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WO1997007242A1 - Procede de detection de la circulation de cellules du cancer du sein - Google Patents

Procede de detection de la circulation de cellules du cancer du sein Download PDF

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Publication number
WO1997007242A1
WO1997007242A1 PCT/US1996/010647 US9610647W WO9707242A1 WO 1997007242 A1 WO1997007242 A1 WO 1997007242A1 US 9610647 W US9610647 W US 9610647W WO 9707242 A1 WO9707242 A1 WO 9707242A1
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WIPO (PCT)
Prior art keywords
psa
polynucleotide
breast cancer
sample
encoding
Prior art date
Application number
PCT/US1996/010647
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English (en)
Inventor
Steven Lehrer
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Steven Lehrer
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
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Publication of WO1997007242A1 publication Critical patent/WO1997007242A1/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
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates to the field of cancer detection. More specifically, the invention provides an effective method for the detection of circulating breast cancer cells in lymph node negative patients.
  • Tumor size, histologic grading, node involvement, and estrogen receptor status of the tumor are the most widely accepted and widely used indicators employed to assess the probability of tumor recurrence and the need for adjuvant therapy.
  • Other markers such as tumor epidermal growth factor receptor, tumor c-erbB-2 level, and tumor angiogenesis, are also used (1) .
  • tumor epidermal growth factor receptor tumor c-erbB-2 level
  • tumor angiogenesis tumor angiogenesis
  • Prostate cancer is one of the most common cancers in men. Since there is no effective way to prevent this cancer, early diagnosis, treatment and monitoring of these patients is necessary. Recently, a new assay has been employed for detecting circulating metastatic prostate cancer cells. These cells produce a protein, prostate specific antigen (PSA) , which is detected by reverse transcription (RT) and polymerase chain reaction (PCR) amplification of the messenger ribonucleic acid (mRNA) encoding the antigen (2-5, 30) .
  • PSA prostate specific antigen
  • RT reverse transcription
  • PCR polymerase chain reaction
  • PSA is produced by the prostate gland only, and therefore only expressed in men.
  • PSA levels in breast tumors were examined using a sensitive immunoassay that was capable of detecting PSA at levels of 0.05 ⁇ g/L or higher.
  • the clinical potential of PSA as a prognostic indicator of recurring breast cancer was explored using this same immunological assay in serum rather than tumor tissue (9) .
  • Serum PSA levels were compared in women with and without breast cancer, between women with PSA-positive and PSA-negative breast cancer and between women with breast cancer before and after surgical removal of the tumor. The data showed that in women over 50 years of age, serum PSA levels did not vary significantly between normal and breast cancer patients. Furthermore, differences were not observed in pre-surgical and post-surgical serum PSA in women with PSA-positive or PSA-negative breast cancer.
  • a polynucleotide amplification assay for PSA-encoding nucleic acids, particularly mRNA, in a blood sample accurately detects circulating breast cancer cells in approximately one-third of the population tested. This proportion reflects the proportion of women expected to possess significant amounts of PSA based on immunological analyses of breast tumors.
  • the success of detecting PSA- encoding nucleic acids from blood samples leads also to the expectation that other antigens associated with breast cancer cells can be detected by polynucleotide amplification in a manner similar to the detection of PSA.
  • a method of detecting breast cancer micrometastases in a patient comprises obtaining a sample of polynucleotides from a patient's blood, and performing a polynucleotide amplification reaction (such as polymerase chain reaction) on the sample.
  • the polynucleotide amplification reaction is performed using nucleotides that specifically hybridize with sequences present in a predetermined polynucleotide encoding a characteristic determinent of breast cancer cells, under conditions causing amplification of the selected polynucleotide. Thereafter, the presence and quantity of amplification products of the predetermined polynucleotide is detected.
  • the term “characteristic determinant” refers to substances such as antigens, haptens and other complex molecules (e.g., carbohydrates, glycoproteins, etc.), which are associated with a cell type of interest.
  • the term “predetermined” denotes that a particular characteristic determinant is known to be associated with breast cancer cells.
  • PSA is a characteristic determinant of breast cancer cells in approximately 30% of breast cancers.
  • polynucleotide generally refers to RNA, specifically mRNA, but is also intended to include DNA.
  • predetermined polynucleotide refers to a polynucleotide that encodes a characteristic determinant of breast cancer cells.
  • the method comprises the detection of mRNA encoding PSA from the blood of a breast cancer patient.
  • An RNA sample is obtained from the patient's blood and the PSA-specific RNA is reverse transcribed and then amplified using a pair of primers which are complementary to separate regions of the PSA DNA. After amplification, the presence or absence of amplified DNA is detected, wherein the presence of amplified DNA indicates micrometastasis of PSA-expressing breast cancer cells.
  • RT- PCR is used to amplify the target polynucleotide.
  • any polynucleotide amplification method known in the art may be employed.
  • the method entails pretreating blood cells from a patient with compounds that induce PSA production. Enhancing production of PSA mRNA prior to the RT-PCR amplification step increases the sensitivity of the assay.
  • steroid hormones capable of inducing PSA production may be administered to the patient for a suitable period prior to obtaining the blood sample.
  • Characteristic determinants for breast cancer cells other than PSA may be detected with the above mentioned methodology. These include, but are not limited to, estrogen receptor (ER) , progesterone receptor (PR) (20) , epidermal growth factor receptor (EGFR) (21) , Bcl-2 (22) , and erbB-2 protein (23) . These antigens are all associated with breast cancer cells, so the polynucleotides encoding these proteins are expected to be detectable with the same assays.
  • ER estrogen receptor
  • PR progesterone receptor
  • EGFR epidermal growth factor receptor
  • Bcl-2 Bcl-2
  • erbB-2 protein erbB-2 protein
  • a new method for detecting metastases in breast cancer patients is disclosed.
  • PSA prostate specific antigen
  • the present inventor surmised that the reverse transcriptase- polymerase chain reaction for PSA, used for detecting metastases in prostate cancer, might also be used for detecting breast cancer metastases.
  • the PSA assay detects circulating breast cancer cells in approximately one third of the population tested. This assay is expected to be useful as an additional independent predictor of tumor recurrence in node negative breast cancer patients.
  • An exemplary embodiment of the clinical test employs an enhanced reverse transcriptase (RT) polymerase chain reaction (PCR) assay utilizing oligonucleotide primers specific for nucleic acids encoding the human prostate-specific antigen (PSA) .
  • This assay identifies PSA-synthesizing cells from reverse transcribed mRNA.
  • the assay is applied to RNAs extracted from the peripheral blood of breast cancer patients.
  • the RT-PCR assay for PSA can recognize one PSA-expressing metastatic breast cancer cell diluted into one hundred thousand white blood cells.
  • the sensitivity of the assay may also be enhanced by the addition of digoxigenin-modified nucleotides to the PCR reaction.
  • the sensitivity of the assay is enhanced by pretreating the blood sample to be tested with steroid hormones. It has been discovered that androgens, progestins, mineralocorticosteroids, glucocorticosteroids and antiestrogens upregulate PSA production in receptor positive breast carcinoma cell lines, T-47D and MCF-7. In the alternative, steroid hormones may also be administered to a patient for a suitable period before obtaining the blood sample to be tested.
  • targeted polynucleotide amplification methods such as self-sustained sequence replication (3SR) and strand-displacement amplification (SDA) ; 2) methods based on amplification of a signal attached to the target polynucleotide, such as "branched chain” DNA amplification (Chiron Corp.); 3) methods based on amplification of probe or primer DNA, such as ligase chain reaction (LCR) and QB replicase amplification (QBR) ; and 4) various other methods, such as ligation activated transcription (LAT) , nucleic acid sequence- based amplification (NASBA) , repair chain reaction (RCR) and cycling probe reaction (CPR) .
  • LCR ligase chain reaction
  • QBR QB replicase amplification
  • LAT ligation activated transcription
  • NASBA nucleic acid sequence- based amplification
  • RCR repair chain reaction
  • CPR cycling probe reaction
  • breast cancer cells produce progesterone receptor (PR) (20) , epidermal growth factor receptor (EGFR) (21) , Bcl-2 (22) , and erbB- 2 protein (23) , the mRNAs encoding these proteins should be detectable with the same assay.
  • PR progesterone receptor
  • EGFR epidermal growth factor receptor
  • Bcl-2 Bcl-2
  • erbB- 2 protein erbB- 2 protein
  • oligonucleotides are designed to hybridize specifically with sequences on the target polynucleotide.
  • a "specifically hybridizing" oligonucleotide is one of sufficient complementarity to a specified region of the target polynucleotide (i.e., the predetermined polynucleotide) to hybridize substantially exclusively with that region under standard hybridization conditions (i.e., conditions normally used for a given polynucleotide amplification reation) .
  • standard hybridization conditions i.e., conditions normally used for a given polynucleotide amplification reation.
  • Fully complementary oligonucleotides are preferred.
  • these oligonucleotides may be utilized in a variety of polynucleotide amplification reactions, for example, primers for polymerase chain reaction, oligonucleotides that bind contiguous stretches of the DNA encoding the targeted determinant, for ligase chain reaction, etc.
  • Other uses for oligonucleotides in targeted polynucleotide amplification reactions will be apparent to those skilled in the art.
  • Synthetic oligonucleotides may be prepared by standard methods, such as the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC) . General procedures to synthesize and purify oligonucleotide, are set forth in Sambrook et al., Molecular Cloning. Cold Spring Harbor Laboratory (1989) .
  • the methodology described herein can also be used to detect micrometastases in regional lymph nodes, as a breast cancer screening technique, or for evaluating the results of adjuvant therapy (hormone therapy, chemotherapy, etc.).
  • the PSA mRNA assay may be used to detect circulating metastatic cells in patients with other tumor types that express PSA.
  • peripheral venous blood Six to eight milliliters of peripheral venous blood was obtained using heparinized tubes and placed immediately on ice. The whole blood was then subjected to a gradient isolation of nucleated cells using Ficoll (Accurate Chemical and Scientific Corp, estbury, NY) (4) . The mononuclear cell layer was aspirated, re- diluted in phosphate-buffered saline, and then centrifuged as previously described (4) . After the supernatant was discarded, the pellet was stored at -70°C or used directly for RNA extraction.
  • RNAzol B Biotecx Laboratories, Houston, TX
  • chloroform 0.2 mL of chloroform
  • the preparation was mixed vigorously and put on ice for 5 minutes. The suspension was then centrifuged at 12,000 x g (4°C) for 15 minutes. The aqueous phase was transferred to a fresh tube and mixed with an equal volume of isopropanol. The samples were then put at -20°C for at least 2 hours. This was followed by centrifugation at 12,000 x g (4°C) for 15 minutes. After the supernatant was discarded, the RNA pellet was washed with 100% ethanol and subsequently centrifuged at 12,000 x g (4°C) for 15 minutes. This washing step was repeated using 75% ethanol. The dry RNA pellet was finally dissolved in 50 ⁇ L of diethylpyrocarbonate-treated water.
  • RNA from each sample were subjected to an RT-PCR using primers PSA3 ' and PSA5' as previously described (5) .
  • the 18 base pair primers were designed to span across three exons; from exon 3 and extending into exon 5 with the following sequences:
  • PSA3 1 5'-CACAGACACCCCATCCTATC-3' (Sequence I.D. No. 1)
  • PSA5' 5'-GATGACTCCAGCCACGACCT-3• (Sequence I.D. No. 2)
  • the entire PCR products were run on a 2% ethidium bromide-stained agarose gel, then transferred to a nylon membrane using the Oncor Probe Tech 2 system (Oncor, Gaithersburg, MD) .
  • the membranes were pre-hybridized at 42°C using Hybrisol I (Oncor) as a pre-hybridization mixture.
  • Hybridization was performed at 42°C for 16 hours with a 32p end-labeled probe internal to the PCR primers: R2: 5 • -CTACGCCTCAGGCTGGGGCAGCATTGAACCAGAGGAGTTCTTGACC-3' (Sequence I.D. No. 3) . This was followed by washes of increasing stringency (final, 52° to 54°C) with 0.1% sodium dodecyl sulfate (SDS)/0.1% sodium chloride-sodium citrate. The blots were exposed to X-OMAT films (Eastman Kodak, Rochester, NY) at -70°C for 48 hours using intensifying screens.
  • SDS sodium dodecyl sulfate
  • PSA prostate specific antigen
  • Estrogens failed to induce such stimulation in both cell lines and, in addition, Yu et al. were able to block the induction by androgens in the cell line T-47D.
  • PSA can also be induced in prostate cancer cell lines by incubation with androgen (32) .
  • the sensitivity of the PSA-RT-PCR assay for detecting circulating breast tumor cells or prostate tumor cells can be increased by incubation of the blood specimen or white blood cells with the following steroids: androgens, progestins, mineralo- corticosteroids, glucocorticosteroids, or antiestrogens before the PSA RT-PCR is performed.
  • the steroids are dissolved to make stock solutions of IO" 2 to 10" 3 M in absolute ethanol.
  • White blood cells from a patient sample are aliquoted in 24 well tissue culture plates and 2 ⁇ L of each steroid, dissolved in absolute ethanol, added to each well and incubated at 37.6° from one hour to eight days. Because the assay is performed on a multiwell plate, samples from several patients may be tested simultaneously.
  • androgens, progestins, mineralocorticosteroids, glucocorticosteroids, or antiestrogens could be administered directly to the patient and blood drawn for analysis after 24-48 hours.
  • Suitable androgens for the above assay are testosterone, dihydroandrosterone, androsterone, R1881 (MethyItrienolone) , R5020, dihydrotestosterone.
  • Suitable glucocorticoids are corticosterone, hydrocortisone, betamethasone-17-valerate, dexamethasone and triamcinolone acetonide.
  • Progestins such as progesterone, norethynodrel, norethindrone, norgestrel, depo-provera, norgestimate may also be used.
  • a mineralocorticoid that could be used is aldosterone. Cyproterone acetate, an antiandrogen/progestin may also be utilized. Other antiandrogens that may prove to be useful are RU56,187, mifepristone, cortexolone, spironolactone. An antiestrogen that may stimulate PSA-mRNA production is tamoxifen.
  • Prostate-specific antigen messenger RNA is expressed in non-prostate cells: implications for detection of micrometastases. Cancer Res 1995;55:2640-2644.
  • Croce CM Fischer R
  • Gomella L Moreno JG
  • Diamandis EP Detection of prostate specific antigen in breast tumors: presence is associated with earlier stage disease, younger women, and better survival.
  • International Publication Number (PCT) WO 94/27152, 13 May 1994.
  • MOLECULE TYPE other nucleic acid
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE other nucleic acid
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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  • 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

Cette invention porte sur un procédé efficace de détection de la circulation de cellules du cancer du sein. L'observation établissant qu'un tiers des cellules du cancer du sein produisent un antigène spécifique du prostatique (PSA) a conduit à une réalisation préférée de l'essai clinique faisant intervenir un dosage de réaction de polymérisation en chaîne (PCR) de la transcriptase inverse (RT) utilisant des amorces oligonucléotidiques spécifiques d'acides nucléiques codant le PSA. On soumet à ce dosage, grâce auquel sont identifiées des cellules synthétisant le PSA à partir d'un acide ribonucléique messager transcrit inverse (ARNm), des ARN tirés du sang périphérique de patientes atteintes d'un cancer du sein, ce qui permet de déceler la présence d'une cellule du cancer du sein métastatique exprimant le PSA dans une dilution de cent mille leucocytes. L'invention a également trait à d'autres procédés d'amplification permettant de déceler un ARN messager du PSA ou, dans une variante, des acides nucléiques codant d'autres antigènes exprimés par des cellules du cancer du sein.
PCT/US1996/010647 1995-08-16 1996-06-20 Procede de detection de la circulation de cellules du cancer du sein WO1997007242A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US246895P 1995-08-16 1995-08-16
US60/002,468 1995-08-16
US56195295A 1995-11-22 1995-11-22
US08/561,952 1995-11-22

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998059073A1 (fr) * 1997-06-20 1998-12-30 Mayo Foundation For Medical Education And Research Procede de detection du cancer du sein
WO1999010528A1 (fr) * 1997-08-22 1999-03-04 Michael Giesing Procede permettant de caracteriser des cellules cancereuses disseminees et micrometastasees
WO2000044940A3 (fr) * 1999-01-28 2000-12-07 Gen Probe Inc Sequences d'acide nucleique permettant de detecter des marqueurs genetiques pour le cancer dans un echantillon biologique
US6479263B1 (en) 1996-11-14 2002-11-12 Baylor College Of Medicine Method for detection of micrometastatic prostate cancer
DE10143776A1 (de) * 2001-09-06 2003-04-03 Adnagen Ag Verfahren und Kit zur Diagnostik oder Behandlungskontrolle von Brustkrebs
US7507528B2 (en) 2001-09-06 2009-03-24 Adnagen Ag Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5344757A (en) * 1988-01-12 1994-09-06 Boehringer Mannheim Gmbh Process for the detection of nucleic acids

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5344757A (en) * 1988-01-12 1994-09-06 Boehringer Mannheim Gmbh Process for the detection of nucleic acids

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, 15 December 1994, Vol. 54, MONNE et al., "Molecular Characterization of Prostate-Specific Antigen Messenger RNA Expressed in Breast Tumors", pages 6344-6347. *
CANCER RESEARCH, June 1995, Vol. 55, SMITH et al., "Prostate-specific Antigen Messenger RNA is Expressed in Non-Prostate Cells: Implications for Detection of Micrometastases", pages 2640-2644. *
UROLOGY, June 1994, Vol. 43, Number 6, KATZ et al., "Molecular Staging of Prostate Cancer with the Use of an Enhanced Reverse Transcriptase-PCR Assay", pages 765-775. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6479263B1 (en) 1996-11-14 2002-11-12 Baylor College Of Medicine Method for detection of micrometastatic prostate cancer
WO1998059073A1 (fr) * 1997-06-20 1998-12-30 Mayo Foundation For Medical Education And Research Procede de detection du cancer du sein
US6235486B1 (en) 1997-06-20 2001-05-22 Mayo Foundation For Medical Education & Research Method for detection of breast cancer
WO1999010528A1 (fr) * 1997-08-22 1999-03-04 Michael Giesing Procede permettant de caracteriser des cellules cancereuses disseminees et micrometastasees
WO2000044940A3 (fr) * 1999-01-28 2000-12-07 Gen Probe Inc Sequences d'acide nucleique permettant de detecter des marqueurs genetiques pour le cancer dans un echantillon biologique
US6551778B1 (en) 1999-01-28 2003-04-22 Gen-Probe Incorporated Nucleic acid sequences for detecting genetic markers for cancer in a biological sample
US6811985B2 (en) 1999-01-28 2004-11-02 Gen-Probe Incorporated Nucleic acid sequences for detecting genetic markers for cancer in a biological sample
US7267956B2 (en) 1999-01-28 2007-09-11 Gen-Probe Incorporated Nucleic acid sequences for detecting genetic markers for cancer in a biological sample
DE10143776A1 (de) * 2001-09-06 2003-04-03 Adnagen Ag Verfahren und Kit zur Diagnostik oder Behandlungskontrolle von Brustkrebs
US7507528B2 (en) 2001-09-06 2009-03-24 Adnagen Ag Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells

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