WO2001034837A1 - Diagnostic et pronostic de cancer par detection de l'antigene tumoral 17-1a par rt-pcr - Google Patents

Diagnostic et pronostic de cancer par detection de l'antigene tumoral 17-1a par rt-pcr Download PDF

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Publication number
WO2001034837A1
WO2001034837A1 PCT/GR2000/000034 GR0000034W WO0134837A1 WO 2001034837 A1 WO2001034837 A1 WO 2001034837A1 GR 0000034 W GR0000034 W GR 0000034W WO 0134837 A1 WO0134837 A1 WO 0134837A1
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PCT/GR2000/000034
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English (en)
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Eleftherios Georgakopoulos
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Limeart Limited
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Priority to AU12916/01A priority Critical patent/AU1291601A/en
Publication of WO2001034837A1 publication Critical patent/WO2001034837A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to a novel method for the detection of the cancer antigen 17-lA in peripheral blood, tissue and urine samples, as well as methods for the diagnosis and prognosis of cancer and the monitoring of cancer therapy.
  • cancer cells may be differentiated from non-cancer cells by the detection of cancer-related proteins that are abnormally expressed in cancer cells relative to the expression in non-cancer cells.
  • cancer-related proteins are known as cancer-related antigens, or tumor antigens. These proteins belong to the glycoprotein group and are found on the outer membrane of cancer cells. Many of the tumor antigens have been isolated and characterised. One of these is the cancer antigen 17-lA (GA 733-1 and GA-733-2)
  • Antigen 17-lA is a 40 kDa glycoprotein found on the surface of epithelial and cancer cells. It is found in the outer membrane of the cell, and multiple molecules of glucose are connected to its protein chain (Varki et al . , 1984, Bumol et al . , 1988, Perez and Walker, 1989). The levels of expression of antigen 17-lA in cancer cells is higher than in non-cancer cells.
  • 17-lA The function of 17-lA is not known. Its high expression in cancer cells and it homogeneous expression in epithelial tissues lead us to the conclusion that it should play some significant role in cell function.
  • Antigen 17-lA is expressed 'at high levels in several different types of cancer. Its detection has been achieved with immunochemical methods. The table that follows shows the detection of antigen 17-lA and its percentile expression for various types of cancer.
  • Figure 1 depicts the results of detecting the level of 17-lA mRNA in samples from subjects treated with 17-lA antibody.
  • the lanes labeled 14T2 and 32T2 are the PCR products generated from samples taken immediately after antibody treatment, the lanes labeled 14T3 and 12T3 are from 24 hours after treatment, and the lanes labeled 14T4 and 31T4 are from samples taken 48 hours after antibody treatment.
  • the lane labeled "+” contains the PCR products obtained from a clinical sample known to be positive for the presence of prostate specific antigen (PSA) mRNA and 17-lA mRNA.
  • PSA prostate specific antigen
  • MW contains standard molecular weight markers .
  • the detection of the cancer antigen 17-1 A in the peripheral blood and the urine of the patient may be used to determine the therapeutic protocol to be used and can be used to determine the degree of therapy necessary and to monitor the effectiveness of the chosen therapy.
  • the method of the invention may also be used as surveillance for the detection and control of micro-metastases, which are difficult to detect by conventional means.
  • the method of the present invention consists of the isolation of nucleic acids, (e.g., mRNA) from a biological sample (e.g., whole blood, a pathological tissue sample, a urine sample) and the production therefrom of complementary DNA (cDNA) with the use of antisense primers or random hexamers .
  • a sample of the cDNA is used for the amplification and detection of a nucleic acid fragment coding for the tumor antigen 17-lA through the method of
  • the amplified fragment is then detected by any means known to one of skill in the art.
  • the amplified fragment may be detected by separating the PCR reaction products on an agarose gel and visualizing the fragment encoding 17-lA by, for example, staining with ethidium bromide and exposing the gel to ultraviolet light.
  • the agarose gel is preferably a 2% agarose gel.
  • the method of the invention has three major components: isolation of cDNA from a biological sample, amplification of a nucleic acid fragment encoding 17-lA, and detection of said fragment.
  • Urine Sample Procedure 1 Collection of 50 ml of urine in a sterilized container
  • cDNA is carried out as follows:
  • a small portion of the complementary DNA is used for the PCR reaction.
  • the PCR reaction is performed such that a portion of a nucleic acid coding for 17-lA is amplified from the cDNA produced from the biological sample. If the cDNA was produced using a specific primer, as opposed to random hexamer primers, the primers used in the PCR step are chosen such that they are complementary to the portion of the 17-lA nucleic acid sequence that was produced by the reverse transcriptase used during the cDNA production step.
  • the primers may be labeled or unlabeled.
  • the primers used are the following: 5'- ATG GCG CCC CCG CAG GTC CTC GCG T -3' (SEQ ID NO 1) 5'- TTC TCG GGC GAG AGT AGC GTC AGT CC -3 ' (SEQ ID NO 27)
  • the PCR reaction is carried out under the following conditions:
  • thermo cycler is a Perkin Elmer cycler, most preferably a Perkin Elmer 2400 model thermal cycler.
  • the thermal protocol is the following: Initial conditions: 93-95 °C, 3-7 min
  • the thermal profile is the following: Initial conditions: 94 °C, 5 min
  • thermal protocol Another example of a thermal protocol that can be used to carry out the method of the invention is as follows: Initial conditions 93-95 °C, 3-7 min
  • the thermal profile is the following: Initial conditions 94 °C, 5 min
  • the quantity of the amplified 17-lA encoding nucleic acid fragment is detected by any means known to one of skill in the art.
  • the PCR products are separated on an agarose gel and visualized with ethidium bromide staining followed by exposure to UV light.
  • the agarose gel is a 2% gel, however the correct agarose concentration may be readily determined by one of skill in the art given the size of the fragment one desires to visualize. For example, a 2% gel may be used to visualize an approximately 500-600 base pair fragment.
  • compositions and methods of the invention are described.
  • SUBST ⁇ UTE SHEET useful for the diagnosis, prognosis and monitoring of cancer or cancer therapy.
  • methods and compositions of the invention can be used for: 1) patients diagnosed with cancer at any clinical stage of the disease, 2) the prognosis of cancer in patients with symptoms or signs of cancer, and 3) the monitoring of the effectiveness of therapy, 4) the monitoring of the progression of cancer.
  • the formulations of the invention are useful for monitoring, diagnosis or prognosis of cancers of the blood, esophagus, gastric tube, intestine, breast, lung, ovary, prostate, head, neck, brain, testes, kidney, pancreas, bone, spleen, liver, and bladder; AIDS-related cancers, such as Kaposi's sarcoma; leukemia (e.g., acute leukemia such as acute lymphocytic leukemia and acute myelocytic leukemia) ; non-small cell lung cancer, and the like.
  • leukemia e.g., acute leukemia such as acute lymphocytic leukemia and acute myelocytic leukemia
  • the effectiveness of antibody therapy is monitored.
  • the methods of the invention may be used to monitor the effectiveness of anti-17-lA antibodies in the treatment of cancer.
  • the methods are also useful for post-operative monitoring of the effectiveness of surgical procedures such as the surgical removal of a tumor .
  • the detection of 17-lA using the methods of the invention can be be used for detection, prognosis, diagnosis, or monitoring of cancer or for drug development.
  • a biological sample from a subject e . g. , a subject suspected of having cancer
  • An increased abundance of 17-lA mRNA in the biological sample from the subject relative to a similar biological sample from a subject or subjects free from cancer indicates the presence of cancer.
  • an increased abundance of 17-lA mRNA in a first biological sample from the subject relative to a second biological sample from the same subject, wherein the second biological sample is from a tissue the same as or similar to the tissue from which the first sample was taken, and wherein the second biological sample is free, or substantially free, from cancerous tissue and/or cells indicates the presence of cancer.
  • the subject, or patient, from which the biological sample is taken and analyzed using the methods of the invention is an animal, e.g., a mammal, and is preferably human, and can be a fetus, child, or adult.
  • the signal obtained upon analyzing a biological sample from a subject having cancer relative to the signal obtained upon analyzing a sample from subjects free from cancer, or from a biological sample from the same subject that does not include cancerous cells, will depend upon the particular analytical protocol and detection technique that is used. Accordingly, the present invention contemplates that each laboratory will, based on the present description, establish a reference range for 17-lA expression in subjects or biological samples free from cancer according to the method of the invention, as is conventional in the diagnostic art.
  • at least one positive control sample from a subject known to have cancer or at least one negative control sample from a subject known to be free from cancer are included in each batch of test samples analyzed.
  • SUBST ⁇ UTE SHEET RULE26 biological sample of a subject (e.g., a subject suspected of having or known to have cancer) is normalized with reference to the level of expression of one or more other reference mRNAs encoding protein known to be invariant, or substantially invariant, with respect to the cancer of interest.
  • a suitable reference mRNA is the mRNA coding for ⁇ 2 microglobulin, which is the preferred reference mRNA for use in breast cancer, but may be used for other cancers as well .
  • Detection of 17-lA Encoding PCR Products 1. Prepared a 2% agarose gel 2. Loaded the PCR products and a standard ladder of DNA fragments of known molecular weight on gel and ran at a constant voltage of 10 volts/cm for 45 minutes 3. Stained separated PCR products with ethidium bromide and visualized with ultraviolet light 4. Detected 17-lA band of approximately 530-540 bp .
  • the intensity of the 17-lA band was quantified via densitometric analysis.
  • Two subjects suffering from metastatic prostate cancer were treated with Enderocolomab, an anti-17-lA antibody.
  • the effect of such antibody treatment on the levels of 17-lA mRNA detectable in the blood by the method of Example 1 was determined.
  • the two subjects are referred to as PS-8 and PS-9.
  • a blood sample from PS-8 was tested after a single 500 mg dose of anti-17-lA antibody, while a blood sample from PS-9 was tested after a third 500 mg dose of anti-17-lA antibody.
  • Samples were taken immediately at the end of the infusion of 17-lA antibody (indicated in Figure 1 as 14T2 for the 1st infusion in patient PS-8 and 32T2 for the 3rd infusion in patient PS-9) , 24 hours after infusion (indicated as 14T3 and 12T3) and 48 hours (indicated as 14T4 and 31T4) .
  • the lane labeled " + " contains the PCR products obtained from a clinical sample known to be positive for the presence of prostate specific antigen (PSA) mRNA and 17-lA mRNA.
  • PSA prostate specific antigen
  • MW contains standard molecular weight markers.
  • Figure 1 shows that at time point T2 the mRNA level of 17-lA is high.
  • the level of 17-lA mRNA is decreased 24 hours (T2) following the 17-lA antibody infusion and increases from the 24 hour levels at 48 hours. However, even at 48 hours, the levels of the 17-lA mRNA remains less than the levels observed at the initial time point .

Abstract

La présente invention concerne une nouvelle technique de détection de l'antigène 17-1A du cancer dans le sang périphérique, dans des tissus et dans des prélèvements d'urine, de même que des techniques permettant d'établir le diagnostic et le pronostic d'un cancer ainsi que la surveillance d'une thérapie de lutte contre le cancer.
PCT/GR2000/000034 1999-11-12 2000-11-13 Diagnostic et pronostic de cancer par detection de l'antigene tumoral 17-1a par rt-pcr WO2001034837A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU12916/01A AU1291601A (en) 1999-11-12 2000-11-13 Diagnosis and prognosis of cancer through detection of the tumor antigen 17-1a by rt-pcr

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GR990100391 1999-11-12
GR99100391 1999-11-12
GR20000100159 2000-08-05
GR2000100159 2000-08-05

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GR1004303B (el) * 2002-11-05 2003-07-30 Ελευθεριος Γεωργακοπουλος ΝΕΑ ΜΕΘΟΔΟΣ ΑΝΙΧΝΕΥΣΗΣ ΤΟΥ ΚΑΡΚΙΝΙΚΟΥ ΜΕΜΒΡΑΝΙΚΟΥ ΑΝΤΙΓΟΝΟΥ Ep-Cam ΚΑΙ ΠΡΟΣΔΙΟΡΙΣΜΟΣ ΚΥΚΛΟΦΟΡΟΥΝΤΩΝ ΜΙΚΡΟΜΕΤΑΣΤΑΤΙΚΩΝ ΚΑΡΚΙΝΙΚΩΝ ΚΥΤΤΑΡΩΝ ΣΤΟ ΠΕΡΙΦΕΡΙΚΟ ΑΙΜΑ ΑΣΘΕΝΩΝ (ΚΑΙ ΣΕ ΙΣΤΟΥΣ) ΜΕ ΚΑΡΚΙΝΟ ΕΠΙΘΗΛΙΑΚΗΣ ΠΡΟΕΛΕΥΣΗΣ

Citations (3)

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EP0376746A2 (fr) * 1988-12-29 1990-07-04 The Wistar Institute Famille de gènes pour des antigènes associés à des tumeurs
EP0520794A1 (fr) * 1991-06-26 1992-12-30 F. Hoffmann-La Roche Ag Méthodes de détection de métastases d'un carcinome par amplification d'acides nucléiques
DE19825082C1 (de) * 1998-06-05 2000-02-24 Gabriele Jaques Nachweisverfahren für epitheliale Tumorzellen mittels Reverse-Transkriptase-Polymerase-Kettenreaktion (RT-PCR)

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0376746A2 (fr) * 1988-12-29 1990-07-04 The Wistar Institute Famille de gènes pour des antigènes associés à des tumeurs
EP0520794A1 (fr) * 1991-06-26 1992-12-30 F. Hoffmann-La Roche Ag Méthodes de détection de métastases d'un carcinome par amplification d'acides nucléiques
DE19825082C1 (de) * 1998-06-05 2000-02-24 Gabriele Jaques Nachweisverfahren für epitheliale Tumorzellen mittels Reverse-Transkriptase-Polymerase-Kettenreaktion (RT-PCR)

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EL SEWEDY T ET AL: "Cloning of the murine Trop2 gene: conservation of a Pip2-binding sequence in the cytoplasmic domain of Trop-2", INTERNATIONAL JOURNAL OF CANCER, vol. 75, 1998, pages 324 - 30, XP000929390 *
HELFRICH W ET AL: "A quantitative reverse transcriptase polymerase chain reaction-based assay to detect carcinoma cells in peripheral blood", BRITISH JOURNAL OF CANCER, vol. 76, no. 1, 1997, pages 29 - 35, XP000929384 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GR1004303B (el) * 2002-11-05 2003-07-30 Ελευθεριος Γεωργακοπουλος ΝΕΑ ΜΕΘΟΔΟΣ ΑΝΙΧΝΕΥΣΗΣ ΤΟΥ ΚΑΡΚΙΝΙΚΟΥ ΜΕΜΒΡΑΝΙΚΟΥ ΑΝΤΙΓΟΝΟΥ Ep-Cam ΚΑΙ ΠΡΟΣΔΙΟΡΙΣΜΟΣ ΚΥΚΛΟΦΟΡΟΥΝΤΩΝ ΜΙΚΡΟΜΕΤΑΣΤΑΤΙΚΩΝ ΚΑΡΚΙΝΙΚΩΝ ΚΥΤΤΑΡΩΝ ΣΤΟ ΠΕΡΙΦΕΡΙΚΟ ΑΙΜΑ ΑΣΘΕΝΩΝ (ΚΑΙ ΣΕ ΙΣΤΟΥΣ) ΜΕ ΚΑΡΚΙΝΟ ΕΠΙΘΗΛΙΑΚΗΣ ΠΡΟΕΛΕΥΣΗΣ

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