WO2006084387A1 - Kallikrem 6 utilise dans le diagnostic de cancer du sein - Google Patents

Kallikrem 6 utilise dans le diagnostic de cancer du sein Download PDF

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Publication number
WO2006084387A1
WO2006084387A1 PCT/CA2006/000216 CA2006000216W WO2006084387A1 WO 2006084387 A1 WO2006084387 A1 WO 2006084387A1 CA 2006000216 W CA2006000216 W CA 2006000216W WO 2006084387 A1 WO2006084387 A1 WO 2006084387A1
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Prior art keywords
kallikrein
breast cancer
sample
polynucleotides
detecting
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PCT/CA2006/000216
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English (en)
Inventor
Chee-Wui Chu
Eleftherios Diamandis
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Mount Sinai Hospital
Ibex Technologies Inc.
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Publication of WO2006084387A1 publication Critical patent/WO2006084387A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • TITLE Kallikrem 6 in the diagnosis of breast cancer
  • the invention relates to compositions, kits, and methods for detecting, diagnosing, monitoring, and characterizing, diseases and/or conditions of the breast such as breast cancer.
  • breast cancer is the most frequently diagnosed cancer in women. Early detection and monitoring of breast cancer following therapy are critical to the outcome of a patient. The most widely utilized diagnostic procedures for breast cancer include mammography, bone scans, chest radiographs, and liver function tests.
  • the serum markers carcinoembryonic antigen (CEA) and CA 15-3 are also used to monitor patients. However, these markers have limitations. For example, CA15-3 and CEA can be negative in many patients with breast cancer and they can be elevated in nonmalignant conditions giving rise to false positives. The low clinical sensitivity for breast cancer makes them unsuitable for use in early detection of breast cancer.
  • Kallikreins are a subgroup of secreted serine proteases, encoded by highly conserved and tightly clustered multigene families in humans, rats and mice.
  • the human kallikrein gene family resides on chromosome 19ql3.4 and is comprised of 15 members, whose genes are designated as KLKl to KLK15 and the corresponding proteins as hKl to hK15 (Yousef GM, Diamandis EP., Endocr Rev .2001;22:184-2041; Yousef GM, et al., Biochem BiophysRes Commun. 2000;276:125-133; Diamandis EP, et al. CHn Chem .2000;46:1855- 1858).
  • the KLK6 gene encodes for a secreted trypsin-like serine protease, human kallikrein 6 (hK6; also known as zyme/protease M/neurosin), which comprises 244 amino acids, of which 16 amino acids constitute the signal peptide and five amino acids the activation peptide.
  • the mature enzyme consists of 223 amino acids.
  • Kallikrein 6 has been found in various biological fluids, including cerebrospinal fluid, breast milk, nipple aspirate fluid, breast cyst fluid, male and female serum, seminal plasma, amniotic fluid and breast cancer cytosols (Diamandis et al. , J. Histochem Cytochem 2001 49(11): 1431-41).
  • the present invention relates to novel markers of diseases and/or conditions of the breast such as breast cancer.
  • kallikrein 6 polypeptides and polynucleotides encoding kallikrein 6 (also referred to as "KLK6"), optionally in combination with CA 15-3 and polynucleotides encoding CA 15-3, constitute biomarkers for diseases and/or conditions of the breast such as breast cancer, and have superior clinical sensitivity in the detection of breast cancer compared to conventional biomarkers, such as CAl 5-3 alone.
  • kallikrein 6, KLK6, and portions or fragments thereof, agents that interact with kallikrein 6 or KLK6, optionally in combination with CA15-3, polynucleotides encoding CA15-3, and portions or fragments thereof, and agents that interact with CA 15-3 or CA 15-3 polynucleotides may be used to detect diseases and/or conditions of the breast, including without limitation breast cancer, and they can be used in detecting, diagnosing, characterizing, and monitoring breast cancer (i.e., monitoring progression of the cancer or the effectiveness of a therapeutic treatment), and the identification of subjects with a predisposition to breast cancer.
  • kallikrein 6 or KLK6 markers preferably in combination with CAl 5-3 or CAl 5-3 polynucleotide markers, in a sample can be assessed by detecting the presence in the sample of (a) a polypeptide or polypeptide fragment corresponding to the marker; (b) a transcribed nucleic acid or fragment thereof having at least a portion with which the marker is substantially identical; and/or (c) a transcribed nucleic acid or fragment thereof, wherein the nucleic acid hybridizes with the marker.
  • a method for detecting kallikrein 6 or KLK6, and preferably CA15-3 and CA15-3 polynucleotides, associated with a disease or condition of the breast (e.g. breast cancer) in a patient comprising or consisting essentially of:
  • the clinical sensitivity of a method of the invention may be greater than about 80% to 95%. In embodiments of the invention where kallikrein 6 is detected in a biological fluid such as serum, the clinical sensitivity can be greater than about 80 to 90%, more particularly greater than about 80 to 85%, most particularly greater than about 83%, 84%, or 85%.
  • the specificity of a method of the invention may be greater than about 80 to 90%. In embodiments of the invention where kallikrein 6 is detected in a biological fluid such as serum, the specificity can be greater than about 85 to 95%, more particularly greater than about 85 to 90%, most particularly greater than about 88%, 89%, or 90%.
  • a method for detecting kallikrein 6 or KLK6 and CA15-3 or CA15-3 polynucleotides associated with breast cancer in a patient comprising or consisting essentially of: (a) obtaining a sample, preferably a physiological fluid, from a patient;
  • kallikrein 6 and CA15-3 are detected in the physiological fluid, in particular serum.
  • the term “detect” or “detecting” includes assaying, or otherwise establishing the presence or absence of the target kallikrein 6, KLK6, CA 15-3 and/or CA15-3 polynucleotides, subunits thereof, or combinations of reagent bound targets, and the like, or assaying for ascertaining, establishing, or otherwise determining one or more factual characteristics of a disease or condition, in particular, cancer, or metastasis.
  • a standard may correspond to levels quantitated for samples from control subjects with no disease or early stage disease or from other samples of the subject.
  • the invention provides a method of assessing whether a patient is afflicted with a disease and/or condition of the breast, in particular breast cancer, the method comprising comparing:
  • levels of kallikrein 6 or KLK6 polynucleotides and preferably levels of CAl 5-3 and CAl 5-3 polynucleotides, associated with the disease and/or condition in a sample from the patient; and (b) normal levels of kallikrein 6 or KLK6 polynucleotides, and preferably normal levels of CA15-
  • kallikrein 6 or KLK6 polynucleotides in an aspect of a method of the invention for assessing whether a patient is afflicted with breast cancer, higher levels of kallikrein 6 or KLK6 polynucleotides, and optionally CA15-3 and CA15-3 polynucleotides, in a sample relative to corresponding normal levels is an indication that the patient is afflicted with breast cancer.
  • methods of the invention are used to diagnose Stage I, Stage II or Stage III/IV breast cancer.
  • the methods of the invention are used to diagnose Stage II or Stage III/IV breast cancer.
  • the invention further provides a non-invasive non-surgical method for detection or diagnosis of breast cancer in a subject comprising: obtaining a sample of blood, plasma, serum, urine or saliva from the subject, preferably blood, plasma, or serum; subjecting the sample to a procedure to detect kallikrein 6 or KLK6 polynucleotides, and optionally CA15-3 or CA15-3 polynucleotides, preferably kallikrein 6 and CA15-3; detecting or diagnosing breast cancer by comparing the levels of kallikrein 6 or KLK6 polynucleotides, and optionally CA15-3 or CA15-3 polynucleotides, to the levels of kallikrein 6 or KLK6 polynucleotides, and optionally CA15-3 or CA15-3 polynucleotides, obtained from a control subject with no breast cancer.
  • breast cancer is detected or diagnosed by determination of increased levels of kallikrein 6 or KLK6 polynucleotides, preferably in combination with increased levels of CAl 5-3 or CA15-3 polynucleotides, when compared to such levels obtained from a control. Different levels as compared to the control are indicative of breast cancer.
  • protein based methods are provided for diagnosing and monitoring breast cancer in a subject comprising detecting kallikrein 6 and preferably CAl 5-3 in a sample from the subject.
  • Kallikrein 6 and CA15-3 may be detected using a binding agent for kallikrein 6 and a binding agent for CA15-3, preferably antibodies specifically reactive with kallikrein 6, CA15-3, or parts thereof.
  • the level of kallikrein 6 in a sample is compared with the normal levels of kallikrein 6 in samples of the same type obtained from controls (e.g. samples from individuals not afflicted with breast cancer).
  • controls e.g. samples from individuals not afflicted with breast cancer.
  • Significantly different levels in the sample of the marker e.g. higher levels relative to the normal levels in a control are indicative of breast cancer.
  • levels of kallikrein 6 in a serum sample from a patient are compared with normal levels of kallikrein 6, and kallikrein 6 levels greater than about 1.5 to 10 ng/ml, 1.5 to 5 ng/ml, 1.5, 2, 4, 5, or 10 ng/ml, in particular greater than about 2 ng/ml are indicative of breast cancer.
  • levels of kallikrein 6 and CA 15-3 in a serum sample from a patient are compared with normal levels of kallikrein 6 and CAl 5-3, and kallikrein 6 levels greater than about
  • the invention provides a method of assessing whether a patient is afflicted with breast cancer, the method comprising comparing:
  • levels of kalllikrein 6 in a serum sample from a patient that are greater than about 1.5 to 10 ng/ml, 1.5 to 5 ng/ml, 1.5, 2, 4, 5, or 10 ng/ml, in particular greater than about 2 ng/ml, are indicative of breast cancer.
  • higher levels of a kallikrein 6 and a CA15-3 in a sample relative to the corresponding normal levels is an indication that the patient is afflicted with breast cancer.
  • levels of kalllikrein 6 greater than about 1.5 to 10 ng/ml, 1.5 to 5 ng/ml, 1.5, 2, 4, 5, or 10 ng/ml, in particular greater than about 2 ng/ml, and CA15-3 levels greater than about 30 to 50U/ml or greater than about 30, 35, 40 or 50U/ml, in particular greater than about 35 U/ml, are indicative of breast cancer.
  • the invention also provides a method of assessing whether a patient is afflicted with breast cancer, in particular Stage II or Stage III/IV breast cancer, which comprises comparing:
  • the invention provides methods for determining the presence or absence of breast cancer, in particular a Stage II or Stage III/IV breast cancer, in a patient, comprising the steps of (a) contacting a biological sample obtained from a patient with a binding agent that specifically binds to kallikrein 6 and preferably a binding agent that specifically binds to CA15-3; and (b) detecting in the sample an amount of kallikrein 6, and preferably CA15-3, that binds to the binding agent(s), relative to a predetermined standard or cut-off value, and therefrom determining the presence or absence of breast cancer in the patient.
  • the invention relates to a method for detecting, diagnosing and monitoring breast cancer in a subject by quantitating kallikrein 6, and preferably CA15-3, in a biological sample from the subject comprising (a) reacting the biological sample with an antibody specific for kallikrein 6, and preferably an antibody for CA15-3, which is directly or indirectly labeled with a detectable substance; and (b) detecting the detectable substance.
  • the invention provides a method of using antibodies to detect expression of kallikrein 6, and preferably CA15-3, in a sample, the method comprising: (a) combining an antibody specific for kallikrein 6, and preferably an antibody specific for CA15-3, with a sample under conditions which allow the formation of antibodyrprotein complexes; and (b) detecting complex formation, wherein complex formation indicates expression of kallikrein 6, and preferably CA15-3, in the sample. Expression may be compared with standards and is diagnostic of breast cancer.
  • the invention provides a method for monitoring the progression of breast cancer in a patient, the method comprising:
  • step (b) repeating step (a) at a subsequent point in time; and (c) comparing the levels detected in (a) and (b), and thereby monitoring the progression of breast cancer in the patient.
  • a method of the invention may employ one or more polynucleotides, oligonucleotides, or nucleic acids capable of hybridizing to polynucleotides encoding kallikrein 6 ("KLK6"), and preferably polynucleotides encoding CAl 5-3.
  • KLK6 kallikrein 6
  • methods for detecting KLK6 and optionally polynucleotides encoding CAl 5-3 can be used to monitor a disease and/or condition of the breast, such as breast cancer, by detecting KLK6 and optionally
  • CA15-3 polynucleotides In an aspect of the invention, KLK6 mRNA, and optionally CA15-3 mRNA is detected.
  • the present invention relates to a method for diagnosing and monitoring a disease and/or condition of the breast, in particular breast cancer, more particularly Stage II or Stage IIWV breast cancer, in a sample from a subject comprising isolating nucleic acids, preferably mRNA, from the sample, and detecting KLK6 and preferably CA15-3 polynucleotides, in the sample.
  • the presence of increased levels of KLK6 polynucleotides and CA15-3 polynucleotides in the sample compared to a standard or control is indicative of breast cancer.
  • the invention also provides methods for determining the presence or absence of breast cancer, more particularly Stage II or Stage III/IV breast cancer, in a subject comprising detecting in the sample a level of nucleic acids that hybridize to a KLK6, and preferably a CA 15-3 polynucleotide, comparing the level(s) with a predetermined standard or cut-off value, and therefrom determining the presence or absence of breast cancer in the subject.
  • a method for determining the presence or absence of breast cancer in a subject comprising (a) contacting a sample taken from the subject with oligonucleotides that hybridize to a KLK6 polynucleotide and oligonucleotides that hybridize to a CA15-3 polynucleotide; and (b) detecting in the sample a level of nucleic acids that hybridize to the oligonucleotides relative to a predetermined standard or cutoff value, and therefrom determining the presence or absence of breast cancer in the subject.
  • the amount of nucleic acid that is mRNA is detected via amplification reactions such as polymerase chain reaction (PCR) using, for example, at least one oligonucleotide primer that hybridizes to a KLK6 polynucleotide and at least one oligonucleotide primer that hybridizes to a CA15-3 polynucleotide or a complement of such polynucleotide.
  • PCR polymerase chain reaction
  • the amount of mRNA is detected using a hybridization technique, employing an oligonucleotide probe that hybridizes to KLK6, or a complement thereof, and an oligonucleotide probe that hybridizes to CA15-3, or a complement thereof.
  • the 1 method may be carried out by combining isolated mRNA with reagents to convert to cDNA according to standard methods; treating the converted cDNA with amplification reaction reagents along with an appropriate mixture of primers to produce amplification products; and analyzing the amplification products to detect the presence of KLK6 polynucleotides and CA15-3 polynucleotides, in the sample.
  • the analyzing step may be accomplished using Northern Blot analysis to detect the presence of KLK6 and CA15-3 polynucleotides.
  • the analysis step may be further accomplished by quantitatively detecting the presence of KLK6 and CA15-3 polynucleotides, in the amplification product, and comparing the quantity of KLK6 and CA 15-3 polynucleotides, detected against a panel of expected values for known presence or absence in normal and malignant tissue derived using similar primers.
  • the invention provides a method wherein mRNA is detected by (a) isolating mRNA from a sample and combining the mRNA with reagents to convert it to cDNA; (b) treating the converted cDNA with amplification reaction reagents and nucleic acid primers that hybridize to a KLK6 polynucleotide, and preferably a CA 15-3 polynucleotide, to produce amplification products; (d) analyzing the amplification products to detect an amount of mRNA KLK.6 polynucleotide, and preferably mRNA CAl 5-3 polynucleotide; and (e) comparing the amount of mRNA to an amount detected against a panel of expected values for normal and malignant tissue derived using similar nucleic acid primers.
  • polynucleotides are assayed or detected in samples from a patient, preferably serum samples.
  • a method of the invention wherein kallikrein 6 or KLK6 in combination with CA15-3 or CA15-3 polynucleotides are assayed can have enhanced sensitivity and/or specificity relative to a method assaying kallikrein 6 or CA 15-3 alone.
  • a method of the invention wherein kallikrein 6 and CA 15-3 are assayed in serum can have enhanced sensitivity relative to a method assaying kallikrein 6 or CA 15-3 alone.
  • the enhanced clinical sensitivity may be about a 5-10% increase, in particular 6-9% increase, more particularly 8% increase in sensitivity.
  • a method of the invention where both kallikrein 6 and CA15-3 are detected in serum provides a breast cancer clinical sensitivity of at least about 85 to 99%, in particular 90 to 95%, more particularly 91%, 92%, 93%, or 94% breast cancer clinical sensitivity.
  • Particular methods of the invention where both kallikrein 6 and CA15-3 are detected can also have a specificity of at least about 85 to 95%, in particular 85 to 90%, more particularly 86% specificity.
  • Clinical sensitivity and specificity may be determined using methods known to persons skilled in the art, in particular using the methods illustrated in the Example herein.
  • the amount of kallikrein 6 or KLK6 and the amount of CA 15-3 or CA 15-3 polynucleotides may be mathematically combined.
  • the amount of kallikrein 6 and the logCA15-3 are combined (e.g. hK6 + logCA15-3)
  • step (c) mathematically combining the results of step (a) and step (b) to provide a mathematical combination
  • the invention provides a method for detecting or diagnosing breast cancer by determining the combination of hK6 + logCA15-3 in a sample from a subject.
  • the invention further relates to a method of assessing the efficacy of a therapy for inhibiting a disease or condition of the breast, in particular breast cancer, more particularly Stage II or Stage III/IV breast cancer, in a patient.
  • This method comprises comparing: (a) levels of kallikrein 6 or KLK6 and preferably CA15-3 or CA15-3 polynucleotides, in a first sample obtained from the patient prior to providing at least a portion of the therapy to the patient; and
  • kallikrein 6 or KLK6, and preferably CA15-3 or CAl 5-3 polynucleotides, in the second sample, relative to the first sample is an indication that the therapy is efficacious for inhibiting breast cancer, more particularly Stage II or Stage III/IV breast cancer.
  • the method is used to assess the efficacy of a therapy for inhibiting breast cancer, more particularly Stage II or Stage III/IV breast cancer, and lower levels of kallikrein 6 or KLK6, and preferably CA15-3 or CA15-3 polynucleotides, in the second sample relative to the first sample, is an indication that the therapy is efficacious for inhibiting the cancer.
  • the therapy may be any therapy for treating cancer including but not limited to chemotherapy, immunotherapy, gene therapy, radiation therapy, and surgical removal of tissue. Therefore, the method can be used to evaluate a patient before, during, and after therapy, for example, to evaluate the reduction in tumor burden.
  • the invention also provides a diagnostic composition comprising kallikrein 6 or KLK6, or agents that interact with kallikrein 6 and/or KLK6, and CA 15-3 and/or CAl 5-3 polynucleotides, or agents that interact with CA15-3 and/or CA15-3 polynucleotides.
  • the invention provides a diagnostic composition comprising kallikrein 6 or KLK6, or agents that bind to kallikrein 6 or hyb, ridize to or amplify KLK6, and CAl 5-
  • CA15-3 polynucleotides or agents that bind to CA15-3, or hybridize to or amplify CA15-3 polynucleotides.
  • the composition comprises a probe that specifically hybridizes to KLK6 or a fragment thereof, and a probe that specifically hybridizes to CAl 5-3 polynucleotide or a fragment thereof.
  • a composition comprising a KLK6 specific primer pair capable of amplifying KLK6 using polymerase chain reaction methodologies, and a specific primer pair capable of amplifying CA15-3 polynucleotides using polymerase chain reaction methodologies.
  • the composition comprises a binding agent (e.g. antibody) that binds to kallikrein 6 or a fragment thereof, and an agent that binds to CA 15-3 or fragment thereof. Probes, primers, and binding agents can be labeled with a detectable substance.
  • kits for carrying out methods of the invention also includes kits for carrying out methods of the invention.
  • the invention provides a kit for detecting and/or diagnosing breast cancer comprising kallikrein 6 or polynucleotides encoding kallikrein 6 and CA15-3 or polynucleotides encoding CA15-3, or agents that bind to kallikrein 6 and CA15-3 or hybridize to or amplify polynucleotides encoding kallikrein 6 and CA 15-3.
  • the invention provides a test kit for diagnosing a disease and/or condition of the breast in a subject which comprises an agent that interacts with kallikrein 6 and/or KLK6, and an agent that interacts with CAl 5-3 and/or a CA 15-3 polynucleotide.
  • the kit is for assessing whether a patient is afflicted with breast cancer and it comprises reagents for identifying and/or assessing levels of kallikrein 6, or kallikrein 6 and CA15-3.
  • the invention further provides a diagnostic composition for detecting and/or diagnosing breast cancer comprising kallikrein 6 or polynucleotides encoding kallikrein 6 and CA15-3 or polynucleotides encoding CA15- 3, or agents that bind to kallikrein 6 and CA15-3 or hybridize to or amplify polynucleotides encoding kallikrein 6 and CA15-3.
  • a diagnositic composition of the invention comprises antibodies to kallikrein 6 and antibodies to CA15-3.
  • the invention relates to use of an agent that interacts with kallikrein 6 and/or KLK6, and optionally an agent that interacts with CA15-3 and/or a CA15-3 polynucleotide, in the manufacture of a composition for detecting a disease and/or condition of the breast disclosed herein, in particular breast cancer, more particularly Stage II or Stage III/IV breast cancer.
  • FIG. 1 Serum hK6 concentrations of breast cancer patients with Stage I-IV disease and normal females (same age group as patients). 40 Stage I, 40 Stage II, 20 Stage III/IV, 80 Normal Female.
  • FIG. 3 Correlation between serum hK6 and CAl 5-3 in 100 breast cancer patients with the respective cut-off of 2ng/mL and 35U/mL.
  • Figure 4 Receiver Operating Characteristic (ROC) Curve-hK6 in breast cancer. (100 cancer patients and 80 normal females.)
  • the invention relates to newly discovered correlations between expression of kallikrein 6 and KLK6, and optionally CA15-3 and CA15-3 polynucleotides, and diseases and/or conditions of the breast, in particular breast cancer.
  • the markers described herein provide sensitive and/or specific methods for detecting breast cancer.
  • Methods are provided for detecting the presence or absence of breast cancer in a sample, and for monitoring the progression of breast cancer, as well as providing information about characteristics of breast cancer, that are relevant to the diagnosis and characterization of breast cancer in a patient.
  • the invention particularly provides methods and kits for detecting and/or diagnosing breast cancer in a subject by determining amounts of kallikrein 6 alone, or kallikrein 6 and CAl 5-3 in serum samples from the subjects.
  • sample and the like mean a material known or suspected of expressing or containing kallikrein 6, KLK6, CA15-3 and/or CA15-3 polynucleotides, or binding agents such as antibodies specific for kallikrein 6 and CAl 5-3.
  • the sample may be derived from a biological source ("biological sample”), such as tissues, extracts, or cell cultures, including cells (e.g. tumor cells), cell lysates, and biological or physiological fluids, such as, for example, whole blood, plasma, serum, saliva, cerebral spinal fluid, sweat, urine, milk, peritoneal fluid and the like.
  • biological sample such as tissues, extracts, or cell cultures, including cells (e.g. tumor cells), cell lysates, and biological or physiological fluids, such as, for example, whole blood, plasma, serum, saliva, cerebral spinal fluid, sweat, urine, milk, peritoneal fluid and the like.
  • a sample may be used directly as obtained from the source or following a pretreatment to modify the character of the sample, such as preparing plasma from blood, diluting viscous fluids, and the like.
  • the sample is a human physiological fluid, such as human serum.
  • the samples that may be analyzed in accordance with the invention include polynucleotides from clinically relevant sources, preferably expressed RNA or a nucleic acid derived therefrom (cDNA or amplified RNA derived from cDNA that incorporates an RNA polymerase promoter).
  • the target polynucleotides can comprise RNA, including, without limitation total cellular RNA, poly(A) + messenger RNA (mRNA) or fraction thereof, cytoplasmic mRNA, or RNA transcribed from cDNA (i.e., cRNA).
  • Target polynucleotides can be detectably labeled at one or more nucleotides using methods known in the art.
  • the label is preferably uniformly incorporated along the length of the RNA, and more preferably, is carried out at a high degree of efficiency.
  • the detectable label can be a luminescent label, fluorescent label, bio- luminescent label, chemi-luminescent label, radiolabel, and colorimetric label.
  • the label is a fluorescent label, such as a fluorescein, a phosphor, a rhodamine, or a polymethine dye derivative, many of which are commercially available
  • Target polynucleotides from a patient sample can be labeled differentially from polynucleotides of a standard.
  • the standard can comprise target polynucleotides from normal individuals (e.g. those not afflicted with or pre-disposed to breast cancer, in particular pooled from samples from normal individuals).
  • the target polynucleotides can be derived from the same individual, but taken at different time points, and thus indicate the efficacy of a treatment by a change in expression of the markers, or lack thereof, during and after the course of treatment.
  • subject refers to a warmblooded animal such as a mammal that is afflicted with, or suspected of having, being pre-disposed to, or being screened for a disease and/or condition of the breast, in particular breast cancer.
  • the term includes but is not limited to domestic animals, sports animals, primates and humans.
  • the terms refer to a human.
  • Diseases and/or conditions of the breast refer to any disease or condition of the breast, including without limitation typical hyperplasia, fibroadenoma, cystic breast disease, and breast cancer.
  • breast cancer refers to any malignant disease of the breast including without limitation, ductal carcinoma in situ, lobular carcinoma in situ, infiltrating ductal carcinoma, medullary carcinoma, tubular carcinoma, mucinous carcinoma, infiltrating lobular carcinoma, infiltrating comedocarcinoma and inflammatory carcinoma.
  • Polypeptide and “protein” are used interchangeably herein and indicate at least one molecular chain of amino acids linked through covalent and/or non-covalent bonds.
  • the terms include peptides, oligopeptides, and proteins, and post-translational modifications of the polypeptides, e.g. glycosylates, acetylations, phosphorylations, and the like. Protein fragments, analogs, mutated or variant proteins, fusion proteins, and the like, are also included within the meaning of the terms.
  • kallikrein 6 refers to a trypsin-like serine protease, also known as zyme, protease M and neurosin which comprises 244 amino acids, of which 16 amino acids constitute the signal peptide and five amino acids the activation peptide.
  • the mature enzyme consists of 223 amino acids.
  • the term includes native-sequence polypeptides, isoforms, precursors, and chimeric or fusion proteins of kallikrein 6, in particular human kallikrein 6.
  • Kallikrein 6 polypeptides that can be employed in the present invention include, without limitation, polypeptides comprising the sequences found in Accession Nos.
  • CA15-3 or "carbohydrate antigen 15-3” refers to a cell surface mucin glycoprotein also known as mucin 1 and polymorphic epithelial mucin (Zotter, S et al, Cancer Rev 1988: 11/12L55-101; Gendler, S. et al, J Biol Chem 1990; 265:5573-8).
  • CA15-3 polypeptides that can be employed in the present invention include, without limitation, polypeptides comprising the sequences found in Accession Nos.
  • a “native-sequence polypeptide” comprises a polypeptide having the same amino acid sequence of a polypeptide derived from nature. Such native-sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term specifically encompasses naturally occurring truncated or secreted forms of a polypeptide, polypeptide variants including naturally occurring variant forms (e.g. alternatively spliced forms or splice variants), and naturally occurring allelic variants.
  • polypeptide variant means a polypeptide having at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity, particularly at least about 70-80%, more particularly at least about 85%, still more particularly at least about 90%, most particularly at least about 95%, 97%, or 99% amino acid sequence identity with a native-sequence polypeptide.
  • Particular polypeptide variants have at least 70-80%, 85%, 90%, 95%, 97% or 99% amino acid sequence identity to the sequences identified in the Accession Nos. set out herein and shown in SEQ ID NOs: 1 and 5.
  • variants include, for instance, polypeptides wherein one or more amino acid residues are added to, or deleted from, the N- or C-terminus of the full-length or mature sequences of the polypeptide, including variants from other species, but exclude a native-sequence polypeptide.
  • variants retain the immunogenic activity of the corresponding native-sequence polypeptide.
  • Sequence identity of two amino acid sequences or of two nucleic acid sequences is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues in a polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be achieved in various conventional ways, for instance, using publicly available computer software including the GCG program package (Devereux J. et al., Nucleic Acids Research 12(1): 387, 1984); BLASTP, BLASTN, and FASTA (Atschul, S.F. et al. J.
  • BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al. NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al. J. MoI. Biol. 215: 403-410, 1990). Skilled artisans can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Methods to determine identity and similarity are codified in publicly available computer programs.
  • Polypeptide variants include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of a native polypeptide which includes fewer amino acids than the full- length polypeptides.
  • a portion or fragment of a polypeptide can be a polypeptide which is for example, 3-5, 8-
  • Portions or fragments in which regions of a polypeptide are deleted can be prepared by recombinant techniques and can be evaluated for one or more functional activities such as the ability to form antibodies specific for a polypeptide.
  • allelic variant may also be created by introducing substitutions, additions, or deletions into a nucleic acid encoding a native polypeptide sequence such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations may be introduced by standard methods, such as site-directed mutagenesis and PCR-mediated mutagenesis. In an embodiment, conservative substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue with a similar side chain, several of which are known in the art.
  • allelic variant may contain conservative amino acid substitutions from the native polypeptide sequence or it may contain a substitution of an amino acid from a corresponding position in a kallikrein 6 homolog, for example, a murine kallikrein 6.
  • a kallikrien 6 or CAl 5-3 polypeptide includes chimeric or fusion proteins.
  • a "chimeric protein” or “fusion protein” comprises all or part (preferably biologically active) of a kallikrein 6 or CA15-3 polypeptide operably linked to a heterologous polypeptide (i.e., a polypeptide other than the same kallikrein 6 or CA15-3 polypeptide).
  • a heterologous polypeptide i.e., a polypeptide other than the same kallikrein 6 or CA15-3 polypeptide.
  • the term "operably linked” is intended to indicate that the kallikrein 6 or CA15-3 polypeptide and the heterologous polypeptide are fused in-frame to each other.
  • the heterologous polypeptide can be fused to the N-terminus or C-terminus of the kallikrein 6 or CA 15-3 polypeptide.
  • a useful fusion protein is a GST fusion protein in which a kallikrein 6 or CA15-3 polypeptide is fused to the C-terminus of GST sequences.
  • Another example of a fusion protein is an immunoglobulin fusion protein in which all or part of a kallikrein 6 or CA15-3 polypeptide is fused to sequences derived from a member of the immunoglobulin protein family.
  • Chimeric and fusion proteins can be produced by standard recombinant DNA techniques.
  • Kallikrein 6 and CA 15-3 polypeptides may be isolated from a variety of sources, such as from human tissue types or from other sources, or prepared by recombinant or synthetic methods, or by any combination of these and similar techniques.
  • Polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The term includes double- and single-stranded DNA and RNA, modifications such as methylation or capping and unmodified forms of the polynucleotide.
  • the terms "polynucleotide” and “oligonucleotide” are used interchangeably herein.
  • a polynucleotide may, but need not, include additional coding or non-coding sequences, or it may, but need not, be linked to other molecules and/or carrier or support materials.
  • Polynucleotides for use in the methods of the invention may be of any length suitable for a particular method. In certain applications the term refers to antisense nucleic acid molecules (e.g. an mRNA or DNA strand in the reverse orientation to a sense KLK6 or CA15-3 polynucleotides).
  • KLK6 or “KLK6 polynucleotide(s)” refer to polynucleotides encoding kallikrein 6 including a native-sequence polypeptide, a polypeptide variant including a portion of a kallikrein 6 polypeptide, an isoform, precursor, and a chimeric polypeptide.
  • a polynucleotide encoding a kallikrein 6 polypeptide that can be employed in the present invention includes, without limitation, nucleic acids comprising a sequence of GenBank Accession Nos.
  • CA15-3 polynucleotide(s) refers to polynucleotides encoding CA15-3 including a native-sequence polypeptide, a polypeptide variant including a portion of a CA 15-3 polypeptide, an isoform, precursor, and a chimeric polypeptide.
  • a polynucleotide encoding a CA125-3 polypeptide that can be employed in the present invention includes, without limitation, nucleic acids comprising a sequence of GenBank Accession Nos.
  • KLK6 or CA 15-3 polynucleotides include complementary nucleic acid sequences, and nucleic acids that are substantially identical to these sequences (e.g. at least about 45%, preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity).
  • KLK6 or CAl 5-3 polynucleotides also include sequences that differ from a nucleic acid sequence of an Accession No. referenced herein and SEQ ID Nos. 2, 3, 4, ,6, and 7 due to degeneracy in the genetic code.
  • DNA sequence polymorphisms within the nucleotide sequence of a kallikrein 6 or CA15-3 polypeptide may result in silent mutations that do not affect the amino acid sequence. Variations in one or more nucleotides may exist among individuals within a population due to natural allelic variation. DNA sequence polymorphisms may also occur which lead to changes in the amino acid sequence of a kallikrein polypeptide.
  • KLK6 and CA15-3 polynucleotides also include nucleic acids that hybridize under stringent conditions, preferably high stringency conditions to a nucleic acid sequence of the Accession Nos described herein and SEQ ID NOs: 2, 3, 4, 6, and 7.
  • Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or can be found in Ausubel et al, (eds) Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6.
  • stringent conditions may be selected that are about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to a target sequence hybridize at equilibrium to the target sequence.
  • stringent conditions will be those in which the salt concentration is less than about l.OM sodium ion or other salts (e.g. about 0.01 to l.OM sodium ion) and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g. 10-50 nucleotides) and at least 60°C for longer probes, primers and oligonucleotides.
  • a hybridization may be conducted at 6.0 x sodium chloride/sodium citrate (SSC) at about 45 0 C, followed by a wash of 2.0 x SSC at 50 0 C, or at 42 0 C in a solution containing 6xSCC, 0.5% SDS and 50% formamide followed by washing in a solution of O.lx SCC and 0.5% SDS at 68°C.
  • SSC sodium chloride/sodium citrate
  • KLK6 and CA 15-3 polynucleotides also include truncated nucleic acids or nucleic acid fragments and variant forms of the nucleic acids that arise by alternative splicing of an mRNA corresponding to a DNA.
  • a fragment of a polynucleotide includes a polynucleotide sequence that comprises a contiguous sequence of approximately at least about 6 nucleotides, in particular at least about 8 nucleotides, more particularly at least about 10-12 nucleotides, and even more particularly 15-20 nucleotides that correspond to (i.e. identical or complementary to), a region of the specified nucleotide sequence.
  • “Significantly different” levels of markers or a "significant difference” in marker levels in a patient sample compared to a control or standard may represent levels that are higher or lower than the standard error of the detection assay, preferably the levels are at least about 1.5, 2, 3, 4, 5, or 6 times higher or lower, respectively, than the control or standard.
  • “Microarray” and “array,” refer to nucleic acid or nucleotide arrays or protein or peptide arrays that can be used to detect biomolecules associated with a disease and/or condition of the breast, for instance to measure gene expression.
  • Binding agent refers to a substance such as a polypeptide, antibody, ribosome, or aptamer that specifically binds to a kallikrein 6 or CA15-3.
  • a substance "specifically binds" to kallikrein 6 or CA15-3 if it reacts at a detectable level with a kallikrein 6 or CA15-3, and does not react detectably with peptides containing unrelated sequences or sequences of different polypeptides.
  • a binding agent may be a ribosome, with or without a peptide component, an RNA or DNA molecule, or a polypeptide.
  • a binding agent may be a polypeptide that comprises a kallikrein 6 or CA15-3 polypeptide sequence, a peptide variant thereof, or a non-peptide mimetic of such a sequence.
  • a kallikrein 6 or CA15-3 sequence may be a peptide portion of a kallikrein 6 or CAl 5-3 that is capable of modulating a function mediated by the kallikrein 6 or CA15-3.
  • An aptamer includes a DNA or RNA molecule that binds to nucleic acids and proteins.
  • An aptamerthat binds to a kallikrein 6 or CA15-3 polypeptide or a KLK6 or CA15-3 polynucleotide can be produced using conventional techniques, without undue experimentation.
  • Klug et al. MoI. Biol. Reports 20:97-107 (1994); Wallis et al., Chem. Biol. 2:543-552 (1995); Ellington, Curr. Biol. 4:427-429 (1994); Lato et al., Chem. Biol. 2:291-303 (1995); Conrad et al., MoI. Div. 1:69-78 (1995); and Uphoff et al., Curr. Opin. Struct. Biol. 6:281-287 (1996)].
  • Antibodies for use in the present invention include but are not limited to monoclonal or polyclonal antibodies, immunologically active fragments (e.g. a Fab or (Fab) 2 fragments), antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single chain F v molecules, chimeric antibodies, for example, antibodies which contain the binding specificity of murine antibodies, but in which the remaining portions are of human origin, or derivatives, such as enzyme conjugates or labeled derivatives.
  • Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods well known to those skilled in the art. Isolated native or recombinant kallikrein 6 and CA 15-3 may be utilized to prepare antibodies. See, for example, Kohler et al. (1975) Nature 256:495-497; Kozbor et al. (1985) J. Immunol Methods 81:31-42; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030; and Cole et al. (1984) MoI Cell Biol 62:109-120 for the preparation of monoclonal antibodies; Huse et al.
  • Antibodies specific for kallikrein 6 and CA15-3 may also be obtained from scientific or commercial sources. In an embodiment of the invention, antibodies are reactive against kallikrein 6 or CA15-3 if they bind with a K a of greater than or equal to 10 "7 M.
  • ROC curve refers to a Receiver Operating Characteristics curve. ROC curves are used to evaluate the power of a classification method for different asymmetric weights of false negative versus false positive errors (or sensitivity versus specificity). A ROC curve plots the tradeoff between false positive and false negative errors as the classification threshold varies. For each potential threshold, the rate of true positives is plotted against the rate of false positives. Accuracy (A) is indexed by the area under the curve (AUC). An AUC is preferably greater than 0.7, 0.8, 0.88, 0.9, 0.92, 0.95, 0.98, or 0.99. Methods for obtaining an ROC curve and AUC are well known to the skilled artisan and are illustrated in the Example.
  • a variety of methods can be employed for the diagnostic and prognostic evaluation of diseases and/or conditions of the breast such as breast cancer involving kallikrein 6 and KLK6, and optionally CAl 5-3 and CA15-3 polynucleotides, and the identification of subjects with a predisposition to such disorders.
  • Such methods may, for example, utilize KLK6 polynucleotides and preferably CAl 5-3 polynucleotides, and fragments thereof, and binding agents (e.g. antibodies) directed against kallikrein 6 and preferably CA15-3, including peptide fragments.
  • the polynucleotides and antibodies may be used, for example, for (1) the detection of the presence of KLK6 or CAl 5-3 polynucleotide mutations, or the detection of either over- or under-expression of KLK6 mRNA or CA15-3 mRNA, relative to a non-disorder state or the qualitative or quantitative detection of alternatively spliced forms of KLK6 or CA15-3 polynucleotide transcripts which may correlate with certain conditions or susceptibility toward such conditions; and (2) the detection of either an over- or an under- abundance of kallikrein 6 or CA15-3, relative to a non- disorder state or the presence of a modified (e.g., less than full length) kallikrein 6 or CA 15-3, which correlates with a disorder state, or a progression toward a disorder state.
  • a modified (e.g., less than full length) kallikrein 6 or CA 15-3 which correlates with a disorder state, or a progression toward a disorder state.
  • the invention contemplates a method for detecting breast cancer comprising producing a profile of levels of kallikrein 6 and preferably CAl 5-3, and other markers associated with breast cancer, in cells from a patient, and comparing the profile with a reference to identify a profile for the test cells indicative of disease.
  • the methods described herein may be used to evaluate the probability of the presence of malignant or pre-malignant cells, for example, in a group of cells freshly removed from a host. Such methods can be used to detect tumors, quantitate and monitor their growth, and help in the diagnosis and prognosis of disease. For example, higher levels of kallikrein 6 and CA15-3 may be indicative of advanced disease, e.g. advanced breast cancer, more particularly Stage II or Stage III/IV breast cancer.
  • levels of kalllikrein 6 greater than about 1.5 to 10 ng/ml, 1.5 to 5 ng/ml, 1.5, 2, 4, 5, or 10 ng/ml, in particular greater than about 2ng/ml, and CA15-3 levels greater than about 30 to 50U/ml or greater than about 30, 35, 40 or 50U/ml, in particular greater than about 35 U/ml, are indicative of Stage I, Stage II, or Stage III/IV breast cancer, more particularly Stage II or Stage III/IV breast cancer.
  • the methods described herein can be adapted for diagnosing and monitoring cancer by detecting kallikrein 6 or KLK6 polynucleotides and optionally CA15-3 or CA15-3 polynucleotides, in samples, in particular physiological or biological fluids, from a subject.
  • These applications require that the amount of kallikrein 6 or KLK6 polynucleotides and CA 15-3 or CA 15-3 polynucleotides, quantitated in a sample from a subject being tested be compared to a predetermined standard or cut-off value.
  • a standard may correspond to levels quantitated for another sample or an earlier sample from the subject, or levels quantitated for a control sample.
  • Levels for control samples from healthy subjects or cancer subjects may be established by prospective and/or retrospective statistical studies.
  • Healthy subjects who have no clinically evident disease or abnormalities may be selected for statistical studies. Diagnosis may be made by a finding of statistically different levels of kallikrein 6 or KLK6, and optionally CA15-3 or CA15-3 polynucleotides, compared to a control sample or previous levels quantitated for the same subject.
  • the invention also contemplates the methods described herein using multiple markers for breast cancer. Therefore, the invention contemplates a method for analyzing a biological sample for the presence of kallikrein 6 or KLK6, CA15-3 or CA15-3 polynucleotides, and other markers that are specific indicators of breast cancer.
  • the methods described herein may be modified by including reagents to detect the markers or polynucleotides encoding the markers.
  • Binding agents may be used for a variety of diagnostic and assay applications. There are a variety of assay formats known to the skilled artisan for using a binding agent to detect a target molecule in a sample. (For example, see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1988).
  • the presence or absence of a disease and/or condition of the breast, in particular breast cancer, in a subject may be determined by (a) contacting a sample from the subject with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined standard or cut-off value.
  • the binding agent is an antibody.
  • the invention provides a diagnostic method for monitoring or diagnosing breast cancer in a subject by quantitating kallikrein 6 and optionally CA15-3 in a biological sample from the subject comprising reacting the sample with antibodies specific for kallikrein 6 and optionally CA15-3, which are directly or indirectly labeled with detectable substances and detecting the detectable substances.
  • a method for detecting or diagnosing breast cancer comprising or consisting essentially of:
  • the invention contemplates a method for monitoring the progression of breast cancer in an individual, comprising:
  • step (d) comparing the result of step (b) with the result of step (c), wherein a difference in the amount of complex formation is indicative of disease, disease stage, and/or progression of the cancer in said individual.
  • the amount of complexes may also be compared to a value representative of the amount of the complexes from an individual not at risk of, or afflicted with breast cancer at different stages.
  • Antibodies specifically reactive with kallikrein 6 and CA15-3, or derivatives, such as enzyme conjugates or labeled derivatives, may be used to detect kallikrein 6 and CAl 5-3 proteins in various samples (e.g. biological materials). They may be used as diagnostic or prognostic reagents and they may be used to detect abnormalities in the level of kallikrein 6 and CA 15-3 expression, or abnormalities in the structure, and/or temporal, tissue, cellular, or subcellular location of kallikrein 6 and CA15-3. Antibodies may also be used to screen potentially therapeutic compounds in vitro to determine their effects on disorders (e.g. breast cancer) involving a kallikrein 6 and CAl 5-3 protein, and other conditions.
  • disorders e.g. breast cancer
  • In vitro immunoassays may also be used to assess or monitor the efficacy of particular therapies.
  • Antibodies may be used in any immunoassay that relies on the binding interaction between antigenic determinants of kallikrein 6 and CA15-3 and the antibodies.
  • Immunoassay procedures for in vitro detection of antigens in fluid samples are also well known in the art. [See for example, Paterson et al., Int. J. Can. 37:659 (1986) and Burchell et al., Int. J. Can. 34:763 (1984) for a general description of immunoassay procedures].
  • kallikrein 6 and CA15-3 in a sample may be accomplished by competitive or non-competitive immunoassay procedures in either a direct or indirect format. Detection of kallikrein 6 and CA15-3 using antibodies can, for example involve immunoassays which are run in either the forward, reverse or simultaneous modes. Examples of immunoassays are radioimmunoassays (RIA), enzyme immunoassays (e.g. ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination, histochemical tests, and sandwich (immunometric) assays.
  • RIA radioimmunoassays
  • enzyme immunoassays e.g. ELISA
  • immunofluorescence immunoprecipitation
  • latex agglutination hemagglutination
  • histochemical tests hemagglutination
  • sandwich (immunometric) assays sandwich assays.
  • the binding of antibodies to kallikrein 6 and CA 15-3 can be detected directly using, for example, a surface plasmon resonance (SPR) procedure such as, for example, Biacore ® , microcalorimetry or nano-cantilivers.
  • SPR surface plasmon resonance
  • the antibodies may be used in immunohistochemical analyses, for example, at the cellular and sub- subcellular level, to detect kallikrein 6 and CA 15-3 , to localize them to particular breast tumor cells and tissues, and to specific subcellular locations, and to quantitate the level of expression.
  • tissue samples obtained from a subject suspected of having a breast-related problem is contacted with antibodies, preferably monoclonal antibodies recognizing kallikrein 6.
  • the site at which the antibodies are bound is determined by selective staining of the sample by standard immunohistochemical procedures. The same procedure may be repeated on the same sample using other antibodies that recognize CAl 5-3.
  • a sample may be contacted with antibodies against kallikrein 6 and antibodies against CA15-3 simultaneously, provided that the antibodies are labeled differently or are able to bind to a different label.
  • the tissue sample is obtained from the breast of a patient.
  • the breast tissue sample may be a normal breast tissue, a cancer breast tissue or a benign breast tissue.
  • Antibodies specific for kallikrein 6 and CA15-3 may be labeled with a detectable substance and localised in biological samples based upon the presence of the detectable substance.
  • detectable substances include, but are not limited to, the following: radioisotopes (e.g., 3 H, 14 C, 35 S, 125 1, 131 I), fluorescent labels, (e.g., FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; and enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase), biotinyl groups (which can be detected by marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), and predetermined polypeptide epitopes recognized by a secondary reporter (e.g
  • an antibody can be detectably labeled is to link it directly to an enzyme.
  • the enzyme when later exposed to its substrate will produce a product that can be detected.
  • detectable substances that are enzymes are horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase, malate dehydrogenase, ribonuclease, urease, catalase, glucose-6-phosphate, staphylococcal nuclease, delta-5-steriod ⁇ somerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, triose phosphate isomerase, asparaginase, glucose oxidase, and acetylcholine esterase.
  • a fluorescence-emitting metal atom such as Eu
  • neuropeptide and other lanthanides can be used. These can be attached to the desired molecule by means of metal- chelating groups such as DTPA or EDTA.
  • a bioluminescent compound may also be used as a detectable substance.
  • bioluminescent detectable substances are luciferin, luciferase and aequorin.
  • Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against kallikrein 6 or CA15-3.
  • the second antibody may be goat anti-rabbit IgG, Fc fragment specific antibody labeled with a detectable substance as described herein.
  • Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect a kallikrein 6 and CAl 5-3 protein.
  • an antibody may be labeled with a detectable substance and a kallikrein 6 and CA15-3 protein may be localized in tissues and cells based upon the presence of the detectable substance.
  • the sample, binding agents e.g. antibodies
  • kallikrein 6, or CA15-3 may be immobilized on a carrier or support, such as, for example, agarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose, polyacrylamides, polystyrene, filter paper, ion-exchange resin, plastic film, nylon or silk.
  • the support material may have any possible configuration including spherical cylindrical or flat.
  • the carrier may be in the shape of, for example, a tube, test plate, well, beads, disc, sphere, etc.
  • the immobilized material may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
  • Binding agents e.g. antibodies
  • binding agents may be indirectly immobilized using second binding agents specific for the first binding agent.
  • mouse antibodies specific for kallikrein 6 or CA 15-3 may be immobilized using sheep anti- mouse IgG Fc fragment specific antibody coated on the carrier or support.
  • a kallikrein 6 and CA 15-3 protein may be localized by radioautography.
  • the results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains.
  • Time-resolved fluorometry may be used to detect a fluorescent signal, label, or detectable substance.
  • the method described in Christopoulos TK and Diamandis EP Anal. Chem., 1992:64:342-346 may be used with a conventional time-resolved fluorometer.
  • an immunoassay for detecting kallikrein 6 in a biological sample comprises contacting an amount of a first binding agent that specifically binds to kallikrein 6 in the sample under conditions that allow the formation of a first complex comprising the first binding agent and kallikrein 6 and determining the presence or amount of the complex as a measure of the amount of the kallikrein 6 contained in the sample.
  • an immunoassay for detecting kallikrein 6 and CAl 5-3 in a sample comprising the additional step of contacting an amount of a second binding agent which specifically binds to CA15-3 with the sample under conditions that allow the formation of a second complex comprising the second binding agent and CA15-3, and determining the presence or amount of the second complex as a measure of the amount of the CA15-3 contained in the sample.
  • a sample may be contacted with the first and the second binding agents simultaneously, provided that the agents are labeled differently or are capable of binding to different labels.
  • a method wherein kallikrein 6 and CA15-3 antibodies are directly or indirectly labeled with enzymes, substrates for the enzymes are added wherein the substrates are selected so that the substrates, or a reaction product of an enzyme and substrate, form fluorescent complexes with lanthanide metals, preferably europium and terbium.
  • a lanthanide metal(s) is added and kallikrein 6 and CA 15-3 are quantitated in the sample by measuring fluorescence of the fluorescent complexes.
  • Enzymes are selected based on the ability of a substrate of the enzyme, or a reaction product of the enzyme and substrate, to complex with lanthanide metals.
  • the fluorescence intensity of the complexes is typically measured using a time-resolved fluorometer.
  • Antibodies specific for kallikrein 6 and CA15-3 may also be indirectly labeled with enzymes.
  • an antibody may be conjugated to one partner of a ligand binding pair, and the enzyme may be coupled to the other partner of the ligand binding pair.
  • Representative examples include avidin-biotin, and riboflavin- riboflavin binding protein.
  • antibodies specific for kallikrein 6 and CA 15-3 are labeled with enzymes.
  • aspects of the methods of the invention involve (a) reacting a biological sample from a subject with antibodies specific for kallikrein 6 and preferably antibodies specific for CA15-3, wherein the antibodies are directly or indirectly labeled with enzymes; (b) adding substrates for the enzymes wherein the substrates are selected so that the substrates, or reaction products of the enzymes and substrates form fluorescent complexes; (c) quantitating kallikrein 6, and preferably CA15-3, in the sample by measuring fluorescence of the fluorescent complexes; and (d) comparing the quantitated levels to levels obtained for other samples from the subject patient, or control subjects.
  • the quantitated levels are compared to levels quantitated for normal subjects wherein an increase in kallikrein 6, and optionally CAl 5-3, levels compared with the control subjects is indicative of disease and/or poor prognosis.
  • a particular embodiment of the invention comprises the following steps:
  • the standard may correspond to levels quantitated for samples from control subjects with no disease or early stage disease or from other samples of the subject. Increased levels of kallikrein 6 as compared to the standard may be indicative of breast cancer, and /or decreased progression-free disease and overall survival.
  • kallikrein 6 in combination with CA15-3 leads to improved identification of breast cancer.
  • the mathematical combination of the amount of kallikrein 6 and CA 15-3 may be used as a serum marker or as an immunohistological marker to help identify early to late stage breast cancer, in particular Stage II or Stage
  • matrix combination or “mathematically combining” as used herein refers to any mathematical calculation of the amount of kallikrein 6 and CA15-3.
  • the mathematical combination is hK6 + logCA15-3.
  • the combination of kallikrein 6 and CAl 5-3 may be analyzed with the aid of a ROC curve.
  • an aspect of the present invention provides a method for determining the presence of breast cancer in a subject comprising the steps of:
  • the agents comprise antibodies.
  • the first and the second agents are directly or indirectly labeled with different detectable substances to form respective complexes that may be detected separately.
  • the sample may be contacted with one agent first so that kallikrein 6 may be detected first, then the same sample may be contacted with another agent in order to detect CAl 5-3.
  • the sample used in the diagnostic method may be a sample of human physiological fluid such as, but not limited to, serum, seminal plasma, urine and plasma, preferably serum.
  • the presence of breast cancer can be identified in patients with kallikrein 6 levels greater than about 1.5 to 10 ng/ml, 1.5 to 5 ng/ml, 1.5, 2, 4, 5, or 10 ng/ml, in particular greater than about 2ng/ml.
  • the presence of breast cancer can be identified in patients with CA15-3 greater than about 30 to 50U/ml, 30, 35, 40 or 50U/ml, in particular greater than about 35 U/ml.
  • the presence of breast cancer can be identified in patients with kallikrein 6 levels greater than about 1.5 to 10 ng/ml, 1.5 to 5 ng/ml, 1.5, 2, 4, 5, or 10 ng/ml, preferably greater than about 2 ng/ml, and CA15-3 levels greater than about 30 to 50 U/ml, 30, 35, 40 or 50U/ml, preferably greater than about 35 U/ml.
  • kits for detecting or quantifying CA15-3 in a sample include Immulite BR-MA (CA15-3) (Diagnostic Products Corp., CA), Bayer Diagnostics ADVIA CentaurTMCA15-3 (Tarrytown, NY), Fujirebio Diagnostics, Inc. CA 15- 3TM RIA (Malvern, PA), Abbott IMx® CA 15-3 (Abbott Park, IL), Boehringer Mannheim Enzymun-Test® CA 15-3 (Tutzing, Germany), CA15-3TM and Roche Modular E170 CA15-3 (Roche Diagnostics Corporation, USA).
  • Immulite BR-MA CA15-3
  • CA Diagnostic Products Corp., CA
  • Bayer Diagnostics ADVIA CentaurTMCA15-3 Tarrytown, NY
  • CA 15- 3TM RIA Mealvern, PA
  • Abbott IMx® CA 15-3 Abbott Park, IL
  • Boehringer Mannheim Enzymun-Test® CA 15-3
  • the present invention provides means for determining kallikrein 6 and CA15-3 in a serum sample by measuring kallikrein 6 and CA15-3 by immunoassay. It will be evident to a skilled artisan that a variety of competitive or non-competitive immunoassay methods can be used to measure kallikrein 6 and CA 15-3 in serum.
  • Competitive methods typically employ immobilized or immobilizable antibodies to kallikrein 6 and CA15-3 (anti-kallikrein 6 and anti-CA15-3) and labeled forms of kallikrein 6 and CA15-3.
  • Sample kallikrein 6 and CA15-3 and labeled kallikrein 6 and CA15-3 compete for binding to anti- kallikrein 6 and CA 15-3.
  • the amount of the label in either bound or unbound fraction is measured and may be correlated with the amount of kallikrein 6 and CA15-3 in the test sample in any conventional manner, e.g., by comparison to a standard curve.
  • a non-competitive method is used for the determination of kallikrein 6 and CA 15-3, with the most common method being the "sandwich” method.
  • two anti-kallikrein 6 and two CA15- 3 antibodies are employed.
  • One of the anti- kallikrein 6 and one of the CA 15-3 antibodies is directly or indirectly labeled (the "detection antibody"), and the other is immobilized or immobilizable (the "capture antibody").
  • the capture and detection antibodies can be contacted simultaneously or sequentially with the test sample. Sequential methods can be accomplished by incubating the capture antibody with the sample, and adding the detection antibody at a predetermined time thereafter or the detection antibody can be incubated with the sample first and then the capture antibody added.
  • the capture antibody may be separated from the liquid test mixture, and the label may be measured in at least a portion of the separated capture antibody phase or the remainder of the liquid test mixture. Generally it is measured in the capture antibody phase since it comprises kallikrein 6 and CA15-3 ("sandwiched" between) the capture and detection antibodies. In another embodiment, the label may be measured without separating the capture antibody and liquid test mixture.
  • sandwich immunoassays of the invention mouse polyclonal/monoclonal antibodies specific for kallikrein 6 or CA15-3 and rabbit polyclonal/monoclonal antibodies specific for kallikrein 6 or CA15-3 are utilized.
  • one or both of the capture and detection antibodies are polyclonal antibodies or one or both of the capture and detection antibodies are monoclonal antibodies (i.e. polyclonal/polyclonal, monoclonal/monoclonal, ormonoclonal/polyclonal).
  • the label used in the detection antibody can be selected from any of those known conventionally in the art.
  • the label may be an enzyme or a chemiluminescent moiety, but it can also be a radioactive isotope, a fluorophor, a detectable ligand (e.g., detectable by a secondary binding by a labeled binding partner for the ligand), and the like.
  • the antibody is labeled with an enzyme which is detected by adding a substrate that is selected so that a reaction product of the enzyme and substrate forms fluorescent complexes.
  • the capture antibody may be selected so that it provides a means for being separated from the remainder of the test mixture. Accordingly, the capture antibody can be introduced to the assay in an already immobilized or insoluble form, or can be in an immobilizable form, that is, a form which enables immobilization to be accomplished subsequent to introduction of the capture antibody to the assay.
  • An immobilized capture antibody may comprise an antibody covalently or noncovalently attached to a solid phase such as a magnetic particle, a latex particle, a microtiter plate well, a bead, a cuvette, or other reaction vessel.
  • an immobilizable capture antibody is antibody which has been chemically modified with a ligand moiety, e.g., a hapten, biotin, or the like, and which can be subsequently immobilized by contact with an immobilized form of a binding partner for the ligand, e.g., an antibody, avidin, or the like.
  • the capture antibody may be immobilized using a species specific antibody for the capture antibody that is bound to the solid phase.
  • a particular sandwich immunoassay method of the invention employs antibodies reactive against kallikrein 6 and CA15-3, antibodies having specificity against antibodies reactive against kallikrein 6 and CA15- 3 labeled with enzymatic labels, and fluorogenic substrates for the enzymes.
  • the enzyme alkaline phosphatase (ALP) and the substrate 5-fluorosalicyl phosphate are utilized.
  • ALP cleaves phosphate out of the fluorogenic substrate, 5-fiuorosalicyl phosphate, to produce 5-fiuorosalicylic acid (FSA).5-Fluorosalicylic acid can then form a highly fluorescent ternary complex of the form FS A-Tb( 3+ )-EDTA, which can be quantified by measuring the Tb 3+ fluorescence for example, using a time-resolved fluorometer.
  • Nucleic Acid Methods As noted herein a disease or condition of the breast such as breast cancer, in particular Stage II or Stage
  • III/IV breast cancer may be detected based on the level of KLK6 and CA15-3 polynucleotides in a sample.
  • Techniques for detecting nucleic acid molecules such as polymerase chain reaction (PCR) and hybridization assays are well known in the art.
  • Probes may be used in hybridization techniques to detect KLK6 and CAl 5-3 polynucleotides.
  • the technique generally involves contacting and incubating nucleic acids obtained from a sample from a patient or other cellular source with a probe under conditions favorable for the specific annealing of the probes to complementary sequences in the nucleic acids (e.g. under stringent conditions as discussed herein). After incubation, the non-annealed nucleic acids are removed, and the presence of nucleic acids that have hybridized to the probe if any are detected.
  • Nucleotide probes for use in the detection of KLK6 and CA15-3 polynucleotide sequences in samples may be constructed using conventional methods known in the art.
  • the probes may comprise DNA or DNA mimics corresponding to a portion of an organism's genome, or complementary RNA or RNA mimics.
  • the nucleic acids can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone.
  • DNA can be obtained using standard methods such as polymerase chain reaction (PCR) amplification of genomic DNA or cloned sequences.
  • PCR polymerase chain reaction
  • a nucleotide probe may be labeled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as 32 P, 3 H, 14 C or the like.
  • detectable substances include antigens that are recognized by a specific labeled antibody, fluorescent compounds, enzymes, antibodies specific for a labeled antigen, and luminescent compounds.
  • An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleic acids to be detected and the amount of nucleic acids available for hybridization.
  • Labeled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.).
  • the nucleic acid probes may be used to detect KLK6 polynucleotides and CA 15-3 polynucleotides, preferably in human cells.
  • the nucleotide probes may also be useful in the diagnosis of a disease and/or condition of the breast, in particular breast cancer, involving KLK6 and CA 15-3 polynucleotides, in monitoring the progression of such disorder, or monitoring a therapeutic treatment.
  • the detection of KLK6 and CA15-3 polynucleotides in a sample may involve the amplification of specific gene sequences using an amplification method such as PCR, followed by the analysis of the amplified molecules using techniques known to those skilled in the art.
  • oligonucleotide primers may be employed in a PCR based assay to amplify a portion of a polynucleotide encoding kallikrein 6 and to amplify a portion of a polynucleotide encoding CA 15-3 derived from a sample, wherein the oligonucleotide primers are specific for (i.e. hybridize to) the polynucleotides.
  • the amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis.
  • primers and probes employed in the methods of the invention generally have at least about 60%, preferably at least about 75% and more preferably at least about 90% identity to a portion of a KLK6 polynucleotide or CA 15-3 polynucleotide; that is, they are at least 10 nucleotides, and preferably at least 20 nucleotides in length. In an embodiment the primers and probes are at least about 10-40 nucleotides in length.
  • Hybridization and amplification reactions may also be conducted under stringent conditions as discussed herein.
  • Hybridization and amplification techniques described herein may be used to assay qualitative and ' quantitative aspects of KLK6 and CA15-3 polynucleotide expression.
  • RNA may be isolated from a cell type or tissue known to express KLK6 and CA 15-3 polynucleotide, and tested utilizing the hybridization (e.g. standard Northern analyses) or PCR techniques.
  • the primers and probes may be used in situ i.e directly on tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections.
  • a method is provided employing reverse transcriptase-polymerase chain reaction (RT-PCR), in which PCR is applied in combination with reverse transcription.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • RNA is extracted from a sample tissue using standard techniques and is reverse transcribed to produce cDNA.
  • the cDNA is used as a template for a polymerase chain reaction.
  • the cDNA is hybridized to primer sets which are specifically designed against a KLK6 sequence or CAl 5-3 polynucleotide.
  • a DNA polymerase is employed to extend from the primer, to synthesize a copy of the template.
  • the DNA strands are denatured, and the procedure is repeated many times until sufficient DNA is generated to allow visualization by ethidium bromide staining and agarose gel electrophoresis.
  • Amplification may be performed on samples obtained from a subject with suspected breast cancer, and an individual who is not afflicted with breast cancer or has early stage disease.
  • the reaction may be performed on several dilutions of cDNA spanning at least two orders of magnitude.
  • a statistically significant difference in expression in several dilutions of the subject sample as compared to the same dilutions of the non-cancerous sample or early-stage cancer sample may be considered positive for the presence of cancer.
  • Oligonucleotides or longer fragments derived from a KLK6 polynucleotide and a CA15-3 polynucleotide may be used as targets in a microarray.
  • the microarray can be used to simultaneously monitor the expression levels of kallikrein 6 and CA15-3 polynucleotides, and to identify genetic variants, mutations, and polymorphisms.
  • the information from the microarray may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
  • the invention also includes an array comprising a KLK6 marker, CA15-3 polynucleotide marker, and optionally other cancer markers.
  • the array can be used to assay expression of KLK6 and CA15-3 polynucleotide in the array.
  • the invention allows the quantitation of expression of kallikrein 6 and CA15-3.
  • the invention provides microarrays comprising KLK6 and CA15-3 polynucleotides.
  • the invention provides a microarray for distinguishing samples associated with diseases and/or conditions of the breast, in particular breast cancer samples, comprising a positionally-addressable array of polynucleotide probes bound to a support, the polynucleotide probes comprising polynucleotide probes comprising a sequence complementary and hybridizable to KLK6 and CAl 5-3 polynucleotides.
  • the array can be used to monitor the time course of expression of KLK6 polynucleotides and CA15-3 polynucleotides in the array. This can occur in various biological contexts such as tumor progression.
  • An array can also be useful for ascertaining differential expression patterns of KLK6 polynucleotides and CAl 5-3 polynucleotides, and optionally other markers (e.g., cancer markers), in normal and abnormal cells. This may provide a battery of nucleic acids that could serve as molecular targets for diagnosis or therapeutic intervention.
  • Computer readable media comprising kallikrein 6, KLK6, CA15-3 and/or CA15-3 polynucleotides, and optionally additional markers, in particular cancer markers, is also provided.
  • Computer readable media refers to any medium that can be read and accessed directly by a computer, including but not limited to magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • the invention contemplates computer readable medium having recorded thereon markers identified for patients and controls.
  • Recorded refers to a process for storing information on computer readable medium.
  • the skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising information on kallikrein 6, KLK6 markers, CA15-3 and/or CA15- 3 polynucleotides, and optionally additional markers (e.g., cancer markers).
  • a variety of data processor programs and formats can be used to store information on kallikrein 6, KLK6 markers, CA15-3 and/or CA15-3 polynucleotides, and optionally additional markers (e.g., cancer markers), on computer readable medium.
  • the information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.
  • Any number of dataprocessor structuring formats e.g., text file or database
  • By providing the marker information in computer readable form one can routinely access the information for a variety of purposes. For example, one skilled in the art can use the information in computer readable form to compare marker information obtained during or following therapy with the information stored within the data storage means.
  • the invention provides a medium for holding instructions for performing a method for determining whether a patient has a disease and/or condition of the breast, in particular breast cancer, comprising determining the presence or absence of a kallikrein 6, KLK6, CA15-3 and/or CA15-3 polynucleotides, and optionally additional markers (e.g. cancer markers), and based on the presence or absence of kallikrein 6 or KLK6 markers, CA 15-3 and/or CAl 5-3 polynucleotides, and optionally additional markers, determining whether the patient has the disease and/or condition, in particular breast cancer, and optionally recommending treatment for the disease and/or condition, in particular breast cancer.
  • additional markers e.g. cancer markers
  • the invention also provides in an electronic system and/or in a network, a method for determining whether a subject has a disease and/or condition of the breast, in particular breast cancer, associated with a kallikrein 6, KLK6, CA15-3 and/or CA15-3 polynucleotides, and optionally additional markers (e.g. cancer markers), comprising determining the presence or absence of a kallikrein 6, KLK6, CA 15-3 and/or CA 15-3 polynucleotides, and optionally additional markers (e.g. cancer markers), and based on the presence or absence of the kallikrein 6, KLK6, CA15-3 and/or CA15-3 polynucleotides, and optionally additional markers (e.g. cancer markers), determining whether the subject has a disease and/or condition of the breast, in particular breast cancer, and optionally recommending treatment for the disease and/or condition, in particular breast cancer or a pre-cancer condition.
  • additional markers e.g. cancer markers
  • the invention further provides in a network, a method for determining whether a subject has a disease and/or condition of the breast, in particular breast cancer, associated with a kallikrein 6, KLK6, CA15-3 and/or CA15-3 polynucleotides, and optionally additional markers (e.g.
  • cancer markers comprising: (a) receiving phenotypic information on the subject and information on kallikrein 6, KLK6, CA15-3 and/or CA15-3 polynucleotides, and optionally additional markers, associated with samples from the subject; (b) acquiring information from the network corresponding to the kallikrein 6, KLK6, CA15-3 and/or CA15-3 polynucleotides, and optionally additional markers; and (c) based on the phenotypic information and information on the kallikrein 6, KLK6, CA15-3 and/or CA15-3 polynucleotides, and optionally additional markers, determining whether the subject has the disease and/or condition , in particular breast cancer; and (d) optionally recommending treatment for the disease and/or condition, in particular breast cancer.
  • a system of the invention generally comprises a digital computer; a database server coupled to the computer; a database coupled to the database server having datastored therein, the data comprising records of data comprising kallikrein 6, KLK6, CA15-3 and/or CA15-3 polynucleotides, and optionally additional markers (e.g. cancer markers), or nucleic acids encoding same, and a code mechanism for applying queries based upon a desired selection criteria to the data file in the database to produce reports of records which match the desired selection criteria.
  • markers e.g. cancer markers
  • step (c) using a code mechanism for applying queries based upon a desired selection criteria to the data file in the database to produce reports of records of step (a) which provide a match of the desired selection criteria of the database of step (b), the presence of a match being a positive indication that the markers of step (a) have been isolated from a cell that is a cancer cell.
  • the invention contemplates a business method for determining whether a subject has a disease and/or condition of the breast, in particular breast cancer, associated with a kallikrein 6, KLK6, CA15-3 and/or CA15-3 polynucleotide, and optionally additional markers (e.g.
  • cancer markers comprising: (a) receiving phenotypic information on the subject and information on a kallikrein 6, KLK6, CA15-3 and/or CA15-3 polynucleotide marker, and optionally additional markers, associated with samples from the subject; (b) acquiring information from a network corresponding to the kallikrein 6, KLK6, CA15-3 and/or CA15-3 polynucleotide markers, and optionally additional markers; and (c) based on the phenotypic information, information on kallikrein 6, KLK6, CA 15-3 and/or CA15-3 polynucleotide markers, and optionally additional markers, and acquired information, determining whether the subject has the disease and/or condition, in particular breast cancer; and (d) optionally recommending treatment for the disease and/or condition, in particular breast cancer.
  • the computer systems, components, and methods described herein are used to monitor a disease and/or condition of the breast, in particular breast cancer.
  • kits for carrying out the methods of the invention typically comprise two or more components required for performing a diagnostic assay.
  • Components include but are not limited to compounds, reagents, containers, and/of equipment.
  • the methods described herein may be performed by utilizing pre-packaged diagnostic kits comprising at least agents (e.g. antibodies, probes, primers, etc) described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients afflicted with a disease and/or condition of the breast, in particular breast cancer, or exhibiting a predisposition to developing a disease and/or condition of the breast, in particular breast cancer.
  • the invention contemplates a container with a kit comprising a binding agent(s) as described herein.
  • the kit may contain antibodies specific for kallikrein 6, and preferably antibodies specific for CA15-3, antibodies against the antibodies labeled with an enzyme(s), and a substrate for the enzyme(s).
  • the kit may also contain microtiter plate wells, standards, assay diluent, wash buffer, adhesive plate covers, and/or instructions for carrying out a method of the invention using the kit.
  • the invention provides a test kit for diagnosing a disease and/or condition of the breast disclosed herein (e.g.
  • breast cancer in a subject which comprises an antibody that binds to kallikrein 6 and/or a polynucleotide that hybridizes to or amplifies KLK6, and preferably an antibody that binds to CA15-3 and/or a polynucleotide that hybridizes to or amplifies a CA15-3 polynucleotide.
  • the invention relates to use of an antibody that binds to kallikrein 6 and/or a polynucleotide that hybridizes to or amplifies KLK6, and preferably an antibody that binds to CA 15-3 and/or a polynucleotide that hybridizes to or amplifies a CA 15-3 polynucleotide, in the manufacture of a composition for detecting a disease and/or condition of the breast disclosed herein.
  • the kit includes antibodies or antibody fragments which bind specifically to an epitope of kallikrein 6 and antibodies or antibody fragments which bind specifically to CA15-3, and means for detecting binding of the antibodies to their epitopes associated with diseased cells, in particular cancer cells, either as concentrates (including lyophilized compositions), which may be further diluted prior to testing.
  • the invention provides a kit for determining the presence of breast cancer comprising a known amount of a first binding agent that specifically binds to kallikrein 6 wherein the first binding agent comprises a detectable substance, or it binds directly or indirectly to a detectable substance.
  • Another aspect of the present invention also provides a kit for determining the presence of breast cancer in a serum sample.
  • the kit includes:
  • the first and the second agents respectively, comprise a detectable substance or bind to a detectable substance.
  • the agents may be antibodies, particularly monoclonal antibodies.
  • the first and the second agents, respectively comprise a different detectable substance or bind to a different detectable substance.
  • kits for diagnosis of breast cancer based on detecting a mathematical combination of kallikrein 6 and CAl 5-3 in a physiological fluid, in particular serum.
  • the invention provides a kit for diagnosis of breast cancer in a patient comprising: a) instructions for determining the presence of breast cancer in the patient; b) reagents for measuring the serum values of the concentrations of kallikrein 6 and CA15-3; and, c) instructions for using a function or mathematical combination that is used to combine the values in order to obtain an end value, wherein analysis of the end value determines the presence of breast cancer in the patient.
  • the mathematical combination is hK6 + logCA15-3.
  • the kit further comprises an ROC curve.
  • the kit comprises tables that allow determination of predictive values depending on the expected prevalence of breast cancer in a patient population.
  • kits may be designed to detect the levels of KLK6 polynucleotides and CA15-3 polynucleotides in a sample.
  • Such kits generally comprise oligonucleotide probes or primers, as described herein, which hybridize to or amplify KLK6 polynucleotides and CA15-3 polynucleotides. Oligonucleotides may be used, for example, within PCR or hybridization procedures.
  • Test kits useful for detecting target KLK6 polynucleotides and CA15-3 polynucleotides are also provided which comprise a container containing a KLK polynucleotide and a CA 15-3 polynucleotide, and fragments or complements thereof.
  • kits of the invention can further comprise containers with tools useful for collecting test samples (e.g. serum) including lancets and absorbent paper or cloth for collecting and stabilizing blood.
  • test samples e.g. serum
  • lancets and absorbent paper or cloth for collecting and stabilizing blood.
  • Example 1 The following non-limiting example is illustrative of the present invention: Example 1
  • Pre-surgery/treatment serum samples were collected from 100 female breast cancer patients. Control serum samples were collected from 80 normal females in the same age range as the patients. The breast cancer patients comprised 80 individuals with early stage and 20 with late stage disease. Serum samples were stored at - 7O 0 C until they were aliquoted and tested for hK6 and CA15-3.
  • the concentration of hK6 was measured in the serum samples with a highly sensitive and specific noncompetitive immunoassay.
  • the assay incorporated two hK6 specific monoclonal antibodies, raised in mouse, one for coating (clone 27-4) and the other for detection (clone E24) in a sequential two site immunometric format with time resolved fluorescence detection.
  • the assay has a detection limit of 0.05 ⁇ g/L and a dynamic range up to 20 ⁇ g/L. Precision was less than 10% within the measurement range.
  • Standards (recombinant hK6) and samples were analyzed in duplicates with inclusion of 3 quality control samples in every run.
  • the assay procedure was as follows. White polystyrene microtiter plates were coated with anti-hk6 (27-
  • the plates were washed six times with the wash buffer, and then 100 ⁇ L of substrate buffer [0.1 mol/L Tris buffer (pH 9.1) containing 1 mrriol/L diflunisal phosphate (DFP), 0.1 mol/L NaCl, and 1 mmol/L MgCl 2 was added to each well and incubated for 10 min with shaking at room temperature. lOO ⁇ l developing solution (1 mol/L Tris base, 0.4 mol/L NaOH, 2
  • CAl 5-3 was measured using CAl 5-3TM from Roche Diagnostics Corporation (IN, USA).
  • Figure 1 shows the distribution of serum hK6 values in breast cancer patients and normal females. Most of the cancer patients had serum hK6 values that were above 2ng/mL whereas most of the normal women had serum hK6 levels that were below 2ng/mL.
  • Scatter plots of hK6 serum levels vs. CA15-3 levels in normal ( Figure 2) and breast cancer patients (Figure 3) demonstrated that hK6 had slightly lower specificity than CAl 5- 3 but more than twice the sensitivity of C Al 5-3 as summarized in Table 1. This study demonstrated the superior clinical sensitivity of serum hK6 for breast cancer detection compared to CA15-3.
  • hK6 Combining hK6 with CA15-3 (a case was considered positive if either marker was positive) increased the clinical sensitivity by 8% but also reduced the specificity by 3%.
  • Kallikrein 6, and a combination of kallikrein 6 and CA15-3 were assessed using the ROC curve analysis.
  • a ROC curve is a graphic presentation of the sensitivity against the false-positive rate (1-specificity).
  • the ROC graphs of Figures 4 and 5 illustrate that the combination of hK6 + logCA15-3 performed better than hK6 alone in detecting breast cancer.

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Abstract

La présente invention concerne des techniques de détection, de diagnostic et de surveillance de maladies et/ou de pathologie du sein tel que le cancer du sein chez une patiente, qui consistent à mesurer le kallikrem 6 ou KLK6, en particulier en combinaison avec des polynucléotides CA15-3 ou CA15-3, dans un prélèvement de patiente. Les moyens de mesures comprennent l'utilisation d'anticorps spécifiques et de réactions d'amplification et d'hybridation impliquant des polynucléotides. Cette invention concerne aussi des kits et des compositions permettant de mettre en oeuvre des techniques de l'invention.
PCT/CA2006/000216 2005-02-14 2006-02-14 Kallikrem 6 utilise dans le diagnostic de cancer du sein WO2006084387A1 (fr)

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CN111721935A (zh) * 2019-03-29 2020-09-29 北京九强生物技术股份有限公司 糖链抗原ca153检测试剂盒

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WO2004021008A2 (fr) * 2002-08-28 2004-03-11 Mount Sinai Hospital Methodes de detection du cancer du sein et des ovaires

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WO2004021008A2 (fr) * 2002-08-28 2004-03-11 Mount Sinai Hospital Methodes de detection du cancer du sein et des ovaires

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DIAMANDIS E.P. ET AL.: "Immunofluorometric Assay of Human Kallikrein 6 (Zyme/Protease M/Neurosin) and Preliminary Clinical Applications", CLINICAL BIOCHEMISTRY, vol. 33, July 2000 (2000-07-01), pages 369 - 375 *
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111721935A (zh) * 2019-03-29 2020-09-29 北京九强生物技术股份有限公司 糖链抗原ca153检测试剂盒

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