtailâ sequence that links the complex to an immobilized sequence by hybridization. That is, the capture oligomer includes a sequence that binds specifically to its PCA3 or to another prostate specific marker (e.g., PSA, hK2/KLK2, PMSA, transglutaminase 4, acid phosphatase, PCGEM1) target sequence and a covalently attached 3â² tail sequence (e.g., a homopolymer complementary to an immobilized homopolymer sequence). The tail sequence which is, for example, 5 to 50 nucleotides long, hybridizes to the immobilized sequence to link the target-containing complex to the solid support and thus purify the hybridized target nucleic acid from other sample components. A capture oligomer may use any backbone linkage, but some embodiments include one or more 2â²-methoxy linkages. Of course, other capture methods are well known in the art. The capture method on the cap structure (Edery et al., 1988, gene 74(2): 517-525, U.S. Pat. No. 5,219,989) or the silica based method are two non-limiting examples of capture methods.
An âimmobilized probeâ or âimmobilized nucleic acidâ refers to a nucleic acid that joins, directly or indirectly, a capture oligomer to a solid support. An immobilized probe is an oligomer joined to a solid support that facilitates separation of bound target sequence from unbound material in a sample. Any known solid support may be used, such as matrices and particles free in solution, made of any known material (e.g., nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane polypropylene and metal particles, preferably paramagnetic particles). Preferred supports are monodisperse paramagnetic spheres (i.e., uniform in size±about 5%), thereby providing consistent results, to which an immobilized probe is stably joined directly (e.g., via a direct covalent linkage, chelation, or ionic interaction), or indirectly (e.g., via one or more linkers), permitting hybridization to another nucleic acid in solution.
The term âalleleâ defines an alternative form of a gene which occupies a given locus on a chromosome.
Gene. A DNA sequence generally related but not necessarely related to a single polypeptide chain or protein, and as used herein includes the 5â² and 3â² untranslated regions. The polypeptide can be encoded by a full-length sequence or any portion of the coding sequence, so long as the functional activity of the protein is retained.
Complementary DNA (cDNA). Recombinant nucleic acid molecules synthesized by reverse transcription of messenger RNA (âRNAâ).
Structural Gene. A DNA sequence that is transcribed into RNA that is then translated into a sequence of amino acids characteristic of a specific polypeptide(s).
As commonly known, a âmutationâ is a detectable change in the genetic material which can be transmitted to a daughter cell. As well known, a mutation can be, for example, a detectable change in one or more deoxyribonucleotide. For example, nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position. Spontaneous mutations and experimentally induced mutations exist. A mutant polypeptide can be encoded from this mutant nucleic acid molecule.
As used herein, the term âpurifiedâ refers to a molecule (e.g. nucleic acid) having been separated from a component of the composition in which it was originally present. Thus, for example, a âpurified nucleic acidâ has been purified to a level not found in nature. A âsubstantially pureâ molecule is a molecule that is lacking in most other components (e.g., 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% free of contaminants). By opposition, the term âcrudeâ means molecules that have not been separated from the components of the original composition in which it was present. For the sake of brevity, the units (e.g. 66, 67 . . . 81, 82, . . . 91, 92% . . . ) have not been specifically recited but are considered nevertheless within the scope of the present invention.
As used herein the terminology âprostate specific markerâ relates to any molecule whose presence in the sample indicates that such sample contains prostate cells (or a marker therefrom). Therefore a âprostate specific sequenceâ refers to a nucleic acid or protein sequence specifically found in prostate cells and usually not in other tissues which could âcontaminateâ a particular sample. For certainty, when a urine sample is used, the second prostate specific marker according to the present invention does not have to be solely expressed in the prostate. In fact markers which are solely expressed in one organ or tissue is very rare. However, should the second prostate specific marker be expressed in non-prostate tissue, this non prostate tissue expression will not jeopardized the specificity of this second marker provided that it occurs in cells of tissues or organs which are not normally present in the urine sample. Thus, when urine is the sample, this second prostate-specific marker is not normally expressed in other types of cells (e.g., cells from the urinary tract system) to be found in the urine sample.
Control sample. By the term âcontrol sampleâ or ânormal sampleâ is meant here a sample that does not contain a specifically chosen cancer. In a particular embodiment, the control sample does not contain prostate cancer or is indicative of the absence of prostate cancer. Control samples can be obtained from patients/individuals not afflicted with prostate cancer. Other types of control samples may also be used. For example, a prostate specific marker can be used as to make sure that the sample contains prostate specific cells (this marker is generally described herein as the second prostate-specific marker). In a related aspect, a control reaction may be designed to control the method itself (e.g., The cell extraction, the capture, the amplification reaction or detection method, number of cells present in the sample, a combination thereof or any step which could be monitored to positively validate that the absence of a signal (e.g., the absence of PCA3 signal) is not the result of a defect in one ore more of the steps).
Cut-off value. The cut-off value for the predisposition or presence of prostate cancer is defined from a population of patients without prostate cancer as the average signal of PCA3 (or other prostate cancer antigen) polynucleotides, polypeptides or fragments thereof plus n standard deviations (or average mean signal thereof). Cut off values indicative of the presence or predisposition to develop prostate cancer may be the same or alternatively, they may be different values.
Variant. The term âvariantâ refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention, to maintain at least one of its biological activities. Thus, provided that two molecules possess a common activity and can substitute for each other, they are considered variants as that term is used herein even if the composition, or secondary, tertiary or quaternary structure of one molecule is not identical to that found in the other, or if the amino acid sequence or nucleotide sequence is not identical.
A âbiological sampleâ or âsample of a patientâ is meant to include any tissue or material derived from a living or dead human which may contain the PCA3 target nucleic acid and second prostate specific marker. Samples include, for example, any tissue or material that may contain cells specific for the PCA3 target (or second specific marker), such as peripheral blood, plasma or serum, biopsy tissue, gastrointestinal tissue, bone marrow, urine, feces, semen or other body fluids, tissues or materials, but preferably is a urine sample following digital rectal examination (or other means which increase the content of prostate cells in urine). The biological sample may be treated to physically disrupt tissue or cell structure, thus releasing intracellular components into a solution which may further contain enzymes, buffers, salts, detergents, and the like which are used to prepare the sample for analysis.
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of illustrative embodiments thereof, given by way of example only with reference to the accompanying drawings.
Having thus, generally described the invention, reference will be made to the accompanying drawings, showing by way of illustration only an illustrative embodiment thereof and in which:
FIG. 1 shows the PCA3 gene structure and location of oligonucleotides and probes for in vitro RNA amplification and amplified product detection. In accordance with one embodiment of the present invention. Panel A. Targeting zone of sense PCA3 primer (SEQ ID NO 4); Panel B. Targeting zone of PCA3 molecular beacon (SEQ ID NO 6); and Panel C. Targeting zone of anti-sense PCA3 primer (SEQ ID NO 3).
FIG. 2 shows a decisional tree used to calculate the positivity of the method in a patient with total blood PSA below 4 ng/ml.
FIG. 3 shows a decisional tree used to calculate the positivity of the method in a patient with total blood PSA between 4-10 ng/ml.
FIG. 4 shows a decisional tree used to calculate the positivity of the method in a patient with total blood PSA above 10 ng/ml.
For purposes of clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections:
The invention encompasses methods for detecting the presence of a PCA3 nucleic acid together with a second prostate specific marker (e.g., PSA, hK2/KLK2, PSMA, transglutaminase 4, acid phophatase, PCGEM1) in a biological sample as well as methods for measuring the level of a PCA3 nucleic acid in the sample. Such methods are useful for the diagnostic of prostate cancers associated with PCA3 overexpression.
The predisposition to develop prostate cancer or the presence of such cancer may be detected based on the presence of an elevated amount of PCA3 nucleic acid in a biological sample (e.g., urine) of a patient. Polynucleotides primers and probes may be used to detect the level of PCA3 RNA present, which is indicative of the predisposition, presence or absence of prostate cancer. In general the elevated amount of PCA3 nucleic acid (e.g., PCA3 mRNA or fragments thereof) in a sample as compared to the amount present in a normal control samples (or a determined cut-off value) indicates that the sample contains prostate cancer or is susceptible to develop prostate cancer. In one embodiment, the detection of a second prostate-specific marker is also performed to serve as a control for the presence of prostate specific cells in the sample as well as to further validate the PCA3 detection results (e.g., a negative result obtained with the detection of PCA3).
Of course, a number of different prostate specific marker can be used as long as they can serve as a control for prostate RNA. Non-limiting examples of such prostate-specific markers include PSA (SEQ ID NO 11) and other Kallikrein family members. In addition and as described above, markers such as hK2/KLK2, PSMA, transglutaminase 4, acid phosphatase, PCGEM1 can also be used in accordance with the present invention.
One non limiting example of a method to detect PCA3 nucleic acid (e.g. PCA3 mRNA) in a biological sample is by (1) contacting a biological sample with at least one oligonucleotide probe or primer that hybridizes to a PCA3 polynucleotide; and (2) detecting in the biological sample a level of oligonucleotide (i.e. probe(s) or primer(s)) that hybridizes to the PCA3 polynucleotide. The sample is also tested for the presence of second prostate-specific marker (e.g., PSA, hK2/KLK2, PSMA, transglutaminase 4, acid phosphatase, PCGEM1 mRNA or fragments thereof) to control for the presence of prostate cells in the sample (or their number) as well as to further control a negative or positive result obtained with the detection of PCA3. The second prostate specific marker may also be a prostate specific PCA3 RNA that is not associated with prostate cancer but is expressed in prostate cells. The amount of PCA3 polynucleotide detected can be compared with a predetermined cut off value, and therefrom the predisposition, presence or absence of a prostate cancer in the patient is determined.
In a related aspect, the methods of the present invention can be used for monitoring the progression of prostate cancer in a patient. In this particular embodiment, the assays described above are performed over time and the variation in the level of PCA3 nucleic acid and of another prostate specific marker (e.g., PSA mRNA) present in the sample (e.g., urine sample) is evaluated. In general, prostate cancer is considered as progressing when the relative (i.e. relatively to the amount of cells or cell components (e.g., protein or nucleic acids present therein) level of PCA3 nucleic acid detected increases with time. In contrast a cancer is not considered as progressing when the relative level of PCA3 nucleic acid either decreases or remains constant over time.
One skilled in the art can select the nucleic acid primers according to techniques known in the art as described above. Samples to be tested include but should not be limited to RNA samples from human tissue.
In a related aspect, it is possible to verify the efficiency of nucleic acid amplification and/or detection only, by performing external control reaction(s) using highly purified control target nucleic acids added to the amplification and/or detection reaction mixture. Alternatively, the efficiency of nucleic acid recovery from cells and/or organelles, the level of nucleic acid amplification and/or detection inhibition (if present) can be verified and estimated by adding to each test sample control cells or organelles (e.g., a define number of cells from a prostate cancer cell line expressing PCA3 and second marker) by comparison with external control reaction(s). To verify the efficiency of both, sample preparation and amplification and/or detection, such external control reaction(s) may be performed using a reference test sample or a blank sample spiked with cells, organelles and/or viral particles carrying the control nucleic acid sequence(s). For example, a signal from the internal control (IC) sequences present into the cells, viruses and/or organelles added to each test sample that is lower than the signal observed with the external control reaction(s) may be explained by incomplete lysis and/or inhibition of the amplification and/or detection processes for a given test sample. On the other hand, a signal from the IC sequences that is similar to the signal observed with the external control reaction(s), would confirm that the sample preparation including cell lysis is efficient and that there is no significant inhibition of the amplification and/or detection processes for a given test sample. Alternatively, verification of the efficiency of sample preparation only may be performed using external control(s) analyzed by methods other than nucleic acid testing (e.g. analysis using microscopy, mass spectrometry or immunological assays).
Therefore, in one particular embodiment, the methods of the present invention uses purified nucleic acids, prostate cells or viral particles containing nucleic acid sequences serving as targets for an internal control (IC) in nucleic acid test assays to verify the efficiency of cell lysis and of sample preparation as well as the performance of nucleic acid amplification and/or detection. More broadly, the IC serves to verify any chosen step of the process of the present invention.
IC in PCR or related amplification techniques can be highly purified plasmid DNA either supercoiled, or linearized by digestion with a restriction endonuclease and repurified. Supercoiled IC templates are amplified much less efficiently (about 100 fold) and in a less reproducible manner than linearized and repurified IC nucleic acid templates. Consequently, IC controls for amplification and detection of the present invention are preferably performed with linearized and repurified IC nucleic acid templates when such types of IC are used.
The nucleic acids, cells, and/or organelles are incorporated into each test sample at the appropriate concentration to obtain an efficient and reproducible amplification/detection of the IC, based on testing during the assay optimization. The optimal number of control cells added, which is dependent on the assay, is preferentially the minimal number of cells which allows a highly reproducible IC detection signal without having any significant detrimental effect on the amplification and/or detection of the other genetic target(s) of the nucleic acid-based assay. A sample to which is added the purified linearized nucleic acids, cells, viral particles or organelles is generally referred to as a âspiked sampleâ.
Within certain embodiments, the amount of mRNA may be detected via a RT-PCR based assay. In RT-PCR, the polymerase chain reaction (PCR) is applied in conjunction with reverse transcription. In such an assay, at least two oligonucleotide primers may be used to amplify a portion of PCA3 cDNA derived from a biological sample, wherein at least one oligonucleotide is specific for (i.e. hybridizes to) a PCA3 RNA. The amplified cDNA may then be separated and detected using techniques that are well known in the art such as gel electrophoresis and ethidium bromide staining. Amplification may be performed on biological samples taken from a test patient and an individual who is not afflicted with a prostate cancer (control sample), or using other types of control samples. The amplification reaction may be performed on several dilutions of cDNA (or directly on several dilutions of the biological sample) spanning, for example, two orders of magnitude. A value above a predetermined cut off value is indicative of the presence or predisposition to develop prostate cancer. In general, the elevated expression of PCA3 nucleic acid in a biological sample as compared to control samples indicates the presence or the predisposition to develop prostate cancer.
In further embodiments, PCA3 RNA is detected in a nucleic acid extract from a biological sample by an in vitro RNA amplification method named Nucleic Acid Sequence-Based Amplification (NASBA). Other mRNA amplification methods well known in the art may also be used and include transcriptase-mediated amplification (TMA), strand displacement amplification (SDA), the Qβ replicase system and Ligase chain reaction (LCR) (see generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25 and (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 2000, Molecular CloningâA Laboratory Manual, Third Edition, CSH Laboratories).
The amplification and/or detection of prostate cancer specific PCA3 RNA sequences and of the prostate specific marker can be carried out simultaneously (e.g., multiplex real-time amplification assays.)
Alternatively, oligonucleotide probes that specifically hybridize under stringent conditions to a PCA3 nucleic acid may be used in a nucleic acid hybridization assay (e.g., Southern and Northern blots, dot blot, slot blot, in situ hybridization and the like) to determine the presence and/or amount of prostate cancer specific PCA3 polynucleotide in a biological sample.
Alternatively, oligonucleotides and primers could be designed to directly sequence and assess the presence of prostate cancer specific PCA3 sequences in the patient sample following an amplification step. Such sequencing-based diagnostic methods are automatable and are encompassed by the present invention.
1. Synthesis of Nucleic Acid
The nucleic acid (e.g. DNA or RNA) for practicing the present invention may be obtained according to well known methods.
Isolated nucleic acid molecules of the present invention are meant to include those obtained by cloning as well as those chemically synthesized. Similarly, an oligomer which corresponds to the nucleic acid molecule, or to each of the divided fragments, can be synthesized. Such synthetic oligonucleotides can be prepared, for example, by the triester method of Matteucci et al., J. Am. Chem. Soc. 103:3185-3191 (1981) or by using an automated DNA synthesizer.
An oligonucleotide can be derived synthetically or by cloning. If necessary, the 5â²-ends of the oligomers can be phosphorylated using T4 polynucleotide kinase. Kinasing of single strands prior to annealing or for labeling can be achieved using an excess of the enzyme. If kinasing is for the labeling of probe, the ATP can contain high specific activity radioisotopes. Then, the DNA oligomer can be subjected to annealing and ligation with T4 ligase or the like. Of course the labeling of a nucleic acid sequence can be carried out by other methods known in the art.
II. Probes and Primers
The present invention relates to a nucleic acid for the specific detection, in a sample, of the presence of PCA3 nucleic acid sequences which are associated with prostate cancer, comprising the above-described nucleic acid molecules or at least a fragment thereof which binds under stringent conditions to PCA3 nucleic acid.
In one preferred embodiment, the present invention relates to oligos which specifically target and enable amplification (i.e. primers) of PCA3 RNA sequences associated with prostate cancer.
In another embodiment, PCA3 RNA can be detected using a specific probe in an hybridization assay (e.g. Northern blot, dot blot, slot blot and the like).
Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted sequences employed. In a preferred embodiment, the oligonucleotide probes or primers are at least 10 nucleotides in length (preferably, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 . . . ) and they may be adapted to be especially suited for a chosen nucleic acid amplification system. Longer probes and primers are also within the scope of the present invention as well known in the art. Primers having more than 30, more than 40, more than 50 nucleotides and probes having more than 100, more than 200, more than 300, more than 500 more than 800 and more than 1000 nucleotides in length are also covered by the present invention. Of course, longer primers have the disadvantage of being more expensive and thus, primers having between 12 and 30 nucleotides in length are usually designed and used in the art. As well known in the art, probes ranging from 10 to more than 2000 nucleotides in length can be used in the methods of the present invention. As for the % of identity described above, non-specifically described sizes of probes and primers (e.g., 16, 17, 31, 24, 39, 350, 450, 550, 900, 1240 nucleotides, . . . ) are also within the scope of the present invention. In one embodiment, the oligonucleotide probes or primers of the present invention specifically hybridize with a PCA3 RNA (or its complementary sequence). More preferably, the primers and probes will be chosen to detect a PCA3 RNA which is associated with prostate cancer. In one embodiment, the probes and primers used in the present invention do not hybridize with the PCA3 gene (i.e. enable the distinction gene and expressed PCA3). Other primers of the present invention are specific for a second prostate-specific marker such as PSA (SEQ ID NO 11). Of course othervariants well known in the art can also be used (U.S. Pat. Nos. 6,479,263 and 5,674,682) as second prostate specific marker. Because of the structural and sequence similarities of the PSA gene with other members of the kallikrein gene family, the appropriate selection of PSA sequences to serve as PSA-specific probes or primers is critical to methods of amplification and/or detection of PSA specific nucleic acids. Examples of suitable primers for PSA, hK2/KLK2, PSMA, amplification and detection (e.g., U.S. Pat. No. 6,551,778) are well known in the art as well as for transglutaminase 4, acid phosphatase and PCGEM1. In one embodiment, the PSA oligonucleotide may also hybridize to other kallikrein family members such as kallikrein 2 (hK2/hKLK2). One example of such oligonucleotide is SEQ ID no 12.
As commonly known in the art, the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (see below and in Sambrook et al., 1989, Molecular CloningâA Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1994, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).
To enable hybridization to occur under the assay conditions of the present invention, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least 70% (at least 71%, 72%, 73%, 74%), preferably at least 75% (75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%) and more preferably at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity to a portion of a PCA3 polynucleotide. Probes and primers of the present invention are those that hybridize to PCA3 nucleic acid (e.g. cDNA or mRNA) sequence under stringent hybridization conditions and those that hybridize to PCA3 gene homologs under at least moderately stringent conditions. In certain embodiments probes and primers of the present invention have complete sequence identity to PCA3 gene sequence (e.g. cDNA or mRNA). However, probes and primers differing from the native PCA3 gene sequence that keep the ability to hybridize to native PCA3 gene sequence under stringent conditions may also be used in the present invention. It should be understood that other probes and primers could be easily designed and used in the present invention based on the PCA3 nucleic acid sequence disclosed herein ( SEQ ID NOs 9, 10 and 13) by using methods of computer alignment and sequence analysis known in the art (cf. Molecular Cloning: A Laboratory Manual, Third Edition, edited by Cold Spring Harbor Laboratory, 2000).
For example, a primer can be designed so as to be complementary to a short PCA3 RNA which is associated with a malignant state of the prostate cancer, whereas a long PCA3 RNA is associated with a non-malignant state (benign) thereof (PCT/CA00/01154 published under No. WO 01/23550). In accordance with the present invention, the use of such a primer with the other necessary reagents would give rise to an amplification product only when a short PCA3 RNA (e.g., SEQ ID NO: 8) associated with prostate cancer is present in the sample. The longer PCA3 (e.g., SEQ ID NO: 7) would not give rise to an amplicon. Of course, the amplification could be designed so as to amplify a short and a long PCA3 mRNA. In such a format, the long PCA3 mRNA could be used as the second prostate specific marker.
In an embodiment as described above, the quantification of the amplification products of short versus long PCA3 could be carried out together with the detection of another prostate specific marker to serve as a molecular diagnostic test for prostate cancer. In another embodiment, primer pairs (or probes) specific for PCA3 could be designed to avoid the detection of the PCA3 gene or of unspliced PCA3 RNA. For example, the primers sequences to be used in the present invention could span two contiguous exons so that it cannot hybridize to an exon/intron junction of the PCA3 gene. The amplification product obtained by the use of such primer would be intron less between two chosen exons (for examples of such primers and probes see table 1 and 2 below). Therefore, unspliced variants and genomic DNA would not be amplified. It will be recognized by the person of ordinary skill that numerous probes can be designed and used in accordance with a number of embodiments of the present invention. Such tests can be adapted using the sequence of PCA3 and that of the second prostate-specific marker. Of course, different primer pairs (and probes) can be designed from any part of the PCA3 sequences (SEQ ID NOs: 7, 8, 9, 10 and 13) as well as from the sequence of PSA (genbank accession number M27274, SEQ ID NO 11) or any other chosen second prostate specific marker (e.g.,KLK2 (genbank acc. No. NM005551), PSMA (genbank acc. No.BC025672), transglutaminase 4 (genbank acc. No.BC007003), acid phosphatase (genbank acc. No. BC016344), PCGEM 1 (genbank acc. No. AF223389)).
Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and α-nucleotides and the like. Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann. Reports Med. Chem. 23:295 and Moran et al., 1987, Nucleic Acids Res., 14:5019. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. Probes can be labeled according to numerous well-known methods (Sambrook et al., 2000, supra). Non-limiting examples of detectable markers and labels include 3H, 14C, 32P, and 35S, ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
As commonly known, radioactive nucleotides can be incorporated into probes of the invention by several methods. Non-limiting examples thereof include kinasing the 5â² ends of the probes using gamma 32P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E. coli in the presence of radioactive dNTP (e.g. uniformly labeled DNA probe using random oligonucleotide primers), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like.
In one embodiment, the label used in a homogenous detection assay is a chemiluminescent compound (e.g., U.S. Pat. Nos. 5,656,207, 5,658,737 and 5,639,604), more preferably an acridinium ester (âAEâ) compound, such as standard AE or derivatives thereof. Methods of attaching labels to nucleic acids and detecting labels-are well known (e.g., see Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Habor, N.Y., 1989), Chapt. 10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174 and 4,581,333; and European Pat. App. No.0 747 706). Preferred methods of labeling a probe with an AE compound attached via a linker have been previously described detail (e.g., see U.S. Pat. No. 5,639,604, Example 8).
Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25. Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Non-limiting examples of amplification techniques include polymerase chain reaction (PCR, RT PCR . . . ), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Qβ replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 2000, supra). Other non-limiting examples of amplification methods include rolling circle amplification (RCA); signal mediated amplification of RNA technology (SMART); split complex amplification reaction (SCAR); split promoter amplification of RNA (SPAR).
Non-limiting examples of suitable methods to detect the presence of the amplified products include the followings: agarose or polyacrylamide gel, addition of DNA labeling dye in the amplification reaction (such as ethidium bromide, picogreen, SYBER green, etc.) and detection with suitable apparatus (fluorometer in most cases). Other suitable methods include sequencing reaction (either manual or automated); restriction analysis (provided restriction sites were built into the amplified sequences), or any method involving hybridization with a sequence specific probe (Southern or Northern blot, TaqMan⢠probes, molecular beacons, and the like). Of course, other amplification methods are encompassed by the present invention. Molecular beacons are exemplified herein as one method for detecting the amplified products according to the present invention (see below).
Of course in some embodiment direct detection (e.g., sequencing) of PCA3 cancer specific sequences as well as that of another prostate specific marker in a sample may be performed using specific probes or primers.
In one embodiment, the present invention has taken advantage of technological advances in methods for detecting and identifying nucleic acids. Therefore, the present invention is suitable for detection by one of these tools called molecular beacons.
Molecular beacons are single-stranded oligonucleotide hybridization probes/primers that form a stem loop structure. The loop contains a probe sequence that is complementary to a target sequence, and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe/primer sequence. A fluorophore is covalently linked to the end of one arm and a quencher is covalently linked to the end of the other arm. Molecular beacons do not fluoresce when they are free in solution. However, when they hybridize to a nucleic acid strand containing a target sequence they undergo comformational change that enables them to fluoresce brightly (see U.S. Pat. Nos. 5,925,517, and 6,037,130). Molecular beacons can be used as amplicon detector probes/primers in diagnostic assays. Because nonhybridized molecular beacons are dark, it is not necessary to isolate the probe-target hybrids to determine for example, the number of amplicons synthesized during an assay. Therefore, molecular beacons simplify the manipulations that are often required when traditional detection and identifications means are used.
By using different colored fluorophores, molecular beacons can also be used in multiplex amplification assays such as assays that target the simultaneous amplification and detection of PCA3 nucleic acid and of the second specific prostate nucleic acid (e.g., PSA, hK2/KLK2, PSMA, transglutaminase 4, acid phosphatase and PCGEM1). The design of molecular beacons probes/primers is well known in the art and softwares dedicated to help their design are commercially available (e.g., Beacon designer from Premier Biosoft International). Molecular beacon probes/primers can be used in a variety of hybridization and amplification assays (e.g., NASBA and PCR).
In accordance with one embodiment of the present invention, the amplified product can either be directly detected using molecular beacons as primers for the amplification assay (e.g., real-time multiplex NASBA or PCR assays) or indirectly using, internal to the primer pair binding sites, a molecular beacon probe of 18 to 25 nucleotides long (e.g., 18, 19, 20, 21, 22, 23, 24, 25) wich specifically hybridizes to the amplification product. Molecular beacons probes or primers having a length comprised between 18 and 25 nucleotides are preferred when used according to the present invention (Tyagi et al., 1996, Nature Biotechnol. 14: 303-308). Shorter fragments could result in a less fluorescent signal, whereas longer fragments often do not increase significantly the signal. Of course shorter or longer probes and primers could nevertheless be used.
Examples of nucleic acid primers which can be derived from PCA3 RNA sequences are shown hereinbelow in Table 1:
TABLE 1 NUCLEIC ACID PRIMERS Size (no. of bases) Nucleotides Exon 1 98 1-98 of SEQ ID NO: 9 Exon 2 165 99-263 of SEQ ID NO: 9 Exon 3 183 264-446 of SEQ ID NO: 9 Exon 4a 539 447-985 of SEQ ID NO: 9 Exon 4b 1052 986-2037 of SEQ ID NO: 9 Exon 1 120 1-120 of SEQ ID NO: 10 Exon 2 165 121-285 of SEQ ID NO: 10 Exon 3 183 286-468 of SEQ ID NO: 10 Exon 4a 539 469-1007 of SEQ ID NO: 10 Exon 4b 1059 1008-2066 of SEQ ID NO: 10 Exon 4c 556 2067-2622 of SEQ ID NO: 10 Exon 4d 960 2623-3582 of SEQ ID NO: 10 Exon junction 1 20 89-108 of SEQ ID NO: 9 Exon junction 1 20 109-128 of SEQ ID NO: 10 Exon junction 2 20 252-271 of SEQ ID NO: 9 Exon junction 2 20 274-293 of SEQ ID NO: 10 Exon junction 3 20 435-454 of SEQ ID NO: 9 Exon junction 3 20 457-476 of SEQ ID NO: 10 Exon junction 4 20 974-993 of SEQ ID NO: 9 Exon junction 4 20 996-1015 of SEQ ID NO: 10 Exon junction 5 20 2055-2074 of SEQ ID NO: 10 Exon junction 6 20 2611-2630 of SEQ ID NO: 10It should be understood that the sequences and sizes of the primers taught in Table 1 are arbitrary and that a multitude of other sequences can be designed and used in accordance with the present invention.
While the present invention can be carried out without the use of a probe which targets PCA3 sequences, such as the exon junctions of PCA3 in accordance with the present invention, such probes can add a further specificity to the methods and kits of the present invention. Examples of specific nucleic acid probes which can be used in the present invention (and designed based on the exonic sequences shown in Table 1) are set forth in Table 2, below:
TABLE 2 NUCLEIC ACID PROBES Size (no. of bases) Nucleotides 1 20 1-20 of SEQ ID NO: 9 Probe 2 30 1-30 of SEQ ID NO: 9 Probe 3 40 1-40 of SEQ ID NO: 9 Probe 4 20 1-20 of SEQ ID NO: 10 Probe 5 30 1-30 of SEQ ID NO: 10 Probe 6 40 1-40 of SEQ ID NO: 10 Probe 7 20 89-108 of SEQ ID NO: 9 Probe 8 30 114-143 of SEQ ID NO: 10 Probe 9 30 257-286 of SEQ ID NO: 9 Probe 10 20 284-303 of SEQ ID NO: 10 Probe 11 20 274-293 of SEQ ID NO: 9Of course, as will be understood by the person of ordinary skill, a multitude of additional probes can be designed from the same or other region of SEQ ID NO. 9 as well as from SEQ ID NO. 10 and 13 and other sequences of the present invention, whether they target exon junctions or not. It will be clear that the sizes of the probes taught in Table 2 are arbitrary and that a multitude of other sequences can be designed and used in accordance with the present invention.
It will be readily recognized by the person of ordinary skill, that the nucleic acid sequences of the present invention (e.g., probes and primers) can be incorporated into anyone of numerous established kit formats which are well known in the art.
In one embodiment of the above-described method, a nucleic acid probe is immobilized on a solid support. Examples of such solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling nucleic acid probes to such solid supports are well known in the art.
The test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids (e.g., urine). The sample used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized. Preferably the sample is a urine sample. When the urine sample is used, it should contain at least one prostate cell in order to enable the identification of the prostate specific marker of the present invention. In fact, assuming that the half-life of PCA3 mRNA in an untreated biological sample is not suitable for easily enabling the preservation of the integrity of its sequence, the collected sample, whether urine or otherwise, should, prior to a treatment thereof contain at least one prostate cell. It will be recognized that the number of cells in the sample will have an impact on the validation of the test and on the relative level of measured PCA3 (or second prostate specific marker).
III. A Kit for Detecting the Presence of PCA3 Nucleic Acid in a Sample
In another embodiment, the present invention relates to a kit for diagnosing prostate cancer in a manner which is both sensitive and specific (i.e lowering the number of false positives). Such kit generally comprises a first container means having disposed therein at least one oligonucleotide probe or primer that hybridizes to a prostate cancer-specific PCA3 nucleic acid sequence. In one embodiment, the present invention also relates to a kit further comprising in a second container means oligonucleotide probes or primers which are specific to a second prostate specific marker, thereby validating a negative result with PCA3.
In a particular embodiment of the present invention, this kit (K) comprises a primer pair which enables the amplification of PSA, hK2/KLK2, PSMA, transglutaminase 4, acid phosphatase and PCGEM1) Of course the present invention also encompasses the use of a third prostate specific marker.
Oligonucleotides (probes or primers) of the kit may be used, for example, within a NASBA, PCR or hybridization assay. Amplification assays may be adapted for real time detection of multiple amplification products (i.e. multiplex real time amplification assays).
In a related particular embodiment, the kit further includes other containers comprising additional components such as additional oligonucleotide or primer and/or one or more of the following: buffers, reagents to be used in the assay (e.g. wash reagents, polymerases or else) and reagents capable of detecting the presence of bound nucleic acid probe or primers. Examples of detection reagents include, but are not limited to radiolabelled probes, enzymatic labeled probes (horse radish peroxidase, alkaline phosphatase), and affinity labeled probes (biotin, avidin, or steptavidin). In one embodiment, the detection reagents are molecular beacon probes which specifically hybridizes to the amplification products. In another embodiment, the detection reagents are chemiluminescent compounds such as Acridinium Ester (AE).
For example, a compartmentalized kit in accordance with the present invention includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample (e.g., an RNA extract from a biological sample or cells), a container which contains the primers used in the assay, containers which contain enzymes, containers which contain wash reagents, and containers which contain the reagents used to detect the extension products. As mentioned above, the separation or combination of reagents can be adapted by the person of ordinary skill to which this invention pertain, according to the type of kit which is preferred (e.g., a diagnostic kit based on amplification or hybridization methods or both), the types of reagents used and their stability or other intrinsic properties. In one embodiment, one container contains the amplification reagents and a separate container contains the detection reagent. In another embodiment, amplification and detection reagents are contained in the same container.
Kits may also contain oligonucleotides that serve as capture oligomers for purifying the target nucleic acids from a sample. Examples of capture oligomers have sequences of at least 15 nucleotides complementary to a portion of the PCA3 target nucleic acid. Embodiments of capture oligomers may have additional bases attached to a 3â² or 5â² end the sequence that is complementary to the PCA3 target sequence which may act functionally in a hybridization step for capturing the target nucleic acid. Such additional sequences are preferably a homopolymeric tail sequence, such as a poly-A or poly-T sequence, although other embodiments of tail sequences are included in capture oligomers of the present invention. In one embodiment, CAP binding protein (e.g., eIF4G-4E) or part thereof may be used to capture cap-structure containing mRNAs (Edery et al., 1987, Gene 74(2): 517-525). In another embodiment, a non specific capture reagent is used (e.g., silica beads).
Kits useful for practicing the methods of the present invention may include those that include any of the amplification oligonucleotides and/or detection probes disclosed herein which are packaged in combination with each other. Kits may also include capture oligomers for purifying the PCA3 target nucleic acid from a sample, which capture oligomers may be packaged in combination with the amplification oligonucleotides and/or detection probes.
In a further embodiment, cells contained in voided urine samples obtained after an attentive digital rectal examination are harvested and lysed in a lysis buffer. Nucleic acids are extracted (e.g., from the lysate by solid phase extraction on silica beads for example). Detection of the presence of RNA encoded by the PCA3 gene in the nucleic acid extract is done by an in vitro specific RNA amplification coupled to real-time detection of amplified products byfluorescent specific probes. In this method, simultaneously to the amplification of the PCA3 prostate cancer specific RNA undergoes the amplification of the second prostate-specific marker (such as the PSA RNA) as a control for the presence in the urine sample of prostate cells.
The screening and diagnostic methods of the invention do not require that the entire PCA3 RNA sequence be detected. Rather, it is only necessary to detect a fragment or length of nucleic acid that is sufficient to detect the presence of the PCA3 nucleic acid from a normal or affected individual, the absence of such nucleic acid, or an altered structure of such nucleic acid (such as an aberrant splicing pattern). For this purpose, any of the probes or primers as described above are used, and many more can be designed as conventionally known in the art based on the sequences described herein and others known in the art.
It is to be understood that although the following discussion is specifically directed to human patients, the teachings are also applicable to any animal that expresses PCA3.
The diagnostic and screening methods of the invention are especially useful for a patient suspected of being at risk for developing a disease associated with an altered expression level of PCA3 based on family history, or a patient in which it is desired to diagnose a PCA3-related disease (ex. prostate cancer). The method of the present invention may also be used to monitor the progression of prostate cancer in patient as described above.
The present invention is illustrated in further details by the following non-limiting example. The examples are provided for illustration only and should not be construed as limiting the scope of the invention.
To estimate the clinical performance of the method, a pilot study was done on 517 patients planned to undergo ultrasound guided needle biopsies coming from five university medical centers located in Montreal and Quebec (Canada) between September 2001 and June 2002. Each sample was processed using the following steps:
Sample Collection
Following an attentive digital rectal examination, the first 20 to 30 ml of voided urine was collected in sterile 80 ml plastic containers (patient urinates directly in the sterile container).
An equal volume of Sample buffer (0.1M phosphate (0.06M Na2HPO4, 0.04M NaH2PO4) 0.3M NaCl, pH 7.0,) was immediately added and the solution mixed by inversion.
If not processed immediately, samples were refrigerated between 2-8° C. for up to three days until further processing. In view of the cell recovery step, freezing should be avoided.
Cell Recovery
The sample was mixed by inversion; and the container gently tapped on the counter in order to detach cells from the inner walls thereof. The sample was then transferred into one or two (if necessary) conical polypropylene tubes (40 ml/tube).
The cells were pelleted by centrifugation in a tabletop centrifuge at 1400 g for 15 minutes. Finally, the supernatant was decanted and the cells were immediately lysed.
Cell Lysis
400 μl of Lysis Buffer (4.68M GUSCN, 20 mM EDTA, 1.2% Triton X-100â¢, 46 mM Tris-HCl, pH 7.2) was added to the urine cell pellet.
The cell pellet was then vigorously vortexed for 20 seconds in order to lyse the cells. It is important to make sure that no particulate matter is left. The lysate was transparent and not too viscous.
The lysate was transferred into a 1.5 ml microtubes and vortexed for 30 seconds.
If desired, the lysed cells can now be stored at â¦â70° C. indefinitely.
Nucleic Acid Extraction
The silica suspension (60 g silica type ±80% particle size 1-5 μm, add MilliQ water at a final volume of 500 ml) was first vigorously vortexed for 30 seconds until an opaque homogeneous suspension was obtained.
200 μl of the suspension was then immediately removed and added to the lysed specimen. All tubes were subsequently vigorously vortexed for 15 seconds to bind nucleic acids to the silica.
On a test tube rack, a series of Microspin⢠Columns identifying each filter unit with the appropriate number of patient were prepared.
The content of each microtube containing the lysed cells and the silica were transferred into the membrane filter unit of one Microspin⢠Column. To facilitate the transfer of the particulate matter, the microtube was vortexed briefly (approximately 5 seconds) in order to resuspend the content. The same was done before transferring. Tips were changed between samples.
The Microspin⢠columns were centrifuged in a non-refrigerated microcentrifuge at 10,000 RPM for 5 minutes at room temperature (18° C.-25° C.). The membrane filter retained silica-bound nucleic acids whereas other cellular components remained in the flow-through.
Meanwhile, a series of 2 ml microtubes corresponding to the number of Microspin⢠columns were prepared.
The membrane filter units containing the silica were transferred to new 2 mL microtubes. 500 μl of Wash Buffer (5.3M GuSCN, 52 mM Tris-HCl pH 6.4) was added to each membrane filter unit. The Microspin⢠columns were then centrifuged in a non-refrigerated microfuge at 10,000 RPM for 5 minutes at room temperature.
On a test tube rack, a new series of 2 ml microtubes were prepared.
The membrane filter units with the silica were transferred to the new 2 ml microtubes. 600 μl ethanol 70% was added to the membrane filter units. The Microspin⢠columns were then centrifuged in a non-refrigerated microfuge at 10,000 RPM for 5 minutes at room temperature.
On a test tube rack, a new series of 2 ml microtubes were prepared.
The membrane filter units with the silica are transferred to a new 2 ml microtube. Discard the microtubes containing the flow-through.
The membrane filter unit containing microtubes were then transferred to a heating block at 65° C.±1° C. installed under a fume hood.
All tubes were opened carefully to ensure evaporation, and incubated for approximately 10 minutes to dry the silica.
200 μl Elution Buffer (Dnase/Rnase-free water) to each membrane filter unit was added.
The membrane filter units were then centrifuged in a microfuge at 10,000 RPM for 5 minutes at room temperature.
The elution steps were repeated once to obtain a second eluate. These steps elute nucleic acids from the silica and concentrate them in the flow-through.
The microfilter units were disposed and the two microtubes containing the nucleic acid elution were kept.
For each eluate, three aliquots of â 50 μl of nucleic acids were stored at â¦â70° C.
In Vitro RNA Amplification and Detection
The nucleic acid eluate sample to test was first thawed on ice. The reaction mix was then prepared according to the number of reactions to be performed. Each sample was made at least in duplicate.
10 μl of the reaction mix was distributed in identified microtubes [80 mM Tris-HCl pH 8.5, 24 mM MgCl2, 180 mM KCl, 10 mM DTT, 2 mM of each dNTP, 4 mM of rATP, rUTP, CTP, 3 mM rGTP, 1 mM ITP, 30% DMSO, 3% sucrose, 1% D-Mannitol, 1% Dextran T-40, 208 nM PSA primers (N2psaP1B, SEQ. ID NO 1 and N2psaP2B, SEQ. ID NO 2), 417 nM PCA-3 primers (N0pcaP1A, SEQ. ID NO 3 and N0pcaP2B, SEQ. ID NO 4), 84 nM PSA beacon (BpsaRD-4, SEQ. ID NO 5), 166 nM PCA-3 beacon (BpcaFD-4, SEQ. ID NO 6).
5 μl of nucleic acid sample eluate was added in each tube and mixed.
Tubes were placed in a thermocyclerâ¢, heated at 65° C.±1° C. for a period of 5 minutes and then the temperature was kept at 41° C. After 5 minutes at 41° C., tubes were retrieved and centrifuged briefly in order to remove the condensation drops from the lids.
The next steps were better carried out quickly, and the tube temperature was preferably kept at 41° C.
5 μl of the enzyme mix (375 mM sorbitol, 0.105 μg/μl BSA, 0.08 units of RnaseH, 32.0 units of T7 RNA polymerase, 6.4 units of AMV-RT) was then quickly added to each tube and the tubes were gently mixed.
The tubes were put back into the EasyQ⢠incubator. When the last tube was in place, the incubator was kept at a temperature of 41° C±0.5° C. for 5 minutes.
The tubes were then briefly centrifuged. Quickly, all tubes were transferred in a thermostated spectrofluorimeter for in vitro RNA amplification and real time amplified product detection with the following characteristics: (1) the light source was a quartz-halogen lamp, (2) the filter used for ROX (6-carboxy-x-rhodamine N-succinimidyl ester) fluorescence was at 550-620 nm and for FAM (6-carboxyfluorescein N-hydroxysuccinimide ester) was at 485-530 nm, (3) the fluorescence integration time per tube was 20 msec; and (4) ROX and FAM emission was read each 30 sec and the tube block was set at the temperature of 41° C.±1° C.
Results
Fluorescence data generated during the two hours of amplification underwent fitting following the approach of Brown [Computer Methods and Programs in Biomedicine 65 (2001) 191-200].
Based on the PSA ratio (fluo maxlfluo min) cut off of 1.3, out of the 517 patients who have been tested, 443 had adequate quantities of prostate cells in the urine.
In this population of patients, 34% (151/443) had prostate cancer confirmed by histology.
TABLE 3 Positive Biopsies versus tPSA Categories tPSA Percentage of Patients Positive Biopsies <4 ng/ml 21% (n = 94) 20% (n = 19) 4-10 ng/ml 55% (n = 243) 35% (n = 85) >10 ng/ml 24% (n = 106) 44% (n = 47)Clinical specificity (Sp) and sensitivity (Se) of the method has been estimated following a tree-structured classification using S-plus⢠software [Insightful Corporation, Seattle, Wash., USA] starting from raw fluorimeter data. Three structured trees have been defined for the three types of patients defined as having a total blood PSA (tPSA) below 4 ng/ml, between 4-10 ng/ml and above 10 ng/ml (see
FIGS. 2-4and TABLE 4.
TABLE 4 Method sensitivity and specificity <4 ng/ml 94 74 (14/19) 91 (68/75) 4-10 ng/ml 243 59 (50/85) 91 (144/158) >10 ng/ml 106 79 (37/47) 80 (47/59) Overall 443 67 (101/151) 89 (259/292)The study demonstrated that the method has a positive predictive value (PPV) of 75%, compared to total PSA (>4.0 ng/ml) with a PPV of only 38%. The negative predictive value of the method is 84%, compared to 81% for tPSA. The overall accuracy of the method is 81%, compared with an accuracy of 47% for tPSA.
Although the present invention has been described hereinabove by way of illustrative embodiments thereof, it will be appreciated by one skilled in the art from reading of this disclosure that various changes in form and detail can be made without departing from the spirit and nature of the invention as defined in the appended claims. For example, various other amplification assays or detection assays, different probes and primers sequences as well as slightly different temperature and time of incubation may be used according to the present invention.
. A method for determining a predisposition, or presence of prostate cancer in a patient comprising:
a) contacting a biological sample of said patient with at least one oligonucleotide that hybridizes to a PCA3 polynucleotide selected from the group consisting of:
i) a polynucleotides according to SEQ ID NOs 9, 10 and 13;
ii) a polynucleotide sequence that hybridizes under high stringency conditions to the polynucleotide sequence in i); and
iii) a polynucleotide sequence fully complementary to i) or ii); and contacting said biological sample with at least one oligonucleotide that hybridizes with a second prostate specific polynucleotide,
b) detecting in said biological sample an amount of PCA3 and second prostate specific polynucleotides; and
comparing the amount of PCA3 polynucleotide that hybridizes to the oligonucleotide to a predetermined cut off value, and therefrom determining the presence or absence of prostate cancer in the biological sample.
2. The method of claim 1 , wherein said second specific prostate specific nucleic acid is selected from the group consisting of: PSA, human kallikrein 2, PSMA, transglutaminase 4, acid phosphatase and PCGEM1 nucleic acid.
3. The method of claim 2 , wherein said prostate specific nucleic acid is PSA.
4. The method of claim 3 , wherein said PSA sequence hybridizes to human kallikrein 2.
5. The method of
claim 1, wherein the amount of PCA3 polynucleotide and of the second specific prostate cancer polynucleotide is determined using an assay selected from the group consisting of:
a) an amplification assay; and
b) a hybridization assay.
6. The method of claim 5 , wherein said amplification assay is an in vitro RNA amplification method.
7. The method of
claim 6, wherein said RNA amplification method is selected from the group consisting of:
a) nucleic acid sequence-based amplification (NASBA);
b) polymerase chain reaction (PCR);
c) transcription mediated amplification assay (TMA); and
d) ligase chain reaction.
8. The method of claim 6 , wherein said amplification of PCA3 and said second prostate specific nucleic acid is performed simultaneously.
9. The method of claim 6 , wherein said amplification of PCA3 is carried out using a primer pair composed of SEQ ID NOs: 3 and 4.
10. The method of claim 6 , wherein said detection is performed by fluorescence, chimiluminescence or colorimetry detection.
11. The method of claim 10 , wherein said detection of PCA3 is carried out using acridinium ester compounds.
12. The method of claim 6 , wherein said detection of PCA3 is carried out using a molecular beacon.
13. The method of claim 12 , wherein said beacon has the sequence set forth in SEQ ID NO: 6.
14. The method of one of claims 6, wherein said second prostate specific nucleic acid is PSA and said amplification thereof is carried out using a primer pair composed of SEQ ID NOs: 1 and 2.
15. The method of claim 14 , wherein said detection of PSA is carried out using acridinium ester compounds.
16. The method of claim 14 , wherein said detection of PSA is carried out using a PSA molecular beacon.
17. The method of claim 16 , wherein said PSA beacon has the sequence set forth in SEQ ID NO: 5.
18. The method of claim 1 , wherein said sample contains at least one prostate cell and said at least one cell is collected from said sample prior to step a).
19. The method of claim 18 , wherein said nucleic acid is extracted from said at least one prostate cell.
20. The method of claim 19 , wherein said nucleic acid is RNA.
21. The method of claim 20 , wherein said RNA is extracted using a silica-based method.
22. The method of claims
1, wherein said sample is selected from the group consisting of:
a) urine;
b) blood or fraction thereof; and
c) prostate biopsy.
23. The method of claim 22 wherein said sample is urine.
24. The method of claim 23 , wherein said urine is collected following a digital rectal examination, thereby increasing the number of prostate cells in said sample.
25. The method of
claim 1, further comprising:
c) repeating steps (a) and (b) using a biological sample from the patient at a subsequent point in time; and
d) comparing the relative amount of said PCA3 polynucleotide detected in step (c) to the relative amount of PCA3 polynucleotide detected in step (b) and therefrom monitoring the progression of the prostate cancer in the patient.
26. The method of claim 1 , wherein the detection of the second prostate specific polynucleotide validates a negative result for PCA3 detection.
27. The methods of
claim 1, wherein the biological sample is spiked with an internal control IC selected from the group consisting of:
a) purified nucleic acid;
b) cells;
c) viral particules containg target nucleic acids; and
d) organelles.
28. The method of claim 6 , wherein RNA is extracted using a target capture method.
29. The method of claim 1 , wherein said detection of PCA3 is carried out using chemiluminescent labels in a homogenous detection method.
30. A diagnostic kit for the detection of prostate cancer or the risk of developing same in a patient comprising:
a) at least one container having disposed therein at least one oligonucleotide probe or primer that hybridizes to one of:
i) a PCA3 nucleic acid sequence according to SEQ ID NO: 9, 10 and 13;
ii) a sequence which is fully complementary to i); and
iii) a sequence which hybridizes under high stringency conditions to i) or ii);
b) at least one oligonucleotide probe or primer that hybridizes with a second prostate specific nucleic acid or complement thereof; and
c) reagents enabling a detection of PCA3 and of said second prostate specific nucleic acid when said PCA3 or second prostate-specific nucleic acid sequence is present.
31. The diagnostic kit according to
claim 30, wherein the detection reagent comprises a reporter group or label selected from the group consisting of:
a) radioisotopes;
b) enzymes;
c) fluorescent groups;
d) biotin;
e) chemiluminescent groups; and
f) dye particles.
32. The kit of claim 30 , wherein said PCA3 nucleic acid and said second prostate specific nucleic acid are amplified simultaneously in the same container.
33. The kit of claim 30 , wherein the detection of said PCA3 nucleic acid and said second prostate specific nucleic acid is performed in the same container.
34. The kit of claim 30 , further comprising an internal control (IC) as well as a primer, and/or probe, and/or reagent for the amplification, and/or hybridization, and/or detection of said internal control.
35. The kit of
claim 34, wherein said IC is selected from the group consisting of:
a) purified nucleic acid;
b) cells;
c) viral particules containg target nucleic acids; and
d) organelles.
36. A kit for assessing the presence of prostate cancer or the risk of developing same in a patient comprising:
a) a first primer pair specific for amplifying a PCA3 nucleic acid associated with prostate cancer present in a patient sample;
b) a second primer pair specific for amplifying a second prostate-specific nucleic acid; and
c) reagents enabling a detection of PCA3 and of said second prostate specific nucleic acid amplification products when said PCA3 or second prostate-specific nucleic acid sequence is present.
37. A method for detecting prostate cancer in a human patient, comprising:
a) performing an in vitro nucleic acid amplification assay on a biological sample of said patient or extract thereof using a first primer pair which is specific to a prostate cancer specific PCA3 sequence and a second primer pair which is specific to a prostate specific nucleic acid sequence; and
b) detecting said PCA3 sequence and said prostate specific nucleic acid sequence,
wherein, a detection of said PCA3 nucleic acid sequence or a level thereof correlates with a risk of developing prostate cancer or to a presence of prostate cancer in said patient, and wherein an absence of detection of said PCA3 nucleic acid sequence or lower level thereof in said sample validates an absence of prostate cancer or a lower risk of developing same, when said second prostate specific nucleic acid is detected.
38. The method of one of claim 1 , wherein said nucleic acid amplification is carried-out in real time.
39. The method of claim 37 , wherein said detection is performed by fluorescence, chimiluminescence or colorimetry detection.
40. The method of claim 8 , wherein said amplification of PCA3 and said second prostate specific nucleic acid is performed simultaneously in one container.
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