WO2005124354A2 - Utilisation de proteine comme marqueur du cancer du sein - Google Patents

Utilisation de proteine comme marqueur du cancer du sein Download PDF

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Publication number
WO2005124354A2
WO2005124354A2 PCT/EP2005/006521 EP2005006521W WO2005124354A2 WO 2005124354 A2 WO2005124354 A2 WO 2005124354A2 EP 2005006521 W EP2005006521 W EP 2005006521W WO 2005124354 A2 WO2005124354 A2 WO 2005124354A2
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Prior art keywords
marker
breast cancer
chio
protein
sample
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PCT/EP2005/006521
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English (en)
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WO2005124354A3 (fr
Inventor
Gabriele Pestlin
Herbert Andres
Peter Berndt
Marie-Luise Hagmann
Johann Karl
Hanno Langen
Michael Thierolf
Werner Zolg
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Roche Diagnostics Gmbh
F. Hoffmann-La Roche Ag
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Publication of WO2005124354A2 publication Critical patent/WO2005124354A2/fr
Publication of WO2005124354A3 publication Critical patent/WO2005124354A3/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

Definitions

  • CHIO human mitochondrial 10 kDa heat shock protein
  • BC breast cancer
  • the staging of cancer is the classification of the disease in terms of extent, progression, and severity. It groups cancer patients so that generalizations can be made about prognosis and the choice of therapy.
  • TNM the most widely used classification of the anatomical extent of cancer. It represents an internationally accepted, uniform staging system. There are three basic variables: T (the extent of the primary tumor), N (the status of regional lymph nodes) and M (the presence or absence of distant metastases).
  • TNM criteria are published by the UICC (International Union against Cancer) (Sobin, L.H., Witte ind, Ch. (eds): TNM Classification of Malignant Tumours, fifth edition, 1997).
  • UICC International Union against Cancer
  • the staging system for breast cancer has recently been revised (Singletary, S.E., et al, J. Clin. Oncol. 20 (2002) 3628-3636).
  • early diagnosis of BC refers to a diagnosis at a pre-cancerous state, ductal carcinoma in situ (DCIS) or at a tumor stage where no metastases at all (neither proximal nor distal), i.e., Ti s , NO, MO or Tl-4; NO; MO are present.
  • T ls denotes carcinoma in situ.
  • the prognosis in advanced stages of tumor is poor. More than one third of the patients will die from progressive disease within five years after diagnosis, corresponding to a survival rate of about 40% for five years.
  • Current treatment is only curing a fraction of the patients and clearly has the best effect on those patients diagnosed in an early stage of disease.
  • BC as a public health problem, it is essential that more effective screening and preventative measures for breast cancer will be developed.
  • the earliest detection procedures available at present for breast cancer involve using clinical breast examination and mammography.
  • significant tumor size must typically exist before a tumor is palpable or can be detected by a mammogram.
  • the densitiy of the breast tissue and the age are important predictors of the accuracy of screening by mammography.
  • the sensitivity ranges from 63% in women with extremely dense breasts to 87% in women with almost entirely fatty breasts.
  • the sensitivity increases with age from 69%) in women of about 40 years of age to 83% in women 80 years and older (Carney, P.A., et al., Ann. Intern. Med.
  • WO 00/60076 shall be mentioned and discussed.
  • This application describes and claims more than two hundred isolated polynucleotides and the corresponding polypeptides as such, as well as their use in the detection of BC.
  • differences on the level of mRNA are not mirrored by the level of the corresponding proteins.
  • a protein encoded by a rare mRNA may be found in very high amounts and a protein encoded by an abundant mRNA may nonetheless be hard to detect and find at all (Chen, G., et al., Mol. Cell. Proteomics 1 (2002) 304-313).
  • This lack of correlation between mRNA-level and protein level is due to reasons like mRNA stability, efficiency of translation, stability of the protein, etc.
  • WO 02/23200 reports about twelve breast cancer-associated spots as found by surface-enhanced laser desorption and ionization (SELDI). These spots are seen more frequently in sera obtained from patients with BC as compared to sera obtained from healthy controls. However, the identity of the molecule(s) comprised in such spot, e.g their sequence, is not known.
  • Nipple aspirate fluid has been used for many years as a potential non- invasive method to identify breast cancer-specific markers.
  • Kuerer et al. compared bilateral matched pair nipple aspirate fluids from women with unilateral invasive breast carcinoma by 2D gel electrophoresis (Kuerer, H.M., et al., Cancer 95 (2002) 2276-2282).
  • 30 to 202 different protein spots were detected in the NAF of breasts suffering from breast carcinoma and not in the matched NAF of the healthy breasts. These spots were detected by a gel image analysis. But the identity of the protein spots is not known.
  • a new diagnostic marker as a single marker should be at least as good as the best single marker known in the art. Or, a new marker should lead to a progress in diagnostic sensitivity and/or specificity either if used alone or in combination with one or more other markers, respectively.
  • the diagnostic sensitivity and/or specificity of a test is best assessed by its receiver-operating characteristics, which will be described in detail below.
  • CA 15-3 cancer antigen 15-3
  • CEA carcinoembryonic antigen
  • CA 15-3 is usually increased in patients with advanced breast cancer.
  • CA 15-3 levels are rarely elevated in women with early stage breast cancer (Duffy, M.J., Crit. Rev. Clin. Lab. Sci. 38 (2001) 225-262). Cancers of the ovary, lung and prostate may also raise CA 15-3 levels. Elevated levels of CA 15-3 may be associated with non- cancerous conditions, such as benign breast or ovary disease, endometriosis, pelvic inflammatory disease, and hepatitis.
  • Pregnancy and lactation can also cause CA 15-3 levels to raise (National Cancer Institute, Cancer Facts, Fact Sheet 5.18 (1998) 1-5).
  • the primary use of CEA is in monitoring colon cancer, especially when the disease has metastasized.
  • a variety of cancers can produce elevated levels of CEA, including breast cancer.
  • the present invention therefore relates to a method for assessing breast cancer comprising the steps of a) providing a liquid sample obtained from an individual, b) contacting said sample with a specific binding agent for CHIO under conditions appropriate for formation of a complex between said binding agent and CHIO, and c) correlating the amount of complex formed in (b) to the assessment of breast cancer
  • Another preferred embodiment of the invention is a method for assessing breast cancer comprising the steps of a) contacting a liquid sample obtained from an individual with a specific binding agent for CHIO under conditions appropriate for formation of a complex between said binding agent and CHIO, and b) correlating the amount of complex formed in (a) to the assessment of breast cancer.
  • Yet another preferred embodiment of the invention relates to a method for assessing breast cancer in vitro by biochemical markers, comprising measuring in a sample the concentration of CHIO and of one or more other marker of breast cancer and using the concentrations determined in the assessment of breast cancer.
  • the present invention also relates to the use of a marker panel comprising at least
  • the present invention also relates to the use of a marker panel comprising at least CHIO and CEA in the assessment of BC.
  • the present invention also relates to the use of a marker panel comprising at least CHIO and CRABP-II in the assessment of BC.
  • the present invention also relates to the use of a marker panel comprising at least CHIO and ASC in the assessment of BC.
  • the present invention also provides a kit for performing the method according to the present invention comprising at least the reagents required to measure CHIO and CA 15-3, respectively, and optionally auxiliary reagents for performing the measurement.
  • the present invention also provides a kit for performing the method according to the present invention comprising at least the reagents required to measure CHIO and CEA, respectively, and optionally auxiliary reagents for performing the measurement.
  • the present invention relates to a method for assessing breast cancer in vitro comprising measuring in a sample the concentration of a) CHIO, b) optionally one or more other marker of breast cancer, and c) using the concentrations determined in step (a) and optionally step (b) in the assessment of breast cancer.
  • a marker means one marker or more than one marker.
  • marker refers to a molecules to be used as a target for analyzing patient test samples.
  • molecular targets are proteins or polypeptides themselves as well as antibodies present in a sample.
  • Proteins or polypeptides used as a marker in the present invention are contemplated to include any variants of said protein as well as fragments of said protein or said variant, in particular, immunologically detectable fragments.
  • proteins which are released by cells or present in the extracellular matrix which become damaged, e.g., during inflammation could become degraded or cleaved into such fragments.
  • Certain markers are synthesized in an inactive form, which may be subsequently activated by proteolysis.
  • proteins or fragments thereof may also be present as part of a complex.
  • Such complex also may be used as a marker in the sense of the present invention.
  • Variants of a marker polypeptide are encoded by the same gene, but differ in their PI or MW, or both (e.g., as a result of alternative mRNA or pre- mRNA processing, e.g. alternative splicing or limited proteolysis) and in addition, or in the alternative, may arise from differential post-translational modification (e.g., glycosylation, acylation, and/ or phosphorylation).
  • assessing breast cancer is used to indicate that the method according to the present invention will (alone or together with other markers or variables, e.g., the criteria set forth by the UICC (UICC (International Union against Cancer), Sobin, L.H., Wittekind, Ch. (eds), TNM Classification of Malignant Tumours, fifth edition, 1997)) e.g., aid the physician to establish or confirm the absence or presence of BC or aid the physician in the prognosis, the detection of recurrence
  • UICC International Union against Cancer
  • Sobin L.H., Wittekind, Ch. (eds), TNM Classification of Malignant Tumours, fifth edition, 1997)
  • sample refers to a biological sample obtained for the purpose of evaluation in vitro.
  • the sample or patient sample preferably may comprise any body fluid.
  • Preferred test samples include blood, serum, plasma, nipple aspirate fluid, urine, saliva, and synovial fluid.
  • Preferred samples are whole blood, serum, plasma or nipple aspirate fluid, with plasma or serum being most preferred.
  • any such assessment is made in vitro.
  • the patient sample is discarded afterwards.
  • the patient sample is solely used for the in vitro method of the invention and the material of the patient sample is not transferred back into the patient's body.
  • the sample is a liquid sample, e.g., whole blood, serum, or plasma.
  • the present invention relates to a method for assessing BC in vitro by biochemical markers, comprising measuring in a sample the concentration of CHIO and using the concentration determined in the assessment ofBC.
  • CHIO mitochondrial 10 kDa heat shock protein, hsplO, 10 kDa chaperonin, CPN10; encoded by the gene HSPE1; Swiss- PROT: P61604
  • CH10 is a eukaryotic CpnlO homologue and together with Cpn60 essential for mitochondrial protein biogenesis.
  • the Cpn-mediated protein-folding apparatus exhibits a high degree of conservation between prokaryotes and mitochondria of higher eukaryotes (Hartman, D.J., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 3394- 3398).
  • CHIO is also known to the art from the following publications: Cappello, F.,
  • the present invention shall not be construed to be limited to the full-length protein CH10 of SEQ ID NO:l.
  • CH10 are also encompassed by the present invention.
  • an "allelic variant" is understood to represent the gene product of one of two or more different forms of a gene or DNA sequence that can exist at a genetic single locus.
  • Artificial fragments preferably encompass a pep tide produced synthetically or by recombinant techniques, which at least comprises one epitope of diagnostic interest consisting of at least 6 contiguous amino acids as derived from the sequence disclosed in SEQ ID NO:l. Such a fragment may advantageously be used for generation of antibodies or as a standard in an immunoassay. More preferred the artificial fragment comprises at least two epitopes of interest appropriate for setting up a sandwich immunoassay.
  • full-length CH10 or a physiological variant of this marker is detected in a method according to the present invention.
  • the assessment method according to the present invention is based on a liquid sample which is derived from an individual. Unlike to methods known from the art CH10 is measured from this liquid sample by use of a specific binding agent.
  • a specific binding agent is, e.g., a receptor for CH10, a lectin binding to CH10 or an antibody to CH10.
  • a specific binding agent has at least an affinity of 10 7 1/mol for its corresponding target molecule.
  • the specific binding agent preferably has an affinity of 10 8 1/mol or even more preferred of 10 9 1/mol for its target molecule.
  • specific is used to indicate that other biomolecules present in the sample do not significantly bind to the binding agent specific for CH10.
  • the level of binding to a biomolecule other than the target molecule results in a binding affinity which is only 10%), more preferably only 5% of the affinity of the target molecule or less.
  • a most preferred specific binding agent will fulfill both the above minimum criteria for affinity as well as for specificity.
  • a specific binding agent preferably is an antibody binding to CHIO.
  • the term antibody refers to a polyclonal antibody, a monoclonal antibody, fragments of such antibodies, as well as to genetic constructs comprising the binding domain of an antibody.
  • Antibodies are generated by state of the art procedures, e.g., as described in Tijssen (Tijssen, P., Practice and theory of enzyme immunoassays 11 (1990) the whole book, especially pages 43-78; Elsevier, Amsterdam).
  • Tijssen Tejssen, P., Practice and theory of enzyme immunoassays 11 (1990) the whole book, especially pages 43-78; Elsevier, Amsterdam.
  • the skilled artisan is well aware of methods based on immunosorbents that can be used for the specific isolation of antibodies. By these means the quality of polyclonal antibodies and hence their performance in immunoassays can be enhanced. (Tijssen, P., supra, pages 108-115).
  • polyclonal antibodies raised in rabbits have been used.
  • polyclonal antibodies from different species e.g. rats or guinea pigs
  • monoclonal antibodies can also be used. Since monoclonal antibodies can be produced in any amount required with constant properties, they represent ideal tools in development of an assay for clinical routine.
  • the generation and use of monoclonal antibodies to CH10 in a method according to the present invention is yet another preferred embodiment.
  • the diagnostic method according to the present invention is based on a liquid sample which is derived from an individual. Unlike to methods known from the art CH10 is measured from this liquid sample by use of a specific binding agent.
  • CH10 has been identified as a marker which is useful in the assessment of BC
  • alternative ways may be used to reach a result comparable to the achievements of the present invention.
  • alternative strategies to generate antibodies may be used.
  • Such strategies comprise amongst others the use of synthetic peptides, representing an epitope of CH10 for immunization.
  • a synthetic peptide comprises a subsequence of SEQ ID NO: 1
  • the synthetic peptide comprises a contiguous subsequence consisting of 5 to 25 amino acid residues of SEQ ID NO:l. More preferred, the peptide comprises a contiguous subsequence consisting of 10 to 15 amino acid residues of SEQ ID NO:l.
  • DNA immunization also known as DNA vaccination may be used.
  • liquid sample obtained from an individual is incubated with the specific binding agent for CHIO under conditions appropriate for formation of a binding agent CHlO-complex.
  • Such conditions need not be specified, since the skilled artisan without any inventive effort can easily identify such appropriate incubation conditions.
  • the amount of complex is measured and correlated to the diagnosis of BC.
  • the skilled artisan will appreciate there are numerous methods to measure the amount of the specific binding agent CHlO-complex, all described in detail in relevant textbooks (cf., e.g., Tijssen P., supra, or Diamandis et al., eds. (1996) Immunoassay,
  • CH10 is detected in a sandwich type assay format.
  • a first specific binding agent is used to capture CH10 on the one side and a second specific binding agent, which is labeled to be directly or indirectly detectable is used on the other side.
  • CH10 can be measured from a liquid sample obtained from an individual sample. No tissue and no biopsy sample is required to apply the marker CH10 in the diagnosis of BC.
  • the method according to the present invention is practiced with serum as liquid sample material.
  • the method according to the present invention is practiced with plasma as liquid sample material.
  • the method according to the present invention is practiced with whole blood as liquid sample material.
  • the method according to the present invention is practiced with nipple aspirate fluid as liquid sample material.
  • the inventors of the present invention have surprisingly been able to detect protein CHIO in a bodily fluid sample. Even more surprising they have been able to demonstrate that the presence of CHIO in such liquid sample obtained from an individual can be correlated to the diagnosis of breast cancer.
  • an antibody to CHIO is used in a qualitative (CHIO present or absent) or quantitative (CHIO amount is determined) immunoassay.
  • BC is one of the most frequent cancers among women in developed countries. Because of its high prevalence, its long asymptomatic phase and the presence of premalignant lesions, BC meets many of the criteria for screening. Clearly, a serum tumor marker which has acceptable sensitivity and specificity would be more suitable for screening than established methods.
  • the diagnostic method according to the present invention is used for screening purposes. I.e., it is used to assess subjects without a prior diagnosis of BC by measuring the level of CHIO and correlating the level measured to the presence or absence of BC.
  • CHIO alone will not suffice to allow for a general screening e.g. of the risk population for BC. Most likely no single biochemical marker in the circulation will ever meet the sensitivity and specificity criteria required for screening purposes. Rather it has to be expected that a marker panel will have to be used in BC screening. Thus, the marker CHIO will form an integral part of a marker panel appropriate for screening purposes.
  • the present invention therefore relates to the use of CHIO as one marker of a BC marker panel for BC screening purposes.
  • the inventors also contemplate CHIO to be used as a diagnostic aid, especially by establishing a baseline value to indicate tumor load before breast surgery.
  • the present invention thus also relates to the use of CH10 for establishing a baseline value before surgery for BC.
  • Antibodies to CH10 with great advantage can also be used in established procedures, e.g., to detect breast cancer cells in situ, in biopsies, or in immunohistological procedures.
  • CHIO alone contributes to the differentiation of BC patients from healthy controls or from healthy controls plus non-malignant diseases, it has to be expected that it will aid in assessing the prognosis of patients suffering from BC.
  • the level of preoperative CHIO will most likely be combined with one or more other marker for BC and/or the TNM staging system.
  • CHIO is used in the prognosis of patients with BC.
  • CHIO will be a clinically useful marker for monitoring of chemotherapy, radiotherapy or immune therapy.
  • Increased levels of CHIO are directly correlated to tumor burden. For example, after chemotherapy a short term (few hours to 14 days) increase in CHIO may serve as an indicator of tumor cell death.
  • the present invention therefore also relates to the use of CHIO in the monitoring of BC patients under chemotherapy.
  • the present invention therefore also relates to the use of CHIO in the monitoring of BC patients under radiotherapy.
  • the present invention relates to the use of CHIO in the monitoring of BC patients under immune therapy.
  • the follow-up of patients with BC after surgery is one of the most important fields of use for an appropriate biochemical marker.
  • an increase of CH10 can be used as an indicator for tumor recurrence. Due to the high sensitivity of CH10 in the BC patients investigated it is expected that CHIO alone or in combination with one or more other marker will be of great help in the follow-up of BC patients, especially in BC patients after surgery.
  • the use of a marker panel comprising CHIO and one or more other marker of BC in the follow-up of BC patients represents a further preferred embodiment of the present invention.
  • the present invention relates to use of protein CHIO as a marker molecule in the diagnosis of breast cancer from a liquid sample obtained from an individual.
  • the ideal scenario for diagnosis would be a situation wherein a single event or process would cause the respective disease as, e.g., in infectious diseases. In all other cases correct diagnosis can be very difficult, especially when the etiology of the disease is not fully understood as is the case of BC. As the skilled artisan will appreciate, no biochemical marker, for example in the field of BC, is diagnostic with 100%) specificity and at the same time 100%) sensitivity for a given disease.
  • biochemical markers are used to assess with a certain likelihood or predictive value the presence or absence of a disease. Therefore, in routine clinical diagnosis various clinical symptoms and biological markers are generally considered together in the diagnosis, treatment, and management of the underlying disease.
  • Biochemical markers can either be determined individually or, in a preferred embodiment of the invention, they can be measured simultaneously using a chip- or a bead-based array technology. The concentrations of the biomarkers are then interpreted independently using an individual cut-off for each marker or they are combined for interpretation.
  • CRABP-II cellular retinoic acid-binding protein II
  • CRABP-II The cellular retinoic acid-binding protein II (CRABP-II) (Swiss-PROT: P29373) is one of two isoforms presently known.
  • the two isoforms (CRABP-I and -II) were first characterized by Siegenthaler et al. 1992.
  • CRABP-II was shown to be the major isoform, highly expressed in human epidermis by fibroblasts and keratinocytes (Siegenthaler, G., Biochemical Journal 287 (1992) 383-389).
  • CRABP-II regulates the intracellular retinoic acid (RA) concentration, transport, and metabolism. It has been demonstrated that RA induced CRABP-II mRNA levels 2 fold in squamous cell cancer by transcriptional upregulation (Vo, H.P., Crowe, D.L., Anticancer Res. 18 (1998) 217-224).
  • CRABP-II The function of CRABP-II in mammary carcinoma cells was described by Budhu, A.S., and Noy, N. (Mol. Cell. Biol. 22 (2002) 2632-2641).
  • the cytosolic CRABP-II undergoes a nuclear localization upon binding RA and interacts with retinoic acid receptor (RAR) by building a short lived CRABP-II - RAR -complex.
  • RAR retinoic acid receptor
  • the overexpression of CRABP-II in MCF7 mammary cell lines enhances their sensitivity to retinoic acid-induced growth inhibition (Budhu, A.S., Noy, N., supra).
  • ASC apoptosis-associated speck-like protein containing a caspase-associated recruitment domain
  • TMS1 target of methylation-induced silencing 1
  • Caspase-associated recruitment domains mediate the interaction between adaptor proteins such as APAF1 (apoptotic protease activating factor 1) and the pro-form of caspases (e.g., CASP 9) participating in apoptosis.
  • ASC is a member of the CARD -containing adaptor protein family.
  • DNMTl DNA cytosine-5-methyltransferase-l
  • McConnell and Vertino showed that inducible expression of ASC inhibits cellular prolifertion and induces DNA fragmentation that can be blocked by caspase inhibitor.
  • Immunofluorescence microscopy demonstrated that induction of apoptosis causes a CARD-dependent shift from diffuse cytoplasmic expression to spherical perinuclear aggregates (McConnell, B.B., and Vertino, P.M., Cancer Res. 60 (2000) 6243-6247).
  • ASC gene in the progression of breast and gastric cancer involving down-regulation of the proapoptotic ASC gene (Moriani, R., et al., Anticancer Res. 22 (2002) 4163- 4168).
  • Virmani et al. examined the methylation status of ASC in lung cancer and breast cancer tissue. They found that aberrant methylation of ASC was present in 46 % of breast cancer cell lines and in 32 % of breast tumor tissue. Methylation was rare in non-malignant breast tissue (7 %) (Virmani, A., et al., Int. J. Cancer 106 (2003) 198-204). Shiohara et al. found out that up-regulation of ASC is closely associated with inflammation and apoptosis in human neutrophils (Shiohara, M., et al., Blood 98 (2001) 229a). Masumoto et al. observed high levels of ASC abundantly expressed in epithelial cells and leucocytes (Masumoto, J., et al., J. Histochem. Cytochem. 49
  • the present invention relates to the use of CH10 as a marker molecule for breast cancer in combination with one or more marker molecules for breast cancer in the diagnosis of breast cancer from a liquid sample obtained from an individual.
  • the expression "one or more” denotes 1 to 10, preferably 1 to 5, more preferred 3 or 4.
  • Preferred selected other BC markers with which the measurement of CH10 may be combined are CEA, CA 15-3, CRABP-II, and ASC.
  • CH10 is used as part of a marker panel at least comprising CH10 and a marker selected from the group consisting of CEA, CA 15-3, CRABP-II, and ASC.
  • the assessment of breast cancer according to the present invention is performed in a method comprising measuring in a sample the concentration of a) CH10, b) optionally one or more other marker of breast cancer, and c) using the concentration determined in step (a) and optionally step (b) in the assessment of breast cancer.
  • the present invention is also directed to a method for assessing BC in vitro by biochemical markers, comprising measuring in a sample the concentration of CH10 and of one or more other marker of BC and using the concentrations determined in the assessment of BC.
  • the method for assessment of BC is performed by measuring the concentration of CH10 and of one or more other marker and by using the concentration of CH10 and of the one or more other marker in the assessment of BC.
  • the method according to the present invention is used with samples of patients suspected to be suffering from breast cancer.
  • An individual suspected of suffering from breast cancer is an individual for which other types of cancers have been excluded.
  • Other cancers include but are not limited to cancers of the colon, lung, stomach, ovary, and prostate.
  • a preferred embodiment of the invention is therefore a method for the diagnosis of breast cancer comprising the steps of a) providing a liquid sample obtained from an individual suspected of suffering from breast cancer, b) contacting said sample with a specific binding agent for CHIO under conditions appropriate for formation of a complex between said binding agent and CHIO, and c) correlating the amount of complex formed in (b) to the diagnosis of breast cancer.
  • Diagnostic reagents in the field of specific binding assays like immunoassays, usually are best provided in the form of a kit, which comprises the specific binding agent and the auxiliary reagents required to perform the assay.
  • the present invention therefore also relates to an immunological kit comprising at least one specific binding agent for CHIO and auxiliary reagents for measurement of CHIO.
  • the ROC graph is a plot of all of the sensitivity/specificity pairs resulting from continuously varying the decision thresh-hold over the entire range of data observed.
  • the clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease or benign versus malignant disease.
  • the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds.
  • sensitivity or the true-positive fraction [defined as (number of true-positive test results) / (number of true-positive + number of false- negative test results)].
  • positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup.
  • false-positive fraction or 1 - specificity [defined as (number of false- positive results) / (number of true-negative + number of false-positive results)]. It is an index of specificity and is calculated entirely from the unaffected subgroup.
  • the ROC plot is independent of the prevalence of disease in the sample.
  • Each point on the ROC plot represents a sensitivity/ 1 -specificity pair corresponding to a particular decision threshold.
  • a test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity).
  • the theoretical plot for a test with no discrimination is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes.
  • One convenient goal to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number.
  • Clinical utility of the novel marker CH10 is best assessed in comparison to and in combination with the established marker CA 15-3 using a receiver operator curve analysis (ROC; Zweig, M. H., and Campbell, G., Clin. Chem. 39 (1993) 561-577).
  • This analysis is based on well-defined patient cohorts consisting of 50 samples each from patients with invasive ductal or lobular carcinoma in Tl-3; NO; M0, more progressed tumor, i.e., T4 and/or various severity of metastasis (N+ and/or M+), medullary, papillary, mucinous and tubular carcinoma, ductal carcinoma in situ, and healthy controls, respectively.
  • tissue specimen from 10 patients suffering from breast cancer are analyzed. From each patient two different tissue types are collected from therapeutic resections: tumor tissue (>80% tumor) (T), and adjacent healthy tissue (N). The latter tissue type serves as matched healthy control sample. Tissues are immediately snap frozen after resection and stored at -80°C before processing. Tumors are diagnosed by histopathological criteria.
  • lysis buffer 40 mM Na-citrate, 5 mM MgCl 2 , 1% Genapol X-080, 0.02% Na-azide, Complete" EDTA-free [Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 1 873 580]
  • a Wheaton® glass homogenizer 20 x loose fitting, 20 x tight fitting.
  • 3 ml of the homogenate are subjected to a sucrose-density centrifugation (10-60% sucrose) for 1 h at 4,500 x g.
  • fractions are obtained.
  • the fraction on top of the gradient contains the soluble proteins and is used for further analysis.
  • Freeze-dried CNBr-activated Sepharose 4B (Amersham Biosciences, 17-0430-01) is reswollen and washed according to the instructions of the manufacturer.
  • Monoclonal antibody directed against human albumin is dissolved in 0.1 M NaHC0 3 , pH 8.3, 0.5 M NaCl, 10 mg/ml.
  • 1 ml antibody solution is mixed with 1 ml reswollen CNBr-activated Sepharose 4B.
  • the reaction time is 1 h. Blocking of the remaining acitve groups and washing of the gel is carried out according to the instructions of the manufacturer.
  • the protein concentration of the soluble protein fraction is determined using Bio- Rad ® protein assay (Cat.No. 500-0006; Bio-Rad Laboratories GmbH, Munchen,
  • the concentrated solution is diluted to 2.5 M urea and incubated with 4 ⁇ g trypsin (Proteomics grade, Roche Diagnostics GmbH, Mannheim, Germany) over night. The digestion is stopped by adding 1 ml 1% formic acid and analyzed.
  • the tryptic digest (500 ⁇ l) is separated on a two-dimensional Nano-HPLC-System
  • the protein CHIO is identified with the sequences given in Table 1.
  • protein CHIO is found specifically expressed or strongly overexpressed in tumor tissue. It therefore - amongst many other proteins - qualifies as a candidate marker for use in the diagnosis of breast cancer.
  • Polyclonal antibody to the breast cancer marker protein CH10 is generated for further use of the antibody in the measurement of serum and plasma and blood levels of CH10 by immunodetection assays, e.g. Western Blotting and ELISA
  • recombinant expression of the protein is performed for obtaining immunogens.
  • the expression is done applying a combination of the RTS 100 expression system and E. coli.
  • the DNA sequence is analyzed and recommendations for high yield cDNA silent mutational variants and respective PCR-primer sequences are obtained using the "ProteoExpert RTS E.coli HY” system. This is a commercial web-based service (www.proteoexpert.com).
  • the "RTS 100 E. coli Linear Template Generation Set, His-tag” (Roche Diagnostics GmbH, Mannheim, Germany, Cat.No.
  • His-CHIO fusion protein Purification of His-CHIO fusion protein is done following standard procedures on a Ni-chelate column. Briefly, 1 1 of bacteria culture containing the expression vector for the His-CHIO fusion protein is pelleted by centrifugation. The cell pellet is resuspended in lysis buffer, containing phosphate, pH 8.0, 7 M guanidium chloride, imidazole and thioglycerole, followed by homogenization using a Ultra-Turrax . Insoluble material is pelleted by high speed centrifugation and the supernatant is applied to a Ni-chelate chromatographic column. The column is washed with several bed volumes of lysis buffer followed by washes with buffer, containing phosphate, pH 8.0 and urea. Finally, bound antigen is eluted using a phosphate buffer containing SDS under acid conditions.
  • Synthesis is carried out using heterobifunctional chemistry (maleimide/SH- chemistry). Selected cysteine containing CHlO-peptides are coupled to 3- maleimidohexanoyl-N-hydroxysuccinimidester (MHS) activated hemocyanin from Concholepas concholepas (Sigma, B-8556).
  • MHS maleimidohexanoyl-N-hydroxysuccinimidester
  • Hemocyanin is brought to 10 mg/ml in 100 mM NaH 2 P0 4 /NaOH, pH 7.2. Per ml hemocyanin 100 ⁇ l MHS ( 12.3 mg in DMSO) are added and incubated for 1 h. The sample is dialyzed over night against 100 mM NaH PO 4 /NaOH, pH 6.5 and adjusted to 6 mg/ml with dialysis buffer. A selected cysteine containing CH10- peptide is dissolved in DMSO (5 mg/ml for a peptide of 1500 Dalton).
  • mice 12 week old A/J mice are initially immunized intraperitoneally with 100 ⁇ g CH10 or hemocyanin-peptide-conjugate (see above). This is followed after 6 weeks by two further intraperitoneal immunizations at monthly intervals. In this process each mouse is administered 100 ⁇ g CH10 or hemocyanin-peptide-conjugate adsorbed to aluminium hydroxide and 10 germs of Bordetella pertussis. Subsequently the last two immunizations are carried out intravenously on the 3rd and 2nd day before fusion using 100 ⁇ g CH10 or hemocyanin-peptide-conjugate in PBS buffer for each.
  • Spleen cells of the mice immunized according to a) are fused with myeloma cells according to Galfre, G., and Milstein, C, Methods in Enzymology 73 (1981) 3-46.
  • ca. lxlO 8 spleen cells of the immunized mouse are mixed with 2xl0 7 myeloma cells (P3X63-Ag8-653, ATCC CRL1580) and centrifuged (10 min at 300 x g and 4°C).
  • the cells are then washed once with RPMI 1640 medium without fetal calf serum (FCS) and centrifuged again at 400 x g in a 50 ml conical tube.
  • FCS fetal calf serum
  • CH10- positive primary cultures are cloned in 96-well cell culture plates by means of a fluorescence activated cell sorter. In this process again interleukin 6 at 100 U/ml is added to the medium as a growth additive.
  • the hybridoma cells obtained are sown at a density of lxl 0 5 cells per ml in RPMI 1640 medium containing 10%) FCS and proliferated for 7 days in a ferm enter (Thermodux Co., Wertheim/Main, Model MCS-104XL, Order No. 144-050).
  • concentrations of 100 ⁇ g monoclonal antibody per ml are obtained in the culture supernatant. Purification of this antibody from the culture supernatant is carried out by conventional methods in protein chemistry (e.g. according to Bruck, C, et al., Methods Enzymol. 121 (1986) 587-695).
  • a fresh emulsion of the protein solution (100 ⁇ g/ml CH10 or hemocyanin-peptide-conjugate) and complete Freund's adjuvant at the ratio of 1:1 is prepared.
  • Each rabbit is immunized with 1 ml of the emulsion at days 1, 7, 14 and 30, 60 and 90. Blood is drawn and resulting anti-CHIO serum used for further experiments as described in Examples 3 and 4.
  • IgG immunoglobulin G
  • rabbit serum is diluted with 4 volumes of acetate buffer (60 mM, pH 4.0). The pH is adjusted to 4.5 with 2 M Tris-base. Caprylic acid (25 ⁇ l/ml of diluted sample) is added drop-wise under vigorous stirring. After 30 min the sample is centrifuged (13,000 x g, 30 min, 4°C), the pellet discarded and the supernatant collected. The pH of the supernatant is adjusted to 7.5 by the addition of 2 M Tris-base and filtered (0.2 ⁇ m).
  • the immunoglobulin in the supernatant is precipitated under vigorous stirring by the drop-wise addition of a 4 M ammonium sulfate solution to a final concentration of 2 M.
  • the precipitated immunoglobulins are collected by centrifugation (8,000 x g, 15 min, 4°C).
  • the supernatant is discarded.
  • the pellet is dissolved in 10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl and exhaustively dialyzed.
  • the dialysate is centrifuged (13,000 x g, 15 min, 4°C) and filtered (0.2 ⁇ m).
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 P0 4 /NaOH, pH 7.5, 30 mM NaCl. Per ml IgG solution 50 ⁇ l Bio tin -N-hydroxysuccinimide (3.6 mg/ml in DMSO) are added. After 30 min at room temperature, the sample is chromatographed on Superdex 200 (10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl). The fraction containing biotinylated IgG are collected. Monoclonal antibodies are biotinylated according to the same procedure.
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 /NaOH, 30 mM NaCl, pH 7.5.
  • Per ml IgG solution 50 ⁇ l digoxigenin-3-O-methylcarbonyl- ⁇ - aminocaproic acid-N-hydroxysuccinimide ester (Roche Diagnostics, Mannheim,
  • the biotinylated primary antibody is diluted in SuperBlock Blocking Buffer (0.01-0.2 ⁇ g/ml) and incubated with the membrane for 1 h. The membranes are washed 3 times in PBS/0.05 % Tween-20.
  • the specifically bound biotinylated primary antibody is labeled with a streptavidin- HRP-conjugate (20 mU AB ⁇ s/ml in SuperBlock Blocking Buffer). After incubation for 1 h, the membranes are washed 3 times in PBS/0.05 % Tween-20.
  • the bound streptavidin-HRP-conjugate is detected using a chemiluminescent substrate (SuperSignal West Femto Substrate, Pierce Biotechnology, Inc., Rockford, IL, USA) and autoradiographic film. Exposure times varies from 10 min to over night.
  • chemiluminescent substrate SuperSignal West Femto Substrate, Pierce Biotechnology, Inc., Rockford, IL, USA
  • a sandwich ELISA For detection of CH10 in human serum or plasma, a sandwich ELISA is developed. For capture and detection of the antigen, aliquots of the anti-CHIO polyclonal antibody (see Example 2) are conjugated with biotin and digoxygenin, respectively.
  • Streptavidin-coated 96-well microwell plates are incubated with 100 ⁇ l biotinylated anti-CHIO polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9%) NaCl and 0.1% Tween-20. After incubation, plates are washed three times with 0.9% NaCl , 0.1% Tween-20. Wells are then incubated for 2 h with either a serial dilution of the recombinant protein (see Example 2) as standard antigen or with diluted plasma samples from patients. After binding of
  • Accuracy is assessed by analyzing individual liquid samples obtained from well- characterized patient cohorts, i.e., 50 patients having undergone mammography and found to be free of BC, 50 patients each diagnosed and staged as invasive ductal and invasive lobular Tl-3, NO, M0 of BC, 50 patients diagnosed with progressed BC, having at least tumor infiltration in at least one proximal lymph node or more severe forms of metastasis, 50 patients each diagnosed with medullary, mucinous, tubular, or papillary breast carcinoma, and 50 patients diagnosed with DCIS, respectively.
  • CA 15-3 as measured by a commercially available assay (Roche)
  • CH10 with the established marker CA 15-3 is calculated by regularized discriminant analysis (Friedman, J. H., Regularized Discriminant Analysis, Journal of the American Statistical Association 84 (1989) 165-175).

Abstract

La présente invention a trait au diagnostic du cancer du sein. L'invention a trait à l'utilisation de la protéine CH10 ( la protéine de choc thermique 10kDa mitochondriale humaine) dans le diagnostic du cancer du sein, L'invention a également trait à un procédé pour le diagnostic du cancer du sein à partir d'un échantillon, prélevé à partir d'un sujet par la mesure de CH10 dans ledit échantillon. La mesure de CH10 peut, par exemple, être utilisé dans la détection précoce ou le diagnostic du cancer du sein.
PCT/EP2005/006521 2004-06-18 2005-06-17 Utilisation de proteine comme marqueur du cancer du sein WO2005124354A2 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005032495A2 (fr) * 2003-10-03 2005-04-14 Bayer Pharmaceuticals Corporation Profils d'expression genique et leurs methodes d'utilisation

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005032495A2 (fr) * 2003-10-03 2005-04-14 Bayer Pharmaceuticals Corporation Profils d'expression genique et leurs methodes d'utilisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KLUGER HARRIET M ET AL: "cDNA microarray analysis of invasive and tumorigenic phenotypes in a breast cancer model." LABORATORY INVESTIGATION, vol. 84, no. 3, March 2004 (2004-03), pages 320-331, XP002368104 ISSN: 0023-6837 *
SAFI F ET AL: "THE VALUE OF THE TUMOR MARKER CA 15-3 IN DIAGNOSING AND MONITORING BREAST CANCER A COMPARATIVE STUDY WITH CARCINOEMBRYONIC ANTIGEN" CANCER, AMERICAN CANCER SOCIETY, PHILADELPHIA, PA, US, vol. 68, no. 3, 1 August 1991 (1991-08-01), pages 574-582, XP000195878 ISSN: 0008-543X *
YANG ERIC C C ET AL: "Protein expression profiling of endometrial malignancies reveals a new tumor marker: chaperonin 10" JOURNAL OF PROTEOME RESEARCH, ACS, WASHINGTON, DC, US, vol. 3, no. 3, May 2004 (2004-05), pages 636-643, XP002307868 ISSN: 1535-3893 *

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