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HK1111721B - Sparc promoter mutations associated with drug resistance, and methods associated therewith - Google Patents

Sparc promoter mutations associated with drug resistance, and methods associated therewith Download PDF

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Publication number
HK1111721B
HK1111721B HK08102424.4A HK08102424A HK1111721B HK 1111721 B HK1111721 B HK 1111721B HK 08102424 A HK08102424 A HK 08102424A HK 1111721 B HK1111721 B HK 1111721B
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sparc
cancer
dna
mutations
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HK08102424.4A
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German (de)
French (fr)
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HK1111721A1 (en
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Isabella T. Tai
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不列颠哥伦比亚大学
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Priority claimed from PCT/CA2005/001819 external-priority patent/WO2006058425A1/en
Publication of HK1111721A1 publication Critical patent/HK1111721A1/en
Publication of HK1111721B publication Critical patent/HK1111721B/en

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Description

FIELD OF THE INVENTION
The field of the invention relates to the assessment and/or treatment of an animal with a neoplastic condition.
BACKGROUND OF THE INVENTION
Resistance to chemotherapy is common to many types of cancer and contributes to the high mortality rates in cancer patients. Many factors can play a part in the initial intrinsic resistance to therapy, such as upregulation of efflux pumps from multidrug resistance (MDR) family P-glycoprotein and other MDR proteins (for example multi-drug resistance-associated protein MRP). Such efflux pumps remove chemotherapeutic agents and their metabolites out of cells, thereby decreasing the efficacy of the chemotherapeutic regimen. However, not all cancers that are resistant to chemotherapy have high expression levels of MDR proteins, suggesting that there may be other mechanisms for therapeutic resistance.
Some cancers are particularly resistant to therapies. For example, colorectal cancer (CRC), often accumulates numerous genetic mutations in the progression to tumorigenesis that can also contribute to drug resistance (mutations in p53; loss of DNA mismatch repair (MMR) genes associated with an increased rate of mutation to drug resistance in hereditary non-polyposis colorectal cancer (HNPCC); K-ras mutations; loss of heterozygosity of chromosomes 18q and 22q; and mutations in cell cycle regulatory genes, p21 and p27, is associated with preventing apoptosis in tumors following some chemotherapeutic treatments).
The secreted protein, acidic, rich in cysteine (SPARC), also known as osteonectin, BM-40 and 43K protein, is a type of extracellular protein, termed a matricellular protein, having counter-adhesive properties shown to disrupt cell-matrix interactions (BORNSTEIN P. J. Cell Biol (1995) 130:503-506; SAGE EH. and BORNSTEIN P. J Biol Chem. (1991) 266:14831-4). SPARC is a Ca2+-binding glycoprotein, which maps to chromosome 5q31-q33 (SWAROOP A. et al. Genomics (1988) 2:37-47), the genomic sequence is reported to have 10 exons and 9 introns (VILLARREAL XC. et al. Biochemistry (1989) 28(15):6483-6491) with the cDNA extending over ~3 kb. Representative Homo sapiens SPARC sequences are listed in GenBank under accession numbers TNM_003118.2 (gi48675809 - mRNA), J03040 (SWAROOP A. et al. (1988) supra) and 102863 (VILLARREAL XC. et al. (1989) supra). A representative sequence containing the SPARC promoter, submitted by JENDRASCHAK E. and KAMINSKI WE. (Genomics (1998) 50(1):53-60), is U65081.1 (gi3253140) containing 1370bp. The SPARC promoter region (accession number X82259) was characterized using various promoter constructs in a luciferase assay by HAFNER M. et al. (Matrix Biology (1994) 14:733-741).
SPARC has been associated with bone mineralization (TERMINE JD. et al. Cell (1981) 26:99-105), tissue remodeling (TREMBLE PM. et al. J. Cell Biol. (1993) 121:1433-1444), endothelial cell migration (SAGE EH. et al. J. Cell Biol. (1989) 109:341-356; HASSELAAR P. and SAGE EH. J. Cell Biochem. (1992) 49(3):272-283), apoptosis (SHI Q. et al. J. Biol. Chem. (2004) 279(50):52200-09), angiogenesis (KUPPRION C. et al. J. Cell Biol. (1998) 273(45):29635-29640; VAJKOCZY P. et al. Int. J. Cancer (2000) 87:261-268), scleroderma (LAGAN AL. et al. Rheumatology (2005) 44(2):197-201; ZHOU X. et al. Arthritis & Rheumatism (2002) 46(11):2990-2999) and adipose tissue accumulation (BRADSHAW AD. et al. PNAS (2003) 100(10):6045-6050). Furthermore; SPARC has also been associated with poor overall survival in head and neck cancer (HNSCC) patients (CHIN D. et al. Int. J. Cancer (2005) 113:789-797), poor prognosis in human breast cancer (JONES C. et al. Cancer research (2004) 64:3037-3045), tumor suppressor activity in human ovarian carcinoma cells (MOK SC. et al. Oncogene (1996) 12:1895-1901), development of squamous cell carcinomas in mice (AYCOCK RL. et al. J. Invest. Dermatol. (2004) 123:592-599), inhibition of Lewis lung carcinoma (LLC) cells (BREKKEN RA. et al. J. Clin. Invest. (2003) 111:487-495) and advanced stage human gastric carcinomas (WANG C-S. et al. British Journal of Cancer (2004) 91:1924-1930).
International application, WO 2004/064785 , describes methods of sensitizing mammalian cancers to a therapeutic treatment through administration of the SPARC family polypeptides or polynucleotides encoding a SPARC polypeptide. International application, WO 00/4671 , describes a method and markers for prognosticating the efficacy of anti-cancer agents by detecting mutant tubulin genes.
SUMMARY OF THE INVENTION
The invention is defined in and by the appended claims.
This invention is based in part on the surprising discovery that SPARC promoter mutations are associated with therapeutic resistance in a variety of cells and to a variety of therapeutic agents.
This invention is also based in part on the identification the particular mutations in (SPARC) sequence associated with resistance to therapeutic regimens.
In accordance with one aspect of the invention, methods are provided for predicting responsiveness of a subject to a therapeutic regimen, the subject having, or suspected of having a cancer, the method including determining a presence or an absence of one or more a secreted protein, acidic, cysteine-rich (SPARC) promoter mutations in the subject's cancer, wherein said presence or absence of one or more SPARC promoter mutations is indicative of a sensitivity of the subject's cancer to the therapeutic regimen.
In accordance with another aspect of the invention, methods are provided for selecting a group of subjects for determining the efficacy of a therapeutic regimen known or suspected of being useful for the treatment of cancer, the method including determining a genotype at one or more mutation sites in a SPARC promoter for each subject, wherein said genotype is indicative of the subject's sensitivity to a therapeutic regimen. The method may further include, administering the therapeutic regimen to the subjects or a subset of subjects and determining each subject's sensitivity to the therapeutic regimen. The method may further include comparing subject response to the therapeutic regimen based on genotype of the subject.
In accordance with another aspect of the invention, methods are provided for selecting a group of subjects to determine the efficacy of a candidate drug known or suspected of being useful for the treatment of a cancer, the method including detecting one or more mutations in a SPARC sequence for each subject, wherein said one or more mutations are indicative of the subject's response to a therapeutic regimen. The method may also include administering the candidate cancer drug to the subjects or a subset of subjects and determining each subject's response to the therapeutic regimen. The method may also include the additional step of comparing subject response to the candidate drug based on the one or more mutations in the subject's SPARC sequence. Response to the candidate drug may be decided by determining each subject's ability to recover from the cancer or by determining the progression of the cancer in response to the therapeutic regimen.
In accordance with another aspect of the invention, methods are provided for identifying a subject with an increased sensitivity to a therapeutic regimen for treating a cancer in a subject in need thereof, including the step of screening a population of subjects to identify those subjects that have a mutation in their SPARC promoter sequence, wherein the identification of a subject with a mutation is predictive of decreased responsiveness to the therapeutic regimen.
The one or more SPARC promoter mutations, may be selected from one or more of the following mutations relative to SEQ ID NO: 1: (a) 295delG and 301 A>CC; (b) 315insA and 316G>A; (c) 341C>T, 342T>C, 344A>C, 347C>A, 354G>A, 356G>A, 359T>G and 363G>A; (d) 395C>T and 396A>T; (e) 488T>C; (f) 533A>G; (g) 734G>A; (h) 734delG; (i) 769T>A, 771G>C and 774G>C; (j) 784T>C, 786T>G and 788T>A; (k) 793T>G, 796T>A and 800T>C; (l) 820C>A and 824T>A; (m) 864T>A and 867T>A; (n) 895T>G, 899T>A, 901G>A and 904G>A; (o) 901G>A; (p) 901G>A, 904G>A, 929A>G and 934delC; (q) 922T>G, 928T>G and 935T>G; (r) 982T>C, 984T>G, 985G>A, 991G>A, 995G>A, 999C>T and 1000T>G; (s) 1063G>C; (t) 1079G>A; (u) 1085delC, 1086delA, 1087deIG, 1089G>A, 1090T>C, 1091T>C and 1092G>T; (v) 1161C>A, 1162T>C, 1163de1A and II64deIT; and (w) 1215C>T, 1216deIG, 1217deIG, 1218delG, 1219delG, 1220delT, 1221delG, 1222delG, 1223delA, 1224delG, 1225delG, 1226delG, 1227delG, 1228dela, 1229delG, 1230delA, 1231delT, 1232delA, 1233delG, 1234delA, 1235delC, 1236delC and 1237C>T. The therapeutic regimen may be chemotherapy or may further comprise radiotherapy. The therapeutic regimen may include the administration of one or more chemotherapeutic agents. The chemotherapeutic agent may be selected from 5-flurouracil, irinotecan, cisplatin, doxorubicin or combinations thereof.
The one or more SPARC promoter mutations may be indicative of the cancer's resistance to the therapeutic regimen. The method may further include obtaining SPARC promoter information for the subject. The one or more SPARC promoter mutations may be determined using a nucleic acid sample from the subject. The nucleic acid sample may have been obtained from the subject. The obtaining of the nucleic acid sample may be from a biological sample taken from the subject's cancer. The biological sample may be obtained from a biopsy. The biological samples may also be repeatedly obtained at various stages during the therapeutic regimen.
Determining the presence or an absence of one or more SPARC promoter mutations in the subject's cancer may be done using one or more of the following techniques: restriction fragment length analysis; sequencing; micro-sequencing assay; hybridization; invader assay; gene chip hybridization assays; oligonucleotide ligation assay; ligation rolling circle amplification; 5' nuclease assay; polymerase proofreading methods; allele specific PCR; matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy; ligase chain reaction assay; enzyme-amplified electronic transduction; single base pair extension assay; and/or reading sequence data.
The cancer may be selected from one or more of the following: carcinoma; melanoma; sarcoma; lymphoma; and leukaemia. The carcinoma may be selected from the following: squamous cell; adeno; small cell; large cell and combinations thereof. The carcinoma may be derived from a primary tumor located in nasopharygeal tissue, head and neck, lung, esophagus, stomach, intestine, rectum, kidney, urinary tract, prostate, testes, ovary, uterus, cervix, vagina, skin, and endocrine glands. The sarcoma may be selected from the following tissues: muscle; adipose; fibrous; vascular; and neural. The lymphoma may be selected from the following: Hodgkin's Disease; and Non-Hodgkins Lymphoma. The leukaemia may be selected from the following: lymphoid; and myeloid. The cancer may be a colorectal carcinoma, prostate carcinoma, ovarian carcinoma or osteosarcoma. The cancer may be a SPARC inhibited cancer. The SPARC inhibited cancer may be a colorectal carcinoma, prostate carcinoma, ovarian carcinoma, osteosarcoma or uterine sarcoma.
DETAILED DESCRIPTION OF THE INVENTION 1. Definitions
In the description that follows, a number of terms are used extensively, the following definitions are provided to facilitate understanding of the invention.
A "nucleic acid sample" as used herein includes any nucleic acid, and may be a deoxyribonucleotide or ribonucleotide polymer in either single or double-stranded form.
A "purine" is a heterocyclic organic compound containing fused pyrimidine and imidazole rings, and acts as the parent compound for purine bases, adenine (A) and guanine (G). "Nucleotides" are generally a purine (R) or pyrimidine (Y) base covalently linked to a pentose, usually ribose or deoxyribose, where the sugar carries one or more phosphate groups. Nucleic acids are generally a polymer of nucleotides joined by 3' 5' phosphodiester linkages. As used herein "purine" is used to refer to the purine bases, A and G, and more broadly to include the nucleotide monomers, deoxyadenosine-5' - phosphate and deoxyguanosine-5' -phosphate, as components of a polynucleotide chain.
A "pyrimidine" is a single-ringed, organic base that forms nucleotide bases, cytosine (C), thymine (T) and uracil (U). As used herein "pyrimidine" is used to refer to the pyrimidine bases, C, T and U, and more broadly to include the pyrimidine nucleotide monomers that along with purine nucleotides are the components of a polynucleotide chain.
An "allele" is defined as any one or more alternative forms of a given gene. In a diploid cell or organism the members of an allelic pair (i.e. the two alleles of a given gene) occupy corresponding positions (loci) on a pair of homologous chromosomes and if these alleles are genetically identical the cell or organism is said to be "homozygous", but if genetically different the cell or organism is said to be "heterozygous" with respect to the particular gene.
A "gene" is an ordered sequence of nucleotides located in a particular position on a particular chromosome that encodes a specific functional product and may include untranslated and untranscribed sequences in proximity to the coding regions (5' and 3' to the coding sequence). Such non-coding sequences may contain regulatory sequences needed for transcription and translation of the sequence or introns etc. or may as yet to have any function attributed to them beyond the occurrence of the mutation of interest. Numerous examples of SPARC gene sequences, both wild type and mutated, are described herein and provided by SEQ ID NO: 1- 55.
A "genotype" is defined as the genetic constitution of an organism, usually in respect to one gene or a few genes or a region of a gene relevant to a particular context (i.e. the genetic loci responsible for a particular phenotype). For example, as described herein the genotype can refer to the presence or absence of a particular mutation within a SPARC promoter sequence indicative of sensitivity or resistance to a therapeutic regimen.
A "promoter" region is 5' or upstream of the translation start site or ATG start codon.
It will be appreciated by a person of skill in the art that the numerical designations of the positions of mutations within a sequence are relative to the specific sequence. Also the same positions may be assigned different numerical designations depending on the way in which the sequence is numbered and the sequence chosen, as illustrated by the alternative numbering in HAFNER et al. (1994) supra, in which a SPARC promoter sequence, represented by accession number X82259 has numerous numbering and sequence differences compared to U65081.1 (JENDRASCHAK E. and KAMINSKI WE. (1998) supra). Furthermore, sequence variations such as insertions or deletions, may change the relative position and subsequently the numerical designations of particular nucleotides at and around a mutational site.
A nucleotide represented by the symbol M may be either an A or C, a nucleotide represented by the symbol W may be either an T/U or A, a nucleotide represented by the symbol Y may be either an C or T/U, a nucleotide represented by the symbol S may be either an G or C, while a nucleotide represented by the symbol R may be either an G or A, and a nucleotide represented by the symbol K may be either an G or T/U. Similarly, a nucleotide represented by the symbol V may be either A or G or C, as shown in SEQ ID NO:2.
A "mutation" as described herein may be the result of a "single nucleotide polymorphism" (SNP) occurring at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. A single nucleotide polymorphism may arise due to substitution of one nucleotide for another at the polymorphic site. A "transition" is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. A "transversion" is the replacement of a purine by a pyrimidine or vice versa. A mutation may also arise from a deletion (represented by "-" or "del") of one or more nucleotides or an insertion (represented by "+" or "ins") of one or more nucleotides relative to a reference sequence or wildtype (wt) sequence (for example SEQ ID NO:1). Alternatively, a mutation may result in a frameshift of the sequence resulting from a deletion or insertion as described above or from an inversion etc. Additionally, a mutation as described herein, may be a multisite mutation, whereby the mutation is comprised of two or more mutations. Examples of multisite mutations are found as described in (a)-(d), (i)-(n), (p)-(r) and (u)-(w). Specifically, (b) is a combination of 315insA and 316G>A relative to SEQ ID NO:1. Furthermore, it would be appreciated by a person of skill in the art, that an insertion or deletion within a given sequence could alter the relative position and therefore the position number of another mutation within the sequence.
SPARC promoter mutations relative to SEQ ID NO: 1 are shown in TABLE 1 below.
(a) 295delG and 301A>CC
(b) 315insA and 316G>A
(c) 341C>T, 342T>C, 344A>C, 347C>A, 354G>A, 356G>A, 359T>G and 363G>A
(d) 395C>T and 396A>T
(e) 488T>C
(f) 533A>G
(g) 734G>A
(h) 734delG
(i) 769T>A, 771G>C and 774G>C
(j) 784T>C, 786T>G and 788T>A
(k) 793T>G, 796T>A and 800T>C
(l) 820C>A and 824T>A
(m) 864T>A and 867T>A
(n) 895T>G, 899T>A, 901G>A and 904G>A
(o) 901G>A
(p) 901G>A, 904G>A, 929A>G and 934delC
(q) 922T>G, 928T>G and 935T>G
(r) 982T>C, 984T>G, 985G>A, 991G>A, 995G>A, 999C>T and 1000T>G
(s) 1063G>C
(t) 1079G>A
(u) 1085delC, 1086delA, 1087delG, 1089G>A, 1090T>C, 1091T>C and 1092G>T
(v) 1161C>A, 1162T>C, 1163delA and 1164deIT
(w) 1215C>T, 1216delG, 1217delG, 1218delG, 1219delG, 1220delT, 1221delG, 1222delG, 1223delA, 1224delG, 1225delG, 1226delG, 1227delG, 1228delA, 1229delG, 1230delA, 1231delT, 1232delA, 1233delG, 1234delA, 1235delC, 1236delC and 1237C>T
A "polymorphic site" or "polymorphism site" or "polymorphism" or "single nucleotide polymorphism site" (SNP site) as used herein is the locus or position with in a given sequence at which divergence occurs. A "Polymorphism" is the occurrence of two or more forms of a gene or position within a gene (allele), in a population, in such frequencies that the presence of the rarest of the forms cannot be explained by mutation alone. The implication is that polymorphic alleles confer some selective advantage on the host. Preferred polymorphic sites have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population. Polymorphic sites may be at known positions within a nucleic acid sequence or may be determined to exist using the methods described herein. Polymorphisms may occur in both the coding regions and the noncoding regions (for example, promoters, enhancers and introns) of genes.
A "SPARC promoter mutation" as used herein refers to any mutation which may be found in a SPARC promoter sequence (for example, SEQ ID NOs: 1-11, but also Accession Not X82259). Such SPARC mutations may be indicative of sensitivity to a therapeutic regimen directed at a cancer whose nucleic acids contain the mutation. For example, SPARC mutations are listed in TABLE 1 ((a)-(w) relative to SEQ ID NO: 1) supra and in SEQ ID NOS:2-33.
A "therapeutic regimen" as described herein may be a chemotherapeutic regimen or a radiotherapy regimen or a combination thereof.
As used herein, a "chemotherapeutic regimen" or "chemotherapy" refers to the use of at least one first line chemotherapy agent to destroy cancerous cells, including leukemia and lymphoma. There are a myriad of such first line chemotherapy agents available to a clinician. Chemotherapy agents may be administered to a subject in a single bolus dose, or may be administered in smaller doses over time. A single chemotherapeutic agent may be used (single-agent therapy) or more than one agent may be used in combination (combination therapy). Chemotherapy may be used alone to treat some types of cancer. Alternatively, chemotherapy may be used in combination with other types of treatment, for example, radiotherapy or "alternative therapies (for example immunotherapy) as described herein.
As used herein, a first line "chemotherapeutic agent" or first line chemotherapy is a medicament that may be used to treat cancer, and generally has the ability to kill cancerous cells directly. Examples of first line chemotherapeutic agents include alkylating agents, antimetabolites, natural products, hormones and antagonists, and miscellaneous agents. Examples of alternate names are indicated in brackets. Examples of alkylating agents include nitrogen mustards such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine and thiotepa; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine (BCNU), semustine (methyl-CCNU), lomustine (CCNU) and streptozocin (streptozotocin); DNA synthesis antagonists such as estramustine phosphate; and triazines such as dacarbazine (DTIC, dimethyltriazenoimidazolecarboxamide) and temozolomide . Examples of antimetabolites include folic acid analogs such as methotrexate (amethopterin); pyrimidine analogs such as fluorouracin (5-fluorouracil, 5-FU, 5FU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside) and gemcitabine; purine analogs such as mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG) and pentostatin (2'-deoxycoformycin, deoxycoformycin), cladribine and fludarabine; and topoisomerase inhibitors such as amsacrine. Examples of natural products include vinca alkaloids such as vinblastine (VLB) and vincristine; taxanes such as paclitaxel and docetaxel (Taxotere); epipodophyllotoxins such as etoposide and teniposide; camptothecins such as topotecan and irinotecan; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, mitomycin (mitomycin C), idarubicin, epirubicin; enzymes such as L-asparaginase; and biological response modifiers such as interferon alpha and interlelukin 2. Examples of hormones and antagonists include luteinising releasing hormone agonists such as buserelin; adrenocorticosteroids such as prednisone and related preparations; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol and related preparations; estrogen antagonists such as tamoxifen and anastrozole; androgens such as testosterone propionate and fluoxymesterone and related preparations; androgen antagonists such as flutamide and bicalutamide; and gonadotropin-releasing hormone analogs such as leuprolide. Examples of miscellaneous agents include thalidomide; platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin and carboplatin; anthracenediones such as mitoxantrone; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH); adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; RXR agonists such as bexarotene; and tyrosine kinase inhibitors such as imatinib. Alternate names and trade-names of these and additional examples of chemotherapeutic agents, and their methods of use including dosing and administration regimens, will be known to a physician versed in the art, and may be found in, for example "The Pharmacological basis of therapeutics", 10th edition. HARDMAN HG., LIMBIRD LE. editors. McGraw-Hill, New York, and in "Clinical Oncology", 3rd edition. Churchill Livingstone/ Elsevier Press, 2004. ABELOFF, MD. editor.
As used herein, the term "radiotherapeutic regimen" or "radiotherapy" refers to the administration of radiation to kill cancerous cells. Radiation interacts with various molecules within the cell, but the primary target, which results in cell death is the deoxyribonucleic acid (DNA). However, radiotherapy often also results in damage to the cellular and nuclear membranes and other organelles. DNA damage usually involves single and double strand breaks in the sugar-phosphate backbone. Furthermore, there can be cross-linking of DNA and proteins, which can disrupt cell function. Depending on the radiation type, the mechanism of DNA damage may vary as does the relative biologic effectiveness. For example, heavy particles (i.e. protons, neutrons) damage DNA directly and have a greater relative biologic effectiveness. Whereas, electromagnetic radiation results in indirect ionization acting through short-lived, hydroxyl free radicals produced primarily by the ionization of cellular water. Clinical applications of radiation consist of external beam radiation (from an outside source) and brachytherapy (using a source of radiation implanted or inserted into the patient). External beam radiation consists of X-rays and/or gamma rays, while brachytherapy employs radioactive nuclei that decay and emit alpha particles, or beta particles along with a gamma ray.
Whether direct or indirect damage from radiotherapy results in the loss of cell division and therefore cell growth. Accordingly, many radiation damaged cells are only detected when they try to undergo cell division and associated processes (for example replication). However, some cells will actually make it through a few rounds of cell division before the damage results in cell death, while others may be recognized a damaged sooner and undergo apoptosis before the cell even attempts division. Therefore depending on the type of radiation applied, the intensity of the radiation, the cell type and other factors, it may be apparent that a cell has been damaged by a radiotherapeutic regimen or it may not, even with histologic review.
Radiotherapy may further be used in combination chemotherapy, with the chemotherapeutic agent acting as a radiosensitizer. The specific choice of radiotherapy suited to an individual patient may be determined by a physician or oncologist, taking into consideration the tissue and stage of the cancer. Examples of radiotherapy approaches to various cancers may be found in, for example. "Clinical Oncology", 3rd edition. Churchill Livingstone/ Elsevier Press, 2004. ABELOFF, MD. editor.
As used herein the term "alternative therapeutic regimen" or "alternative therapy" (not a first line chemotherapeutic regimen as described above) may include for example, SPARC polypeptide (as described in WO 2004/064785 ), receptor tyrosine kinase inhibitors (for example Iressa™ (gefitinib), Tarceva™ (erlotinib), Erbitux™ (cetuximab), imatinib mesilate (Gleevec™), proteosome inhibitors (for example bortezomib, Velcade™); VEGFR2 inhibitors such as PTK787 (ZK222584), aurora kinase inhibitors (for example ZM447439); mammalian target of rapamycin (mTOR) inhibitors, cyclooxygenase-2 (COX-2) inhibitors, rapamycin inhibitors (for example sirolimus, Rapamune™); farnesyltransferase inhibitors (for example tipifarnib, Zarnestra); matrix metalloproteinase inhibitors (for example BAY 12-9566; sulfated polysaccharide tecogalan); angiogenesis inhibitors (for example Avastin™ (bevacizumab); analogues of fumagillin such as TNP-4; carboxyaminotriazole; BB-94 and BB-2516; thalidomide; interleukin-12; linomide; peptide fragments; and antibodies to vascular growth factors and vascular growth factor receptors); platelet derived growth factor receptor inhibitors, protein kinase C inhibitors, mitogen-activated kinase inhibitors, mitogen-activated protein kinase kinase inhibitors, Rouse sarcoma virus transforming oncogene (SRC) inhibitors, histonedeacetylase inhibitors, small hypoxia-inducible factor inhibitors, hedgehog inhibitors, and TGF-β signalling inhibitors. Furthermore, an immunotherapeutic agent would also be considered an alternative therapeutic regimen. For example, serum or gamma globulin containing preformed antibodies; nonspecific immunostimulating adjuvants; active specific immunotherapy; and adoptive immunotherapy. In addition, alternative therapies may include other biological-based chemical entities such as polynucleotides, including antisense molecules, polypeptides, antibodies, gene therapy vectors and the like. Such alternative therapeutics may be administered alone or in combination, or in combination with other therapeutic regimens described herein. Alternate names and trade-names of these agents used in alternative therapeutic regimens and additional examples of agents used in alternative therapeutic regimens, and their methods of use including dosing and administration regimens, will be known to a physician versed in the art. Furthermore, methods of use of chemotherapeutic agents and other agents used in alternative therapeutic regimens in combination therapies, including dosing and administration regimens, will also be known to a physician versed in the art.
The detection of nucleic acids in a sample may rely on the technique of specific nucleic acid hybridization in which the oligonucleotide is annealed under conditions of "high stringency" to nucleic acids in the sample, and the successfully annealed oligonucleotides are subsequently detected (see for example Spiegelman, S., Scientific American, Vol. 210, p. 48 (1964)). Hybridization under high stringency conditions primarily depends on the method used for hybridization, the oligonucleotide length, base composition and position of mismatches (if any). High stringency hybridization is relied upon for the success of numerous techniques routinely performed by molecular biologists, such as high stringency PCR, DNA sequencing, single strand conformational polymorphism analysts, and in situ hybridization. In contrast to northern and Southern hybridizations, these techniques are usually performed with relatively short probes (e.g., usually about 16 nucleotides or longer for PCR or sequencing and about 40 nucleotides or longer for in situ hybridization). The high stringency conditions used in these techniques are well known to those skilled in the art of molecular biology, and examples of them can be found, for example, in AUSUBEL et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998.
A representative of a Homo sapiens SPARC promoter gene sequence is listed in GenBank under accession number U65081 (SEQ ID NO:1). Polymorphic sites in SPARC promoter sequence are identified by their variant (i.e. M, W, Y, S, R, K or V) in SEQ ID NO:2.
34 35-57 of SEQ ID NO: 9 Set 1 fwd
35 546-567 of SEQ ID NO: 9 (on opposite strand) Set 1 rev
36 431-452 of SEQ ID NO: 9 Set 2 fwd
37 1360-1380 of SEQ ID NO: 9(on opposite strand) Set 2 rev
38 GTCATCCCTC CAGGCC 1126-1141 of SEQ ID NO: 9 Set 3 fwd
39 2905-2924 of SEQ ID NO: 9 (on opposite strand) Set 3 rev
40 430-452 of SEQ ID NO: 9 SPARC fwd (with 5' M13 tag in lower case)
41 1359-1379 of SEQ ID NO: 9 (on opposite strand) SPARC rev (with 5' M13 tag in lower case)
42 1338-1354 of SEQ ID NO: 9 sp-seq-1-F (with 5' M13 tag in lower case)
43 2140-2158 of X82259 (on opposite strand) sp seq 1-R (with M13 tag in lower case)
44 430-452 of SEQ ID NO: 9 sp seq-up-1-F (with 5' M13 tag in lower case)
45 1360 -1380 of SEQ ID NO: 9 (on opposite strand) sp seq-up-1-R (with 5' M13 tag in lower case)
46 2097-2116 of X82259 sp seq 2-F (with 5' M13 tag in lower case)
47 Intronic sp seq 2-R (with 5' M13 tag in lower case)
48 31-52 of X82259 sp-seq up-0-F (with 5' M13 tag in lower case)
49 546-567 of SEQ ID NO: 9 (on opposite strand) sp-seq up-0-R (with 5' M13 tag in lower case)
50 1126-1141 of SEQ ID NO: 9 sp-seq up-2-F (with 5' M13 tag in lower case)
51 1527-1512 of X82259 sp-seq up-2-R (with 5' M13 tag in lower case)
52 (1553-1575 of SEQ ID NO: 1) SPARC fwd (TAI 2005)
53 1901-1926 of SEQ ID NO: 1 (opposite strand) SPARC rev (TAI 2005)
54 Not SPARC GAPDH fwd (TAI 2005)
55 Not SPARC GAPDH rev (TAI 2005)
Sparc Promoter Sequencing Primers
Sequencing primers corresponding to SEQ ID NO: 34 and SEQ ID NO: 35 were used to obtain sequence information on both strands of the SPARC promoter (SEQ ID NO: 1) for the region between nucleotide 1 and nucleotide 425.
Sequencing primers corresponding to SEQ ID NO: 36 and SEQ ID NO: 37 were used to obtain sequence information on both strands of the SPARC promoter (SEQ ID NO: 1) for the region between nucleotide 333 and nucleotide 1241.
Sequencing primers corresponding to SEQ ID NO: 38 and SEQ ID NO: 39 were used to obtain sequence information on both strands of the SPARC promoter (SEQ ID NO: 1) for the region between nucleotide 1024 and nucleotide 1370.
The sequences shown in TABLE 3 below (SEQ ID NOS: 11-33) show SPARC promoter mutations (underligned) with flanking sequences. The numbering associated with these sequences represents the mutation positions relative to SEQ ID NO:1 (wild type SPARC promoter sequence).
295deIG and 301A>CC 11
315insA and 316G>A 12
341C>T, 342T>C, 344A>C, 347C>A, 354G>A, 356G>A, 359T>G and 363G>A 13
395C>T and 396A>T 14
488T>C 15
533A>G 16
734G>A , 17
734delG 18
769T>A. 771 G>C and 774G>C 19
784T>C, 786T>G and 788T>A 20
793T>G, 796T>A and 800T>C 21
820C>A and 824T>A 22
864T>A and 867T>A 23
895T>G, 899T>A, 901 G>A and 904G>A 24
901G>A 25
901G>A, 904G>A, 929A>G and 934delC 26
922T>G, 928T>G and 935T>G 27
982T>C, 984T>G, 985G>A, 991G>A, 995G>A, 999C>T and 1000T>G 28
1063G>C 29
1079G>A 30
1085delC, 1086delA, 31
1087delG, 1089G>A, 1099T>C, 109lT>C and 1092G>T
1161C>A, 1162T>C, 1163delA and 1164delT 32
1215C>T, 1216delG, 1217delG, 1218delG, 1219delG, 1220delT, 1221delG, 1222delG, 1223delA, 1224delG, 1225delG, 1226delG, 1227delG, 1228delA, 1229delG, 1230delA, 1231delT, 1232delA, 1233delG, 1234delA, 1235delC, 1236delC and 1237C>T 33
The Sequences given in TABLE 3 (SEQ ID NO: 11-33) above and in SEQ ID NO:1-10 may be useful to a person of skill in the art in the design of further primers, and probes or other oligonucleotides or peptide nucleic acids for the identification of SPARC mutations as described herein.
"Oligonucleotides" as used herein are variable length nucleic acids, which may be useful as probes, primers and in the manufacture of microarrays (arrays) for the detection and/or amplification of specific nucleic acids. Such DNA or RNA strands may be synthesized by the sequential addition (5'-3' or 3'-5') of activated monomers to a growing chain which may be linked to an insoluble support. Numerous methods are known in the art for synthesizing oligonucleotides for subsequent individual use or as a part of the insoluble support, for example in arrays (BERNFIELD MR. and ROTTMAN FM. J. Biol. Chem. (1967) 242(18):4134-43; SULSTON J. et al. PNAS (1968) 60(2):409-415; GILLAM S. et al. Nucleic Acid Res.(1975) 2(5):613-624; BONORA GM. et al. Nucleic Acid Res.(1990) 18(11):3155-9; LASHKARI DA. et al. PNAS (1995) 92(17):7912-5; MCGALL G. et al. PNAS (1996) 93(24):13555-60; ALBERT TJ. et al. Nucleic Acid Res.(2003) 31(7):e35; GAO X. et al. Biopolymers (2004) 73(5):579-96; and MOORCROFT MJ. et al. Nucleic Acid Res.(2005) 33(8):e75). In general, oligonucleotides are synthesized through the stepwise addition of activated and protected monomers under a variety of conditions depending on the method being used. Subsequently, specific protecting groups may be removed to allow for further elongation and subsequently and once synthesis is complete all the protecting groups may be removed and the oligonucleotides removed from their solid supports for purification of the complete chains if so desired.
"Peptide nucleic acids" (PNA) as used herein refer to modified nucleic acids in which the sugar phosphate skeleton of a nucleic acid has been converted to an N-(2-aminoethyl)-glycine skeleton. Although the sugar-phosphate skeletons of DNA/RNA are subjected to a negative charge under neutral conditions resulting in electrostatic repulsion between complementary chains, the backbone structure of PNA does not inherently have a charge. Therefore, there is no electrostatic repulsion. Consequently, PNA has a higher ability to form double strands as compared with conventional nucleic acids, and has a high ability to recognize base sequences. Furthermore, PNAs are generally more robust than nucleic acids.
A "vector" as used herein includes any means for delivery of a nucleic acid sample to a cell. For example, a vector may include a plasmid, artificial chromosome etc.
A "cancer" is defined as including cancers in which mutations can be detected in accordance with the methods described herein and can include but, are not limited to, carcinomas, mealomas, sarcomas, lymphomas, and leukaemias. Carcinomas may include, without limitation, squamous cell, adeno, small cell and large cell carcinomas, and combinations thereof. Carcinomas may have their primary tumor located, for example in the nasopharygeal tissue, head and neck, lung, esophagus, stomach, intestine, rectum, kidney, urinary tract, prostate, testes, ovary, uterus, cervix, vagina, skin, and endocrine glands or may be multicentric. Sarcomas may be, for example of muscle, adipose, fibrous, vascular, or neural origin. Lymphomas may include, without, limitation, for example Hodgkin's Disease and Non-Hodgkins Lymphoma. Leukaemias may be, for example, of lymphoid or myeloid cell origin and may be acute or chronic in presentation and/or course. Furthermore, a cancer may be a "SPARC inhibited cancer", whereby the cancer decreases SPARC expression to facilitate growth of the cancer. The cancer may, for example, also be selected from colorectal carcinomas, prostate carcinomas, ovarian carcinomas, osteosarcomas or combinations thereof.
A cancer may be described as "sensitive to" or "resistant to" a given therapeutic regimen based on the ability of the regimen to kill cancer cells or decrease tumor size, reduce overall cancer growth (i.e. through reduction of angiogenesis), and or inhibit metastasis. Cancer cells that are resistant to a therapeutic regimen would not respond to the regimen and may continue to proliferate. Whereas cancer cells that are sensitive to a therapeutic regimen are those that do respond to the regimen resulting in cell death, a reduction in tumor size, reduced overall growth or inhibition of metastasis. Monitoring of a response may be accomplished by numerous pathological, clinical and imaging methods as described herein and known to persons of skill in the art. below shows correlation of SPARC mutations with values representing resistance (R) or sensitivity (S) to a therapeutic regimen for the treatment of cancer.
295delG and 301A>CC R
315insA and 316G>A R
341C>T, 342T>C, 344A>C, 347C>A, 354G>A, 356G>A, 359T>G and 363G>A R
395C>T and 396A>T R
488T>C R
533A>G R
734G>A R
734delG R
769T>A, 771 G>C and 774G>C R
784T>C, 786T>G and 788T>A R
793T>G, 796T>A and 800T>C R
820C>A and 824T>A R
864T>A and 867T>A R
895T>G, 899T>A. 901G>A and 904G>A R
901G>A R
901 G>A, 904G>A, 929A>G and 934delC R
922T>G, 928T>G and 935T>G R
982T>C, 984T>G, 985G>A, 991G>A, 995G>A, 999C>T and 1000T>G R
1063G>C R
1079G>A R
1085delC, 1086delA, 1087delG, 1089G>A, 1090T>C, 1091T>C and 1092G>T R
1161C>A, 1162T>C, 1163delA and 1164delT R
1215C>T, 1216delG, 1217delG, 1218delG, 1219delG, 1220delT, 1221delG, 1222delG, 1223delA, 1224delG, 1225delG, 1226delG, 1227delG, 1228delA, 1229delG, 1230delA, 1231delT, 1232delA, 1233delG, 1234delA, 1235delC, 1236delC and 1237C>T R
Wild type S
Sensitive (S); Resistant (R).
2.A. General Methods
Once a subject is identified as being at risk for developing or having a cancer or a SPARC-inhibited cancer, genetic sequence information may be obtained from the subject. Various methods for obtaining biological samples from a subject that contain genetic material, are known in the art. For example, tissue samples may be obtained by curettage, needle aspiration biopsy or needle (core) biopsy, incisional biopsy for sampling a tumor, or excisional biopsy, which may entail total removal of the tissue of interest. Alternatively, other bodily samples that contain genetic material, such as hair, sputum, urine, stool, semen or blood may be collected using methods known in the art.
Genetic sequence information may be obtained from a biological sample containing genetic material in numerous different ways, particularly, genetic material containing the sequence or sequences of interest. Many methods are known in the art for extracting genetic material from biological samples. There are many known methods for the separate isolation of DNA and RNA from biological samples. Typically, DNA may be isolated from a biological sample when first the sample is lysed and then the DNA is isolated from the lysate according to any one of a variety of multi-step protocols, which can take varying lengths of time. DNA isolation methods may involve the use of phenol
(SAMBROOK, J. et al. "Molecular Cloning", Vol. 2, pp. 9.14-9.23, Cold Spring Harbor Laboratory Press (1989) and AUSUBEL, FM. et al., "Current Protocols in Molecular Biology", Vol. 1, pp. 2.2.1-2.4.5, John Wiley & Sons, Inc. (1994)). Typically, a biological sample is lysed in a detergent solution and the protein component of the lysate is digested with proteinase for 12-18 hours. Next, the lysate is extracted with phenol to remove most of the cellular components, and the remaining aqueous phase is processed further to isolate DNA. In another method, described in VAN NESS et al. (U.S. Pat. # 5,130,423), non-corrosive phenol derivatives are used for the isolation of nucleic acids. The resulting preparation is a mix of RNA and DNA.
Other methods for DNA isolation utilize non-corrosive chaotropic agents. These methods, which are based on the use of guanidine salts, urea and sodium iodide, involve lysis of a biological sample in a chaotropic aqueous solution and subsequent precipitation of the crude DNA fraction with a lower alcohol. The final purification of the precipitated, crude DNA fraction can be achieved by any one of several methods, including column chromatography (Analects, (1994) Vol 22, No. 4, Pharmacia Biotech), or exposure of the crude DNA to a polyanion-containing protein as described in KOLLER (U.S. Pat. # 5,128,247).
Yet another method of DNA isolation, which is described by BOTWELL, DDL. (Anal. Biochem. (1987) 162:463-465) involves lysing cells in 6M guanidine hydrochloride, precipitating DNA from the lysate at acid pH by adding 2.5 volumes of ethanol, and washing the DNA with ethanol.
Numerous other methods are known in the art to isolate both RNA and DNA, such as the one described by CHOMCZYNSKI (U.S. Pat. # 5,945,515 ), whereby genetic material can be extracted efficiently in as little as twenty minutes. EVANS and HUGH (U.S. Pat. # 5,989,431 ) describe methods for isolating DNA using a hollow membrane filter.
Once a subject's genetic material has been obtained from the subject it may then be further be amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR), Polymerase Chain Reaction (PCR), Transcription Mediated Amplification (TMA), Ligase chain reaction (LCR), Nucleic Acid Sequence Based Amplification (NASBA) or other methods known in the art, and then further analyzed to detect or determine the presence or absence of one or more polymorphisms or mutations in the sequence of interest, provided that the genetic material obtained contains the sequence of interest. Particularly, a person may be interested in determining the presence or absence of a mutation in the SPARC promoter sequence , as described in TABLE 1. The sequence of interest may also include other mutations, or may also contain some of the sequence surrounding the mutation of interest.
Detection or determination of a nucleotide identity, or the presence of one or more single nucleotide polymorphism(s) (SNP typing), may be accomplished by any one of a number methods or assays known in the art. Many DNA typing methodologies are useful detection of SNPs. The majority of SNP genotyping reactions or assays can be assigned to one of four broad groups (sequence-specific hybridization, primer extension, oligonucleotide ligation and invasive cleavage). Furthermore, there are numerous methods for analyzing/detecting the products of each type of reaction (for example, fluorescence, luminescence, mass measurement, electrophoresis, etc.). Furthermore, reactions can occur in solution or on a solid support such as a glass slide, a chip, a bead, etc.
In general, sequence-specific hybridization involves a hybridization probe, which is capable of distinguishing between two DNA targets differing at one nucleotide position by hybridization. Usually probes are designed with the polymorphic base in a central position in the probe sequence, whereby under optimized assay conditions only the perfectly matched probe target hybrids are stable and hybrids with a one base mismatch are unstable. A strategy which couples detection and sequence discrimination is the use of a "molecular beacon", whereby the hybridization probe (molecular beacon) has 3' and 5' reporter and quencher molecules and 3' and 5' sequences which are complementary such that absent an adequate binding target for the intervening sequence the probe will form a hairpin loop. The hairpin loop keeps the reporter and quencher in close proximity resulting in quenching of the fluorophor (reporter) which reduces fluorescence emissions. However, when the molecular beacon hybridizes to the target the fluorophor and the quencher are sufficiently separated to allow fluorescence to be emitted from the fluorophor.
Similarly, primer extension reactions (i.e. mini sequencing, nucleotide-specific extensions, or simple PCR amplification) are useful in sequence discrimination reactions. For example, in mini sequencing a primer anneals to its target DNA immediately upstream of the SNP and is extended with a single nucleotide complementary to the polymorphic site. Where the nucleotide is not complementary, no extension occurs.
Oligonucleotide ligation assays require two sequence-specific probes and one common ligation probe per SNP. The common ligation probe hybridizes adjacent to a sequence-specific probe and when there is a perfect match of the appropriate sequence-specific probe, the ligase joins both the sequence-specific and the common probes. Where there is not a perfect match the ligase is unable to join the sequence-specific and common probes.
Alternatively, an invasive cleavage method requires an oligonucleotide called an Invader™ probe and sequence-specific probes to anneal to the target DNA with an overlap of one nucleotide. When the sequence-specific probe is complementary to the polymorphic base, overlaps of the 3' end of the invader oligonucleotide form a structure that is recognized and cleaved by a Flap endonuclease releasing the 5' arm of the allele specific probe.
5' exonuclease activity or TaqMan™ assay (Applied Biosystems) is based on the 5' nuclease activity of Taq polymerase that displaces and cleaves the oligonucleotide probes hybridized to the target DNA generating a fluorescent signal. It is necessary to have two probes that differ at the polymorphic site wherein one probe is complementary to the 'normal' sequence and the other to the mutation of interest. These probes have different fluorescent dyes attached to the 5' end and a quencher attached to the 3' end when the probes are intact the quencher interacts with the fluorophor by fluorescence resonance energy transfer (FRET) to quench the fluorescence of the probe. During the PCR annealing step the hybridization probes hybridize to target DNA. In the extension step the 5' fluorescent dye is cleaved by the 5' nuclease activity of Taq polymerase, leading to an increase in fluorescence of the reporter dye. Mismatched probes are displaced without fragmentation. The presence of a mutation in a sample is determined by measuring the signal intensity of the two different dyes.
It will be appreciated that numerous other methods for sequence discrimination and detection are known in the art and some of which are described in further detail below. It will also be appreciated that reactions such as arrayed primer extension mini sequencing, tag microarrays and sequence-specific extension could be performed on a microarray. One such array based genotyping platform is the microsphere based tag-it high throughput genotyping array (BORTOLIN S. et al. Clinical Chemistry (2004) 50(11): 2028-36). This method amplifies genomic DNA by PCR followed by sequence-specific primer extension with universally tagged genotyping primers. The products are then sorted on a Tag-It array and detected using the Luminex xMAP system.
Mutation detection methods may include but are not limited to the following:
  • Restriction Fragment Length Polymorphism (RFLP) strategy - An RFLP gel-based analysis can be used to indicate the presence or absence of a specific mutation at polymorphic sites within a gene. Briefly, a short segment of DNA (typically several hundred base pairs) is amplified by PCR. Where possible, a specific restriction endonuclease is chosen that cuts the short DNA segment when one polymorphism is present but does not cut the short DNA segment when the polymorphism is not present, or vice versa. After incubation of the PCR amplified DNA with this restriction endonuclease, the reaction products are then separated using gel electrophoresis. Thus, when the gel is examined the appearance of two lower molecular weight bands (lower molecular weight molecules travel farther down the gel during electrophoresis) indicates that the DNA sample had a polymorphism was present that permitted cleavage by the specific restriction endonuclease. In contrast, if only one higher molecular weight band is observed (at the molecular weight of the PCR product) then the initial DNA sample has the polymorphism that could not be cleaved by the chosen restriction endonuclease. Finally, if both the higher molecular weight band and the two lower molecular weight bands are visible then the DNA sample contained both polymorphisms, and therefore the DNA sample, and by extension the subject providing the DNA sample, was heterozygous for this polymorphism;
Sequencing - For example the Maxam-Gilbert technique for sequencing (MAXAM AM. and GILBERT W. Proc. Natl. Acad. Sci. USA (1977) 74(4):560-564) involves the specific chemical cleavage of terminally labelled DNA. In this technique four samples of the same labeled DNA are each subjected to a different chemical reaction to effect preferential cleavage of the DNA molecule at one or two nucleotides of a specific base identity. The conditions are adjusted to obtain only partial cleavage, DNA fragments are thus generated in each sample whose lengths are dependent upon the position within the DNA base sequence of the nucleotide(s) which are subject to such cleavage. After partial cleavage is performed, each sample contains DNA fragments of different lengths, each of which ends with the same one or two of the four nucleotides. In particular, in one sample each fragment ends with a C, in another sample each fragment ends with a C or a T, in a third sample each ends with a G, and in a fourth sample each ends with an A or a G. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. This technique permits the sequencing of at least 100 bases from the point of labeling. Another method is the dideoxy method of sequencing was published by SANGER et al. (Proc. Natl. Acad. Sci. USA (1977) 74(12):5463-5467). The Sanger method relies on enzymatic activity of a DNA polymerase to synthesize sequence-dependent fragments of various lengths. The lengths of the fragments are determined by the random incorporation of dideoxynucleotide base-specific terminators. These fragments can then be separated in a gel as in the Maxam-Gilbert procedure, visualized, and the sequence determined. Numerous improvements have been made to refine the above methods and to automate the sequencing procedures. Similarly, RNA sequencing methods are also know. For example, reverse transcriptase with dideoxynucleotides have been used to sequence encephalomyocarditis virus RNA (ZIMMERN D. and KAESBERG P. Proc. Natl. Acad. Sci. USA (1978) 75(9):4257-4261). MILLS DR. and KRAMER FR. (Proc. Natl. Acad. Sci. USA (1979) 76(5):2232-2235) describe the use of Qβ replicase and the nucleotide analog inosine for sequencing RNA in a chain-termination mechanism. Direct chemical methods for sequencing RNA are also known (PEATTIE DA. Proc. Natl. Acad. Sci. USA (1979) 76(4):1760-1764). Other methods include those of Donis-Keller et al. (1977, Nucl. Acids Res. 4:2527-2538), SIMONCSITS A. et al. (Nature (1977) 269(5631):833-836), AXELROD VD. et al. (Nucl. Acids Res.(1978) 5(10):3549-3563), and KRAMER FR. and MILLS DR. (Proc. Natl. Acad. Sci. USA (1978) 75(11):5334-5338). Nucleic acid sequences can also be read by stimulating the natural fluoresce of a cleaved nucleotide with a laser while the single nucleotide is contained in a fluorescence enhancing matrix ( U.S. Pat. # 5,674,743 ); In a mini sequencing reaction, a primer that anneals to target DNA adjacent to a SNP is extended by DNA polymerase with a single nucleotide that is complementary to the polymorphic site. This method is based on the high accuracy of nucleotide incorporation by DNA polymerases. There are different technologies for analyzing the primer extension products. For example, the use of labeled or unlabeled nucleotides, ddNTP combined with dNTP or only ddNTP in the mini sequencing reaction depends on the method chosen for detecting the products;
Probes used in hybridization can include double-stranded DNA, single-stranded DNA and RNA oligonucleotides, and peptide nucleic acids. Hybridization methods for the identification of single nucleotide polymorphisms or other mutations involving a few nucleotides are described in the U.S. Pat. 6,270,961 ; 6,025,136 ; and 6,872,530 . Suitable hybridization probes for use in accordance with the invention include oligonucleotides and PNAs from about 10 to about 400 nucleotides, alternatively from about 20 to about 200 nucleotides, or from about 30 to about 100 nucleotides in length.
A template-directed dye-terminator incorporation with fluorescent polarization-detection (TDI-FP) method is described by FREEMAN BD. et al. (J Mol Diagnostics (2002) 4(4):209-215) for large scale screening;
Oligonucleotide ligation assay (OLA) is based on ligation of probe and detector oligonucleotides annealed to a polymerase chain reaction amplicon strand with detection by an enzyme immunoassay (VILLAHERMOSA ML. J Hum Virol (2001) 4(5):238-48; ROMPPANEN EL. Scand J Clin Lab Invest (2001) 61(2):123-9; IANNONE MA. et al. Cytometry (2000) 39(2):131-40);
Ligation-Rolling Circle Amplification (L-RCA) has also been successfully used for genotyping single nucleotide polymorphisms as described in QI X. et al. Nucleic Acids Res (2001) 29(22):E116;
5' nuclease assay has also been successfully used for genotyping single nucleotide polymorphisms (AYDIN A. et al. Biotechniques (2001) (4):920-2, 924, 926-8.);
Polymerase proofreading methods are used to determine SNPs identities, as described in WO 0181631 ;
Detection of single base pair DNA mutations by enzyme-amplified electronic transduction is described in PATOLSKY F et al. Nat Biotech. (2001) 19(3):253-257;
Gene chip technologies are also known for single nucleotide polymorphism discrimination whereby numerous polymorphisms may be tested for simultaneously on a single array ( EP 1120646 and GILLES PN. et al. Nat. Biotechnology (1999) 17(4):365-70);
Matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy is also useful in the genotyping single nucleotide polymorphisms through the analysis of microsequencing products (HAFF LA. and SMIRNOV IP. Nucleic Acids Res. (1997) 25(18):3749-50; HAFF LA. and SMIRNOV IP. Genome Res. (1997) 7:378-388; SUN X. et al. Nucleic Acids Res. (2000) 28 e68; BRAUN A. et al. Clin. Chem. (1997) 43:1151-1158; LITTLE DP. et al. Eur. J. Clin. Chem. Clin. Biochem. (1997) 35:545-548; FEI Z. et al. Nucleic Acids Res. (2000) 26:2827-2828; and BLONDAL T. et al. Nucleic Acids Res. (2003) 31(24):e155).
Sequence-specific PCR methods have also been successfully used for genotyping single nucleotide polymorphisms (HAWKINS JR. et al. Hum Mutat (2002) 19(5):543-553). Alternatively, a Single-Stranded Conformational Polymorphism (SSCP) assay or a Cleavase Fragment Length Polymorphism (CFLP) assay may be used to detect mutations as described herein.
Provided herein are methods of detecting mutations, which result in an altered levels of SPARC polypeptide. "Altered levels" as used herein include, e.g., a change of about 10%, a change of about 20%, a change of about 30%, a change of about 40%, a change of about 50%, a change of about 75%, and a change of about 100% as compared to wild type levels for the same cell or tissue type. "Altered levels" as used herein also include, e.g., an at least about 2 fold, an at least about 5 fold, an at least about 10 fold, an at least about 20 fold, an at least about 50 fold, an at least about 100 fold, an at least about 200 fold, an at least about 500 fold, and an at least about 1,000 fold change in the level as compared to wild type levels for the same cell or tissue type. Accordingly, included is a method of detecting a mutation in a SPARC sequence occurring in an animal, wherein the mutation is in the promoter of the gene and results in an altered intracellular and/or extracellular levels of a polypeptide encoded by the SPARC gene. The method in accordance with the invention further provides for the detection of mutations wherein the mutation is a point mutation, deletion, insertion or frameshift mutation or combinations thereof. In addition, mutations detectable with the methods described include, e.g., deletion and/or insertions of 1 nucleotide, from 1 to about 5 nucleotides, from about 5 to about 10 nucleotides, from about 10 to about 20 nucleotides, from about 20 to about 50 nucleotides, and from about 50 to about 100 nucleotides, from about 100 nucleotides to about 1000 nucleotides. Altered levels as used herein may be a reduction in the intracellular and/or extracellular SPARC polypeptide.
Mutation detection in accordance with the methods described herein may include thy determination of SPARC promoter sequence of at least about 1 nucleotide, alternatively at least about 5 nucleotides, at least about 10 nucleotides, at least about 50 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, or at least about 2,000 nucleotides.
Provided herein are nucleic acid molecules with one or more SPARC gene promoter mutations, further including a reporter sequence and/or further including a vector. Suitable nucleic acid molecules may include molecules of at least about 10 base pairs, at least about 50 base pairs, at least about 100 base pairs, at least about 200 base pairs, at least about 300 base pairs, at least about 500 base pairs, and at least about 1,000 base pairs. In addition, there is provided a cell including the nucleic acid molecule with a mutant SPARC promoter genotype.
Alternatively, if a subject's genetic sequence data is already known, then obtaining may involve retrieval of the subject's nucleic acid sequence data from a database, followed by determining or detecting the identity of a nucleic acid or genotype at a polymorphism site by reading the subject's nucleic acid sequence at the polymorphic site.
Once the identity of a polymorphism(s) or other mutation is determined or detected an indication may be obtained as to resistance of a subject's cancer to a therapeutic regimen based on the based on the presence or absence of one or more mutations in the subject's SPARC promoter sequence. As described herein, polymorphisms or other mutations in the SPARC promoter sequence, may used to obtain a prognosis or to make a determination regarding the ability of the subject to have a favourable outcome following a chemotherapy, radiotherapy or combination therapy regiment for a cancer. Specifically, to determine whether the subject's cancer is likely to be sensitive to a particular therapeutic regimen or whether the cancer is likely to be resistant to a particular therapeutic regimen. Alternatively, polymorphisms or other mutations in the SPARC promoter may be used to screen subjects to determine the ability of a subject to be treated by a chemotherapy, radiotherapy or combination therapy regimen for cancer. Examples may include subjects to be included in a clinical trial or other similar study. Alternatively, mutations may be used as an indication of a subject's ability to respond to a chemotherapy, radiotherapy or combination therapy regimen directed at the subject's cancer. A mutation may be indicative of a subject's cancer being resistant to a chemotherapy, radiotherapy or combination therapy regimen. Accordingly, a decision regarding the subject's chemotherapy, radiotherapy or combination therapy regimen may be made based on the subject's SPARC gene sequence.
Once a subject's SPARC promoter sequence or SPARC mutational status is determined, such information may be of interest to physicians and surgeons to assist in deciding between potential treatment options, to help determine the degree to which subjects are monitored and the frequency with which such monitoring occurs. Ultimately, treatment decisions may be made in response to factors, both specific to the subject and based on the experience of the physician or surgeon responsible for a subject's care. For example, a subject who has one or more SPARC promoter mutations that are indicative of resistance to a particular therapeutic regimen, may be offered an "alternative therapeutic regimen". Accordingly, a subject who is receiving a first line chemotherapeutic regimen (or is about to receive such a regimen) and has one or more SPARC promoter mutations in their cancer may be preferentially administered an alternative therapeutic regimen. For example, SPARC polypeptide (as described in WO 2004/064785 ), receptor tyrosine kinase inhibitors (for example Iressa™ (gefitinib), Tarceva™ (erlotinib), Erbitux™ (cetuximab), imatinib mesilate (Gleeve) or Avastin™ (bevacizumab), proteosome inhibitors (for example bortezomib, Velcade); aurora kinase inhibitors (for example ZM447439); rapamycin inhibitors (for example sirolimus, Rapamune); farnesyltransferase inhibitors (for example tipifarnib, Zarnestra); matrix metalloproteinase inhibitors (for example BAY 12-9566; sulfated polysaccharide tecogalan); angiogenesis inhibitors (for example Avastin™ (bevacizumab); TNP-4, a synthetic analogue of fumagillin; carboxyaminotriazole; BB-94 and BB-2516; thalidomide; interleukin-12; linomide; peptide fragments; and antibodies to vascular growth factors and vascular growth factor receptors); cyclo-oxygenase inhibitors, platelet derived growth factor receptor inhibitors, protein kinase C inhibitors, mitogen-activated protein kinase kinase inhibitors, histonedeacetylase inhibitors, small hypoxia-inducible factor inhibitors, hedgehog inhibitors, and TGF-β signalling inhibitors. Furthermore, "Immunotherapeutic agent", as used herein, would also be considered an alternative therapeutic regimen. For example, agents used to transfer the immunity of an immune donor, e.g., another person or an animal, to a host by inoculation. The term embraces the use of serum or gamma globulin containing performed antibodies produced by another individual or an animal; nonspecific systemic stimulation; adjuvants; active specific immunotherapy; and adoptive immunotherapy. Adoptive immunotherapy refers to the treatment of a disease by therapy or agents that include host inoculation of sensitized lymphocytes, transfer factor, immune RNA, or antibodies in serum or gamma globulin. Such alternative therapeutics may be administered alone or in combination or in combination with other therapeutic regimens described herein.
A subject may be monitored on an ongoing basis for SPARC promoter mutations during the course of chemotherapy, radiotherapy or combination therapy regimen. A response to a selected regimen may be predicted by the presence or absence of one or more SPARC promoter mutations as described herein. An improved response may include an increased likelihood of survival, reduction in tumor size or reduction in the rate of metastasis. Tumor staging provides a method to assess the size and spread, and by extension guide prognosis of a tumor in a subject. The TNM tumor staging system uses three components to express the anatomic extent of disease: T is a measure of the local extend of tumor spread (size), N indicates the presence or absence of metastatic spread to regional lymph nodes, and M specifies the presence or absence of metatstatic spread to distant sites. The combination of these three classifications combine to provide a stage grouping. Clinical TNM (cTNM) defines the tumor based on clinical evidence. Pathologic TNM (pTNM) defines the tumor based on examination of a surgically resected specimen.
Changes in tumor size may be observed by various imaging methods known to physicians or surgeons in the field of oncology therapy and diagnostics. Examples of imaging methods include positron emission tomography (PET) scanning, computed tomography (CT) scanning, PET/CT scanning, magnetic resonance imaging (MRI), chemical shift imaging, radiography, bone-scan, mammography, fiberoptic colonoscopy or ultrasound. Contrast agents, tracers and other specialized techniques may also be employed to image specific types of cancers, or for particular organs or tissues, and will be known to those skilled in the art. Changes in rate of metastasis may also be observed by the various imaging methods, considering particularly the appearance, or frequency of appearance, of tumors distal to the primary site. Alternatively, the presence of tumor cells in lymph nodes adjacent and distal to the primary tumor site may also be detected and used to monitor metastasis.
2.B. Methods and Materials Cell Culture and Maintenance
A human colorectal cancer cell line, MIP-101, and uterine sarcoma cell line, MES-SA were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA) at 37°C and 5% CO2. MIP/5FU (MIP-101 cells resistant to 5-fluorouracil), MIP/CIS (MIP-101 cells resistant to cisplatin) and MIP/CPT (MIP-101 cells resistant to irinotecan) were maintained as described above, but with the inclusion of the respective resistant drug (5-flurouracil 200 µM, cisplatin 10 µM, irinotecan 20 µM). MES-SA cells resistant to doxorubicin (MES-SA/DX5) were acquired from ATCC (# CRL-1977) and maintained according to supplier's recommendations.
Development of Resistant Cell Lines
Resistant MIP101 cells were developed following long-term incubation with incremental concentrations of chemotherapeutic agents 5-fluorouracil (5FU; 10-500 µM) (Sigma, St. Louis, MO), cisplatin (CIS; 1-100 µM) (Sigma, St. Louis, MO) or irinotecan (CPT; 1-50 µM) (Pharmacia, Kalamazoo, MI) over 3 months. The concentration of each chemotherapeutic agent was increased by 10% every 2 weeks for a 6 month period.
Isolation of Cellular DNA
10-20 million colorectal cancer cells from sensitive (MIP101, MES-SA) and resistant (MIP/5FU, MIP/CPT, MIP/CIS, MES-SA/DX5) cells were collected and DNA extracted based on the manufacturer's protocol (DNA Extraction Kit, Gentra Systems, Inc.).
Sequencing Reactions
Overlapping sections of the SPARC promoter were sequenced using the BigDye™ Terminator v 3.1 Cycle Sequencing Kit (BD Biosciences) following manufacturer's instructions. Briefly, 4 µl of reaction premix, 2 µl of sequencing buffer, 3.2 pmol of primer and 50-100 ng of isolated cellular DNA was combined with water to make a final volume of 20 µl. Cycle sequencing was performed using the following parameters: an initial denaturation step at 95°C for 2 minutes, followed by 30 cycles of 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, with a final extension at 72°C for 10 min. Sequencing reaction amplicons were purified by incubation with AMPure magnetic beads (Agencourt Bioscience Corporation), following manufacturer's instructions. Briefly, the sequencing reaction mixture was incubated with AMPure magnetic beads for 10 min, followed by collection of beads with a magnetic plate. The collected beads were washed once with 70% ethanol, and the PCR amplicons eluted with Tris-EDTA, ph 8.0. The eluted sequencing reaction amplicons were sequenced using an ABI Prism 3700 DNA analyzer. Sequence chromatograms generated by ABI Prism 3700 DNA Analyzer were analyzed and assembled by Phred-Phrap programs and assembled by Consed programs sing reference sequences from UCSC Bioinformatics for the identification of mutations.
Analytical RT-PCR.
To determine SPARC mRNA levels Analytical RT-PCR could be performed as described by TAI, IT. et al. (J. Clin Invest. (2005) 115:1493-1502). Briefly, total RNA would be extracted from cultured cells, at 75% confluence and under similar culture conditions, using TRIZOL reagent, following manufacturer's instructions. RT-PCR could be performed using One-Step RT-PCR (BD Biosciences), following manufacturer's instructions. SPARC-specific primers such as SEQ ID NO: 52 and SEQ ID NO: 53. GAPDH-specific primers SEQ ID NO: 54 and SEQ ID NO: 55 could be used. The reaction conditions for such a reaction would be 50°C for 1 hour, followed by 45 cycles at 94°C for 1 minute, 65°C for 1 minute, 72°C for 2 minutes, and 72°C for 10 minutes. PCR products could then be separated on 1% agarose gel electrophoresis.
Protein Extracts and Immunoblots
To determine SPARC protein levels, extracts and immunoblots could be prepared as described by TAI IT et al. 2005, supra. Briefly, total protein could be extracted from cell lysates using CHAPS lysis buffer (50 mM PIPES/HCl, pH 6.5; 2 mM EDTA; 0.1% CHAPS; 20 µg/ml Leupeptin; 10 µg/ml Pepstatin A; 10 µg/ml Aprotinin; 5 mM DTT; 1 mM PMSF). The proteins could be resolved by SDS-PAGE (10% gel) under reducing conditions and electrotransferred onto a PVDF membrane and analyzed by Western blot. Antibodies to SPARC (1 µg/ml; Haematologic Technologies Inc.) and alpha-tubulin (0.2 µg/ml; Sigma-Aldrich) could be used.
SPARC mRNA and protein levels have been reported to be low in cell lines resistant to chemotherapy. TAI IT. et al. 2005, supra describe decreased SPARC mRNA and protein levels in MIP resistant cells (MIP/5FU, MIP/CPT, MIP/CIS and MES-SA/DX5, among others), as evaluated by RT-PCR and western blotting. Significant reductions in SPARC-specific mRNA compared with the sensitive parental lines was observed for all cell lines tested. This result was of particular interest as it was consistent over both colorectal carcinoma and uterine sarcoma cells, resistant to four different chemotherapeutic agents. Protein levels of SPARC were also significantly decreased in the resistant cell lines, again reflected in both colorectal carcinoma and uterine sarcoma cell lines, resistant to flour different chemotherapeutic agents.
2. EXAMPLES Example 1 Mutations in SPARC regulatory regions in MIP/5FU cell lines
A region spanning 2963 base pairs spanning a regulatory region of SPARC∼1400 bp upstream of exon 1, exon 1 and ∼1300 of intron 1 (SEQ ID NO: 9) was sequenced on both strands using primers corresponding to SEQ ID NOs: 34 to 39 in both sensitive (MIP-101) and 5FU resistant (MIP/5FU) cell lines. Several mutations were identified in the regulatory region between nucleotides 1 to 1370 of SEQ ID NO: 1 that were not found in the sensitive cell line. The individual mutations are listed in TABLE 1- mutations specifically identified to date in the MIP/SFCT cell lines are shown in TABLE 5 infra.
Example 2 Mutations in SPARC regulatory regions in MIP/CPT cell lines
A region spanning 2963 base pairs spanning a regulatory region of SPARC∼1400 bp upstream of exon 1, exon 1 and ∼1300 of intron 1 (SEQ ID NO: 9) was sequenced on both strands using primers corresponding to SEQ ID NOs: 34-39 in both sensitive (MIP-101) and 5FU resistant (MIP/5FU) cell lines. Several mutations were identified in the regulatory region between nucleotides 1 to 1370 of SEQ ID NO: 1 that were not found in the sensitive cell line. The individual mutations are listed in TABLE 1- mutations specifically identified to date in the MIP/CPT cell lines are shown in TABLE 5 infra.
Example 3 Mutations in SPARC regulatory regions in MIP/CIS cell lines
A region spanning 2963 base pairs spanning a regulatory region of SPARC∼1400 bp upstream of exon 1, exon 1 and ∼1300 of intron 1 (SEQ ID NO: 9) was sequenced on both strands using primers corresponding to SEQ ID NOs: 34-39 in both sensitive (MIP-101) and 5FU resistant (MIP/5FU) cell lines. Several mutations were identified in the regulatory region between nucleotides 1 to 1370 of SEQ ID NO: 1 that were not found in the sensitive cell line. The individual mutations are listed in TABLE 1- mutations specifically identified to date in the MIP/CIS cell lines are shown in TABLE 5 infra.
Example 4 Mutations in SPARC regulatory regions in MES-SA/DX5 cell lines
A region spanning 2963 base pairs spanning a regulatory region of SPARC ∼1400 bp upstream of exon 1, exon 1 and ∼1300 of intron 1 (SEQ ID NO: 9) was sequenced on both strands using primers corresponding to SEQ ID NOs: 34-39 in both sensitive (MIP-101) and 5FU resistant (MIP/5FU) cell lines. Several mutations were identified in the regulatory region between nucleotides 1 to 1370 of SEQ ID NO: 1 that were not found in the sensitive cell line. The individual mutations are listed in TABLE 1- mutations specifically identified to date in the MES-SA/DX5 cell lines are shown in TABLE 5 infra. The results shown in TABLE 5 below represent mutations identified to date in various drug resistant cell lines and the absence of a mutation (i.e. no √) is not meant to represent that such a mutation is not possible or even likely, only that to date not such mutation has been identified in a given resistant cell line with a given chemotherapeutic regimen.
(a) 295delG and 301A>CC √
(b) 315insA and 316G>A √
(c) 341C>T, 342T>C, 344A>C, 347C>A, 354G>A, 356G>A, 359T>G and 363G>A √
(d) 395C>T and 396A>T √ √ √
(e) 488T>C √
(f) 533A>G √
(g) 734G>A √ √ √ √
(h) 734delG √ √ √ √
(i) 769T>A, 771G>C and 774G>C √
(j) 784T>C, 786T>G and 788T>A √
(k) 793T>G. 796T>A and 800T>C
(l) 820C>A and 824T>A √
(m) 864T>A and 867T>A √
(n) 895T>G, 899T>A, 901G>A and 904G>A √ √ √
(o) 901G>A √ √ √ √
(p) 901G>A, 904G>A, 929A>G and 934delC √ √ √
(q) 922T>G, 928T>G and 935T>G √
(r) 982T>C, 984T>G, 985G>A, 991G>A, 995G>A, 999C>T and 1000T>G √
(s) 1063G>C √ √ √
(t) 1079G>A √ √ √
(u) 1085delC, 1086delA, 1087delG, 1089G>A, 1090T>C, 1091T>C and 1092G>T √ √ √
(v) 1161C>A, 1162T>C, 1163delA and 1164delT √ √ √
(w) 1215C>T, 1216delG, 1217delG, 1218delG, 1219delG, 1220delT, 1221delG, 1222delG, 1223delA, 1224delG, 1225delG, 1226delG, 1227delG, 1228delA, 1229delG, 1230delA, 1231delT, 1232delA, 1233delG, 1234delA, 1235delC, 1236delC and 1237C>T √ √ √
SEQUENCE LISTING
  • <110> The University of British Columbia TAI, Isabella T.
  • <120> SPARC PROMOTER MUTATIONS ASSOCIATED WITH DRUG RESISTANCE, AND METHODS, OLIGONUCLEOTIDES, CELLS AND ARRAYS ASSOCIATED THEREWITH
  • <130> 80021-835
  • <140> NOT YET ASSIGNED <141> 2005-11-30
  • <160> 55
  • <170> PatentIn version 3.3
  • <210> 1 <211> 1370 <212> DNA <213> Homo sapiens
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  • <210> 2 <211> 1370 <212> DNA <213> Homo sapiens
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  • <210> 4 <211> 1371 <212> DNA <213> Homo sapiens
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  • <210> 5 <211> 1369 <212> DNA <213> Homo sapiens
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  • <210> 6 <211> 1369 <212> DNA <213> Homo sapiens
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  • <210> 7 <211> 1367 <212> DNA <213> Homo sapiens
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  • <210> 8 <211> 1368 <212> DNA <213> Homo sapiens
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  • <210> 9 <211> 2963 <212> DNA <213> Homo sapiens
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  • <210> 10 <211> 1349 <212> DNA <213> Homo sapiens
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  • <210> 11 <211> 399 <212> DNA <213> Homo sapiens
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  • <210> 12 <211> 407 <212> DNA <213> Homo sapiens
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  • <210> 13 <211> 400 <212> DNA <213> Homo sapiens
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  • <210> 14 <211> 406 <212> DNA <213> Homo sapiens
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  • <210> 15 <211> 403 <212> DNA <213> Homo sapiens
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  • <210> 16 <211> 408 <212> DNA <213> Homo sapiens
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  • <210> 17 <211> 407 <212> DNA <213> Homo sapiens
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  • <210> 18 <211> 406 <212> DNA <213> Homo sapiens
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  • <210> 19 <211> 402 <212> DNA <213> Homo sapiens
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  • <210> 20 <211> 407 <212> DNA <213> Homo sapiens
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  • <210> 21 <211> 398 <212> DNA <213> Homo sapiens
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  • <210> 22 <211> 401 <212> DNA <213> Homo sapiens
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  • <210> 23 <211> 397 <212>. DNA <213> Homo sapiens
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  • <210> 24 <211> 446 <212> DNA <213> Homo sapiens
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  • <210> 25 <211> 440 <212> DNA <213> Homo sapiens
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  • <210> 26 <211> 439 <212> DNA <213> Homo sapiens
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  • <210> 27 <211> 419 <212> DNA <213> Homo sapiens
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  • <210> 28 <211> 419 <212> DNA <213> Homo sapiens
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  • <210> 29 <211> 418 <212> DNA <213> Homo sapiens
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  • <210> 30 <211> 402 <212> DNA <213> Homo sapiens
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  • <210> 31 <211> 415 <212> DNA <213> Homo sapiens
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  • <210> 32 <211> 408 <212> DNA <213> Homo sapiens
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  • <210> 33 <211> 339 <212> DNA <213> Homo sapiens
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  • <210> 34 <211> 22 <212> DNA
  • <213> Artificial sequence
  • <220> <223> Primer
  • <400> 34 ttctgcactt aactcgtcta aa    22
  • <210> 35 <211> 22 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 35 gctgattcta cgtttcaact tg    22
  • <210> 36 <211> 22 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 36 tgaggacaag gaccaggtat tt    22
  • <210> 37 <211> 21 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 37 aggaaaccac tcagagctct g    21
  • <210> 38 <211> 16 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 38 gtcatccctc caggcc    16
  • <210> 39 <211> 19 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 39 cggggttggt gcaactata    19
  • <210> 40 <211> 40 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 40 tgtaaaacga cggccagttg aggacaagga ccaggtattt    40
  • <210> 41 <211> 38 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 41 caggaaacag ctatgacagg aaaccactca gagctctg    38
  • <210> 42 <211> 37 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 42 tgtaaaacga cggccagtgg tggaggggag atagacc    37
  • <210> 43 <211> 37 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 43 caggaaacag ctatgactcc caaggaactc aggacag    37
  • <210> 44 <211> 40 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 44 tgtaaaacga cggccagttg aggacaagga ccaggtattt    40
  • <210> 45 <211> 38 <212> DNA
  • <213> Artificial sequence
  • <220> <223> Primer
  • <400> 45 caggaaacag ctatgacagg aaaccactca gagctctg 38
  • <210> 46 <211> 38 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 46 tgtaaaacga cggccagtaa atggcaccat cacaacct 38
  • <210> 47 <211> 37 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 47 caggaaacag ctatgacccg aagcctgtgt cctactt 37
  • <210> 48 <211> 40 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 48 tgtaaaacga cggccagttt ctgcacttaa ctcgtctaaa 40
  • <210> 49 <211> 39 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 49 caggaaacag ctatgacgct gattctacgt ttcaacttg 39
  • <210> 50 <211> 34 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 50 tgtaaaacga cggccagtgt catccctcca ggcc    34
  • <210> 51 <211> 36 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 51 caggaaacag ctatgaccgg ggttggtgca actata    36
  • <210> 52 <211> 23 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 52 cgaagaggag gtggtggcgg aaa    23
  • <210> 53 <211> 26 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 53 ggttgttgtc ctcatccctc tcatac    26
  • <210> 54 <211> 24 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 54 ctctctgctc ctcctgttcg acag    24
  • <210> 55 <211> 24 <212> DNA <213> Artificial sequence
  • <220> <223> Primer
  • <400> 55 aggggtctta ctccttggag gcca    24

Claims (23)

  1. A method for predicting responsiveness of a subject to a therapeutic regimen, the subject having, or suspected of having a cancer, the method comprising determining a presence or an absence of one or more a secreted protein, acidic, cysteine-rich (SPARC) promoter mutations in the subject's cancer, wherein said presence or absence of one or more SPARC promoter mutations is indicative of a sensitivity of the subject's cancer to the therapeutic regimen, wherein the one or more SPARC promoter mutations, are selected from the following mutations relative to SEQ will NO: 1:
    (a) 295delG and 301A>CC;
    (b) 315insA and 316G>A;
    (c) 341C>T, 342T>C, 344A>C, 347C>A, 354G>A, 356G>A, 359T>G and 363G>A;
    (d) 395C>T and 396A>T;
    (e) 488T>C;
    (f) 533A>G;
    (g) 734G>A;
    (h) 734delG;
    (i) 769T>A, 771 G>C and 774G>C;
    (j) 784T>C, 786T>G and 788T>A;
    (k) 793T>G, 796T>A and 800T>C;
    (l) 820C>A and 824T>A;
    (m) 864T>A and 867T>A;
    (n) 895T>G, 899T>A, 901G>A and 904G>A;
    (o) 901G>A;
    (p) 901G>A, 904G>A, 929A>G and 934delC;
    (q) 922T>G, 928T>G and 93 5T>G;
    (r) 982T>C, 984T>G, 985G>A, 991G>A, 995G>A, 999C>T and 1000T>G;
    (s) 1063G>C;
    (t) 1079G>A;
    (u) 1085delC, 1086delA, 1087delG, 1089G>A, 1090T>C, 1091T>C and 1092G>T;
    (v) 1161C>A, 1162T>C, 1163delA and 1164delT; and
    (w) 1215C>T, 1216delG, 1217delG, 1218delG, 1219delG, 1220delT, 1221delG, 1222delG, 1223delA, 1224delG, 1225delG, 1226delG, 1227delG, 1228delA, 1229delG, 1230delA, 1231delT, 1232delA, 1233delG, 1234delA, 1235delC, 1236delC and 1237C>T, and
    wherein the therapeutic regimen comprises administration of one or more chemotherapeutic agents.
  2. The method of claim 1, wherein the promoter mutation results in altered intracellular and/or extracellular levels of a polypeptide encoded by the SPARC promoter sequence.
  3. The method of claim 1 or 2, wherein the therapeutic regimen further comprises radiotherapy.
  4. The method of claim 1, wherein the chemotherapeutic agent is selected from 5-flurouracil, irinotecan, cisplatin, doxorubicin or combinations thereof.
  5. The method of claim 1, wherein the one or more SPARC promoter mutations are indicative of the cancer's resistance to the therapeutic regimen.
  6. The method of any one of claims 1-5, further comprising obtaining SPARC promoter information for the subject.
  7. The method of any one of claims 1-6, wherein the one or more SPARC promoter mutations are determined using a nucleic acid sample from the subject.
  8. The method of claim 7, wherein the nucleic acid sample has been obtained from the subject.
  9. The method of claim 8, wherein the nucleic acid sample is from a biological sample taken from the subject's cancer.
  10. The method of any one of claims 1-9, wherein said determining the presence or an absence of one or more SPARC promoter mutations in the subject's cancer is done using one or more of the following techniques:
    (a) restriction fragment length analysis;
    (b) sequencing;
    (c) micro-sequencing assay;
    (d) hybridization;
    (e) invader assay;
    (f) gene chip hybridization assays;
    (g) oligonucleotide ligation assay;
    (h) ligation rolling circle amplification;
    (i) 5' nuclease assay;
    (j) polymerase proofreading methods;
    (k) allele specific PCR;
    (l) matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy;
    (m) ligase chain reaction assay;
    (n) enzyme-amplified electronic transduction;
    (o) single base pair extension assay; and
    (p) reading sequence data.
  11. The method of any one of claims 1-10, wherein the cancer is selected from the following: carcinoma; melanoma; sarcoma; lymphoma; and leukaemia.
  12. The method of claim 11, wherein the carcinoma is selected from the following: squamous cell; adeno; small cell; large cell carcinoma and combinations thereof.
  13. The method of claim 12, wherein the carcinoma is derived from a primary tumor located in nasopharygeal tissue, head and neck, lung, esophagus, stomach, intestine, rectum, kidney, urinary tract, prostate, testes, ovary, uterus, cervix, vagina, skin, or endocrine glands.
  14. The method of claim 11, wherein the sarcoma is selected from the following: muscle; adipose; fibrous; vascular; and neural sarcoma.
  15. The method of claim 11, wherein the lymphoma is selected from the following: Hodgkin's Disease; and Non-Hodgkins Lymphoma.
  16. The method of claim 11, wherein the leukaemia is selected from the following: lymphoid; and myeloid leukaemia.
  17. The method of claim 11, wherein the cancer is a colorectal carcinoma, prostate carcinoma, ovarian carcinoma or osteosarcoma.
  18. The method of any one of claims 1-17, wherein the cancer is a SPARC inhibited cancer.
  19. The method of claim 18, wherein the SPARC inhibited cancer is selected from colorectal carcinoma, prostate carcinoma, ovarian carcinoma, osteosarcoma and uterine sarcoma.
  20. A method for selecting a group of subjects for determining the efficacy of a therapeutic regimen known or suspected of being useful for the treatment of cancer, the method comprising determining a genotype at one or more mutation sites in a SPARC promoter for each subject, wherein said genotype is indicative of the subject's sensitivity to a therapeutic regimen and wherein the one or more SPARC promoter mutations, are selected from the following mutations relative to SEQ ID NO: 1:
    (a) 295delG and 301A>CC;
    (b) 315insA and 316G>A;
    (c) 341C>T, 342T>C, 344A>C, 347C>A, 354G>A, 356G>A, 359T>G and 363G>A;
    (d) 395C>T and 396A>T;
    (e) 488T>C;
    (f) 533A>G;
    (g) 734G>A;
    (h) 734delG;
    (i) 769T>A, 771 G>C and 774G>C;
    (j) 784T>C, 786T>G and 788T>A;
    (k) 793T>G, 796T>A and 800T>C;
    (l) 820C>A and 824T>A;
    (m) 864T>A and 867T>A;
    (n) 895T>G, 899T>A, 901G>A and 904G>A;
    (o) 901G>A;
    (p) 901G>A, 904G>A, 929A>G and 934delC;
    (q) 922T>G, 928T>G and 935T>G;
    (r) 982T>C, 984T>G, 985G>A, 991G>A, 995G>A, 999C>T and 1000T>G;
    (s) 1063G>C;
    (t) 1079G>A;
    (u) 1085delC, 1086delA, 1087delG, 1089G>A, 1090T>C, 1091T>C and 1092G>T;
    (v) 1161C>A, 1162T>C, 1163delta and 1164delT; and
    (w) 1215C>T, 1216delG, 1217delG, 1218delG, 1219delG, 1220delT, 1221delG, 1222delG, 1223delA, 1224delG, 1225delG, 1226delG, 1227delG, 1228delA, 1229delG, 1230delA, 1231delT, 1232delA, 1233delG, 1234delA, 1235delC, 1236delC and 1237C>T, and
    wherein the therapeutic regimen is a chemotherapy.
  21. The method of claim 20, further comprising determining each subject's sensitivity to the therapeutic regimen and comparing subject response to the therapeutic regimen based on the genotype of the subject.
  22. The method of claim 20, wherein the chemotherapy is 5-flurouracil, irinotecan, cisplatin, doxorubicin or combinations thereof.
  23. A method of identifying a subject with an increased sensitivity to a chemotherapy for treating a cancer in a subject in need thereof, comprising the step of screening a population of subjects to identify those subjects that have a mutation in their SPARC promoter sequence, wherein the identification of a subject with a mutation is predictive of decreased responsiveness to the chemotherapy, wherein the SPARC promoter mutation, is selected from one or more of the following mutations relative to SEQ ID NO: 1:
    (a) 295delG and 301 A>CC;
    (b) 315insA and 316G>A;
    (c) 341C>T, 342T>C, 344A>C, 347C>A, 354G>A, 356G>A, 359T>G and 363G>A;
    (d) 395C>T and 396A>T;
    (e) 488T>C;
    (f) 533A>G;
    (g) 734G>A;
    (h) 734delG;
    (i) 769T>A, 771 G>C and 774G>C;
    (j) 784T>C, 786T>G and 788T>A;
    (k) 793T>G, 796T>A and 800T>C;
    (l) 820C>A and 824T>A;
    (m) 864T>A and 867T>A;
    (n) 895T>G, 899T>A, 901 G>A and 904G>A;
    (o) 901G>A;
    (p) 901G>A, 904G>A, 929A>G and 934delC;
    (q) 922T>G, 928T>G and 935T>G;
    (r) 982T>C, 984T>G, 985G>A, 991G>A, 995G>A, 999C>T and 1000T>G;
    (s) 1063G>C;
    (t) 1079G>A;
    (u) 1085delC, 1086delA, 1087delG, 1089G>A, 1090T>C, 1091T>C and 1092G>T;
    (v) 1161C>A, 1162T>C, 1163delta and 1164delT; and
    (w) 1215C>T, 1216delG, 1217delG, 1218delG, 1219delG, 1220delT, 1221delG, 1222delG, 1223delA, 1224delG, 1225delG, 1226delG, 1227delG, 1228delA, 1229delG, 1230delA, 1231delT, 1232delA, 1233delG, 1234delA, 1235delC, 1236delC and 1237C>T.
HK08102424.4A 2004-11-30 2005-11-30 Sparc promoter mutations associated with drug resistance, and methods associated therewith HK1111721B (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US63141404P 2004-11-30 2004-11-30
US60/631,414 2004-11-30
PCT/CA2005/001819 WO2006058425A1 (en) 2004-11-30 2005-11-30 Sparc promoter mutations associated with drug resistance, and methods, oligonucleotides, cells and arrays associated therewith

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HK1111721A1 HK1111721A1 (en) 2008-08-15
HK1111721B true HK1111721B (en) 2012-08-10

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