EP3077812A1 - Méthodes de pronostic du cancer du sein - Google Patents

Méthodes de pronostic du cancer du sein

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Publication number
EP3077812A1
EP3077812A1 EP14867931.9A EP14867931A EP3077812A1 EP 3077812 A1 EP3077812 A1 EP 3077812A1 EP 14867931 A EP14867931 A EP 14867931A EP 3077812 A1 EP3077812 A1 EP 3077812A1
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EP
European Patent Office
Prior art keywords
icd
nuclear
breast cancer
score
amount
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EP14867931.9A
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German (de)
English (en)
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EP3077812A4 (fr
Inventor
Paul Walfish
Ranju Ralhan
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Individual
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Individual
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Priority claimed from US14/099,529 external-priority patent/US20140187438A1/en
Priority claimed from US14/501,020 external-priority patent/US20150094224A1/en
Application filed by Individual filed Critical Individual
Publication of EP3077812A1 publication Critical patent/EP3077812A1/fr
Publication of EP3077812A4 publication Critical patent/EP3077812A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present description relates generally to the field of prognosing cancer. More particularly, the description relates to methods and kits for prognosing breast cancer.
  • Nuclear magnetic resonance (NMR) and mass spectrometry (MS)-based serum metabolite profiling has been shown to accurately identify 80% of breast cancer patients whose tumors failed to respond to chemotherapy (Wei et al., Molecular oncology 2013, 7(3):297-307).
  • a five-gene Integrated Cytokine score (ICS) has been proposed for predicting metastatic outcome from primary HRneg/Tneg breast tumors independent of nodal status, adjuvant chemotherapy use, and Tneg molecular subtype (Yau et al., Breast Cancer Research 2013, 15(5):R103).
  • EpCAM Epithelial cell adhesion molecule
  • EpCAM is comprised of an extracellular domain (EpEx) with epidermal growth factor (EGF) and thyroglobulin repeat-like domains, a single transmembrane domain, and a 26-amino acid intracellular domain called Ep-ICD.
  • EGF epidermal growth factor
  • Ep-ICD 26-amino acid intracellular domain
  • EpCAM is expressed in a majority of human epithelial cancers, including breast, colon, gastric, head and neck, prostate, pancreas, ovarian and lung cancer and is one of the most widely investigated proteins for its diagnostic and therapeutic potential (Spizzo et al. 2004, Breast Cancer Res Treat 86: 207-213; Went et al. 2004, Hum Pathol 35: 122-128; Saadatmand et al. 2013, Br J Surg 100: 252-260; Soysal et al. 2013, Br J Cancer 108: 1480- 1487).
  • An EpCAM expression-based assay is the only FDA-approved test widely used to detect circulating tumor cells in breast cancer patients (Cristofanilli et al. 2004, N Engl J Med 351 : 781 -791).
  • EpCAM-targeted molecular therapies are being studied for several cancers including breast, ovarian, gastric and lung cancer (Baeuerle & Gires 2007, Br. J Cancer 96: 417-423; Simon et al. 2013, Expert Opin Drug Deliv 10: 451 -468). EpCAM expression has been used to predict response to anti-EpCAM antibodies in breast cancer patients (Baeuerle & Gires 2007, Schmidt et al. 2005, Annals of Oncology 23: 2306-2313; Schmidt et al. 2010, Annals of Oncology 21 : 275-282).
  • EpCAM expression has been associated with a favorable prognosis in colorectal and gastric cancers (Songun et al. 2005, Br J Cancer 92:1767-1772; Went et al. 2006, Br J Cancer 94:128-135; Ensinger et al. 2006, J Immunother 29:569-573; Ralhan et al. 2010, BMC Cancer 10:331).
  • increased EpCAM expression is a marker of poor prognosis in breast and gall bladder cancers (Gastl et al. 2000, Lancet 356:1981-1982; Varga et al. 2004, Clin Cancer Res 10:3131-3136).
  • Ep-ICD and EpEx expression analyses were reported on and theirpotential for use as diagnostic markers in ten epithelial cancers, including breast cancer (US Patent Publication No. 201 1/0275530).
  • the presence of nuclear Ep-ICD was found to be a marker of cancerous breast tissue relative to non-cancerous breast tissue (US Patent Publication No. 201 1/0275530).
  • the disclosed invention provides a method for prognosing breast cancer in a subject.
  • the method comprises: (a) measuring an amount of nuclear Ep-ICD in a biological sample from the subject; (b) comparing the amount measured in the biological sample to a control; and prognosing breast cancer based on the comparison between the measured amount of nuclear Ep-ICD and the control.
  • control is: an amount of nuclear Ep- ICD in a non-aggressive breast cancer sample, then a higher measured amount of nuclear Ep-ICD indicates a poor prognosis, and an equal or lower measured amount of nuclear Ep- ICD indicates a favorable prognosis; or an amount of nuclear Ep-ICD in an aggressive breast cancer sample, then an equal or higher measured amount of nuclear Ep-ICD indicates a poor prognosis.
  • the non-aggressive breast cancer sample is known not to progress in disease for at least 40 months following measurement of the nuclear Ep- ICD amount.
  • the aggressive breast cancer sample is known to progress in disease in less than about five years following measurement of the nuclear Ep- ICD amount.
  • the poor prognosis comprises disease free survival of less than five years.
  • the disease free survival is less than or equal to about 41 months.
  • the favorable prognosis comprises disease free survival of at least about five years.
  • the biological sample from the subject is obtained post-therapeutic treatment.
  • the biological sample from the subject comprises one or more of breast epithelial cells, breast tissue, breast tumor tissue, and stage I or II breast cancer tumor cells.
  • the breast cancer prognosed is invasive ductal carcinoma, invasive lobular carcinoma, invasive mucinous carcinoma, ductal carcinoma in situ, or lobular carcinoma in situ.
  • the measured amount of nuclear Ep-ICD is one or more of a quantitative and qualitative amount.
  • the quantitative amount is a percentage of cells in the biological sample that are positive for nuclear Ep-ICD or an absolute quantity of nuclear Ep-ICD.
  • the qualitative amount is an intensity of signal emitted by a label indicative of nuclear Ep- ICD.
  • the method further comprises determining quantitative and qualitative scores for nuclear Ep-ICD and cytoplasmic Ep-ICD, wherein increased quantitative and qualitative nuclear and cytoplasmic Ep-ICD scores are associated with a poor prognosis of breast cancer.
  • the determining of the quantitative and qualitative nuclear Ep-ICD and cytoplasmic Ep-ICD scores comprises: (i) contacting the sample with: a binding agent that specifically binds to Ep-ICD or part thereof and a detectable label for detecting binding of the first binding agent to Ep-ICD, wherein the detectable label emits a detectable signal upon binding of the binding agent to Ep-ICD; (ii) measuring: (a) a first percentage, comprising the percentage of cells in the sample having Ep-ICD in the nucleus bound to the binding agent, and assigning a first quantitative score to the first percentage according to a first scale; and (b) a second percentage, comprising the percentage of cells in the sample having Ep-ICD in the cytoplasm bound to the binding agent, and assigning a second quantitative score to the second percentage according to the first scale; (iii) measuring: (a) a first intensity, comprising the intensity of the signal emitted in the nucleus by the label, and
  • the method further comprises calculating total nuclear Ep-ICD and cytoplasmic Ep-ICD scores, the calculating comprising: (a) adding the first quantitative and qualitative scores to generate the total nuclear Ep-ICD score; and (b) adding the second quantitative and qualitative scores to generate the total cytoplasmic Ep-ICD score.
  • the method further comprises: calculating an Ep-ICD Subcellular Localization Index (ESLI) value for the sample, the ESLI value being a sum of the total nuclear Ep-ICD score and the total cytoplasmic Ep-ICD score, divided by two; comparing the calculated ESLI value to a reference value, wherein the reference value is: (i) an ESLI value indicative of a non-aggressive breast cancer; or (ii)an ESLI value indicative of an aggressive breast cancer; and determining a poor prognosis of breast cancer in the subject when the calculated ESLI value of the sample is greater than the reference value of (i) or is greater than or equal to the reference value of (ii).
  • ESLI Ep-ICD Subcellular Localization Index
  • the binding agent is an antibody.
  • the label is chosen from detectable radioisotopes, luminescent compounds, fluorescent compounds, enzymatic labels, biotinyl groups and predetermined polypeptide epitopes recognizable by a secondary reporter.
  • the quantitative amount is obtained using immunohistochemical (IHC) analysis.
  • the qualitative amount is obtained using immunohistochemical (IHC) analysis.
  • the first scale comprises the following scores: a score of 0 is assigned when less than 10% of the cells are positive; a score of 1 is assigned when 10 - 30% of the cells are positive; a score of 2 is assigned when 31 - 50% the cells are positive; a score of 3 is assigned when 51 - 70% of the cells are positive; and a score of 4 is assigned when more than 70% of the cells are positive, and the second scale comprises the following scores: a score of 0 is assigned when no signal is detected; a score of 1 is assigned when a mild signal is detected; a score of 2 is assigned when a moderate signal is detected; and a score of 3 is assigned when an intense signal is detected.
  • an ESLI value indicative of non-aggressive breast cancer is less than 3 and an ESLI value indicative of non-aggressive breast cancer is less than 3 and an ESLI value indicative of non-ag
  • the measuring of an amount of nuclear Ep-ICD is manual or automated.
  • Figures 1A and 1 B depict immunohistochemical analysis of Ep-ICD and EpEx expression in breast cancer.
  • Figure 1 A depicts representative photomicrographs demonstrating: (I) predominantly cytoplasmic Ep-ICD expression in normal breast tissues; nuclear and cytoplasmic accumulation of Ep-ICD in (II) DCIS; (III) IDC; (IV) ILC; (V) IMC; and (VI) negative control breast cancer tissue incubated with isotype specific IgG showing no detectable immunostaining for Ep-ICD.
  • Figure 1 B depicts expression of EpEx in: (I) normal breast tissues; (II) DCIS; (III) IDC; (IV) ILC; and (V) IMC.
  • Original magnification x 400; arrows labelled N, C and M depict nuclear, cytoplasmic and membrane staining, respectively.
  • Figures 2A and 2B depict Kaplan-Meier curves for disease-free survival (DFS) stratified by nuclear Ep-ICD expression in all breast carcinoma patients and in IDC patients, respectively.
  • Figure 2A shows nuclear accumulation of Ep-ICD was associated with significantly reduced DFS in the entire cohort of breast carcinoma patients (p ⁇ 0.001).
  • Figure 2B shows nuclear accumulation of Ep-ICD was associated with significantly reduced DFS in IDC patients (p ⁇ 0.001).
  • Figures 3A and 3B show Ep-ICD Subcellular Localization Index (ESLI) values and disease free survival in breast cancer patients and IDC patients, respectively.
  • ESLI Ep-ICD Subcellular Localization Index
  • EpCAM refers to the epithelial cell adhesion molecule having the amino acid sequence set forth in SEQ ID NO: 1 (SEQ ID NO: 1 corresponds to Genbank Accession No. NPJD02345).
  • EpCAM comprises an extracellular domain, referred to herein as ⁇ ", that is 265 amino acids in length (amino acids 1-265 in SEQ ID NO: 1), a single transmembrane domain that is 23 amino acids in length (amino acids 266-288 in SEQ ID NO. 1), and an intracellular domain, referred to herein as ⁇ -ICD", that is 26 amino acids in length (amino acids 289-314 in SEQ ID NO. 1).
  • aggressive refers to a type of cancer that forms, grows and/or spreads more quickly than a "non-aggressive" cancer.
  • a subject having an aggressive breast cancer may have an expected disease free survival (DFS) time that is less than a subject having a non-aggressive breast cancer.
  • DFS is the time period until disease recurrence, metastasis and/or death.
  • score refers to a rating or grade provided to a result, wherein the rating or grade is measured on a scale that comprises minimum and maximum possible scores for a result.
  • ESLI algorithm refers to a mathematical formula for numerically characterizing Ep-ICD sub-cellular expression by determining a value (i.e., an Ep-ICD Subcellular Localization Index "ESLI" value).
  • ESLI Ep-ICD Subcellular Localization Index
  • Prognosis refers to a prediction of the probable course and/or outcome of a disease. For example, a poor prognosis may predict a reduced DFS in a patient relative to a patient having a good prognosis. For example, a poor prognosis would predict a DFS of less than about five years and a favourable or good prognosis would predict a DFS of more than about five years.
  • the inventors have found that breast cancer patients having a poor prognosis have breast tissue comprising an increased amount of Ep-ICD, in particular increased nuclear Ep-ICD, relative to breast cancer patients having a favorable prognosis.
  • Methods prognosing breast cancer comprising one or more of detecting, measuring, scoring and evaluating subcellular localization of Ep-ICD are discussed further below.
  • the invention provides a numerical scoring method to quantify prognosis, such scoring method is referred to herein as the Ep-ICD Subcellular Localization Index (ESLI).
  • ESLI Ep-ICD Subcellular Localization Index
  • the present disclosure is generally directed to a method for prognosing cancer, in particular breast cancer, in a subject.
  • the subject also referred to herein as a patient, may be a mammal that is afflicted with, suspected of having, at risk for pre-disposal to, or being screened for breast cancer.
  • the subject is a human.
  • an amount of nuclear and/or cytoplasmic Ep-ICD is measured in a biological sample from the subject.
  • the biological sample comprises breast epithelial cells.
  • the biological sample comprises breast tissue.
  • the biological sample comprises breast cancer tumor cells, such as, for example, stage I and/or II breast cancer tumor cells.
  • Measurement of Ep-ICD may be quantitative and/or qualitative. In one embodiment, measurement may be achieved by contacting the biological sample with a first binding agent and measuring in one or more nuclei and/or cytoplasms of the biological sample the amount of the first binding agent bound to Ep-ICD. In one embodiment, an amount of membranous EpEx is measured in a biological sample from the subject.
  • Measurement of EpEx may be achieved by contacting the biological sample with a second binding agent and measuring in one or more membranes of the biological sample the amount of the second binding agent bound to EpEx.
  • a binding agent refers to a substance that specifically binds to a specific polypeptide.
  • a binding agent may be, for example, an antibody, a ribosome, RNA, DNA, a polypeptide or an aptamer.
  • an antibody specifically reactive with Ep-ICD may be used to detect Ep-ICD in the biological sample and may be used to determine the subcellular localization of Ep-ICD (i.e., nuclear or
  • an Ep-ICD-specific antibody is used to detect Ep-ICD.
  • an EpEx-specific antibody is used to detect EpEx.
  • Binding agents specific for Ep-ICD or EpEx may be labelled with a detectable substance which facilitates identification in biological samples based upon the presence of the detectable substance.
  • detectable substances include, but are not limited to, the following: radioisotopes, fluorescent labels, luminescent labels, bioluminescent labels, enzymatic labels, biotinyl groups, and predetermined polypeptide epitopes recognized by a secondary reporter.
  • Binding agents may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualized by electron microscopy.
  • Indirect methods may also be employed in which a primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against an epitope of the target polypeptide.
  • a primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against an epitope of the target polypeptide.
  • the second antibody may be goat anti-rabbit IgG, Fc fragment specific antibody labelled with a detectable substance, as described herein.
  • Ep-ICD and/or EpEx may be automated or it may be done manually.
  • quantitative and/or qualitative measurement of Ep-ICD may be automated using software, such as, for example, VisiopharmTM software.
  • VisiopharmTM software For example, the inventors have scanned IHC-treated breast cancer tissue samples using a NanoZoomer at 20x magnification. The scanned Images were loaded onto the Visiopharm Integrator System (VIS, version 4.6.3.857; Visiopharm, Hoersholm, Denmark) for digital analysis. Regions-of-interest (ROI) were manually drawn on each digital image. Regions within the ROIs were analyzed by the VIS to measure 3,3'-Diaminobenzidine (DAB) staining in epithelial cells in the nuclei, cytoplasm and/or membrane and to measure the intensity of staining. Results of this analysis were then used to stratify patients based on their risk for disease reoccurrence.
  • DAB 3,3'-Diaminobenzidine
  • Ep-ICD nuclear and cytoplasmic Ep-ICD quantitative and qualitative measurements
  • the control is an amount of nuclear Ep-ICD in a non- aggressive cancerous biological sample, for example, a non-aggressive cancerous breast tissue or a sample comprising non-aggressive cancerous breast epithelial cells.
  • a higher detected amount of nuclear Ep-ICD in the biological sample relative to the control indicates a poor prognosis of breast cancer and an equal or lower detected amount of nuclear Ep-ICD in the biological sample relative to the control indicates a favorable prognosis.
  • the control is the amount of nuclear Ep-ICD in a biological sample known not to progress to breast cancer for at least 40 months following measurement of the control amount.
  • a higher detected amount of nuclear Ep-ICD in the biological sample relative to the control indicates a poor prognosis
  • an equal or lower detected amount of nuclear Ep-ICD in the biological sample relative to the control indicates a favorable prognosis.
  • the control is an amount of nuclear Ep-ICD in an aggressive cancerous biological sample, for example, an aggressive breast tumor or a sample comprising aggressive cancerous breast epithelial cells.
  • an equal or higher detected amount of nuclear Ep-ICD in the biological sample relative to the control indicates a poor prognosis of breast cancer.
  • the control is the amount of nuclear Ep-ICD in a biological sample known to progress to breast cancer in less than about five years following measurement of the control amount.
  • an equal or higher detected amount of nuclear Ep-ICD in the biological sample relative to the control indicates a poor prognosis.
  • the breast cancer prognosed using the method provided herein is invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), invasive mucinous carcinoma (IMC), ductal carcinoma in situ (DCIS) or lobular carcinoma in situ (LCIS).
  • IDC invasive ductal carcinoma
  • IMC invasive lobular carcinoma
  • IMC invasive mucinous carcinoma
  • DCIS ductal carcinoma in situ
  • LCIS lobular carcinoma in situ
  • a method for prognosing breast cancer in a subject comprises determining quantitative and qualitative scores corresponding to the amounts of nuclear Ep-ICD and cytoplasmic Ep-ICD.
  • the quantitative and qualitative nuclear and cytoplasmic Ep-ICD scores are calculated and compared to control values for determining the poor prognosis of breast cancer in a subject.
  • the method may further comprise a step of calculating an Ep-ICD Subcellular Localization Index (ESLI) value for a sample obtained from the subject.
  • ESLI Ep-ICD Subcellular Localization Index
  • the present inventors developed the ESLI algorithm by: i) examining subcellular localization of Ep-ICD in samples from subjects having healthy breasts and various stages of breast cancer; ii) determining associations between Ep-ICD subcellular localization and DFS times in breast cancer patients; iii) determining that both quantitative and qualitative measurement of subcellular localization of Ep-ICD provided useful prognostic information; iv) generating an algorithm for using the quantitative and qualitative data to calculate a value with prognostic significance; and v) generating scales and equations for use in the algorithm, wherein the scales are appropriate for scoring the quantitative and qualitative data and weighting the quantitative and qualitative data with respect to one another.
  • the combination of collecting quantitative and qualitative data regarding Ep-ICD subcellular localization in a breast tissue sample, applying the ESLI algorithm to the collected data to generate and ESLI value for the sample, comparing the ESLI value of the sample to a reference value facilitates prognosis of prognosis of breast cancer in subjects.
  • the quantitative and qualitative data are collected from tissue samples prepared for IHC.
  • Ep-ICD and cytoplasmic Ep-ICD scores are determined for a breast tissue sample obtained from a subject.
  • the breast tissue sample would comprise cells (e.g., epithelial cells), each of such cells having a nucleus and cytoplasm.
  • determination of quantitative and qualitative nuclear Ep-ICD and cytoplasmic Ep-ICD scores is done manually and comprises the following four steps.
  • the sample is contacted with a binding agent that specifically binds to Ep-ICD or part thereof.
  • a detectable label is used to detect binding of the binding agent to Ep-ICD.
  • the detectable label may, for example, emit a detectable signal upon binding of the binding agent to Ep-ICD.
  • the binding agent may be a labelled antibody specific to Ep-ICD.
  • the label may be chosen from, for example, detectable radioisotopes, luminescent compounds, fluorescent compounds, enzymatic labels, biotinyl groups and predetermined polypeptide epitopes recognizable by a secondary reporter.
  • Subcellular localization of Ep-ICD is measured quantitatively and scored based on the percentages of cells in a tissue sample that are positive for Ep-ICD in (a) their nucleus and (b) their cytoplasm.
  • the percentage of cells in a tissue sample that are positive for nuclear Ep-ICD expression is referred to as the "first percentage”.
  • the first percentage is then assigned a score according to a scale that correlates percentage ranges with integer values. Such score and scale are referred to as the "first quantitative score” and "first scale”.
  • the percentage of cells in a measured tissue sample that are positive for cytoplasmic Ep- ICD expression is referred to as the "second percentage”.
  • the second percentage is then assigned a "second quantitative score" according to the first scale.
  • the first percentage i.e., the percentage positive for nuclear Ep- ICD
  • the second percentage i.e., the percentage positive for cytoplasmic Ep-ICD
  • first scale when less than 10% of cells are positive a score of 0 is assigned; when 10 - 30% cells are positive a score of 1 is assigned; when 31 - 50% cells are positive a score of 2 is assigned; when 51 - 70% of cells are positive a score of 3 is assigned; and when more than 70% of cells are positive a score of 4 is assigned.
  • first scale is used only for convenience and is provided as an example. Various other scaling methods can also be used.
  • the first and second percentages are obtained from tissue samples prepared for IHC.
  • Immunohistochemistry is a known method for IHC.
  • IHC comprises fixing and embedding a tissue sample, sectioning the tissue, mounting the tissue section, deparaffinizing and rehydrating the section, antigen retrieval, immunohistochemical staining, optional counterstaining, dehydrating and stabilizing with mounting medium, and viewing the stained section under a microscope.
  • a cell that is positive for nuclear and/or cytoplasmic Ep-ICD is one that is
  • immunopositive i.e., a cell comprising staining or fluorescence that is detectable upon microscopic examination and indicative of the Ep-ICD-specific antibody used in IHC of the sample.
  • (iii)Subcellular localization of Ep-ICD is measured qualitatively and scored based on the intensity of the signals emitted by a detectable label of an Ep-ICD binding agent in (a) the nucleus and (b) the cytoplasm of cells in the tissue sample.
  • the intensity of the signal detected in the nucleus of the cells in the tissue is referred to as the "first intensity”.
  • the first intensity is then assigned a score according to a scale that correlates a categorical assessment of signal intensity (e.g., categories ranging from zero detectable signal to a maximum or near maximum detected signal) with integer values.
  • Such score and scale are referred to as the "first qualitative score” and "second scale”.
  • the intensity of the signal detected in the cytoplasm of the cells in the tissue is referred to as the "second intensity”.
  • the second intensity is then assigned a "second qualitative" score according to the second scale.
  • the first intensity i.e., the categorical assessment of nuclear Ep- ICD binding agent signal emission
  • the second intensity i.e., the categorical assessment of cytoplasmic Ep-ICD binding agent signal emission
  • the first intensity i.e., the categorical assessment of nuclear Ep- ICD binding agent signal emission
  • the second intensity i.e., the categorical assessment of cytoplasmic Ep-ICD binding agent signal emission
  • the first intensity i.e., the categorical assessment of nuclear Ep- ICD binding agent signal emission
  • the second intensity i.e., the categorical assessment of cytoplasmic Ep-ICD binding agent signal emission
  • the first and second intensities are obtained using IHC analysis.
  • the antibody-antigen interaction i.e., the anti-Ep- ICD-Ep-ICD interaction
  • chromogenic detection in which an enzyme conjugated to the antibody cleaves a substrate to produce a colored precipitate at the location of the protein.
  • the antibody-antigen interaction is visualized using fluorescent detection, in which a fluorophore is conjugated to the antibody and the location of the fluorophore can be visualized using fluorescence microscopy.
  • a total nuclear Ep-ICD score and a total cytoplasmic Ep-ICD score are calculated by adding the first quantitative and qualitative scores to generate the total nuclear Ep-ICD score and adding the second quantitative and qualitative scores to generate the total cytoplasmic Ep-ICD score.
  • an Ep-ICD Subcellular Localization Index (ESLI) value for the sample is calculated.
  • the ESLI value is the sum of the total nuclear Ep-ICD score and the total cytoplasmic Ep-ICD score.
  • the ESLI value is the sum of the nuclear Ep-ICD score and the cytoplasmic Ep-ICD score, divided by two (such arithmetic function being for convenience).
  • the calculated ESLI value is then compared to a reference value in order to determine a prognosis of breast cancer in the subject.
  • the reference value is a pre- determined cut-off value, wherein values on one side of the cut off value indicate a poor prognosis of breast cancer and vales on the other side of the cut-off value indicate a favourable prognosis of breast cancer.
  • the reference value is an ESLI value indicative of a non- aggressive cancerous breast tissue.
  • a poor prognosis of breast cancer in the subject is determined when the calculated ESLI value of the sample is greater than the reference value.
  • a favourable prognosis of breast cancer is determined when the calculated ESLI value of the sample is less than or equal to the reference value.
  • the reference value is an ESLI value indicative of an aggressive breast cancer.
  • the sample may be obtained from an aggressive breast tumour tissue.
  • a poor prognosis of breast cancer in the subject is determined when the calculated ESLI value of the sample is greater than or equal to the reference value.
  • the reference value is determined by retrospectively analyzing a plurality of breast cancer patients' tissue samples and corresponding patient clinical data regarding time of DFS.
  • an ESLI value is calculated using total nuclear and cytoplasmic Ep-ICD scores generated according to the aforementioned first and second scales (i.e., 0-4 and 0-3 for percentage positivity and intensity, respectively)
  • a finding of an ESLI value of greater than or equal to 3 is indicative of aggressive breast cancer and a poor prognosis of breast cancer.
  • a method for detecting abnormal subcellular localization of Ep-ICD in a breast tissue sample obtained from a subject comprises measuring an amount of nuclear Ep-ICD in a biological sample from the subject, comparing the amount detected in the biological sample to a control; and detection of abnormal subcellular localization of Ep-ICD in the breast the breast tissue sample is made based on the comparison between the detected amount of nuclear Ep-ICD and the control. Measurement may be quantitative and/or qualitative, as described herein.
  • the control may be a non-aggressive or aggressive breast cancer, as described herein. Detection of an abnormal subcellular localization of Ep-ICD in a breast tissue sample is found when the measured amount of Ep-ICD is greater than that of the non-aggressive control or greater than or equal to that of the aggressive control.
  • the method for detecting abnormal subcellular localization of Ep-ICD in a breast tissue sample obtained from a subject comprises the steps of (A) measuring nuclear and cytoplasmic Ep-ICD scores for the sample, (B) calculating an ESLI value for the sample and (C) comparing the calculated ESLI value to a reference value.
  • the measuring and calculating steps may be carried out as discussed above with respect to breast cancer prognosis.
  • abnormal subcellular localization of Ep-ICD in the breast tissue sample is detected when the calculated ESLI value of the sample is greater than a reference value corresponding to an ESLI value indicative of a non-aggressive cancerous breast tissue; or when the calculated ESLI value of the sample is greater than or equal to a reference value corresponding to an ESLI value indicative of an aggressive breast cancer.
  • kits for carrying out the methods disclosed herein.
  • Such kits typically comprise two or more components required for performing a prognostic breast cancer assay.
  • Components include but are not limited to one or more of compounds, reagents, containers, equipment and instructions for using the kit. Accordingly, the methods described herein may be performed by utilizing pre-packaged prognostic kits provided herein.
  • the kit comprises one or more of binding agents, standards, stains, fixatives and instructions.
  • the instructions comprise one or more reference values for use as controls.
  • the kit comprises one or more binding agents as described herein for prognosing breast cancer.
  • the kit may contain antibodies specific for Ep-ICD, antibodies against the Ep-ICD antibodies labelled with an enzyme(s), and a substrate for the enzyme(s).
  • the kit may further contain antibodies specific for EpEX, antibodies against the EpEX antibodies labelled with an enzyme(s), and a substrate for the enzyme(s).
  • the kit may also contain one or more of microtiter plates, reagents (e.g., standards, buffers), adhesive plate covers, and instructions for carrying out a method using the kit.
  • the kit comprises antibodies or antibody fragments which bind specifically to epitopes of Ep-ICD and means for detecting binding of the antibodies to their epitopes associated with breast cancer cells, either as concentrates (including lyophilized compositions), which may be further diluted prior to testing.
  • a kit for prognosing breast cancer may contain a known amount of a first binding agent that specifically binds to Ep-ICD, wherein the first specific binding agent comprises a detectable substance or has the capacity to bind directly or indirectly to a detectable substance.
  • the kit further comprises antibodies or antibody fragments which bind specifically to epitopes of EpEX and means for detecting binding of the EpEX-specific antibodies to their epitopes associated with breast cancer cells, either as concentrates (including lyophilized compositions), which may be further diluted prior to testing.
  • the kit comprises one or more binding agents, standards, stains, fixatives and instructions for measuring nuclear Ep-ICD and optionally membrane EpEX.
  • a kit comprising such binding agents, standards, stains fixatives and instructions may be used to practice methods disclosed herein.
  • the kit can be used to practice a method disclosed herein that comprises IHC.
  • the kit may further comprise tools useful for collecting biological samples (e.g. a breast tissue sample).
  • biological samples e.g. a breast tissue sample.
  • the patient cohort consists of 266 breast cancer patients treated at Mount Sinai Hospital (MSH) between 2000 and 2007.
  • the cohort consists of patients who had mastectomy or lumpectomy.
  • Inclusion criteria Breast cancer tissue samples of patients who had up to 60 months follow-up with or without an adverse clinical event; availability of clinical, pathological and treatment data in the clinical database.
  • Ep-ICD monoclonal antibody from Epitomics Inc. (Burlingame, CA) was used in this study.
  • the a-Ep-ICD antibody 1 144 recognizes the cytoplasmic domain of human EpCAM and has been used in our previous study of Ep-ICD expression in thyroid carcinoma and other epithelial cancers [Ralhan et al., BMC Cancer 2010].
  • Anti-EpCAM monoclonal antibody EpEx (MOC-31 , AbD Serotec, Oxford, UK) recognizes an extracellular component (EGF1 domain- aa 27-59) in the amino-terminal region (Chaudry et al., Br J Cancer 2007 , 96(7):1013-1019).
  • the sections were incubated with either a-Ep-ICD rabbit monoclonal antibody 1 144 (dilution 1 :1500) or mouse monoclonal antibody MOC-31 (dilution 1 :200) for 60 minutes, followed by biotinylated secondary antibody (goat anti-rabbit or goat anti-mouse) for 20 minutes.
  • the sections were finally incubated with VECTASTAIN Elite ABC Reagent (Vector Laboratories, Burlington, ON, Canada) and diaminobenzidine was used as the chromogen.
  • Tissue sections were then counterstained with hematoxylin.
  • Negative controls comprised of breast tissue sections incubated with isotype specific IgG in place of the primary antibody, and positive controls (colon cancer tissue sections known to express Ep- ICD) were included with each batch of staining for both Ep-ICD and EpEx.
  • Sections were also scored qualitatively on the basis of intensity of staining as follows: 0, none; 1 , mild; 2, moderate; and 3, intense.
  • a total score (ranging from 0 to 7) for each tissue section was obtained by adding the scores of percentage positivity and intensity for each of the breast cancer tissue sections. The average total score from the five areas was used for further statistical analysis. Each tissue section was scored for cytoplasmic and nuclear Ep-ICD as well as for membrane EpEx following the aforementioned percentage positivity and intensity scales.
  • Statistical analysis of IHC data [00100] The IHC data were subjected to statistical analysis with SPSS 21.0 software (SPSS, Chicago, IL) and GraphPad Prism 6.02 software (GraphPad Software, La Jolla, CA) as described previously (Ralhan et al., Mol Cell Proteomics 2008, 7(6) :1 162-1 173].
  • ESLI 1 ⁇ 2 x (%positivity score of nuclear Ep-ICD + intensity score of nuclear Ep-ICD + %positivity score of cytoplasm Ep-ICD + intensity score of cytoplasm Ep-ICD).
  • the %positivity score comprises a score on a scale of 0 to 4 and the intensity score comprises a score on a scale of 0 to 3.
  • An ESLI cutoff value of 3 was found to be useful for distinguishing between samples from patients having good and poor prognoses. For example, an ESLI value of > 3 was considered a "positive" result and indicative of a poor breast cancer prognosis and an ESLI value of ⁇ 3 was considered a "negative" result and indicative of a good prognosis of breast cancer.
  • the clinicopathological parameters and treatment details of 266 breast carcinomas, including 180 IDC cases and 45 normal controls are summarized in Table 1.
  • the median age of patients was 59.9 years (range 30.6 - 89.8 years).
  • AJCC pTNM Stage I (35.3%) and II (32.7%) comprised a large proportion of tumors in this cohort.
  • Tumor grades distribution was Grade I - 21.1%; II - 39.8%, and III - 32.0%.
  • the IDC cases comprised of Grade I - 23.3%; Grade II - 36.7%; and Grade III - 36.1 % tumors.
  • Table 1 Clinicopathological characteristics of breast cancer patients.
  • IDC 180 75 (42%) 145 (81 %) 128 (71 %) 52 (29%)
  • Nuclear Ep-ICD was more frequently expressed in breast cancers as compared to normal tissues. Significant association was observed between increased nuclear Ep-ICD expression and reduced disease-free survival in patients with ductal carcinoma in situ
  • Ep-ICD nuclear and cytoplasmic Ep-ICD expression in ten different epithelial cancers, including breast cancers.
  • the previous report did not examine the correlation of nuclear Ep-ICD expression with clinical parameters or its prognostic utility in the ten epithelial cancers, including breast cancer.
  • the current study assessed the suitability of Ep-ICD as a marker for predicting prognosis of breast cancer.
  • expression of the full length EpCAM protein has been widely investigated in human malignancies, the expression and subcellular localization of its intracellular domain, Ep-ICD, has not been well-characterized in clinical specimens. The present data indicate that there are significant differences in Ep-ICD expression in normal relative to malignant breast tissues and in non-aggressive relative to aggressive breast cancers.

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Abstract

L'invention concerne des méthodes et des trousses pour le pronostic du cancer du sein consistant à mesurer les polypeptides Ep-ICD. La mesure peut être quantitative et/ou qualitative. L'invention concerne également un système de génération d'une valeur-indice de localisation infracellulaire d'EP-ICD (ESLI), qui peut être utilisée pour pronostiquer un cancer du sein chez un sujet.
EP14867931.9A 2013-12-06 2014-12-05 Méthodes de pronostic du cancer du sein Withdrawn EP3077812A4 (fr)

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US14/099,529 US20140187438A1 (en) 2010-05-04 2013-12-06 Methods and Compositions for the Diagnosis and Treatment of Epithelial Cancers
US14/501,020 US20150094224A1 (en) 2010-05-04 2014-09-29 Methods for the diagnosis or prognosis of breast cancer
PCT/CA2014/051176 WO2015081446A1 (fr) 2013-12-06 2014-12-05 Méthodes de pronostic du cancer du sein

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