WO2012152800A1 - Marqueurs moléculaires dans le cancer de la prostate - Google Patents

Marqueurs moléculaires dans le cancer de la prostate Download PDF

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WO2012152800A1
WO2012152800A1 PCT/EP2012/058473 EP2012058473W WO2012152800A1 WO 2012152800 A1 WO2012152800 A1 WO 2012152800A1 EP 2012058473 W EP2012058473 W EP 2012058473W WO 2012152800 A1 WO2012152800 A1 WO 2012152800A1
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prostate cancer
expression
prostate
hoxc4
patients
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PCT/EP2012/058473
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Franciscus Petrus Smit
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Noviogendix Research B.V.
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Priority to US14/116,548 priority Critical patent/US20140106363A1/en
Priority to EP12720161.4A priority patent/EP2707500A1/fr
Priority to CA2835728A priority patent/CA2835728A1/fr
Priority to AU2012252426A priority patent/AU2012252426A1/en
Publication of WO2012152800A1 publication Critical patent/WO2012152800A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to methods for diagnosing prostate cancer and especially diagnosing low grade (LG) prostate cancer, i.e., individuals with good prognosis; high grade (HG) prostate cancer, i.e.,
  • PrCa Met i.e., individuals with poor prognosis and metastasis
  • CRPC castration resistant prostate cancer
  • the present invention further relates to the use of the expression of the indicated genes for
  • prostate cancer In the Western male population, prostate cancer has become a major public health problem. In many developed countries, it is not only the most commonly diagnosed malignancy, but prostate cancer is also the second leading cause of cancer related deaths in males as well. Because the incidence of prostate cancer increases with age, the number of newly diagnosed cases continues to rise as the life expectancy of the general population increases. In the
  • prostate cancer is an indolent disease and more men die with prostate cancer than from it.
  • a significant fraction of the tumours behave aggressively and, as a result, approximately 35,800 American men and approximately 80,000 European men die from this disease on an annual basis.
  • Radical prostatectomy and radiotherapy are curative therapeutic options for prostate cancer, but their curative potential is limited to anatomically localized disease.
  • PSA prostate specific antigen
  • PSA prostate specific antigen
  • biopsy specimens may not be representative, also attributing to the failure to detect some cancers, or, in other words, false negative diagnosis.
  • biopsies are proposed which have at least 10% probability of demonstrating cancer. Moreover, if the combined use of serum PSA, DRE and TRUS biopsy indicates clinically confined cancer, 40% of these men are found to have already extra ⁇ capsular disease upon radical prostatectomy.
  • non-invasive molecular tests capable of identifying those men having an early stage, clinically localized prostate cancer are urgently needed thereby providing through early radical intervention a prolonged survival and quality of life.
  • biomarkers for prostate cancer. These biomarkers may be instrumental in the
  • a suitable biomarker preferably fulfils the
  • biomarkers are tested in terms of tissue-specificity and discrimination potential between prostate cancer, normal prostate and BPH. Furthermore, it can be expected that (multiple) biomarker-based assays enhance the specificity for cancer detection.
  • chromosomal abnormalities like changes in chromosome number, translocations, deletions, rearrangements and duplications in cells
  • FISH fluorescence in situ hybridization
  • CGH comparative genomic hybridization
  • SAGE serial analysis of gene expression
  • oligonucleotide arrays oligonucleotide arrays and cDNA arrays characterize gene expression profiles.
  • tissue microarray TMA
  • Disease-related proteins can be directly sequenced and identified in intact whole tissue sections using the matrix-assisted laser desorption- ionization time-of-flight mass spectrometer (MALDI-TOF) .
  • MALDI-TOF matrix-assisted laser desorption- ionization time-of-flight mass spectrometer
  • SMDI surface-enhanced laser desorption-ionization
  • MS mass spectroscopy
  • prostate cancer is a very heterogeneous disease .
  • Biomarkers can be classified into four different prostate cancer-specific events: genomic alterations, prostate cancer-specific biological processes, epigenetic modifications and genes uniquely expressed in prostate cancer .
  • HPC1 (lq24), CAPB (lp36), PCAP (lq42), ELAC2 (17pll), HPC20 (20ql3), 8p22-23 and HPCX (Xq27-28).
  • CAPB lp36
  • PCAP lq42
  • ELAC2 (17pll
  • HPC20 (20ql3)
  • 8p22-23 HPCX (Xq27-28).
  • Three candidate hereditary prostate cancer genes have been mapped to these loci, HPC1 /2 ' -5 ' -oligoadenylate dependent
  • RNASEL ribonuclease L
  • MSR1 macrophage scavenger 1 gene located on chromosome 8p22-23
  • HPC2/ELAC2 chromosome 17pll.
  • prostate cancer susceptibility genes probably account for only 10% of hereditary prostate cancer cases. The other 30% of familial prostate cancers are most likely associated with shared environmental factors or more common genetic variants or polymorphisms. Since such variants may occur at high
  • oncogenes and tumour suppressor genes is probably very small in primary prostate cancer.
  • the frequency of p53 mutations in primary prostate cancer is reported to be low but have been observed in almost 50% of advanced
  • Mitochondrial DNA is present in approximately 1,000 to 10,000 copies per cell. Because of these quantities, mitochondrial DNA mutations have been used as target for the analysis of plasma and serum DNA from prostate cancer patients. Mitochondrial DNA mutations were detected in three out of three prostate cancer patients having the same mitochondrial DNA mutations in their primary tumour.
  • Microsatellite Critical alterations in gene expression can lead to the progression of prostate cancer.
  • microsatellite instability Defined microsatellite
  • DNA methylation status may be influenced in early life by environmental exposures, like nutritional factors or stress, and that this leads to an increased risk for cancer in adults. Changes in DNA methylation patterns have been observed in many human tumors.
  • MSP methylation-specific PCR
  • this technique requires a tumour to normal ratio of only 0.1- 0.001%. This means that using this technique,
  • hypermethylated alleles from tumour DNA can be detected in the presence of 10 4 -10 5 excess amounts of normal alleles.
  • DNA methylation can serve as a useful marker in cancer detection.
  • hypermethylated genes in human prostate cancer Two of these genes are RASSFIA (ras association domain family protein isoform A) and GSTP1.
  • RASSFIA hypermethylation is a common phenomenon in breast cancer, kidney cancer, liver cancer, lung cancer and prostate cancer. In 60-70% of prostate tumours, RASSFIA hypermethylation has been found, showing a clear association with aggressive prostate tumors. No RASSFIA hypermethylation has been detected in normal prostate tissue. These findings suggest that RASSFIA hypermethylation may distinguish the more aggressive tumours from the indolent ones. Further studies are needed to assess its diagnostic value.
  • GSTP1 Glutathione S-transferase PI (GSTP1) promoter.
  • GSTP1 belongs to the cellular protection system against toxic effects and as such this enzyme is involved in the detoxification of many xenobiotics.
  • hypermethylation of the GSTP1 gene has been detected in 50% of ejaculates from prostate cancer patients but not in men with BPH. Because of the fact that ejaculates are not always easily obtained from prostate cancer patients, hypermethylation of GSTP1 was determined in urinary sediments obtained from prostate cancer patients after prostate massage. In 77% of these sediments cancer could be detected.
  • GSTPl hypermethylation has been detected in 40 to 50% of urinary sediments that were obtained from patients who just underwent prostate biopsies. GSTPl hypermethylation was detected in urinary sediments of patients with negative biopsies (33%) and patients with atypia or high-grade PIN (67%) . Because hypermethylation of GSTPl has a high
  • prostate cancer specificity for prostate cancer, it suggests that these patients may have occult prostate cancer. This indicates that the test could also be used as indicator for a second biopsy.
  • Other cancer associated genes are also know to be methylated such as APC and Cox 2.
  • Micro-array studies have been useful and informative to identify genes that are consistently up- regulated or down-regulated in prostate cancer compared to benign prostate tissue. These genes can provide prostate cancer-specific biomarkers and provide insight into the etiology of the disease.
  • genes that are highly up-regulated in prostate cancer compared to low or normal expression in normal prostate tissue are of special interest. Such genes could enable the detection of one tumour cell in a large background of normal cells, and could therefore be applied as a diagnostic marker in
  • TMPRSS2 serine protease
  • TMPRSS2 has been found in colon, lung, kidney, and pancreas.
  • a 492 amino acid protein has been predicted for
  • TMPRSS2 This predicted protein is a type II integral membrane protein, most similar to the hepsin family. These proteins are important for cell growth and maintenance of cell morphology. It is proposed that TMPRSS2 could be an activator of the precursor forms of PSA and hK2 and that TMPRSS2, like other serine proteases, may play a role in prostate carcinogenesis. Since TMPRSS2 has a low prostate cancer-specificity, it cannot be applied in the detection of prostate cancer cells in urinary sediments.
  • AMACR chromosome 5pl3
  • AMACR expression has not been detected in atrophic glands, basal cell hyperplasia and urothelial epithelium or metaplasia. IHC studies also showed that AMACR expression in needle biopsies had a 97%
  • AMACR greatly facilitated the identification of malignant prostate cells. Its high expression and cancer-cell specificity implicate that AMACR may also be a candidate for the development of molecular probes which may facilitate the identification of prostate cancer using non-invasive imaging modalities.
  • hepsin a type II transmembrane serine protease
  • hepsin a type II transmembrane serine protease
  • hepsin is up- regulated in advanced and more aggressive tumours, it suggests a role as a prognostic tissue marker to determine the aggressiveness of a tumour.
  • telomeres a ribonucleoprotein
  • the human telomeres consist of tandem repeats of the TTAGGG sequence as well as several different binding proteins. During cell division telomeres cannot be fully replicated and will become shorter.
  • Telomerase can lengthen the telomeres and thus prevents the shortening of these structures.
  • Cell division in the absence of telomerase activity will lead to shortening of the telomeres.
  • the lifespan of the cells becomes limited and this will lead to senescence and cell death.
  • telomeres are significantly shorter than in normal cells.
  • cancer cells with short telomeres telomerase activity is required to escape senescence and to allow immortal growth. High telomerase activity has been found in 90% of prostate cancers and was shown to be absent in normal prostate tissue .
  • telomerase activity has been used to detect prostate cancer cells in voided urine or urethral washing after prostate massage. This test had a sensitivity of 58% and a specificity of 100%. The negative predictive value of the test was 55%. Although it has been a small and preliminary study, the low negative predictive value indicates that telomerase activity measured in urine samples is not very promising in reducing the number of unnecessary biopsies.
  • hTERT The quantification of the catalytic subunit of telomerase, hTERT, showed a median over-expression of hTERT mRNA of 6-fold in prostate cancer tissues compared to normal prostate tissues. A significant relationship was found between hTERT expression and tumour stage, but not with Gleason score. The quantification of hTERT using real-time PCR showed that hTERT could well discriminate prostate cancer tissues from non-malignant prostate tissues. However, hTERT mRNA is expressed in leukocytes, which are regularly present in body fluids such as blood and urine. This may cause false positivity. As such, quantitative measurement of hTERT in body fluids is not very promising as a diagnostic tool for prostate cancer.
  • PSMA Prostate-specific membrane antigen
  • PSMA mRNA expression in primary prostate cancers and metastasis correlated with PSMA protein over- expression. Its clinical utility as a diagnostic or
  • prognostic marker for prostate cancer has been hindered by the lack of a sensitive immunoassay for this protein.
  • SELDI-TOF MS has provided the introduction of a protein biochip immunoassay for the quantification of serum PSMA. It was shown that the average serum PSMA levels for prostate cancer patients were significantly higher compared to those of men with BPH and healthy controls.
  • PSMA in combination with its splice variant PSMA' could be used as a prognostic marker for prostate cancer.
  • PSMA' expression is higher than PSMA expression.
  • PSMA expression is more dominant. Therefore, the ratio of PSMA over PSMA' is highly indicative for disease progression. Designing a quantitative PCR analysis which discriminates between the two PSMA forms could yield another application for PSMA in diagnosis and prognosis of prostate cancer.
  • Delta-catenin (pl20/CAS), an adhesive junction-associated protein, has been shown to be highly
  • DD3 PCA3 has been identified using differential display analysis. DD3 PCA3 was found to be highly over- expressed in prostate tumours compared to normal prostate tissue of the same patient using Northern blot analysis.
  • DD3 PCA3 was found to be strongly over-expressed in more than 95% of primary prostate cancer specimens and in prostate cancer metastasis. Furthermore, the expression of DD3 PCA3 is restricted to prostatic tissue, i.e., no expression has been found in other normal human tissues.
  • the gene encoding for DD3 PCA3 is located on chromosome 9q21.2.
  • the DD3 PCA3 mRNA contains a high density of stop-codons. Therefore, it lacks an open reading frame resulting in a non-coding RNA.
  • a time-resolved quantitative RT-PCR assay (using an internal standard and an external calibration curve) has been developed. The accurate quantification power of this assay showed a median 66-fold up-regulation of DD3 PCA3 in prostate cancer tissue compared to normal prostate tissue. Moreover, a median-up-regulation of 11-fold was found in prostate tissues containing less than
  • DD3 PCA3 was capable to detect a small number of tumour cells in a large background of normal cells.
  • PSA mRNA expression was shown to be relatively constant in normal prostate cells and only a weak down-regulation (-1.5- fold) of PSA expression has been reported in prostate cancer cells. Therefore, PSA mRNA has been used as a housekeeping gene' to correct for the number of prostate cells present in urinary sediments.
  • These urine samples were obtained after extensive prostate massage from a group of 108 men who were indicated for prostate biopsies based on a total serum PSA value of more than 3 ng/ml. This test had 67% sensitivity and 83% specificity using prostatic biopsies as a gold- standard for the presence of a tumour.
  • this test had a negative predictive value of 90%, which indicates that the quantitative determination of DD3 PCA3 transcripts in urinary sediments obtained after extensive prostate massage bears great potential in the reduction of the number of invasive TRUS guided biopsies in this population of men.
  • DD3 PCA3 is the most prostate cancer-specific gene described so far. Therefore, validated DD3 PCA3 assays could become valuable in the detection of disseminated prostate cancer cells in serum or plasma.
  • Multicenter studies using the validated DD3 PCA3 assay can provide the first basis for the molecular diagnostics in clinical urological practice.
  • Modulation of expression has clearly identified those cancers that are aggressive - and hence those that may require urgent treatment, irrespective of their morphology. Although not widely employed, antibodies to these proteins are authenticated, are available commercially, and are straightforward in their application and interpretation, particularly in conjunction with other reagents as double- stained preparations.
  • E2F transcription factors including E2F3 located on chromosome 6p22, directly modulate expression of EZH2.
  • Overexpression of the EZH2 gene has been important in development of human prostate cancer.
  • EZH2 was identified as a gene overexpressed in castration resistant and metastatic prostate cancer and showed that patients with clinically localized prostate cancers that express EZH2 have a worse progression than those who do not express the protein.
  • the prime challenge for molecular diagnostics is the identification of clinically insignificant prostate cancer, i.e., separate the biologically aggressive cancers from the indolent tumours. Furthermore, markers predicting and monitoring the response to treatment are urgently needed .
  • AMACR immunohistochemistry is widely used in the identification of malignant processes in the prostate thereby contributing to the diagnosis of prostate cancer.
  • introduction of molecular markers on tissue as prognostic tool has not been validated for any of the markers discussed.
  • tissue biorepositories of well documented specimens including clinical follow up data, play a pivotal role in the validation process.
  • hypermethylation and the gene DD3 PCA3 which is highly over- expressed in prostate cancer, enabled the detection of prostate cancer in non-invasively obtained body fluids such as urine or ejaculates.
  • the up-regulation of these genes in cancer should be more than 10% in prostate cancer compared to normal prostate to enable the detection of a single prostate cancer cell in a large background of normal cells in body fluids such as urine or ejaculates.
  • tumour markers and especially prostate tumour markers, there is a need in the art for tumour markers, and especially prostate tumour markers, there is a need in the art for tumour markers, and especially prostate tumour markers, there is a need in the art for tumour markers, and especially prostate tumour markers, there is a need in the art for tumour markers, and especially prostate tumour markers, there is a need in the art for tumour markers, and especially prostate tumour markers, there is a need in the art for tumour markers, and especially prostate tumour markers, there is a
  • tumour markers and especially markers indicative of the clinical course and outcome of the disease.
  • tumour markers providing a reliable identification of prostate cancer in a tissue specimen, and especially a reliable predictive value of the clinical course and outcome of the disease.
  • Such tumour markers will provide a tool aiding a trained physician to decide on a suitable treatment protocol of individuals diagnosed either using tumour markers, or any other indication, with prostate cancer.
  • the above object is met by the provision of a novel tumour marker and methods as outlined in the appended claims .
  • the above object, amongst other objects is met by a method for establishing the presence, or absence, of prostate cancer in a human individual
  • HOXC4 compared to expression of said HOXC4 in a sample originating from said human individual not comprising prostate tumour cells or prostate tumour tissue, or from an individual not suffering from prostate cancer;
  • prostate cancer based on the established up- or down regulation of HOXC4.
  • prostate cancer preferably
  • expression analysis comprises establishing an increased or decreased expression of a gene as compared to expression of said respective one or more genes in a sample originating from said human individual not comprising prostate tumour cells or prostate tumour tissue, or from an individual not
  • an increased or decreased expression of a gene according to the present invention is a measure of gene expression relative to a non- disease standard. For example, establishing an increased expression of HOXC4, as compared to expression of this gene under non-prostate cancer conditions, allows establishing the presence, or absence, of prostate cancer, preferably diagnosis, prognosis and/or prediction of disease survival, according to the present invention.
  • the HOXC4 gene belongs to the homeobox family of genes.
  • the homeobox genes encode a highly conserved family of transcription factors that play an important role in morphogenesis in all multicellular organisms. Mammals possess four similar homeobox gene clusters, HOXA, HOXB, HOXC and HOXD, which are located on different chromosomes and consist of 9 to 11 genes arranged in tandem.
  • This gene, HOXC4 is one of several homeobox HOXC genes located in a cluster on chromosome 12.
  • Transcripts may include the shared exon spliced to the gene-specific exons, or they may include only the gene-specific exons. Two alternatively spliced variants that encode the same protein have been described for HOXC4. Transcript variant one includes the shared exon, and
  • transcript variant two includes only gene-specific exons.
  • determining the expression comprises determining mRNA expression of HOXC4.
  • mRNA analysis based on mRNA is generally known in the art and routinely practiced in diagnostic labs world-wide.
  • suitable techniques for mRNA analysis are Northern blot hybridisation and amplification based techniques such as PCR, and especially real time PCR, and NASBA.
  • expression analysis comprises high-throughput DNA array chip analysis not only allowing the simultaneous analysis of multiple samples but also automatic analysis processing.
  • determining the expression comprises determining protein levels of the genes.
  • Suitable techniques are, for example, matrix-assisted laser desorption- ionization time-of-flight mass spectrometer (MALDI-TOF) based techniques, ELISA and/or immunohistochemistry .
  • MALDI-TOF matrix-assisted laser desorption- ionization time-of-flight mass spectrometer
  • the present method is preferably carried out using, in addition,
  • the present method is carried out by additional expression analysis of at least HOXC6.
  • the present presence, or absence, of prostate cancer in a human individual further comprises identification, establishing and/or diagnosing low grade PrCa (LG) , high grade PrCa (HG) , PrCa Met and/or CRPC .
  • LG low grade PrCa
  • HG high grade PrCa
  • PrCa Met and/or CRPC .
  • LG indicates low grade PrCa (Gleason Score equal or less than 6) and represent patients with good prognosis.
  • HG indicates high grade PrCa (Gleason Score of 7 or more) and represents patients with poor prognosis.
  • PrCa Met represents patients with poor prognosis.
  • CRPC indicates castration resistant prostate cancer and represents patients with aggressive localized disease.
  • the present invention provides
  • the present invention also relates to the use of expression analysis of HOXC4, optionally in combination with one or more of the other genes indicated above, and especially HOXC6, for establishing the presence, or absence, of
  • prostate cancer in a human individual prostate cancer in a human individual.
  • the present invention also relates to a kit of parts for establishing the presence, or absence, of prostate cancer in a human individual comprising:
  • the present kit of parts comprises mRNA expression analysis means, preferably suitable for expression analysis by, for example, PCR, rtPCR and/or NASBA.
  • the present kit of parts additionally comprises means for expression analysis of one or more or two or more, three or more, four or more, five or more, six ore more, or seven of the genes HOXC6, sFRP2, HOXD10, RORB, RRM2, TGM4, and SNAI2.
  • the present kit of parts additionally comprises means for expression analysis of at least HOXC6.
  • the appended figures provide the cDNA sequence of these genes as also their accession number, thereby allowing the skilled person to develop expression analysis assays based on analysis techniques commonly known in the art.
  • analysis techniques can, for example, be based on the genomic sequence of the gene, the provided cDNA or amino acid sequences. This sequence information can either be derived from the provided sequences, or can be readily obtained from the public databases, for example by using the provided accession numbers.
  • HOXC6 gene (NM_004503.3, NP_004494.1) ; the SFRP2 gene (NM_003013.2, NP_003004.1) ; the HOXD10 gene (NM_002148.3, NP_002139.2) ; the RORB gene (NM_006914.3, NP_008845.2) ; the RRM2 gene (NM_001034.2, NP_001025.1) ; the TGM4 gene
  • Figures 8-14 show boxplot TLDA data based on group LG (low grade) , HG (high grade) , CRPC (castration resistant) and PrCa Met (prostate cancer metastasis) expression analysis of HOXC6 gene
  • NP indicates no prostate cancer, i.e., normal or standard expression levels.
  • Figure 15 shows the mRNA and amino acid sequence of the
  • Figure 16 shows show boxplot data based on group LG (low grade) , HG (high grade) , CRPC (castration resistant) , PrCa Met (prostate cancer metastasis) , normal prostate and BPH expression analysis of the HOXC4 gene
  • LG low grade PrCa (Gleason Score equal or less than 6) . This group represents patients with good prognosis;
  • HG high grade PrCa (Gleason Score of 7 or more) .
  • This group represents patients with poor prognosis; sample type, mRNA from primary tumor;
  • PrCa Met This group represents patients with poor prognosis; sample type; mRNA from PrCa metastasis;
  • CRPC castration resistant prostate cancer
  • mRNA from primary tumor material from patients that are progressive under endocrine therapy This group represents patients with aggressive localized disease.
  • the expression analysis is performed according to standard protocols. Briefly, from patients with prostate cancer (belonging to one of the four previously mentioned categories) tissue was obtained after radical prostatectomy or TURP . The tissues were snap frozen and cryostat sections were H.E. stained for classification by a pathologist.
  • Single-stranded cDNA was regenerated through a random-primed reverse transcription using a dNTP mix containing dUTP.
  • the RNA was hydrolyzed with RNase H and the cDNA was purified.
  • the cDNA was then fragmented by incubation with a mixture of UDG (uracil DNA glycosylase) and APE1 (apurinic/apyrimidinic endonuclease 1) restriction endonucleases and, finally, end-labeled via a terminal transferase reaction incorporating a biotinylated
  • transcript clusters on the array of which 90,000 contain more than one exon. Of these 90,000 there are more than 17,000 high confidence (CORE) genes which are used in the default analysis. In total there are more than 5.5 million features per array.
  • CORE high confidence
  • the array was washed and stained according to the Affymetrix protocol.
  • the stained array was scanned at 532 nm using an Affymetrix GeneChip Scanner 3000, generating CEL files for each array.
  • Exon-level expression values were derived from the CEL file probe-level hybridization intensities using the model-based RMA algorithm as implemented in the Affymetrix Expression ConsoleTM software.
  • RMA Robot Multiarray
  • the target identification is biased since clinically well defined risk groups were analyzed.
  • the markers are categorized based on their role in cancer biology. For the identification of markers the PrCa Met group is compared with ' HG ' and ' LG ' .
  • biomarkers were identified based on 30 tumors.
  • PCA3 prostate cancer gene 3 AF 103907 80-103
  • LG Low grade prostate cancer
  • HG High grade prostate cancer
  • HG tissue specimens from primary tumors with a Gleason Score ⁇ 7 obtained after radical prostatectomy. This group represents patients with poor prognosis.
  • Prostate cancer metastases tissue specimens are obtained from positive lymfnodes after LND or after autopsy. This group represents patients with poor prognosis
  • CRPC Castration resistant prostate cancer
  • specimens are obtained from patients that are
  • H.E. hematoxylin and eosin
  • NanoDrop 1000 spectrophotometer
  • Agilent 2100 Bioanalyzer Agilent 2100 Bioanalyzer
  • TaqMan® Low Density Arrays (TLDA; Applied Biosystems) .
  • a list of assays used in this study is given in Table 3.
  • 3 ⁇ is added to 50 ⁇ Taqman® Universal Probe Master Mix (Applied Biosystems ) and 47 ⁇ milliQ.
  • One hundred ⁇ of each sample was loaded into 1 sample reservoir of a TaqMan® Array (384-Well Micro Fluidic Card) (Applied Biosystems) .
  • the TaqMan® Array was
  • phosphoribosyltransferase 1 endogenous control gene
  • TLDAs TaqMan® Low Density arrays
  • expression levels were determined for the 33 genes of interest.
  • the prostate cancer specimens were put in order from low Gleason scores, high Gleason scores, CRPC and finally prostate cancer metastasis.
  • Both GeneChip® Human Exon 1.0 ST Array and TLDA data were analyzed using scatter- and box plots.
  • HOXC6 ( Figure 8) The present GeneChip® Human Exon 1.0 ST Array data showed that HOXC6 was upregulated in prostate cancer metastases compared with primary high and low grade prostate cancers. Validation experiments using TaqMan® Low Density arrays confirmed this upregulation .
  • HOXC6 was found to be upregulated in all four groups of prostate cancer compared with normal prostate. Therefore, HOXC6 has diagnostic potential.
  • clinical follow-up data it was observed that all patients with progressive disease and 50% of patients with biochemical recurrence within 3 years after initial therapy had a higher upregulation of HOXC6 expression compared with patients who had biochemical recurrence after 5 years and patients with curative treatment.
  • the patients with biochemical recurrence within 3 years after initial therapy who had higher HOXC6 expression also had a worse prognosis compared with patients with lower HOXC6
  • HOXC6 expression is correlated with prostate cancer progression.
  • SFRP2 ( Figure 9) : The present GeneChip® Human Exon 1.0 ST Array data showed that SFPR2 was downregulated in prostate cancer metastases compared with primary high and low grade prostate cancers. Validation experiments using TaqMan® Low Density arrays confirmed this downregulation . Furthermore, SFRP2 was found to be downregulated in all four groups of prostate cancer compared with normal prostate.
  • SFRP2 has diagnostic potential.
  • SFRP2 can be used for the detection of patients with
  • a ratio of that marker and SFRP2 could be used for the detection of circulating tumor cells.
  • HOXD10 ( Figure 10) : The present GeneChip® Human Exon 1.0 ST Array data showed that HOXD10 was downregulated in prostate cancer metastases compared with primary high and low grade prostate cancers. Validation experiments using TaqMan® Low Density arrays confirmed this downregulation. Furthermore, HOXDIO was found to be downregulated in all four groups of prostate cancer compared with normal
  • HOXDIO has diagnostic potential.
  • HOXDIO can be used for the detection of patients with progression under endocrine therapy (CRPC) and patients with prostate cancer metastases.
  • RORB ( Figure 11) : The present GeneChip® Human Exon 1.0 ST Array data showed that RORB was upregulated in prostate cancer metastases and CRPC compared with primary high and low grade prostate cancers. Validation experiments using TaqMan® Low Density arrays confirmed this
  • RORB was found to be
  • RORB has diagnostic potential.
  • RRM2 ( Figure 12) : Experiments using TaqMan® Low Density arrays showed upregulation of RRM2 in all four groups of prostate cancer compared with normal prostate.
  • RRM2 has diagnostic potential. Moreover, the expression of RRM2 is higher in CRPC and metastasis showing that it may be involved in the invasive and metastatic potential of prostate cancer cells. Therefore, RRM2 can be used for the detection of circulating prostate tumor cells.
  • TGM4 (Figure 13) : The present GeneChip® Human Exon 1.0 ST Array data showed that TGM4 was downregulated in prostate cancer metastases compared with primary high and low grade prostate cancers. Validation experiments using TaqMan® Low Density arrays confirmed this downregulation .
  • TGM4 was found to be extremely downregulated in all four groups of prostate cancer compared with normal prostate. Therefore, TGM4 has diagnostic potential.
  • TGM4 downregulation of TGM4 (subgroup of patients) compared with patients with curative treatment and biochemical recurrence after initial therapy. In metastases the TGM4 expression is completely downregulated. Therefore, TGM4 has prognostic potential.
  • SNAI2 ( Figure 14) : The present GeneChip® Human Exon 1.0 ST Array data showed that SNAI2 was downregulated in prostate cancer metastases compared with primary high and low grade prostate cancers. Validation experiments using TaqMan® Low Density arrays confirmed this downregulation . Furthermore, SNAI2 was found to be downregulated in all four groups of prostate cancer compared with normal prostate. Therefore, SNAI2 has diagnostic potential.
  • HOXC4 revealed for prostate cancer (LG, HG, CRPC and Meta) an upregulation of expression of at least 2 to 3 fold. More striking, expression analysis of HOXC4 revealed not only an upregulation in prostate cancer but a clear discrimation between LG, HG and CRPC on one hand and Meta on the other hand .

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Abstract

La présente invention concerne des procédés pour diagnostiquer le cancer de la prostate et en particulier diagnostiquer LG, c'est-à-dire des individus avec un bon pronostic ; HG, c'est-à-dire des individus avec un pronostic médiocre de tumeur primaire ; PrCa Met, c'est-à-dire des individus avec un pronostic médiocre et des métastases ; et CRPC, c'est-à-dire des individus avec un pronostic médiocre souffrant d'une maladie localisée agressive. Spécifiquement, la présente invention concerne un procédé pour établir la présence, ou l'absence, d'un cancer de la prostate chez un individu humain comprenant : a) la détermination de l'expression de HOXC4 dans un échantillon provenant dudit individu humain ; b) l'établissement d'une régulation à la hausse ou à la baisse de l'expression de HOXC4 par rapport à l'expression de HOXC4 dans un échantillon provenant dudit individu humain ne comprenant pas de cellules cancéreuses de prostate ou de tissu de tumeur de prostate, ou d'un individu ne souffrant pas de cancer de la prostate ; et c) l'établissement de la présence, ou l'absence, d'un cancer de la prostate sur la base de la régulation à la hausse ou à la baisse établie de HOXC4.
PCT/EP2012/058473 2011-05-12 2012-05-08 Marqueurs moléculaires dans le cancer de la prostate WO2012152800A1 (fr)

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Cited By (11)

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Publication number Priority date Publication date Assignee Title
US9976188B2 (en) 2009-01-07 2018-05-22 Myriad Genetics, Inc. Cancer biomarkers
US10519513B2 (en) 2009-01-07 2019-12-31 Myriad Genetics, Inc. Cancer Biomarkers
US10954568B2 (en) 2010-07-07 2021-03-23 Myriad Genetics, Inc. Gene signatures for cancer prognosis
US9605319B2 (en) 2010-08-30 2017-03-28 Myriad Genetics, Inc. Gene signatures for cancer diagnosis and prognosis
US10876164B2 (en) 2012-11-16 2020-12-29 Myriad Genetics, Inc. Gene signatures for cancer prognosis
WO2015022164A1 (fr) * 2013-08-13 2015-02-19 Noviogendix Research B.V. Combinaisons de marqueurs moléculaires du cancer de la prostate, permettant d'obtenir un outil de diagnostic ayant une sensibilité/spécificité améliorées
EP3090265A4 (fr) * 2013-12-30 2018-03-21 The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. Profils de gènes du cancer de la prostate et procédés de leur utilisation
AU2014373927B2 (en) * 2013-12-30 2018-03-22 Genomatix Prostate cancer gene profiles and methods of using the same
US11174517B2 (en) 2014-05-13 2021-11-16 Myriad Genetics, Inc. Gene signatures for cancer prognosis
US10329625B2 (en) 2015-11-20 2019-06-25 Mdxhealth Research B.V. Method for detecting DLX1 mRNA, HOXC6 mRNA, and KLK3 mRNA in a urine sample and predicting and treating clinically significant prostate cancer
US11767563B2 (en) 2015-11-20 2023-09-26 Mdxhealth Sa Method for predicting and treating clinically significant prostate cancer

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