[go: up one dir, main page]

WO1993004200A1 - Procedes et compositions pour le depistage et la prevision du cancer du sein - Google Patents

Procedes et compositions pour le depistage et la prevision du cancer du sein Download PDF

Info

Publication number
WO1993004200A1
WO1993004200A1 PCT/US1992/006701 US9206701W WO9304200A1 WO 1993004200 A1 WO1993004200 A1 WO 1993004200A1 US 9206701 W US9206701 W US 9206701W WO 9304200 A1 WO9304200 A1 WO 9304200A1
Authority
WO
WIPO (PCT)
Prior art keywords
breast cancer
nucleic acid
dna
allele
fragment
Prior art date
Application number
PCT/US1992/006701
Other languages
English (en)
Inventor
William L. Mcguire
Suzanne A. Fuqua
Original Assignee
Board Of Regents, The University Of Texas System
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 Board Of Regents, The University Of Texas System filed Critical Board Of Regents, The University Of Texas System
Publication of WO1993004200A1 publication Critical patent/WO1993004200A1/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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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 methods for detecting, as well as predicting, persons at risk of developing breast cancer. More specifically, the present invention provides a method for characterizing a person as having breast cancer, or as at risk of developing breast cancer, on the basis of a particular allelic profile manifest in persons with a particular nucleic acid genetic abnormality. Such a genetic abnormality is detectable in a ffindlll RFLP.
  • the RFLP of the present invention is further defined as being in close proximity or within to the progesterone receptor gene, most specifically on the long arm of the chromosome 11.
  • the RFLP of the present invention is identified in tfindl I enzyme generated DNA fragments employing particularly defined nucleic acid segments suitable for use as DNA probes.
  • the inventors have discovered that a certain percentage of persons whom have breast cancer or whom are characterized as "at risk" of developing this condition are found to be homozygous for the H2 allele (H2/H2) or are absent the HI allele. Thus, it is either the presence of both H2 alleles, or the absence of the HI allele, that serves to identify persons susceptible to breast cancer or persons who already have breast cancer.
  • the present invention also relates to the field of diagnostic tests and kits for breast cancer.
  • the steroid hormones have profound effects upon the differentiation and proliferation of a number of target tissues, such as uterus and breast, and their actions are mediated by specific, low abundance intracellular receptors.
  • the receptors for these hormones are known to modulate the transcription of specific genes, and are members of a superfamily of ligand-inducible enhancer factors 1 .
  • the PgR (progesterone receptor) in addition to being a gene regulator, is itself regulated by estrogen through the estrogen receptor (ER) , thereby adding to the complexity of hormonal regulation in target tissues.
  • PgR content has been found useful for predicting disease-free survival in human breast cancer, and is routinely measured in the clinical setting 2 ⁇ .
  • ER estrogen receptor
  • Several groups have now cloned the gene for PgRs from several species, and have demonstrated that human PgR is encoded by a single gene residing on chromosome ll "8 . This localization is characterized by frequent alterations and loss of chromosome 11 sequences in human breast cancer 9 " 11 .
  • Chromosomal alterations and/or deletions are aberrations which have been proposed as mechanisms underlying oncogenesis. Indeed, amplification of specific oncogenes, such as c-erbB-2 and int-2, or alterations to c-H-ras-1, c-myc, and c-myb, may all contribute to either the genesis or the progression of human breast cancer 12-17 . Progesterone and breast cancer have been linked since the concept of hormone-dependent cancers was first elaborated 27 " 28 . In regard to biological factors, the literature reveals a wide range of potential physiological and biochemical factors as well as oncogene linked mechanisms, which result in the development of breast cancer.
  • Restriction fragment length polymorphism offers a powerful molecular genetic tool for the direct analysis of the human genome to determine elements that signal predisposition to genetic diseases 29 .
  • RFLP Restriction fragment length polymorphism
  • the technique of RFLP has been used to obtain information necessary to create genetic probes (isolated from chromosome-specific phage libraries) which contain a specific DNA portion of human DNA of interest. 30 With this tool in hand, the analysis of human gene segments is possible.
  • restriction fragment analysis has been employed to examine relatively large segments of the human PgR gene which resides on chromosome ll. 1
  • the absence of PgR in a breast tumor could be due to a molecular alteration specifically in the PgR gene, or a consequence of defective regulation of the PgR gene by ER, or even to the concentration of steroid ligands themselves.
  • These theories are supported by observations of others which report that PgR gene expression is under the dual control of both estrogen and progesterone 18 * 19 .
  • a specific method for genetically identifying or predicting patients at genetic risk for developing breast cancer employing a part specific RFLP and specific patient allelic profile has not yet been reported.
  • the present invention provides a powerful specific method for the identification of particularly defined allelic polymorphisms useful in the diagnosis of human breast cancer.
  • the present invention also provides methods whereby persons at risk for developing breast cancer may also be identified, i.e. in the diagnosis of occult breast cancer. These methods may be used in the analysis of nucleic acid obtained from tu orous tissue, such as from a breast tumor biopsy, as well as from non- tumorous tissues, such as blood, tissues adjacent a malignant breast tumor mass, skin, hair, buccal smear and thy us tissue.
  • the present inventors have discovered that the technique of RFLP may be employed in a method to detect the presence of a specific polymorphism(s) which indicates the presence of or a predisposition to breast cancer.
  • This polymorphism may potentially be present in the DNA found in cells of many tissue types. It is postulated that the presence of the specific polymorphism described herein may also be involved in the mechanism whereby a particular oncogene(s) is activated during the malignant disease process, or in the alternative, be involved in the suppression of a tumor suppressor gene.
  • the present inventors have discerned the existence of particular chromosome alterations in DNA obtained from breast tumor tissue.
  • This particular RFLP is identifiable using a nucleic acid segment having a sequence defined in Figure 1.
  • Test sample nucleic acid obtained from a patient which is found to include sequences hybridizable to a sequence or part of a sequence which corresponds to the Hindlll RFLP of breast cancer, as defined in Figure 1, or a fragment thereof, are identified as positive for breast cancer.
  • the particular HindiII RFLP for human breast cancer employed in the disclosed methods and kits are further defined as being located in close proximity to or within the progesterone receptor (PgR) gene.
  • the PgR gene is located at chromosome 11.
  • a method employing a specific nucleic acid segment characteristic of human breast cancer is defined.
  • the particular nucleic acid segment thus constitutes a genetic marker detectable as a RFLP (restriction fragment length polymorphism) comprising a ffindlll polymorphism.
  • the described genetic marker of the present invention includes a Hindlll polymorphism further defined by a particular allelic profile, being either an absence of an HI allele or an H2/H2 homozygous allelic profile condition.
  • the RFLP of the present invention may be defined in terms of hybridization probe sequences that will bind to, and therefore identify, the RFLP.
  • the HI or H2 is defined as a nucleic acid segment that will hybridize specifically with a nucleic acid segment prepared with sequence characteristics of an HI or H2 polymorphism. Nucleic acid segments of about 1.23 kb or less are preferred because of enhanced specificity and decreased monetary expense for the production of a shorter nucleic acid segment. More specifically, it is generally recognized that the longer a particular "identifying" (i.e. "probe") nucleic acid sequence is, the greater the possibility that hybridization will occur between parts of the "identifying" sequence unrelated to the particular genetic disorder of interest.
  • nucleic acid segment As an "identifying" nucleic acid sequence, the inventors provide a diagnostic test having enhanced specificity for human breast cancer and having a decreased probability of reporting a "false positive" result for breast cancer or susceptibility thereto.
  • An example of such a nucleic acid segment is the 1.23 kb nucleic acid sequence set forth in Figure 1. Even more preferred, however, will be shorter sequences that maintain specificity for both the HI and H2 alleles. For example, such may be defined in the inventors prophetic
  • a method for diagnosing breast cancer or susceptibility to breast cancer in a patient comprises obtaining a tissue or blood specimen from a patient, processing the specimen to obtain DNA, subjecting the DNA to a restriction enzyme capable of defining regions of a Hin ⁇ lll polymorphism, so as to provide DNA restriction fragments, and diagnosing a patient homozygous for the H2 allele or absent a HI allele as having breast cancer or as being susceptible to breast cancer. More specifically, the DNA of the patient used in the described method is genomic DNA.
  • the DNA restriction fragments as prepared in the process of the claimed method are separated by length to provide isolated DNA restriction fragments.
  • the isolated DNA restriction fragments so obtained are then most preferably probed to locate segments of a HindiII RFLP therein, said segments of the RFLP being hybridizable to a nucleic acid segment having an HI or H2 allele.
  • the inventors have found the HindiII RFLPs to reside at those patient DNA fragments which have a length of about 2.8 kb (HI allele) and about 2.5 kb (H2 allele).
  • the isolated DNA restriction fragments are probed with a nucleic acid segment suitable for use as a probe and having a sequence defined in Figure 1 or a fragment thereof.
  • a prophetic embodiment of the method employs a nucleic acid segment suitable for use as a probe and having a sequence as defined in Figure 2 or a fragment thereof.
  • the DNA restriction fragments are separated by the process of electrophoresis.
  • the inventors propose to discern smaller nucleic acid fragments which include even more narrowly defined nucleic acid sequences characteristic of the HI and H2 allele.
  • the nucleic acid fragments of a patient DNA sample need not first be separated by electrophoresis. Instead, the presence of the HI and the H2 alleles would be determined, and the allelic profile of the patient discerned and classified as either characteristic of breast cancer or susceptibility thereto, or not.
  • invariant PgR bands which appear at 3.6, 4.7 and 9.0 kb on the Southern Blot of Figure 3, need not be considered.
  • probes employed for detection purposes will be labeled to provide for their ready detection following hybridization.
  • radioactive labels such as ⁇ P are generally the most sensitive for identification purposes, through, e.g., autoradiography.
  • radioactive labels suffer from disadvantages due to the need for disposal of radioactive wastes and the short half-life of, e.g., 32 P. Therefore, it may be desirable to employ non-radioactive labels such as avidin/biotin or enzyme ligands such as alkaline phosphatase, horseradish peroxidase, etc., which may be detected through the use of colorimetric substrates.
  • restriction enzymes While it is proposed that any of a variety of restriction enzymes may be used in the practice of the present invention, the restriction enzyme most particularly preferred is the Hindlll restriction enzyme.
  • a restriction enzyme capable of defining regions of a Hindlll polymorphism may be used in conjunction with any and all of the described aspects of the present invention. Restriction enzymes found not to provide these requisites include EcoRI and Pvu II. In a most preferred embodiment of the claimed method, the restriction enzyme is Hindlll restriction enzyme.
  • isolated DNA restriction fragments obtained from the patient's DNA include the HI allele to be located in a restriction fragment which has a length of about 2.8 kb.
  • the H2 allele is to be found in a DNA restriction fragment which has a length of about 2.5 kb.
  • the HI allele or the H2 allele may be located according to the present methods by probing the isolated DNA restriction fragments with a nucleic acid segment having the sequence defined in Figure 1 or a fragment thereof.
  • the presence of an H2 allele or an HI allele is identifiable in a DNA restriction fragment which hybridizes with a probe having a sequence defined in Figure 1, or a fragment thereof.
  • the presence of an H2 allele or an HI allele may be identified in a DNA restriction fragment which hybridizes with a nucleic acid a sequence defined in Figure 2 or a fragment thereof.
  • a nucleic acid probe preferably DNA, having a sequence as defined in Figure 1 may be prepared as an AccI/BamHI generated fragment of a PgR cDNA.
  • This particular embodiment of the probe is a 1.23 kb DNA fragment of the PgR cDNA.
  • a method of detecting a Hindlll RFLP in DNA comprises treating the DNA sample with a restriction enzyme capable of producing a DNA restriction fragment having a Hindlll polymorphism, to produce DNA restriction fragments, probing the DNA restriction fragments with a nucleic acid segment capable of identifying a Hindlll RFLP, and identifying a Hindlll RFLP in a segment of the separated DNA restriction fragment which hybridizes with the nucleic acid segment or a fragment thereof.
  • the polymorphism for breast cancer or susceptibility to breast cancer is a deletion of an HI allele.
  • the polymorphism for breast cancer or susceptibility to breast cancer discernible with the described method is a H2/H2 allelic profile.
  • the particular allelic profile characteristic of breast cancer or susceptibility thereto is defined in an HI PgR gene-free allelic profile or in an H2/H2 PgR gene allelic profile.
  • a nucleic acid segment suitable for use as probe and capable of identifying a Hindlll RFLP characteristic of human breast cancer or susceptibility thereto is provided.
  • the particular nucleic acid segment most preferably has 1.23 kb or less.
  • the RFLP identifiable using the described nucleic acid segment in turn identifies an H2/H2 allelic condition or identifies the absence of an HI allele in the allelic profile of the patient.
  • the nucleic acid segment has a sequence as defined in Figure 1 or a fragment thereof.
  • the segment is defined as having a sequence as defined in Figure 2, or a fragment thereof. Even more specifically this particular prophetic embodiment of the nucleic acid fragment has a length of about 0.48 kb.
  • kits of the present invention is directed to kits for the prediction of breast cancer or breast cancer susceptibility in a patient.
  • Kits of the present invention may be defined generally as including a hybridization probe capable of hybridizing to an HI and H2 RFLP, wherein the probe is comprised in a suitable container, such as a test tube or vial.
  • the probe whether it be an RNA or DNA segment, will also preferably be suitably aliquoted to render it ready for use with little or no pre-experimentation.
  • the probe container will generally be packaged in a larger container or box for easy transportation, shipping, etc.
  • the nucleic acid probe will generally comprise a segment of about 1.23 kb or less that will nevertheless faithfully hybridize, and therefore identify, HI and H2 alleles. Probes of this size are preferred in that they provide greater diagnostic specificity for the diagnosis of breast cancer and are economical to prepare. For this reason, nucleic acid probes that comprise the sequence of figure 1, or a fragment comprising a hybridizable portion of the sequence, will be particularly preferred. As used herein, the term "hybridizable" portion, is intended to refer to nucleic acid segments that are long enough to form specific hybrids with the HI or H2 allele. In a prophetic embodiment, the nucleic acid probe comprises that sequence of Figure 2.
  • the size of the particular nucleic acid fragment employed in the described methods and kits is not to be limited to those of 1.23 kb or less in size.
  • Hind III RFLP described herein, and being hybridizable at least in part to the nucleic acid segment defined in Figure 1 may be employed in the described methods.
  • kits of the present invention will include a means for detecting hybridization between the probe and an HI or H2 RFLP, typically a label located either on the probe, or contained on a separate molecule that can be made to bind specifically to the probe (such as a second nucleic acid probe, and avidin/biotin binding pair, etc.).
  • Preferred labels comprise an enzyme or radioactive label.
  • Figure 2 SEQUENCE OF A 0.48 kb NUCLEIC ACID SEGMENT PROPOSED AS SUITABLE FOR USE AS A PROBE FOR THE PROPOSED IDENTIFICATION OF A BREAST CANCER POLYMORPHISM PRESENT IN A Hindlll RFLP.
  • FIG. 3 SOUTHERN HYBRIDIZATION ANALYSIS OF DNA FROM BREAST TUMORS, PLACENTAS AND NORMAL LYMPHOCYTES DIGESTED WITH Hindlll AND PROBED WITH A NUCLEIC ACID SEGMENT OF 1.23 kb CLONE.
  • the presented Hindlll RFLP provides a representative Southern blot of genomic DNA from human placenta, breast tumor and peripheral blot lymphocytes digested with the restriction enzyme Hindlll.
  • the two Hindlll alleles HI about 2.8 kb
  • H2 about 2.5-2.6 kb
  • Invariant PgR bands are demonstrated at 9.0, 4.7, and 3.6 kb.
  • the nucleic acid segment sequence of Figure 1 was employed as the probe.
  • the presently described invention provides a specific genetic diagnostic test for the identification of patients who have breast cancer as well as those persons genetically susceptible to the development thereof.
  • the trait observed by the inventors is highly correlatable to the incidence of breast cancer in humans. This trait is identified in particularly described DNA restriction fragments of the patient DNA, which reveal either a homozygous condition for the H2 allele or an absence of the HI allele. Thus, HI could be or could be revealing a tumor suppressor gene.
  • the method for detecting the polymorphism diagnostic for breast cancer includes a nucleic acid segment which identifies the particular genetic abnormality in a
  • Hindlll restriction fragment prepared from a DNA patient sample. Accordingly, included in the present invention disclosure is information which may be used to prepare a wide variety of nucleic acid fragments having a number of potential utilities, such as the preparation of DNA and RNA sequences in PCR and hybridization studies as probes for in vitro detection, as well as other useful medical and biochemical applications related to the research, diagnosis and treatment of breast cancer.
  • the nucleic acid fragment for the purposes of the present invention is defined as a polymer of nucleic acids. More specifically, the nucleic acid segment is defined as a polymer of nucleic acids suitable for use as a probe and sufficient to provide for the hybridization of the nucleic acid segment with segments of a patient DNA sequence which include a complementary base sequence thereto, thus identifying a Hindlll RFLP characteristic of breast cancer or a susceptibility to breast cancer. Measurement of any resulting double-helix formation
  • hybridization provides a relative measurement of the relatedness between the patient DNA and the nucleic acid segment of the disclosed method.
  • tissue of the patient could be used to obtain a DNA sample suitable for analysis for the herein described genetic polymorphism.
  • tissue include both tumorous and nontumorous tissues.
  • Tissues may also be used from a variety of tissue types, such as breast tissue, tissue adjacent breast tissue, blood cells, placental tissue, thymus tissue (thymus is a lymphocyte-rich tissue important in the production and maintenance of immune cells) , skin, buccal smear and hair, among others.
  • tissue type of choice for detection of a polymorphism characteristic of breast cancer or susceptibility thereto is breast tissue or blood cells.
  • Blood cells such as granulocytes and lymphocytes are most particularly preferred.
  • the present example is provided to demonstrate several preferred methods by which the nucleic acid segments for identifying human breast cancer or susceptibility thereto may be prepared. However, any of a variety of methods other than the specific methods described herein, as well as a variety of variations from the methods specifically described herein, may be used in the practice of the present invention.
  • the particular methods outlined herein are the synthesis of a cDNA clone, synthesis of a sequence using PCR, and synthesis of a sequence using a synthetic oligonucleotide synthesizer.
  • cDNA probe A DNA probe useful for the identification of a genetic polymorphism characteristic of breast cancer or susceptibility thereto in a particularly preferred embodiment is prepared using a nucleic acid sequence isolatable from a human PgR clone.
  • PgR clone which was used in the preparation of a DNA probe suitable for use in the present invention was the human PgR clone hPR-56.
  • This particular PgR clone, hPR-56 was isolated from a T47D- pCD library.
  • the T47D-pCD library comprises a variety of clones containing the PgR gene sequence, and is described in Okayama et al. (1983) . 32
  • the particular hPR-56 clone consists of a 2.7 kb BamHI fragment of the human PgR, representing nucleotides 70-7835. The authenticity of the fragment sequence was confirmed by DNA sequence analysis.
  • the hPR-56 nucleic acid was digested with Accl to cleave the PgR sequence at nucleotide 1609, followed by digestion with BamHI, and a 1.23 kb Accl/BamHI fragment subcloned into pGEM (Promega, Inc.). This fragment was found by the present inventors to include a sequence useful in the identification of the genetic polymorphism for breast cancer and susceptibility to breast cancer. b . PCR amplified probe
  • GCAAGCTTAAGAGAAGGGGTTTCACCATC-3' can be used to amplify by polymerase chain reaction (PCR) the 1.23 kb fragment from PgR cDNA.
  • the fragment was purified by agarose gel electrophoresis and labeled with 32 P-dNTP by random prime labeling using a commercially available kit (Boehringer Mannheim) .
  • these labeling components may also be prepared individually following standard procedures (Maniatus Molecular Biology Manual) .
  • a prophetic nucleic acid segment having a length of about 1.23 kb may be prepared by PCR for use in conjunction with the described methods and kits.
  • a nucleic acid subfragment of the 1.23 kb PgR cDNA may be prepared using two oligonucleotide sense and antisense primer pairs chosen within the 1.23 kb cDNA. These internal PgR cDNA primers should consist of 20 to 30 oligonucleotides and contain approximately 50 to 65% C/G content. The distance between the two pairs may vary, but lengths between 100 to 200 may provide optimum ease of PCR amplification and specificity for detection of the Hindlll RFLP.
  • the primers can be used to PCR amplify PgR cDNA and the amplified fragment used as a probe as outlined in Example 1.
  • the labeled form of the fragment may be prepared using the radioisotopic label 32 P and used in a Southern analysis with geno ic DNA from tissue specimens.
  • the PCR-generated probe may then also be used in hybridization reactions with the Southern nitrocellulose membranes as described in Example 2.
  • the nucleic acid segment employed in the diagnostic methods and compositions herein may be synthesized as a 20 - 30 bp (base pair) synthetic oligonucleotide.
  • the oligonucleotides may be prepared via automated synthesizers, desalted, and used as probes. Most preferably, these oligonucleotides are to be labeled with a radioisotope, such as 32 P, using 32 P-dNTP's and T4 polynucleotide kinase. These labeled nucleotides may then be used in hybridization reactions with the Southern nitrocellulose membranes as described in Example 2.
  • the present example is provided to demonstrate the first molecular genetic evidence, through the use of RFLP analysis, that an allelic polymorphism exists which is highly correlated to the occurrence and/or subsequent development of human breast cancer.
  • the present example is also provided to demonstrate the utility of employing the methods described herein for the detection of particular restriction fragment length polymorphisms (RFLP) indicative or predictive of human breast cancer, as identified in digested and isolated DNA restriction fragments of a patient's DNA.
  • RFLP restriction fragment length polymorphisms
  • the data herein indicate an identifiable polymorphism at the HI and/or the H2 allele is present in a statistically significant number of tissues obtained from persons with breast cancer, compared to non-tumorous human tissues obtained from persons without breast cancer.
  • a total of 132 breast tumors were examined for the presence of the Hindlll RFLP as an indicator of breast cancer, and established that an allelic profile of H2/H2 or an allelic profile absent the HI allele was present in tissues obtained from patients diagnosed or later diagnosed to have breast cancer.
  • Hindlll RFLP was found by the inventors to not display typical Mendelian distribution in the breast tumors. In the ligand-binding assays conducted by the inventors, the Hindlll RFLP did not correlate with the PgR expression. This suggests to the present inventors that the RFLP is not related to the heterogeneity of PgR expression seen in breast tumors.
  • the present examples provide an examination of the genomic status of the PgR gene in a total of 132 breast tumor biopsies using Southern hybridization analysis.
  • T47D (ATCC No.HTB 133): ZR75 (ATCC NO.CRL1500) : MDA-231 (ATCC NO.HTB26) : MDA-468 (ATCC No.HTB132) : MCF-7 (ATCC NO.HTB22) :
  • MDA MB-330 (ATCC NO.HTB127) :
  • Human placental tissues were obtained from a local medical center hospital. These tissues were collected immediately after parturition, trimmed free of excess connective tissue, rinsed in sterile phosphate buffered saline, frozen in liquid nitrogen, and stored at -70°C.
  • Human breast tumor specimens maintained at -70°C, consist of tissue remaining after routine estrogen and progesterone receptors assays and were collected from throughout the United States.
  • Human peripheral blood leukocyte DNA was provided from local sources. Normal breasts and breast tumor pairs were obtained also from local sources and consisted of primary breast carcinoma specimens with adjacent benign breast tissue.
  • High molecular weight genomic DNA was isolated on a Model 340A Nucleic Acid Extractor (Applied Biosystem, Inc., Foster City, CA) according to manufacturer's recommendations, and quantitated by diphenylamine assay 25 .
  • Ten micrograms of DNA was digested with the appropriate restriction enzyme, separated by a electrophoresis on a 1% agarose gel, and transferred onto nitrocellulose by the method of Southern 20 .
  • the nitrocellulose filters were hybridized to a ⁇ P-labelled 26 AccI/BamHI fragment of the human hPR-56 PgR cDNA clone which corresponds to the hormone binding and 3'-untranslated domains of the receptor mRNA 7 at 42°C for 16 hours in the presence of dextran sulfate.
  • blots were stained both before and after transfer with ethidium bromide.
  • ⁇ 2 analysis was performed to compare allele distribution between PR+ and PR- tumors.
  • the frequency of the HI allele in breast tumors was 77% and 70% in PgR-positive and PgR-negative tumors, respectively, and the Hindlll alleles were not associated with PgR expression levels in these tumors.
  • the frequency of the Hindlll RFLP in tumors did not exhibit a typical Mendelian distribution.
  • the inventors hypothesize whether a genetic selection is occurring which accounts for the observed disequilibrium of these Hindlll alleles in breast tumors.
  • p 0.06
  • the present example is provided to demonstrate that the H2 homozygotes or the HI allele free patient profiles in the breast tumor population may represent a specific loss of the HI allele as a marker in tumor tissue.
  • the Hindlll RFLP was used to determine the presence of breast cancer or the susceptibility to breast cancer in lymphocyte samples. A group of 10 lymphocyte DNAs were examined: The H2 homozygote was not present in this series. These gene frequency differences reflect the three populations selected for analysis. A larger series of lymphocyte samples would determine the incidence and significance of the H2 homozygote in normal tissues. **
  • H2/H2 genotype has been reported by others in lymphocyte DNA 8 , this genotype per se, or the absence of the HI genotype, has not been described as part of a method for cancer prediction or susceptibility. Note that the present inventors have found that the HI/HI or H1/H2 genotype is not a useful predictor for this condition.
  • Example 2 Ten breast tumors and adjacent normal breast tissue were examined according to the protocol outlined in Example 2. Five informative normal/tumor pairs heterozygous for the Hindlll allele were detected, but a loss of the HI allele in these tumors was not seen. Thus, while it appears that not all breast tumors will show an H2/H2 genotype, the H2/H2 genotype has been shown only in individuals having a breast tumor (or at risk) . A previous report has detected a loss of heterozygosity at one or more loci on the short arm of chromosome ll 10 .
  • the present example is provided to demonstrate the use of the currently defined location of the RFLP for breast cancer to isolate and prepare smaller subsets of the 1.23 kb probe which recognize the Hindlll RFLP.
  • a more narrowly defined, smaller nucleic acid fragment which identified the polymorphism of a patient DNA characteristic of breast cancer or susceptibility to breast cancer provides several practical and clinical advantages not provided by the use of a larger nucleic acid segment for DNA analysis among the practical advantages are the economical savings for preparing a smaller nucleic acid segment vs. a larger nucleic acid segment such as in the amount of nucleotides and reagents necessary for synthesis, as well as the time required to prepare, process and verify authenticity of a small verses large nucleic acid fragment.
  • the clinical advantages associated with employing a smaller nucleic acid include a reduced risk of identifying a change in the DNA (of the patient) which perhaps overlaps the region of DNA polymorphism specific for breast cancer susceptibility, as indicative of breast cancer, but which instead reflects either a different clinical pathology, or at the very least, not be indicative of breast cancer or susceptibility thereto (false positive) .
  • a smaller fragment would also be more readily utilizable in routine testing in the clinical laboratory of patient samples for testing for the presence of the RFLP described herein.
  • the smaller fragment may be employed in a technique for the rapid diagnosing of breast cancer development, prognosis and susceptibility.
  • a smaller diagnostic nucleic acid fragment sequence would facilitate a more sensitive assay for detection of the characteristic polymorphism, as "background" bands would be eliminated.
  • a nucleic acid fragment including a smaller fragment will be prepared as described in Example 1.
  • the labeled form of the oligonucleotide was prepared using the radioisotopic label, 32 P.
  • this prophetic probe may be prepared by PCR. More specifically, a prophetic synthetic oligonucleotide primer corresponding to nucleotides 2301 to 2320 of the human PgR plus an added EcoRI restriction site (5'- CGGAATTCCTACAAACACGTCAGTGGGC-3') and an antisense primer corresponding to nucleotides 2769 to 2788 plus an added Hindlll restriction site (5'-
  • GCAAGCTTAAGAGAAGGGGTTTCACCATC-3' may be used to amplify by polymerase chain reaction (PCR) a 0.48 kb fragment of the 1.23 kb PgR.
  • PCR polymerase chain reaction
  • the PCR-amplified 0.48 kb fragment may then be subcloned into pGEM7zf+ and its sequence confirmed by dideoxysequence analysis.
  • the present prophetic example is provided to demonstrate the proposed use of PCR to detect the Hindlll RFLP in small sample specimens.
  • the amount of clinical specimen available is sometimes limiting and isolation of genomic DNA and traditional Southern analysis are not always feasible.
  • the inventors propose to determine the nucleotide sequence of the about 2.8 and the about 2.5-2.6 kb Hindlll alleles using techniques currently available for sequence determinations in the laboratory. These sequences can be amplified from small amounts of tissue or isolated genomic DNA using an oligonucleotide primer to the 5' end and an antisense oligonucleotide primer to the 3' end of the 2.8 kb and/or 2.5 kb Hindlll allele. The PCR products may then be digested with Hindlll or directly run on a standard Southern gel and hybridized with probes recognizing the RFLPs by standard hybridization techniques. This proposed method obviates the requirement for sufficient tissue for genomic DNA isolation and will be possible once the sequences of the 2.8 and 2.5 kb Hindlll RFLPs are delineated.
  • the sequence of the 2.8 and 2.5 kb Hindlll RFLPs are to be determined, more specifically, by obtaining a frozen normal placental tissue, obtaining genomic DNA therefrom, isolating the 2.8 kb band (HI allele) or the 2.5 kb band (H2 allele) therefrom, preparing a subgenomic clone containing the 2.8 kb fragment or the 2.5 kb fragment, and employing the respective subgenomic clone to analyze patient sample DNA for the presence of sequences hybridizable thereto.
  • the placental tissue will first be analyzed for its allelic profile. A placental tissue DNA which has a discernable Hl/Hl allelic profile will then be selected for use in determining a nucleotide sequence for HI allele.
  • the 28 kb DNA containing the HI allele will be excised from a mock Southern Blot and the DNA cloned into a pGEM vector.
  • the Hindlll HI allele insert will be identified by hybridization with the 1.23 kb probe, as described in Example 1. Dideoxy sequence analysis of the Hl/Hl clones that hybridized with the 1.23 kb probe will then be conducted to determine the nucleotide sequence for HI.
  • the same procedure will be repeated with the 2.5 kb fragment obtained from placental DNA found to have an H1/H2 allelic profile. Specifically, the 2.5 kb region will be excised from a Southern Blot of DNA from placental tissue known to have an H1/H2 allelic profile.
  • the inventors plan to prepare a separate subgenomic clone library for HI and H2. More specifically, a subgenomic library out of the H2 excised region sequence and of the HI excised region of the described placental Southern Blot analysis of DNA will be prepared. Subgenomic cloning is to be conducted according to that protocol outlined by Struhl, 32 which reference is specifically incorporated herein by reference for this purpose.
  • the presently proposed method will eliminate the need for tissue amounts sufficient to obtain genomic DNA isolation in a method for analyzing genetic abnormalities in a patient DNA.
  • Watkins PC Restriction fragment length polymorphism (RFLP) : Applications in human chromosome mapping and genetic disease research. Biotechni ues 6:310-319, 1988

Landscapes

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

Abstract

On décrit un polymorphisme de longueur de fragment de restriction (PLFS) d'HindIII d'un acide nucléique caractéristique du cancer du sein chez l'être humain. Les PLFSs sont détectés à l'aide d'une endonucléase capable de produire des fragments de restriction d'ADN HindIII. Le profil allélique HindIII le plus fréquent chez une personne atteinte du cancer du sein ou susceptible d'en être atteinte est déterminé comme étant homozyge H2/H2 ou ne possédant pas d'allèle H1. On peut utiliser la technique du PLFS HindIII pour le dépistage ou la prévision des personnes à risque ou susceptibles de développer le cancer du sein en pratiquant l'analyse d'une grande variété de tissus, comprenant les tissus du sein, les tissus situés autour d'une tumeur du sein, les tissus et les cellules sanguines. On décrit également un segment d'acide nucléique, notamment de 1,23 kb ou moins, destiné à être utilisé comme sonde pour l'identification de régions d'ADN spécifiques du polymorphisme PLFS. Ledit segment d'acide nucléique constitue une partie de la trousse diagnostique permettant d'identifier les patients atteints du cancer du sein ou les personnes à risque.
PCT/US1992/006701 1991-08-12 1992-08-10 Procedes et compositions pour le depistage et la prevision du cancer du sein WO1993004200A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US74360691A 1991-08-12 1991-08-12
US743,606 1991-08-12

Publications (1)

Publication Number Publication Date
WO1993004200A1 true WO1993004200A1 (fr) 1993-03-04

Family

ID=24989431

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/006701 WO1993004200A1 (fr) 1991-08-12 1992-08-10 Procedes et compositions pour le depistage et la prevision du cancer du sein

Country Status (2)

Country Link
AU (1) AU2479292A (fr)
WO (1) WO1993004200A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994011531A1 (fr) * 1992-11-13 1994-05-26 California Institute Of Biological Research Identification de neoplasmes par detection des insertions et deletions genetiques
WO1999015701A1 (fr) * 1997-09-23 1999-04-01 Oncormed, Inc. MUTATION DE SUSCEPTIBILITE 6495delGC DE BRCA2
EP1170364A1 (fr) * 2000-07-07 2002-01-09 ARTEMIS Pharmaceuticals GmbH Modification des sites d'épissages cryptiques dans des gènes Cre et des protéines de fusion de Cre pour améliorer l'expression inductible
US6686163B2 (en) 1998-05-06 2004-02-03 Gene Logic Inc. Coding sequence haplotype of the human BRCA1 gene
US6838256B2 (en) 1996-02-12 2005-01-04 Gene Logic Inc. Coding sequences of the human BRCA1 gene

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2628441A1 (fr) * 1988-03-11 1989-09-15 Ici Plc Methode et kit de diagnostic d'une maladie maligne du sein

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2628441A1 (fr) * 1988-03-11 1989-09-15 Ici Plc Methode et kit de diagnostic d'une maladie maligne du sein

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. vol. 143, no. 2, 1987, DULUTH, MINNESOTA US pages 740 - 748 MISRAHI M. ET AL. 'Complete amino acid sequence of the human progesterone receptor deduced from cloned DNA' cited in the application *
BREAST CANCER RESEARCH AND TREATMENT vol. 14, no. 1, 1989, THE HAGUE,THE NETHERLANDS pages 57 - 64 F.F.PARL ET AL. 'Genomic DNA analysis of the estrogen receptor gene in breast cancer' *
CANCER RESEARCH vol. 48, no. 14, July 1988, PHILADELPHIA,USA pages 4045 - 4048 J.S. LEE ET AL. 'Multiple restriction fragment length polymorphism of the human epidermal growth factor receptor gene' *
SCIENCE vol. 238, 9 October 1987, LANCASTER, PA US pages 185 - 188 I.U. ALI ET AL. 'Reduction to homozygosity of genes on chromosome 11 in human breast neoplasia' cited in the application *
SURGICAL FORUM vol. 38, 1987, CHICAGO,USA pages 402 - 405 A.-M. MARCOUX ET AL. 'Restriction fragment length polymorphisms of type I collagen as genetic markers for breast cancer' *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994011531A1 (fr) * 1992-11-13 1994-05-26 California Institute Of Biological Research Identification de neoplasmes par detection des insertions et deletions genetiques
US6566053B1 (en) 1992-11-13 2003-05-20 Stratagene Identification of neoplasms by detection of genetic insertions and deletions
US7090978B2 (en) 1992-11-13 2006-08-15 Stratagene California Identification of neoplasms by detection of genetic insertions and deletions
US6838256B2 (en) 1996-02-12 2005-01-04 Gene Logic Inc. Coding sequences of the human BRCA1 gene
WO1999015701A1 (fr) * 1997-09-23 1999-04-01 Oncormed, Inc. MUTATION DE SUSCEPTIBILITE 6495delGC DE BRCA2
US6492109B1 (en) 1997-09-23 2002-12-10 Gene Logic, Inc. Susceptibility mutation 6495delGC of BRCA2
US6686163B2 (en) 1998-05-06 2004-02-03 Gene Logic Inc. Coding sequence haplotype of the human BRCA1 gene
EP1170364A1 (fr) * 2000-07-07 2002-01-09 ARTEMIS Pharmaceuticals GmbH Modification des sites d'épissages cryptiques dans des gènes Cre et des protéines de fusion de Cre pour améliorer l'expression inductible
WO2002004609A3 (fr) * 2000-07-07 2002-05-23 Artemis Pharmaceuticals Gmbh Mutations de sites cryptriques d'epissage en cre et proteines hybrides cre permettant une amelioration de l'expression et de l'inductibilite

Also Published As

Publication number Publication date
AU2479292A (en) 1993-03-16

Similar Documents

Publication Publication Date Title
EP0390324B1 (fr) Procédé général pour l'évaluation de cancer
Guo et al. Allelic deletion at 11q23 is common in MYCN single copy neuroblastomas
US5272057A (en) Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase
US5994057A (en) Method of detecting aneuploidy by amplified short-tandem repeats
US6797471B2 (en) Detection and diagnosis of smoking related cancers
Mackay et al. Partial deletion of chromosome 11p in breast cancer correlates with size of primary tumour and oestrogen receptor level
US20020001800A1 (en) Diagnostic methods using serial testing of polymorphic loci
AU675774B2 (en) Detecting digeorge syndrome mutations
US6083698A (en) Cancer susceptibility mutations of BRCA1
WO2003068054A2 (fr) Identification de marqueurs de tumeur du cancer de l'ovaire et cibles therapeutiques
JP2000511433A (ja) 前立腺癌に関連する遺伝子の変化
WO2007009755A2 (fr) Compositions et methodes de diagnostic du cancer contenant des marqueurs pantumoraux
Harada et al. Genetic studies of 457 breast cancers. Clinicopathologic parameters compared with genetic alterations
CA2939539A1 (fr) Survie au cancer de la prostate et recurrence de ce dernier
EP0943017B1 (fr) Methode diagnostique pour determiner la predisposition d'un sujet a developper un carcinome mammaire
US6063567A (en) Method, reagents and kit for diagnosis and targeted screening for retinoblastoma
Ganly et al. PCR-based RFLP analysis allows genotyping of the short arm of chromosome 3 in small biopsies from patients with lung cancer
NZ331041A (en) In vitro method for diagnosis of osteoporosis, atherosclerosis, cancer and immune disorders and medicaments
US5912127A (en) Method and kit for evaluating risk of ovarian cancer in carriers of a BRCA1 mutation
JP2007530075A (ja) 乳がんの予後診断のための組成物および方法
Silverman et al. The 18q-syndrome: analysis of chromosomes by bivariate flow karyotyping and the PCR reveals a successive set of deletion breakpoints within 18q21. 2-q22. 2
US5552282A (en) Diagnosis of myotonic muscular dystrophy
EP2046984B1 (fr) Détection de l'amplification du esr1 dans le cancer du sein
WO1993004200A1 (fr) Procedes et compositions pour le depistage et la prevision du cancer du sein
SOMMER et al. Application of DNA-based diagnosis to patient care: the example of hemophilia A

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR CA CH CS DE DK ES FI GB HU JP KP KR LK LU MG MN MW NL NO PL RO RU SD SE

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL SE BF BJ CF CG CI CM GA GN ML MR SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase