WO2001083816A2 - Procedes de detection de tumeurs - Google Patents

Procedes de detection de tumeurs Download PDF

Info

Publication number
WO2001083816A2
WO2001083816A2 PCT/IB2001/000899 IB0100899W WO0183816A2 WO 2001083816 A2 WO2001083816 A2 WO 2001083816A2 IB 0100899 W IB0100899 W IB 0100899W WO 0183816 A2 WO0183816 A2 WO 0183816A2
Authority
WO
WIPO (PCT)
Prior art keywords
pcr
mrna
domain
extracellular matrix
cells
Prior art date
Application number
PCT/IB2001/000899
Other languages
English (en)
Other versions
WO2001083816A3 (fr
Inventor
Dario Neri
Francesca Viti
Original Assignee
Philogen S.R.L.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Philogen S.R.L. filed Critical Philogen S.R.L.
Priority to AU58679/01A priority Critical patent/AU5867901A/en
Publication of WO2001083816A2 publication Critical patent/WO2001083816A2/fr
Publication of WO2001083816A3 publication Critical patent/WO2001083816A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method for the detection of tumors, including occult tumors (i.e. tumors which are not detectable by standard procedures or careful examination using techniques such as CT scan or surgery) .
  • occult tumors i.e. tumors which are not detectable by standard procedures or careful examination using techniques such as CT scan or surgery
  • the invention provides a method which is performed on bodily fluids, e.g., the peripheral blood of the individual.
  • the methods of the present invention are based on the detection of mRNA coding for a component of the extracellular matrix which accumulates around the neo-vasculature during the angiogenic processes which are involved in tumor formation.
  • the methods use RT-PCR to detect the mRNA encoding said component.
  • a preferred technique uses RT-PCR to detect mRNA containing the ED-B domain.
  • the present invention is based on the inventors' surprising discovery that mRNA encoding certain extracellular matrix components is actually present in the tumor cells themselves, and can be detected in the peripheral blood of patients with tumors, but not in healthy blood.
  • Monoclonal antibodies have been used to identify occult single cells derived from solid malignancies in the bone marrow and peripheral blood of many individuals (Moss and Sanders, (1990) J. Clin. Oncol. 8 736-740).
  • this method has two main limitations: poor sensitivity and false positivity due to cross-reaction of the monoclonal antibodies with normal cells.
  • RNA targets include the cytokeratins 18, 19 and 20 (CK18 CK19, CK20) , tyrosinase, prostate-specific antigen (PSA) , prostate-specific membrane antigen (PSMA) , carcinoembrionic antigen (CEA) , neuroendocrine protein gene product (PGP 9.5) (Kvalheim (1998) Acta Oncol., 37, 455-462) .
  • RT-PCR is a highly sensitive technique, for example the detection of one tumor cell in more than 10 7 mononucleated cells has been achieved (Keilholz (1998) Eur. J. Cancer, 34, 837-841) .
  • a problem with RT-PCR based techniques is that the high sensitivity means that it is difficult to discriminate between positive and negative samples. There could be background detection leading to false positives results, for example due to the presence of genomic DNA in the samples, or due to the presence in occasional cells of an mRNA with the same or similar sequence.
  • the present invention provides a method for the broad diagnosis of malignancies and residual disease by assaying the bodily fluids to detect an mRNA which is found in a broad range of tumor cells.
  • the present invention is based on the inventors' experimental work using RT-PCR to detect the presence of the ED-B and ED-A domains of fibronectin, and tenascin-C large isoform. These represent pan-tumor markers, since their presence in extracellular matrix associated with a tumor is not constrained to a particular type of tumor.
  • Tumors cannot grow beyond a certain mass without the formation of new blood vessels (angiogenesis) , and a correlation between microvessel density and tumor invasiveness has been reported for a number of tumors (Folkman, Nat. Med. 1: 27, 1995).
  • the ED-B domain of fibronectin a sequence of 91 amino acids, which is inserted by alternative splicing into the fibronectin molecule, specifically accumulates around neovascular structures (Zardi et al . (1987) EMBO J, 6, 2337-2342; Carnemolla et al . (1992) J. Cell Biol., 108, 1139-1148).
  • the domain is usually absent from normal adult tissues (Zardi et al. (1987) EMBO J, 6, 2337-2342; Carnemolla et al . (1996) Int. J. Cancer 68 397-405; Tarli et al. (1999) Blood 94 192- 198; Castellani et al . (1994) Int. J. Cancer 59 612-618).
  • ED-B-containing fibronectin Teavian et al . (1994) Int. J. Cancer, 56, 820-825
  • Carnemolla et al . (1989) J. Cell Biol, 108 1139-1148; Mariani et al . (1997) Cancer, 80, 2378-2384; Pujuguet et al.,(1996) Am. J. Pathol. 148 579-592).
  • the invention is based in various aspects upon the inventors' surprising finding that mRNA containing the ED- B domain is found in the vast majority of tumor cells themselves and is not limited, as previously thought, to the neovasculature associated with the tumors. They show that tumor cells can be detected by RT-PCR of the ED-B+ mRNA, when diluted in blood. The inventors also show that mRNA containing the ED-B domain can be detected in the peripheral blood of tumor bearing animals, but, importantly for discrimination, not healthy animals. The inventors also show that the mRNA of extracellular domains ED-A, tenascin C large isoform (particularly the repeat D) can be detected in transformed cells but not in the blood of healthy donors.
  • RT-PCR directed at extracellular matrix mRNA is able to discriminate between the blood of healthy individuals and the blood of individuals with tumors. This is particularly true for Aonco-fetal markers ⁇ of which ED-B is one.
  • the present invention provides a method of determining the presence or absence of tumors in an individual, comprising: performing reverse transcription PCR (RT-PCR) on a sample of bodily fluid taken from an individual to determine the presence or absence of a messenger RNA encoding an extracellular matrix component, which mRNA is present in tumors, said RT-PCR reaction comprising the use of at least one primer specific for said mRNA, thereby determining the presence or absence of tumors in the individual.
  • RT-PCR reverse transcription PCR
  • the sample Before performing the RT-PCR, the sample may be treated, e.g. concentrated, by methods known in the art.
  • the extracellular matrix component is selected from the group: ED-B domain of fibronectin; the large isoform of tenascin C; repeat C of tenascin C; repeat D of tenascin-C; or the ED-A domain of fibronectin.
  • the bodily fluid may be blood, plasma, serum, cerebro-spinal fluid, needle aspirates, tissue samples, tumor samples, saliva and urine.
  • the bodily fluid is blood, e.g., peripheral blood.
  • the primers used in the PCR reaction are specific for the extracellular matrix component mRNA which is present in tumors. Where the extracellular matrix component is present in tumors as a result of splicing, it is preferred that at least one of the primers overlaps either the 5' or the 3' splice junction, preferably a first PCR primer overlaps the 5' junction and a second PCR primer overlaps the 3' splice junction.
  • the forward primer may comprise sequence of the 5' extremity of the tumor specific exon and sequence of the 3' extremity of the exon which is 5' to the tumor specific exon.
  • the reverse primer may comprise sequence of the 3' extremity of the tumor specific exon and sequence of the 5' extremity of the exon which is 3 ' to the tumor specific exon .
  • the mRNA is the fibronectin ED-B domain mRNA.
  • a preferred forward primer comprises sequence of the 5' extremity of the ED-B exon and sequence of the 3' extremity of the fibronectin repeat 7 (i.e. overlaps the 5' splice junction) .
  • a preferred reverse primer comprises sequence of the 3' extremity of the ED-B exon and sequence of the 5' extremity of the fibronectin repeat 8 (i.e. overlaps the 3' splice junction).
  • the preferred PCR primers are FN7-EDB and EDB-FN8 described herein. In accordance with the requirements of PCR, the reverse primer EDB-FN8 has the same sequence as the target cDNA and the forward primer FN7-EDB is complementary to the target cDNA.
  • a typical procedure may include: contacting said sample with a first nucleic acid primer hybridisable to an mRNA encoding an ED-B domain of fibronectin; synthesising a DNA molecule complementary to said mRNA; mixing said complementary DNA with at least a second nucleic acid primer hybridisable to said DNA; performing a polymerase chain reaction on said DNA; and analysing the PCR product to determine the presence or absence in said sample of an mRNA encoding said ED-B domain, by analysis of the PCR product.
  • the PCR product may be analysed by a technique known in the art, for example by examining the size of the product, by restriction digest of the product, probing the product, or sequence analysis of the product. Exemplary methods are discussed further below.
  • nucleic acid amplification or detection techniques may also be used (see for example Nolte, (1998) Adv. Clin. Chem. 33 201- 235
  • Figure 1 shows alternative pre-mRNA splicing of the ED-B exon in normal and tumoral cells.
  • FN fibronectin
  • FN primary transcript two introns separate ED-B exon from the sequences coding for domains 7 and 8 of fibronectin.
  • normal cells such as blood leukocytes
  • the exon of ED-B is removed together with the adjacent introns during pre-mRNA splicing. Therefore, the resulting mRNA is devoid of ED-B exon and the sequences coding for repeat 7 and repeat 8 are joined together .
  • the splicing machinery of tumoral cells does not remove ED-B exon from the primary transcript but only the flanking introns.
  • the exons coding for repeat 7, ED-B and repeat 8 of fibronectin follow each other.
  • Figure 2 shows the primers used for the selective amplification of the ED-B exon from cDNA and a schematic description of the amplification of the ED-B exon from cDNA, but not from genomic DNA or primary transcript, which can be obtained by RT-PCR.
  • the forward primer overlaps the 5" extremity of ED-B exon and part of the 3' extremity of the exon coding for the adjacent repeat 7 of FN.
  • the reverse primer overlaps the 3' end of ED-B exon and the 5' extremity of the exon coding for the adjacent repeat 8 of fibronectin. Only in mRNA of tumoral cells, where the exons are adjacent, the primers find the correct annealing sequence .
  • the methods make use of bodily fluid samples from individuals which may for example, have or be suspected of having tumors.
  • bodily fluids include blood, plasma, serum, tissue samples, tumor samples, saliva and urine, blood is the preferred bodily fluid, in particular peripheral blood. Comparison may be made with samples taken from normal individuals, or with samples previously taken from an individual under scrutiny.
  • mRNA is detected using RT-PCR.
  • RT-PCR a cDNA is first transcribed from the mRNA.
  • the primer used to initiate transcription may be specific for the mRNA of interest, or could be hybridisable to the polyA tail of mRNA, i.e. could be an "oligo dT" primer, as is understood by the person skilled in the art.
  • PCR is then performed on the resultant cDNA.
  • At least one additional primer is used in the PCR reaction, which is complementary to the mRNA of interest. In this case the primer used to prime the cDNA synthesis acts as the second PCR primer.
  • primers are added in the PCR reaction, one primer being the forward primer to the cDNA of interest and one being the reverse primer, e.g. this could be specific for the mRNA of interest, or could be an "oligodT"- based primer. It is preferred that both primers in the PCR reaction are specific for the cDNA of interest. Where the mRNA is present in tumors as a result of a splicing event, it is preferred that at least one of the primers overlaps a splice junction as discussed elsewhere herein.
  • PCR techniques for the amplification of nucleic acid are described in US Patent No. 4,683,195. In general, such techniques require that sequence information from the ends of the target sequence is known to allow suitable forward and reverse oligonucleotide primers to be designed to be identical or similar to the polynucleotide sequence that is the target for the amplification.
  • References for the general use of PCR techniques include Mullis et al, Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed) , PCR technology, Stockton Press, NY, 1989, Ehrlich et al, Science, 252:1643- 1650, (1991) , "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, Academic Press, New York, (1990) .
  • oligonucleotide probes or primers may be designed, taking into account the degeneracy of the genetic code, and, where appropriate, codon usage of the organism from the candidate nucleic acid is derived.
  • An oligonucleotide for use in nucleic acid amplification may have about 10 or fewer codons (e.g. 6, 7 or 8), i.e. be about 30 or fewer nucleotides in length (e.g. 18, 21 or 24) .
  • specific primers are upwards of 14 nucleotides in length, but not more than 18-20.
  • the amplified nucleic acid may then be sequenced, and/or tested in any other way to determine the presence or absence of a particular sequence, for example by determining the size of the PCR product on an agarose gel, by probing the product with a suitably labelled probe specific for the sequence of interest, or by restriction enzyme digest and electrophoresis .
  • Sequencing of a PCR product may involve precipitation with isopropanol, resuspension and sequencing using a TaqFS+ Dye terminator sequencing kit. Extension products may be electrophoresed on an ABI 377 DNA sequencer and data analysed using Sequence Navigator software.
  • a variant form of the mRNA may contain one or more insertions, deletions, substitutions and/or additions of one or more nucleotides compared with the wild-type sequences. Differences at the nucleic acid level are not necessarily reflected by a difference in the amino acid sequence of the encoded polypeptide.
  • Nucleic acid according to the present invention such as a full-length coding sequence or oligonucleotide probe or primer, may be provided as part of a kit, e.g. in a suitable container such as a vial in which the contents are protected from the external environment.
  • the kit may include instructions for use of the nucleic acid, e.g. in PCR and/or a method for determining the presence of nucleic acid of interest in a test sample.
  • a kit wherein the nucleic acid is intended for use in PCR may include one or more other reagents required for the reaction, such as polymerase, nucleosides, buffer solution etc.
  • the nucleic acid may be labelled.
  • a kit for use in determining the presence or absence of nucleic acid of interest may include one or more articles and/or reagents for performance of the method, such as means for providing the test sample itself, e.g. a syringe for removing a blood sample (such components generally being sterile) .
  • the mRNA coding for the extracellular matrix domain which is the target of the PCR reaction is one which is present in tumors. It is preferred that the mRNA is one which is expressed to an enhanced level in tumor cells compared with normal adult cells.
  • the extracellular domain may be spliced out in adult cells but spliced into mRNA in tumor cells.
  • suitable mRNAs include fibronectin ED-B domain; fibronectin ED-A domain, tenascin C large isoform; repeat D of tenascin C large isoform; MUC1 (Hartman et al . (1999) Int. J. Cancer 82 256-267); CD 44 (Naot et al . (1997) Adv.
  • the preferred extracellular matrix domain is the fibronectin ED-B domain.
  • the mRNA encoding the domain is spliced out in almost all normal adult tissues.
  • Other examples include the large isoform of Repeat D of tenascin C, and the ED-A domain of fibronectin, as discussed further below.
  • the insertion (by a mechanism of alternative splicing) of the ED-A domain into fibronectin, and/or of a set of domains into the tenascin-C molecule is associated to pathological conditions, including malignancies (Castellani et al., (1986) J. Cell Biol. 104 595-600; Borsi et al.(1987) J. Cell Biol. 104 595-600; Carnemolla et al . (1999) Am. J. Pathol. 154 1345-1352) .
  • the Alarge ⁇ tenascin isoforms (which include a number of extra domains inserted by alternative splicing) are abundant components of the extracellular matrix of many tumors, such as breast and brain tumors (Borsi et al . (1992) Int. J. Cancer 52 688-692), but are undetectable in many normal tissues.
  • the extra-domain C of tenascin-C is undetectable in virtually all normal adult tissues, but extremely abundant in high grade astrocytoma, especially around vascular structures and proliferating cells (Carnemolla, 1999) .
  • Other domains of interest include MUC1, CD 44, Versican, and the IIICS domain of fibronectin as described above .
  • the invention also encompasses the development of a multiple marker RT-PCR test, based on the detection of the insertion of domains into components of the modified extracellular matrix. This should further increase the reliability and sensitivity of tumor cell detection in blood, and possibly other body fluids (Sauter et al. (1999) British J. Cancer 81 1222-1227; Nakanishi et al . (1999) Cancer Chemother Pharmacol. 43 (Suppl.) S32-S36) .
  • EXAMPLE 1 ED-B+ mRNA CAN BE DETECTED BY RT-PCR IN TUMOR AND TRANSFORMED CELL LINES.
  • RT-PCR using primers specific for the ED-B mRNA was performed on total RNA isolated from 9 different tumoral cell lines .
  • RT-PCR oligonucleotide primers which anneal to ED- B only when it is inserted in mRNA. These primers therefore avoid false positives due to amplification of genomic DNA or primary transcript.
  • the forward primer overlaps the 5' extremity of the ED-B exon and part of the 3' extremity of the exon coding for repeat 7 of fibronectin.
  • the reverse primer overlaps the 3' extremity of ED-B exon and the 5' extremity of the exon coding for repeat 8 of fibronectin. Since the exons ED-B/FN7 and the exons EDB/FN8 are separated by an intron in genomic DNA and in the primary transcript these primers will anneal only to in mRNA, because it has undergone alternative splicing.
  • Amplification of the ED-B exon from cDNA but not from genomic DNA or primary transcript can be obtained by RT-PCR performed with the primers schematically represented in Figure 1.2.
  • the forward primer overlaps the 5' extremity of ED-B exon and part of the 3' extremity of the exon coding for repeat 7 of FN.
  • the reverse primer overlaps the 3' end of ED-B exon and the 5' extremity of the exon coding for repeat 8 of fibronectin.
  • the primers only find the correct annealing sequence in those cells where the exons are adjacent.
  • ED-B+mRNA seems therefore a suitable pan-tumor marker for RT-PCR based tumor diagnosis .
  • DMEM Dulbecco Modified Eagle's Medium
  • FCS Fetal Calf Serum
  • the EA.hy 926 human lung cancer cell line, the C6 rat glial tumor cells, and the T-24 human bladder carcinoma cells were cultured respectively in: Dulbecco' s HAT medium, 10% FCS; H199 medium, 10% FCS; McCoy's medium 10%FCS.
  • RNA concentration and purity of RNA was determined by measuring the absorbance at 260 nm and 280 nm in a spectrophotometer (an adsorbance of 1 unit at 260 nm corresponds to a RNA concentration of 40 ⁇ g/ml whereas the ratio between the absorbance values at 260 and 280 nm gives an estimate of RNA purity) . Quality of total RNA was checked by formaldehyde- agarose gel electrophoresis and ethidium bromide staining.
  • RT-PCR was performed with the two-enzymes Access RT-PCR System (Promega) , following the manufacturer's instructions. lOOpg- l ⁇ g of total RNA were used as template.
  • the primers FN7-EDB (5' tct gat ace ate ate cca gag gtg ccc 3' - SEQ ID NO. 1) and
  • EDB-FN8 (5' ggg agg agg aac age cgt ttg ttg 3' - SEQ ID NO. 2) were used.
  • RT-PCR with primers specific for the housekeeping gene ⁇ -microglobulin, was performed.
  • RT-PCR reactions were performed on a thermal cycler running the program: 48°C (45' ) - [94°C ( 2' ) -60°C ( 1' ) -68°C (2' ) ] 40c ycies- 68°C(7') .
  • RT-PCR products were analyzed by electrophoresis on 1.4% agarose gel.
  • RT-PCR with primers FN7-EDB and EDB-FN8 was performed on RNA isolated from different cell lines: WI-38-VA human transformed fibroblasts, F9 murine teratocarcinoma cells, FE-8 cells, FE-8 ras-transformed rat fibroblasts, C-51 murine colon stirnoma, SK-MEL28 human melanoma, LOVO colorectal cancer cell line, T24 human bladder carcinoma, C6 rat glial tumor cells, EA.hy 926 human lung cancer cell line.
  • the PCR products were run on an agarose gel. For all the tested cell lines, amplification of the ED-B exon from the mRNA was detected as a band of 280 bp. All the tested cell lines contain a detectable amount of mRNA which is positive for the presence of ED-B exon.
  • RT-PCR performed with primers ⁇ -micro-f and ⁇ - micro-r, gave amplification of the housekeeping gene ⁇ - microglobulin, confirming that the isolated RNA could be retrotranscribed and further amplified.
  • EXAMPLE 2 ED-B+ mRNA CANNOT BE DETECTED BY RT-PCR IN PERIPHERAL BLOOD FROM HEALTHY DONORS UNLESS TUMORAL CELLS ARE PRESENT IN THE BLOOD
  • RT-PCR PERIPHERAL BLOOD FROM HEALTHY DONORS UNLESS TUMORAL CELLS ARE PRESENT IN THE BLOOD
  • RNA from fresh whole human peripheral blood was isolated using the QIAamp RNA Blood Mini Kit (QIAGEN, Basel, Switzerland) . The same kit was used for the isolation of RNA from blood added with cultured WI-38-VA cells. Concentration, purity, quality of the isolated RNA was determined as reported in Example 1.
  • ED-B+mRNA cannot be detected by RT-PCR in blood cells from healthy donors, and that tumor cells can be detected by RT-PCR of the ED-B+ mRNA, when diluted in blood.
  • Peripheral blood was obtained from human healthy donors .
  • different amounts of F9 cells ranging from 0 to 10 5 , were diluted.
  • RNA was isolated from the different samples and RT-PCR performed with primers FN7-EDB and EDB-FN8. The products were analysed on agarose gels.
  • ED- B+mRNA was undetectable in the samples with no F9 cells, 10 2 WI-38-VA cells and 10 3 WI-38-VA cells were added to 1 ml of blood.
  • 10 4 WI-38-VA cells were added to the blood
  • ED- B+mRNA was detectable as a weak amplification signal. The intensity of the signal increased proportionally with the increase of the number of WI38VA cells diluted into the blood.
  • EXAMPLE 3 ED-B+ mRNA can be detected in peripheral blood from tumor bearing mice but not in peripheral blood of healthy mice .
  • ED-B+mRNA was detected by the RT-PCR approach described in this application. In contrast, no detection of ED-B+mRNA was possible, when RT- PCR was performed on peripheral blood isolated from healthy mice.
  • mice (12-week-old Swiss nudes, females) were subcutaneously implanted with FE-8 rat carcinoma cells.
  • tumor size was approx. 500 mm 3 (tumors are measured with a dial-caliper and the volume determined using the formula: width 2 x Length x 0.52), 1 ml of peripheral blood was extracted from each mouse.
  • RT-PCR was performed as described in Example 1, using primers FN7-EDB and EDB-FN8.
  • Tumoral cells circulating in peripheral blood were identified in a murine model by RT-PCR detecting ED-B+mRNA.
  • RNA isolated from peripheral blood of 5 tumor bearing mice and 5 healthy mice was the FE-8 rat sarcoma, which is positive for the presence of ED-B+mRNA (see Example 1) .
  • EXAMPLE 4 mRNA CONTAINING THE SEQUENCE CODING FOR REPEAT D OF TENASCIN-C AND FOR EXTRA DOMAIN A OF FIBRONECTIN CAN BE DETECTED BY RT-PCR IN WI-38-VA CELLS, BUT NOT IN BLOOD CELLS FROM HEALTHY DONORS.
  • ED-A Extra Domain A
  • fibronectin a mechanism of pre-mRNA alternative splicing gives rise to protein isoforms differentially expressed in normal and tumoral tissues.
  • ED-A Extra Domain A
  • fibronectin a mechanism of pre-mRNA alternative splicing gives rise to protein isoforms differentially expressed in normal and tumoral tissues.
  • ED-A Extra Domain A
  • fibronectin isoforms containing ED-A associated with transformed cells and malignancies, compared to normal cells and tissues
  • Tenascin-C is another extracellular protein which exists in several polymorphic isoforms due to alternative splicing of nine fibronectin-like type III repeats.
  • Tenascin-C isoforms are present in some normal adult tissues but are upregulated in neoplastic tissues (Borsi et al . , 1992) . Most large tenascin-C isoforms contain the extra domain D (Borsi et al . , 1992) .
  • a combined RT-PCR test which detects several alternatively spliced exons of mosaic proteins of the extracellular matrix (e.g., the ED-B domain of FN, the ED-A domain of FN, the IIICS domain of FN, the D domain of TN-C, the C domain of TN-C) may provide a more informative identification of circulating cells in the blood of individuals with cancerous lesions.
  • the extracellular matrix e.g., the ED-B domain of FN, the ED-A domain of FN, the IIICS domain of FN, the D domain of TN-C, the C domain of TN-C
  • the SV-40 transformed WI-38-VA cell line was grown as reported in Example 1.
  • RT-PCR was performed as described in Example 1 .
  • the primers FNll-EDA (5 ' gtt cag act gca gta ace gac att gat 3' - SEQ ID NO. 3) and EDA-FN12 (5' tgg tgc agg aat age tgt gga ctg 3' - SEQ ID NO. 4) were used.
  • primers TNDba (5' age cga ace gga agt tga ca 3' - SEQ ID NO. 5) and TN6D (5' ttt ggg gag ccc atg get gtt gtt get 3' - SEQ ID NO. 6) were synthesised.
  • the oligonucleotide primer TNDba for amplification of the repeat D from the tenascin trascript, does not discriminate between transcripts containing or devoid of the adjacent repeat C, which undergoes additional splicing control (Carnemolla et al . , 1999). Choosing other backward primers would allow the discrimination betweeen tenascin transcripts containing the repeat D, in the presence or the absence of the adjacent repeat C.
  • ED-A+ mRNA and large form of TN-C mRNA were used the RT-PCR approach designed for the detection of ED-B+ mRNA in tumor cells.
  • RT-PCR For the detection of ED-A+mRNA we performed RT-PCR with primers annealing to ED-A exon and to the exon coding for the flanking FN repeats (the forward primer overlaps the 5' extremity of ED-A exon and the 3' extremity of the exon coding for the adjacent repeat 11; the reverse primer overlaps the 3' end of ED-A exon and the 5' extremity of the adjacent exon coding for repeat 12 of FN) .
  • a forward primer [oligo TN6D] which anneals to the exon coding for repeat D of TN-C and to the the following exon (which codes for a repeat which is not subjected to alternative splicing) .
  • oligo TNDba backward primer
  • PSA Prostate Prostate Specific Antigen
  • PSM Prostate Specific Membrane Antigen
  • PGP 9.5 Neuroblastoma Neuroendocrine Proteing Gene Product 9.5

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne la détection de tumeurs, y compris de tumeurs latentes, par analyse du liquide organique d'un individu. En particulier, l'invention concerne la détermination de la présence ou de l'absence, dans un échantillon de liquide organique, d'un ARN messager (mRNA) codant pour un constituant de matrice extracellulaire dans cet échantillon, lequel constituant est trouvé en particulier dans les tumeurs. Ce constituant peut être de la fibronectine ED-A, ED-B, ou une isoforme de grande taille de tenascine C.
PCT/IB2001/000899 2000-05-04 2001-05-03 Procedes de detection de tumeurs WO2001083816A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU58679/01A AU5867901A (en) 2000-05-04 2001-05-03 Method for detecting tumors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US20186400P 2000-05-04 2000-05-04
US60/201,864 2000-05-04

Publications (2)

Publication Number Publication Date
WO2001083816A2 true WO2001083816A2 (fr) 2001-11-08
WO2001083816A3 WO2001083816A3 (fr) 2003-03-13

Family

ID=22747609

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2001/000899 WO2001083816A2 (fr) 2000-05-04 2001-05-03 Procedes de detection de tumeurs

Country Status (2)

Country Link
AU (1) AU5867901A (fr)
WO (1) WO2001083816A2 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7115374B2 (en) 2002-10-16 2006-10-03 Gen-Probe Incorporated Compositions and methods for detecting West Nile virus
US7927840B2 (en) 2006-09-11 2011-04-19 Gen Probe Incorporated Method for detecting West Nile Virus nucleic acids in the 3′ non-coding region
US8222377B2 (en) 2007-10-30 2012-07-17 Philogen, S.P.A. Antigen associated with rheumatoid arthritis
US8263041B2 (en) 2007-04-02 2012-09-11 Philogen, S.P.A. Antigen associated with the neovasculature of tumour metastases
US8679488B2 (en) 2009-08-05 2014-03-25 Philogen S.P.A. Targeting of bone marrow neovasculature
US9527907B2 (en) 2009-01-07 2016-12-27 Philogen S.P.A. Antigens associated with endometriosis, psoriatic arthritis and psoriasis
WO2017084017A1 (fr) * 2015-11-16 2017-05-26 合肥立方制药股份有限公司 Utilisation de protéine ed-b dans le diagnostic d'une hyperplasie tissulaire
US9695232B2 (en) 2012-10-03 2017-07-04 Philogen S.P.A. Anti-ED-A immunoconjugates for inflammatory bowel disease
US10202442B2 (en) 2007-07-25 2019-02-12 Philogen S.P.A. Antigen associated with lung cancers and lymphomas

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2299275B1 (fr) 2004-07-30 2018-03-07 Adeza Biomedical Corporation Classification de la teneur en fibronectine oncofoetale pour les indications associées à la grossesse

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992015602A1 (fr) * 1991-02-28 1992-09-17 Dana-Farber Cancer Institute, Inc. Diagnostic et therapie du cancer
US5981218A (en) * 1995-08-09 1999-11-09 Bristol-Myers Squibb Company Isolated nucleic acid molecules useful as leukemia markers and in breast cancer prognosis and encoded polypeptides
WO2000052204A2 (fr) * 1999-02-22 2000-09-08 Orntoft Torben F Expression genique dans les tumeurs de la vessie

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992015602A1 (fr) * 1991-02-28 1992-09-17 Dana-Farber Cancer Institute, Inc. Diagnostic et therapie du cancer
US5981218A (en) * 1995-08-09 1999-11-09 Bristol-Myers Squibb Company Isolated nucleic acid molecules useful as leukemia markers and in breast cancer prognosis and encoded polypeptides
WO2000052204A2 (fr) * 1999-02-22 2000-09-08 Orntoft Torben F Expression genique dans les tumeurs de la vessie

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALBRECHT MARTIN ET AL: "Fibronectin in human prostatic cells in vivo and in vitro: Expression, distribution, and pathological significance." HISTOCHEMISTRY AND CELL BIOLOGY, vol. 112, no. 1, July 1999 (1999-07), pages 51-61, XP002218305 ISSN: 0948-6143 *
MICHELL A J ET AL: "HUMAN TENASCIN-C: IDENTIFICATION OF A NOVEL TYPE III REPEAT IN ORAL CANCER AND OF NOVEL SPLICE VARIANTS IN NORMAL, MALIGNANT AND REACTIVE ORAL MUCOSAE" INTERNATIONAL JOURNAL OF CANCER, NEW YORK, NY, US, vol. 72, 1997, pages 236-240, XP000937664 ISSN: 0020-7136 *
NICOLO G ET AL: "EXPRESSION OF TENASCIN AND OF THE ED-B CONTAINING ONCOFETAL FIBRONECTIN ISOFORM IN HUMAN CANCER" CELL DIFFERENTIATION AND DEVELOPMENT, vol. 32, no. 3, 1990, pages 401-408, XP008009720 ISSN: 0922-3371 *
OYAMA FUMITAKA ET AL: "Coordinate oncodevelopmental modulation of alternative splicing of fibronectin pre-messenger RNA at ED-A, ED-B, and CS1 regions in human liver tumors." CANCER RESEARCH, vol. 53, no. 9, 1993, pages 2005-2011, XP002218306 ISSN: 0008-5472 *
VOLLMER G ET AL: "EXPRESSION OF TENASCIN-C BY HUMAN ENDOMETRIAL ADENOCARCINOMA AND STROMA CELLS: HETEROGENEITY OF SPLICE VARIANTS AND INDUCTION BY TGF-BETA" BIOCHEMISTRY AND CELL BIOLOGY. BIOCHIMIE ET BIOLOGIE CELLULAIRE, XX, XX, vol. 75, 1997, pages 759-769, XP000937670 ISSN: 0829-8211 *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8759003B2 (en) 2002-10-16 2014-06-24 Gen-Probe Incorporated Detection of West Nile virus nucleic acids in the viral 3′ non-coding region
US7732169B2 (en) 2002-10-16 2010-06-08 Gen-Probe Incorporated Method for detecting West Nile virus nucleic acids in the 5′ non-coding/capsid region
US10781495B2 (en) 2002-10-16 2020-09-22 Gen-Probe Incorporated Detection of West Nile virus nucleic acids in the viral 3′ non-coding region
US7115374B2 (en) 2002-10-16 2006-10-03 Gen-Probe Incorporated Compositions and methods for detecting West Nile virus
US9580762B2 (en) 2002-10-16 2017-02-28 Gen-Probe Incorporated Detection of west nile virus nucleic acids in the viral 3′ non-coding region
US7927840B2 (en) 2006-09-11 2011-04-19 Gen Probe Incorporated Method for detecting West Nile Virus nucleic acids in the 3′ non-coding region
US9896503B2 (en) 2007-04-02 2018-02-20 Philogen S.P.A. Antigen associated with the neovasculature of tumour metastases
US8481684B2 (en) 2007-04-02 2013-07-09 Philogen S.P.A. Antigen associated with the neovasculature of tumour metastases
US9181347B2 (en) 2007-04-02 2015-11-10 Philogen S.P.A. Antigen associated with the neovasculature of tumour metastases
US8263041B2 (en) 2007-04-02 2012-09-11 Philogen, S.P.A. Antigen associated with the neovasculature of tumour metastases
US10202442B2 (en) 2007-07-25 2019-02-12 Philogen S.P.A. Antigen associated with lung cancers and lymphomas
US9556257B2 (en) 2007-10-30 2017-01-31 Philogen S.P.A. Antigen associated with rheumatoid arthritis
US10385121B2 (en) 2007-10-30 2019-08-20 Philogen S.P.A. Antigen associated with rheumatoid arthritis
US8222377B2 (en) 2007-10-30 2012-07-17 Philogen, S.P.A. Antigen associated with rheumatoid arthritis
US9527907B2 (en) 2009-01-07 2016-12-27 Philogen S.P.A. Antigens associated with endometriosis, psoriatic arthritis and psoriasis
US10112992B2 (en) 2009-01-07 2018-10-30 Philogen S.P.A. Antigens associated with endometriosis, psoriatic arthritis and psoriasis
US8679488B2 (en) 2009-08-05 2014-03-25 Philogen S.P.A. Targeting of bone marrow neovasculature
US9446124B2 (en) 2009-08-05 2016-09-20 Philochem Ag Targeting of bone marrow neovasculature
US9695232B2 (en) 2012-10-03 2017-07-04 Philogen S.P.A. Anti-ED-A immunoconjugates for inflammatory bowel disease
US10239939B2 (en) 2012-10-03 2019-03-26 Philogen S.P.A. Anti-ED-A immunoconjugates for inflammatory bowel disease
WO2017084017A1 (fr) * 2015-11-16 2017-05-26 合肥立方制药股份有限公司 Utilisation de protéine ed-b dans le diagnostic d'une hyperplasie tissulaire
CN108291915A (zh) * 2015-11-16 2018-07-17 合肥立方制药股份有限公司 Ed-b蛋白在诊断组织增生中的应用
CN108291915B (zh) * 2015-11-16 2021-01-05 合肥立方制药股份有限公司 Ed-b蛋白在诊断组织增生中的应用

Also Published As

Publication number Publication date
AU5867901A (en) 2001-11-12
WO2001083816A3 (fr) 2003-03-13

Similar Documents

Publication Publication Date Title
ES2281416T3 (es) Metodos, composiciones y sistemas para la deteccion y monitorizacion del cancer de mama.
USRE49542E1 (en) Method for the detection of cancer
Smith et al. Prostate-specific antigen messenger RNA is expressed in non-prostate cells: implications for detection of micrometastases
US6607898B1 (en) Method for detection of hTR and hTERT telomerase-associated RNA in plasma or serum
US6303318B1 (en) Methods for the early diagnosis of ovarian cancer
Laurell et al. Identification of biomarkers of human pancreatic adenocarcinomas by expression profiling and validation with gene expression analysis in endoscopic ultrasound-guided fine needle aspiration samples
JP2003523524A5 (fr)
CA2318354A1 (fr) Marqueurs biochimiques et cibles pour le diagnostic, le pronostic et le traitement des affections de la prostate
CA2467629A1 (fr) Procedes pour evaluer une expression genique de resistance aux medicaments chez un patient atteint d'un cancer
Wong et al. Detection of circulating tumour cells and nodal metastasis by reverse transcriptase-polymerase chain reaction technique
EP1080227B1 (fr) Procédé de diagnostic, de surveillance et de détermination du stade du cancer du colon
WO1998050404A1 (fr) Procede de detection de cancer de la thyroide metastatique
US5858673A (en) Method for detecting prostate cells
WO2001083816A2 (fr) Procedes de detection de tumeurs
Fabjani et al. HMGA2 is associated with invasiveness but not a suitable marker for the detection of circulating tumor cells in breast cancer
Koenig et al. Urinary markers of malignancy
Nezos et al. Molecular markers detecting circulating melanoma cells by reverse transcription polymerase chain reaction: methodological pitfalls and clinical relevance
Miyake et al. Highly specific and sensitive detection of malignancy in urine samples from patients with urothelial cancer by CD44v8–10/CD44v10 competitive RT‐PCR
US5830646A (en) Diagnostic method
CA2328377A1 (fr) Nouveau procede de diagnostic, de surveillance et de stadification du cancer de la prostate
US20040248142A1 (en) Method for detecting hepatocellular carcinoma
Ghossein et al. Reverse transcriptase polymerase chain reaction (RT-PCR) detection of melanoma-related transcripts in the peripheral blood and bone marrow of patients with malignant melanoma. What have we learned?
de la Taille et al. Blood-based RT-PCR assays of MN/CA9 or PSMA: clinical application in renal cancer patients
AU2004224448A1 (en) Detection and monitoring of lung cancer
KR20070116567A (ko) 위 암종으로부터의 림프절 전이의 검출법

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP