WO2015010596A1 - 一种乳腺癌预后诊断的方法 - Google Patents

一种乳腺癌预后诊断的方法 Download PDF

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WO2015010596A1
WO2015010596A1 PCT/CN2014/082680 CN2014082680W WO2015010596A1 WO 2015010596 A1 WO2015010596 A1 WO 2015010596A1 CN 2014082680 W CN2014082680 W CN 2014082680W WO 2015010596 A1 WO2015010596 A1 WO 2015010596A1
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score
breast cancer
staining
cell membrane
cells
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French (fr)
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孟坤
王兆一
陈凤
白伟
王培培
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北京盛诺基医药科技有限公司
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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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • 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 invention relates to a method for prognosis diagnosis of breast cancer, belonging to the field of medical diagnosis. Background technique
  • ER-a36 is a novel estrogen receptor discovered in recent years, mainly in the cell membrane and cytoplasm, which can initiate a rapid estrogen signaling pathway.
  • the rapid response of estrogen typically activates, for example, MARK/ERK, phosphatidylinositol-3-kinase, and protein kinase C.
  • MARK/ERK phosphatidylinositol-3-kinase
  • protein kinase C protein kinase C.
  • ER-a36 is highly expressed in breast cancer cells and promotes the proliferation of breast cancer cells.
  • ER-a36 has recently been found to be highly expressed in various tumor cells and plays an important role in the development of tumors.
  • Tamoxifen has been used as a standard drug for endocrine therapy of estrogen receptor (ER-a66)-positive breast tumors for many years. However, most patients with advanced breast cancer eventually have a poor prognosis after receiving tamoxifen. In “Journal of Clinical Oncology”, the title “Expression of ER-a36, a Novel Variant of Estrogen Receptor, and Resistance to Tamoxifen Treatment in Breast Cancer” was published. In this article, it is proposed that in patients with breast cancer who are positive for both estrogen receptors ER-a36 and ER-a66, treatment with tamoxifen leads to a significant decrease in survival.
  • One aspect of the present invention provides a method for prognosis diagnosis of breast cancer, which comprises detecting the expression level of ER-a36 in a tumor specimen of a breast cancer patient, and when the ER-a36 is highly expressed, indicating a poor prognosis of the breast cancer patient.
  • the poor prognosis of the breast cancer patient indicates a breast cancer patient is at risk of breast cancer recurrence and death.
  • the risk of breast cancer recurrence comprises in situ recurrence and metastasis recurrence of breast cancer.
  • the expression level of ER-a36 in the tumor specimen tissue of the breast cancer patient is detected by an immunohistochemical method.
  • ER-a36 is highly expressed such that ER-a36 has an expression value of 5 or more by immunohistochemical scoring criteria.
  • the expression value of ER-a36 is obtained by the following immunohistochemical scoring criteria: cell membrane staining intensity score multiplied by cell membrane staining score + cytoplasmic staining intensity score multiplied by cytoplasmic staining score;
  • the cell membrane staining score is 0% corresponding to the cell membrane staining cell ratio of 0; the cell membrane staining cell ratio 1-24% corresponds to a score of 1, and the cell membrane staining cell ratio is 25-49% corresponding to a score of 2
  • the ratio of cell membrane staining cells to 50-74% corresponds to a score of 3; the ratio of cell membrane stained cells to 75-100% corresponds to a score of 4;
  • the cytoplasmic staining score is 0% corresponding to the proportion of cytoplasmic staining cells; the score corresponding to cytoplasmic staining cells is 1-24%, and the score of cytoplasmic staining cells is 25-49%.
  • the cytoplasmic staining cell ratio of 50-74% corresponds to a score of 3; the cytoplasmic stained cell ratio of 75-100% corresponds to a score of 4.
  • the high expression of ER-a36 indicates that the expression amount of ER-a36 in the tumor tissue of the patient exceeds the expression level of the tumor tissue intermediate cells or the cancer surrounding normal tissues.
  • the tumor specimen tissue is obtained after treatment of the surgical or perforated breast tumor tissue.
  • the antibody that specifically recognizes ER-a36 interacts with the tumor specimen tissue to detect The expression level of ER-a36 in tumor specimen tissues.
  • the ER-a36-recognizing antibody is a murine or rabbit-derived antibody.
  • the tumor specimen tissue contains malignant tumor cells.
  • the breast cancer patient comprises a drug-treated breast cancer patient.
  • said drug-treated breast cancer patient comprises a breast cancer patient treated with tamoxifen.
  • the prognosis diagnosis method for breast cancer of the present invention proves that the detection method of the present invention has high accuracy by detecting the marker ER-a36 in the tumor specimen tissue through more than 1000 clinical cases.
  • the method for prognosis diagnosis of breast cancer of the present invention can help determine the prognosis of breast cancer patients by the expression level of ER-a36 in breast cancer patients or breast cancer patients treated with tamoxifen, and provides guidance for clinical treatment.
  • Figure 1 shows a comparison of overall survival and disease-free survival in patients with positive or negative breast cancer after ER-a36 expression, respectively.
  • Figure 1A shows ER-a36, ER-a66 double positive patients after surgery. Overall survival in patients with ER-a66-positive and ER-a36-negative patients;
  • Figure 1B shows disease-free survival in patients with ER-a66, ER-a36 double-positive and ER-a66-positive, ER-a36-negative patients after surgery
  • Figure 1C shows the overall survival of patients with ER-a66, ER-a36 double-negative and ER-a66-positive ER-a36-negative after surgery;
  • Figure 1D shows ER-a66, ER-a36 after surgery Disease-free survival in negative patients and E-a66-negative, ER-a36-positive patients.
  • Figure 2 shows the effect of tamoxifen on the proliferation of breast cancer cells with different expression of ER-a36, wherein Figure 2A shows that MCF-7 breast cancer cells with high expression of ER-a36 are relatively blank under the action of tamoxifen. The relative growth rate of breast cancer cells in the control group; Figure 2B shows the relative growth rate of MCF-7 breast cancer cells with low expression of ER-a36 relative to the blank control group under the action of tamoxifen.
  • Tumor refers to the abnormal regulation of the clonal dysplasia caused by the loss of normal regulation of the growth of a certain cell in the local tissue at the gene level under various factors. Academics generally divide tumors into benign and malignant categories.
  • patient refers to a patient from China who has undergone radical mastectomy or partial eradication mastectomy.
  • prognosis of breast cancer refers to predicting the condition progression of breast cancer patients.
  • metastasis recurrence means that cancer cells that are identical to the original cancer cells are again found in areas other than the region where the breast cancer was originally diagnosed during the recovery or remission phase of the breast cancer.
  • tumor specimen tissue refers to breast tumor tissue obtained by surgery or perforation, which is then fixed, dehydrated, transparent and immersed in wax, embedded in paraffin, and then paraffin sectioned for tumors for immunohistochemistry. Specimen organization.
  • immunohistochemistry refers to the application of the basic principle of immunology, that is, the principle of specific binding of an antigen to an antibody.
  • the chemical reaction of the coloring agent of the labeled antibody is used to determine the antigen in the tissue cell, and to locate and characterize the antigen. the study.
  • immunohistochemical scoring criteria refers to the first method by immunohistochemistry according to the following formula
  • ER-a36 expression cell membrane staining intensity score multiplied by cell membrane staining score + cytoplasmic staining intensity score multiplied by cytoplasmic staining score;
  • the score is the score of the intensity of staining on the cytoplasm;
  • the cell membrane staining score is 0% corresponding to the cell membrane staining cell ratio, 0; the cell membrane staining cell ratio 1-24% corresponds to a score of 1; the cell membrane staining cell ratio is 25-49%, and the corresponding score is 2
  • the proportion of cell membrane stained cells is 50-74% corresponding to a score of 3; the proportion of cell membrane stained cells is 75-100%, and the corresponding score is 4;
  • the cytoplasmic staining score is 0% corresponding to the proportion of cytoplasmic staining cells; the score corresponding to 1-24% of cytoplasmic staining cells is 1; the ratio of cytoplasmic staining cells is 25-49% corresponding to 2
  • the proportion of cytoplasmic stained cells is 50-74% corresponding to a score of 3; the proportion of cytoplasmic stained cells is 75-100%, and the corresponding score is 4.
  • metastasis refers to the invasion of tumor cells from the primary site into lymphatic vessels, blood vessels or other organs, forming tumors of the same type in the primary site of the tumor, a process known as metastasis.
  • piercing means that a suspicious lesion of the breast (eg, a mass, a thickened area, a calcified foci, etc.) is removed from the breast by means of a hollow needle.
  • a suspicious lesion of the breast eg, a mass, a thickened area, a calcified foci, etc.
  • ER-a36 positive expression of ER-a36 means that the ER-a36 value calculated by the formula (I) is 5 or more by immunohistochemistry.
  • ER-a36 negative expression of ER-a36 means that the ER-a36 value calculated by the formula (I) is less than 5 and does not contain 5 by immunohistochemistry.
  • ER-a66 expression is negative can be determined by immunohistochemistry according to the existing WHO breast cancer scoring standard.
  • malignant tumor cell is a variant cell. Unlike normal cells, it is destroyed by normal tissue cells and destroys the function of normal tissues due to its infinite growth, transformation and metastasis.
  • drug-treated breast cancer patients refers to breast cancer patients who have been treated with breast cancer by oral, intramuscular, or intravenous drug therapy.
  • mouse-derived antibody is an antibody obtained by collecting purified cells from the ascites by transplanting hybridoma cells into the peritoneal cavity of a mouse and producing ascites.
  • rabbit-derived antibody is an antibody obtained by collecting purified cells from the ascites by transplanting hybridoma cells into the abdominal cavity of a rabbit and producing ascites.
  • all survival rate refers to the proportion of patients who survive in a given time period.
  • disease-free survival refers to the proportion of patients who have not relapsed within a specified time period.
  • the ER-a36 antibody provided below includes at least one of SNGmBl antibodies, for example, 2, 3, 4, 5 or all 6 CDR regions.
  • the "SNGmBl antibody” provided herein is a murine monoclonal antibody against ER-a36.
  • the amino acid sequence includes the light chain of SEQ ID: 1 and the heavy chain sequence of amino acid sequence SEQ ID NO: 2.
  • the nucleotide sequences encoding the light and heavy chains of the SNGmB1 antibody are SEQ ID NO: 17 antibody and SEQ ID NO: 19, respectively, and the amino acid sequences of the light and heavy chains are shown below, wherein the CDR regions are italicized and underlined, and The fixed area is an italic shaded portion.
  • amino acid sequence of the SNGmBl heavy chain (SEQ ID NO: 2):
  • the ER-a36 antibody herein comprises SEQ ID NO: 8, (B: heavy chain CDR3 sequence of SNGmBl antibody:).
  • the heavy chain CDR3 region is located at the center of the antigen binding region and is therefore the easiest to bind antigen.
  • the antibody of ER-a36 comprises the heavy chain variable region of the SNGmB1 antibody, ie, the heavy chain SEQ ID NO: 10 ; and/or comprises the light chain variable region of the SNGmB1 antibody, gpSEQ ID NO: 9.
  • the tumor tissue obtained by surgery or perforation is immersed in a fixative, and then the tissue is fully dehydrated with a concentration gradient ethanol.
  • the dehydrated tissue is then paraffin-embedded by xylene transparent and paraffin wax, and the wax block is solidified. After that, cut into pathological sections of 4 ⁇ m thick.
  • the slices were baked in an oven at 65 ° C for 2 hours, soaked in xylene twice for 10 minutes to remove paraffin, then soaked in 100% ethanol for 3 minutes twice, 95% ethanol for 3 minutes and Soak in 75% ethanol for 3 minutes, and finally wash 5 times with phosphate buffer (PBS) for 2 minutes each time.
  • PBS phosphate buffer
  • the antigen was repaired by high pressure repair method, and the section was completely immersed in 1 mmol of disodium edetate (EDTA) pH 8.0 antigen repair solution, heated to boiling in high pressure, and the time was started after the pressure cooker was added to the pressure cooker, 2 minutes 30 After the second, the autoclave was naturally cooled to room temperature, and the sections were taken out, and the phosphate buffer (PBS) was washed 5 times for 2 minutes each time. The sections were placed in a reaction dish containing a freshly prepared peroxidase blocking solution and incubated at 37 ° C for 30 minutes in an oven to eliminate endogenous peroxidase activity.
  • EDTA disodium edetate
  • PBS phosphate buffer
  • phosphate buffer 5% goat serum was added to the tissue area of the immunohistochemical pen for 30 minutes in a 37 ° C oven to block non-specific antigen. Carefully remove and wipe off excess liquid on and around the tissue, add primary antibody working solution (primary antibody is Example 1 antibody, concentration 0.02mg/ml) 4 °C overnight (preferably 16 hours to 18 hours), Rewarrate at room temperature for 30 minutes. Rinse in phosphate buffer (PBS) 5 times for 2 minutes each time. Add appropriate amount of horseradish peroxidase-labeled secondary antibody working solution and incubate at 37 °C for 30 minutes. Rinse the phosphate buffer (PBS) 5 times for 2 minutes each time.
  • ER-a36 of the tumor specimen of the patient was detected by the immunohistochemical routine method using the antibody of Example 1, and the expression value of ER-a36 in each pathological slice was calculated according to the formula (I).
  • ER-a36 expression cell membrane staining intensity score multiplied by cell membrane staining score + cytoplasmic staining intensity score multiplied by cytoplasmic staining score;
  • the cell membrane staining score is 0% corresponding to the cell membrane staining cell ratio of 0; the cell membrane staining cell ratio 1-24% corresponds to a score of 1, and the cell membrane staining cell ratio is 25-49% corresponding to a score of 2
  • the ratio of cell membrane staining cells to 50-74% corresponds to a score of 3; the ratio of cell membrane stained cells to 75-100% corresponds to a score of 4;
  • the cytoplasmic staining score is 0% corresponding to the proportion of cytoplasmic staining cells; the score corresponding to cytoplasmic staining cells is 1-24%, and the score of cytoplasmic staining cells is 25-49%.
  • the proportion of cytoplasmic stained cells is 50-74% corresponding to a score of 3; the proportion of cytoplasmic stained cells is 75-100%, and the corresponding score is 4.
  • the expression of ER-a36 was positive in patients with breast cancer with ER-a36 expression above 5, and the expression of ER-a36 was less than 5 (excluding 5), indicating that the patient's breast cancer ER-a36 expression was negative.
  • Example 4 Examination of the expression of ER-a36 in tumor tissues metastasized from breast cancer patients
  • Experimental method Tumor tissues of breast cancer patients obtained by surgery or perforation were selected, and the patients were determined by the method of epidemic and the method of Example 3. Expression of ER-a36 in breast cancer tissues. And to test whether these patients have lymph node metastasis.
  • Fig. 1A It can be seen from Fig. 1A that, in the ER-a66-positive patients after surgery, the overall survival rate of ER-a36 and ER-a66 positive patients is less than ER-a66-positive, ER with the increase of time after surgery. Survival rate of -a36 negative patients.
  • MCF-7 breast cancer cells were selected and obtained from the American Type Culture Collection (ATCC). Through the gene stabilization technology, the exogenous ER-a36 gene was transferred, and MCF-7 breast cancer cells were highly expressed ER-a36. Tamoxifen was added to the cell culture medium and cultured for 6 days. As can be seen from Fig. 2A, tamoxifen promoted the proliferation of MCF-7 breast cancer cells, and the MCF-7 mammary gland was promoted by tamoxifen. The relative growth rate of cancer cells is much greater than the relative growth rate of MCF-7 breast cancer cells in the blank control group.
  • MCF-7 breast cancer cells not transfected with ER-a36 were selected, which underexpressed ER-a36.
  • Tamoxifen was added to the cell culture medium and cultured for 6 days. As can be seen from Fig. 2B, tamoxifen did not promote the proliferation of MCF-7 breast cancer cells. Under the action of tamoxifen, the relative growth rate of MCF-7 breast cancer cells was smaller than that of the blank control group MCF-7 breast cancer cells.
  • tamoxifen does not inhibit the ER-a36 high expression of breast cancer cells, but promotes the proliferation of ER-a36 breast cancer cells.
  • tamoxifen does not promote proliferation of breast cancer cells with low expression of ER-a36. Therefore, tamoxifen may increase the risk of breast cancer cell proliferation and metastasis if used in the treatment of breast cancer with high expression of ER-a36.

Abstract

本发明提供了一种乳腺癌患者预后诊断的方法，该方法包括检测乳腺癌患者肿瘤标本组织中ER-α36的表达量，当ER-α36高表达时，表示患者具有乳腺癌原位复发或转移的风险。通过本发明方法，为乳腺癌患者的预后判断提供了指导意义。

Description

一种乳腺癌预后诊断的方法
技术领域
本发明涉及一种乳腺癌预后诊断的方法， 属于医学诊断学领域。 背景技术
ER-a36是近年来发现的一种新型***受体， 主要存在于细胞膜和细胞浆， 可以启动快速***信号通路。 ***的快速应答通常激活例如 MARK/ERK, 磷脂酰肌醇 -3-激酶以及蛋白激酶 C。 实验表明 ER-a36在乳腺癌细胞中高表达， 并且促进乳腺癌细胞的增殖。 此外， 近来发现 ER-a36在多种肿瘤细胞中高表达， 在肿瘤的发生发展中发挥重要作用。
在 WO2005087811专利文件中， 对于 ER-a36的结构、 获得方法及其用途做了 详细的描述。
他莫西芬多年来一直被用于***受体（ER-a66 )阳性的乳腺肿瘤的内分泌 治疗的标准药物。 然而大多数中晚期乳腺癌患者在接受他莫西芬治疗后最终会 出现预后不良。 在《Journal Of Clinical Oncology》杂志中发表了题为" Expression of ER-a36， a Novel Variant of Estrogen Receptor, and Resistance to Tamoxifen Treatment in Breast Cancer"。 在该文章中提出对于***受体 ER-a36和 ER-a66 同时为阳性的乳腺癌患者， 他莫西芬的治疗导致其生存期的明显下降。
因此， ER-a36在乳腺癌患者体内起什么作用， 成为了亟待解决的问题。 发明内容
本发明一个目的是提供一种乳腺癌预后诊断的方法。
本发明一方面提供了一种乳腺癌预后诊断的方法，该方法包括检测乳腺癌患 者肿瘤标本组织中 ER-a36的表达量， 当 ER-a36高表达时， 表示乳腺癌患者预后 不良。 优选地，其中所述的乳腺癌患者预后不良表示乳腺癌患者存在乳腺癌复发和 死亡的风险。
优选地， 其中所述的乳腺癌复发风险包括乳腺癌的原位复发和转移复发。 优选地， 其中通过免疫组化的方法检测乳腺癌患者肿瘤标本组织中 ER-a36 的表达量。
优选地， 其中所述的 ER-a36高表达为通过免疫组化评分标准得到 ER-a36的 表达值在 5以上。
优选地，其中 ER-a36的表达值为通过以下免疫组化的评分标准得出：细胞膜 染色强度分值乘以细胞膜染色的分值 +细胞质染色强度分值乘以细胞质染色的 分值；
所述的染色强度分值为未染色 =0; 弱染色 =1 ; 中等染色 =2; 强染色 =3， 并 且所述的细胞膜染色强度分值为在细胞膜上染色强度的分值， 细胞质染色强度 分值为在细胞质上染色强度的分值；
所述的细胞膜染色分值为细胞膜染色细胞比例 0%对应的分值为 0; 细胞膜染 色细胞比例 1-24%对应的分值为 1， 细胞膜染色细胞比例 25-49%对应的分值为 2; 细胞膜染色细胞比例 50-74%对应的分值为 3 ; 细胞膜染色细胞比例 75-100%对应 的分值为 4;
所述的细胞质染色分值为细胞质染色细胞比例 0%对应的分值为 0; 细胞质染 色细胞比例 1-24%对应的分值为 1， 细胞质染色细胞比例 25-49%对应的分值为 2; 细胞质染色细胞比例 50-74%对应的分值为 3 ; 细胞质染色细胞比例 75-100%对应 的分值为 4。
优选地， 其中所述的 ER-a36高表达表示患者肿瘤标本组织中的 ER-a36的表 达量超过肿瘤组织中***或肿瘤周围癌旁正常组织的表达量。
优选地，其中所述的肿瘤标本组织为手术或穿剌的乳腺肿瘤组织经处理后得 到。
优选地，其中通过特异性识别 ER-a36的抗体与肿瘤标本组织相互作用，检测 肿瘤标本组织中 ER-a36的表达量。
优选地， 其中所述的识别 ER-a36抗体为鼠源或兔源抗体。
优选地， 其中所述的肿瘤标本组织中含有恶性肿瘤细胞。
优选地， 其中所述的乳腺癌患者包括经过药物治疗的乳腺癌患者。
优选地，其中所述的经过药物治疗的乳腺癌患者包括经过他莫西芬治疗的乳 腺癌患者。
本发明的有益效果在于：本发明乳腺癌预后诊断方法通过检测肿瘤标本组织 中的标志物 ER-a36， 经过 1000例以上的临床病例证明本发明的检测方法准确率 高。
本发明乳腺癌预后诊断的方法通过乳腺癌患者或者他莫西芬治疗后乳腺癌 患者中 ER-a36的表达水平， 从而辅助判断乳腺癌患者的预后， 为临床治疗提供 了指导意义。 附图说明
图 1表示手术后的 ER-a36表达分别为阳性或者阴性乳腺癌病人的总体生存 率和无病生存率的比较， 其中图 1A表示在手术结束后， ER-a36、 ER-a66双阳 性患者和 ER-a66阳性、 ER-a36阴性患者的总体生存率； 图 1B表示在手术结束后， ER-a66、 ER-a36双阳性患者和 ER-a66阳性、 ER-a36阴性的患者的无病生存率； 图 1C表示在手术结束后， ER-a66、 ER-a36双阴性患者和 ER-a66阳性 ER-a36阴性 的患者的总体生存率； 图 1D表示手术结束后， ER-a66、 ER-a36双阴性患者和 E -a66阴性、 ER-a36阳性患者的无病生存率。
图 2表示他莫西芬对 ER-a36表达不同的乳腺癌细胞增殖的促进作用， 其中， 图 2A表示在他莫西芬作用下， ER-a36高表达的 MCF-7乳腺癌细胞相对于空白对 照组的乳腺癌细胞的相对生长率； 图 2B表示他莫西芬作用下， ER-a36低表达的 MCF-7乳腺癌细胞相对于空白对照组的相对生长率。 具体实施方式
以下实施例仅用于对本发明进行示例性说明， 不用于限制本发明， 在本发 明保护范围内所做的修改、 改变、 变型等都在本发明的保护范围内。
术语： "肿瘤 "指的是机体在各种因素作用下， 局部组织的某一个细胞在基 因水平上失去对其生长的正常调控， 导致其克隆性异常增生而形成的异常病变。 学界一般将肿瘤分为良性和恶性两大类。
术语"患者"指的是来自中国国内的经过根除性***切除术或者部分根除性 ***切除术的病人。
术语"乳腺癌预后"表示预测乳腺癌患者的病情发展状况。
术语： "乳腺癌的原位复发 "表示在乳腺癌初愈或缓解阶段， 在乳腺癌最初 患病的区域再次出现与原有癌细胞相同的癌症细胞。
术语"转移复发"表示在乳腺癌初愈或缓解阶段， 在乳腺癌最初患病的区域 以外的其他区域再次发现与原有癌细胞相同的癌细胞。
术语"肿瘤标本组织 "指的是通过手术或穿剌获得的乳腺肿瘤组织， 该组织 接着进行固定、 脱水、 透明与浸蜡、 石蜡包埋和再经石蜡切片获得用于免疫组 化检测的肿瘤标本组织。
术语"免疫组化"指的是应用免疫学基本原理， 即抗原与抗体特异性结合的 原理， 通过化学反应使标记抗体的显色剂显色来确定组织细胞内抗原， 对其进 行定位、 定性研究。
术语"免疫组化的评分标准 "指的是首先通过免疫组化的方法根据下述公式
(I) 计算本发明 ER-a36的表达值。
ER-a36的表达值=细胞膜染色强度分值乘以细胞膜染色的分值 +细胞质染 色强度分值乘以细胞质染色的分值 ；
所述的染色强度分值为未染色 =0; 弱染色 =1 ; 中等染色 =2; 强染色 =3， 并 且所述的细胞膜染色强度分值为在细胞膜上染色强度的分值， 细胞质染色强度 分值为在细胞质上染色强度的分值； 所述的细胞膜染色分值为细胞膜染色细胞比例 0%对应的分值为 0; 细胞膜 染色细胞比例 1-24%对应的分值为 1 ; 细胞膜染色细胞比例 25-49%对应的分值为 2； 细胞膜染色细胞比例 50-74%对应的分值为 3 ; 细胞膜染色细胞比例 75-100% 对应的分值为 4;
所述的细胞质染色分值为细胞质染色细胞比例 0%对应的分值为 0; 细胞质 染色细胞比例 1-24%对应的分值为 1 ; 细胞质染色细胞比例 25-49%对应的分值为 2； 细胞质染色细胞比例 50-74%对应的分值为 3 ; 细胞质染色细胞比例 75-100% 对应的分值为 4。
术语"转移"指的是肿瘤细胞从原发部位侵入***、 血管或其它器官， 形 成于原发部位肿瘤相同类型的肿瘤， 这个过程称为转移。
术语"穿剌"指的是就是借助空心针这一器械， 对乳腺的可疑病灶 （如： 肿 块、 增厚区域、 钙化灶等） 穿剌取出部分腺体组织进行检査。
术语" ER-a36表达为阳性"表示通过免疫组化的方法， 通过式 （I) 计算得到 的 ER-a36值在 5以上。
术语" ER-a36表达为阴性"表示通过免疫组化的方法， 通过式 （I) 计算得到 的 ER-a36值小于 5， 不包含 5。
术语" ER-a66表达为阳性"根据现有的 WHO乳腺癌评分标准， 通过免疫组化 方法可以确定。
术语" ER-a66表达为阴性"根据现有的 WHO乳腺癌评分标准， 通过免疫组化 方法可以确定。
术语"恶性肿瘤细胞"是一种变异的细胞， 与正常细胞不同， 由于它无限增 长、 转化、 转移三大特点， 因此破坏正常组织细胞， 进而破坏正常组织的功能。
术语"经过药物治疗的乳腺癌患者 "指的是通过口服、 肌肉注射、 静脉注射 治疗药物等方式进行过乳腺癌治疗的乳腺癌患者。
术语"鼠源抗体"是通过将杂交瘤细胞移植在老鼠腹腔内并产生腹水， 从腹 水中收集纯化得到的抗体。 术语"兔源抗体"是通过将杂交瘤细胞移植在兔子腹腔内并产生腹水， 从腹 水中收集纯化得到的抗体。
术语"总体生存率"指的是特定时间内， 生存患者的比例。
术语"无病生存率"指的是特定时间内， 疾病未复发的患者比例。 实施例
实施例 1
参见 PCT/CN2013/072844， 该专利申请引入本文作为参考。
以下提供的 ER-a36抗体包括 SNGmBl抗体的至少一个，例如可以为 2、 3、 4、 5或全部 6个 CDR区。
本文提供的" SNGmBl抗体"是一种鼠源的 ER-a36的单克隆抗体。 包括氨基 酸序列为 SEQ ID: 1的轻链和氨基酸序列 SEQ ID NO:2的重链序列。编码 SNGmBl 抗体轻链和重链的核苷酸序列分别为 SEQ ID NO:17抗体和 SEQ ID NO:19,轻链 和重链的氨基酸序列如下所示， 其中 CDR区为斜体并有下划线， 并且固定区为 阴影部分的斜体。
SNGmBl （SEQ ID NO: l )
LCDR2 LCDR3
KDEYEBHNSYTCEATHKTSTSPIVKSFNRSJEC
SNGmBl重链的氨基酸序列 （SEQ ID NO:2) ：
Figure imgf000007_0002
HCDR2 HCDR3
Figure imgf000008_0001
ς
Mmm (Π Μ αι as) iamoMS#¾ri ^¾¾ f?
Figure imgf000008_0002
d KSAA ID j ISAaymSl SY dllXAO Y A TO I SI 3IdS lQ^MNA D>L33iDS JiQ0H
Figure imgf000008_0003
089Z80/M0ZN3/X3d 96S0T0/S10Z OAV 表 1为 SNGmB 1抗体的 CDR序列
表 1
Figure imgf000009_0001
在某些实施例中， ER-a36的抗体和包括至少一个选自 SEQ ID:3-8的 CDR区， CDR区用于与抗原的结合， 然而并不是所有的 6个 CDR均不可缺少地与抗原结 合， 可以替换 SNGmBl抗体的一个或者更多 CDRs仍然保持与 ER-a36抗原结合的 选择性和亲合性。
在某些实施例中， 本文的 ER-a36抗体包括 SEQ ID NO:8， (B : SNGmBl抗 体的重链 CDR3序列：)。 重链 CDR3区位于抗原结合区域的中心， 因此最易于结合 抗原。
在某些实施例中， ER-a36的抗体包括 SNGmBl抗体的重链可变区， 即重链 SEQ ID NO:10; 和 /或包括 SNGmBl抗体的轻链可变区， gpSEQ ID NO:9。
SEQ ID NO:9
Glu Thr Thr Val Thr Gin Ser Pro Ala Ser Leu Ser Met Thr lie Gly Glu Lys Val Thr lie Arg Cys lie Thr Ser Thr Asp lie Asp Asp Asp Met Asn Trp Tyr Arg Lys Lys Pro Gly Gin Pro Pro Lys Leu Leu lie Ser Glu Gly Asn Thr Leu Arg Pro Gly Val Pro Ser Arg Phe Ser Ser Ser Gly Tyr Gly Thr Asp Phe Val Phe Thr lie Glu Asn Met Leu Ser Glu Asp Val Ala Asp Tyr Tyr Cys Leu Gin Ser Asp Asn Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
SEQ.ID NO: 10
Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His Asn Met His Trp lie Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp lie Gly Ala lie His Pro Val Asn Gly Asp Thr Ala Tyr Asn Gin Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Tyr Gly Ser Val Asp Tyr Trp Gly Gin Gly Thr Thr Leu Thr Val Ser Ser 实施例 2
通过免疫组化的方法获得可供读取的病理切片
首先将手术或者穿剌获得的肿瘤组织浸泡在固定剂中， 接着用浓度梯度乙 醇使组织进行充分脱水， 脱水后的组织再经二甲苯透明和石蜡透蜡后用石蜡包 埋， 待蜡块凝固后， 切成 4μπι厚的病理切片。切片在 65°C的烘箱中烤片 2小时后， 在二甲苯中浸泡 10分钟 2次，从而去除石蜡，然后依次在 100%乙醇内浸泡 3分钟 2 次， 95%的乙醇内浸泡 3分钟和在 75%的乙醇内浸泡 3分钟， 最后用磷酸盐缓冲液 (PBS ) 清洗 5次， 每次 2分钟。
采用高压修复法对抗原进行修复， 将切片完全浸没于 lmmol 乙二胺四乙酸 二钠 (EDTA) pH8.0抗原修复液中， 高压加热至沸腾， 待高压锅冒气加阀开始计 时， 2分 30秒后将高压锅离火自然冷却至室温后取出切片， 磷酸盐缓冲液（PBS ) 清洗 5次， 每次 2分钟。 将切片放入装有新鲜配置的过氧化物酶封闭液的反应皿 中， 37°C烘箱孵育 30分钟，以消除内源性过氧化物酶的活性。磷酸盐缓冲液 (PBS ) 冲洗干净后， 在免疫组化笔圈定的组织区域内加入 5%山羊血清 37°C烘箱孵育 30 分钟， 以封闭非特异性抗原。 小心甩掉并拭去组织上及周围多余的液体， 滴加一抗工作液 （一抗抗体为 实施例 1抗体， 浓度 0.02mg/ml) 4°C过夜 （最好 16小时至 18小时） ， 室温复温 30 分钟。 磷酸盐缓冲液（PBS )冲洗 5次， 每次 2分钟。 滴加适量辣根过氧化物酶标 记二抗工作液， 37 °C孵育 30分钟。 磷酸盐缓冲液 （PBS ) 冲洗 5次， 每次 2分钟。 经显色剂显色 1-2分钟， 镜下观察适时终止显色， 苏木素染核 2分钟， 自来水冲洗 多余的苏木素后， 为了使核染色持久， 再置于盐酸酒精中 1秒， 磷酸盐缓冲液 (PBS )或自来水冲洗返蓝， 再经脱水， 透明， 封片。制成可供读取的病理片子。 实施例 3
诊断标准的确定
检测 1068乳腺癌患者，通过免疫组化常规方法使用实施例 1中的抗体检测患 者肿瘤标本的 ER-a36， 根据公式 （I) 计算出每个病理片中 ER-a36的表达值。
ER-a36的表达值=细胞膜染色强度分值乘以细胞膜染色的分值 +细胞质染 色强度分值乘以细胞质染色的分值 ；
所述的染色强度分值为未染色 =0; 弱染色 =1 ; 中等染色 =2; 强染色 =3 ; 并 且所述的细胞膜染色强度分值为在细胞膜上染色强度的分值， 细胞质染色强度 分值为在细胞质上染色强度的分值。
所述的细胞膜染色分值为细胞膜染色细胞比例 0%对应的分值为 0; 细胞膜染 色细胞比例 1-24%对应的分值为 1， 细胞膜染色细胞比例 25-49%对应的分值为 2; 细胞膜染色细胞比例 50-74%对应的分值为 3 ; 细胞膜染色细胞比例 75-100%对应 的分值为 4;
所述的细胞质染色分值为细胞质染色细胞比例 0%对应的分值为 0; 细胞质 染色细胞比例 1-24%对应的分值为 1， 细胞质染色细胞比例 25-49%对应的分值为 2； 细胞质染色细胞比例 50-74%对应的分值为 3 ; 细胞质染色细胞比例 75-100% 对应的分值为 4。 以 ER-a36的表达值在 5 以上确定为患者乳腺癌 ER-a36表达为阳性， 当 ER-a36的表达值小于 5 (不包含 5 ) ， 表示患者的乳腺癌 ER-a36表达为阴性。
实施例 4 检验乳腺癌患者转移的肿瘤组织中 ER-a36的表达情况 实验方法： 选取经过手术或者穿剌获得的乳腺癌患者的肿瘤组织， 通过 疫组化方法和实施例 3方法确定这些患者的乳腺癌组织中 ER-a36的表达。并且 测这些患者是否带有***转移。 实验结果：
考察指标 数量 （人数） E -a36 表达 P值 总体 1068 阳性 阴性
(512, 47.8% ) (556, 52.2%)
年龄
<50 648 (60.7%) 307 (60.0%) 341 (61.3%) 0.661
^50 420 (39.3%) 205 (40.0%) 215 (38.7%)
肿瘤大小 (cm)
2 276 (25.8%) 114 (22.3%) 162 (29.1%)
2-5 653 (61.1%) 319 (62.3%) 334 (60.1%) 0.009
^5 139 (13.0%) 79 (14.4%) 60 (10.8%)
组织学情况
I 348 (32.6%) 74 (14.5%) 274 (49.3%)
II 595 (55.7%) 368 (71.9%) 227 (40.8%) <0.001
III 125 (11.7%) 70 (13.7%) 55 (9.9%)
***转移情况
0 465 (43.5%) 151 (29.5%) 314 (56.5%)
1-3 242 (22.7%) 147 (28.7%) 95 (17.1%) <0.001
^4 203 (19.0%) 134 (26.2%) 69 (12.4%)
未知 158 (14.8%) 80 (15.6%) 78 (14.0%) 从以上分析可以看出，在以上的 1068位乳腺癌患者中，在肿瘤大小超过 2cm 的患者中， ER-a36阳性的患者比例高于 ER-a36阴性的患者； 在组织学为 I期以上 的患者中， ER-a36阳性的患者比例高于 ER-a36阴性的患者； 在***转移的病 人中， ER-a36阳性的患者比例大于 ER-a36阴性的患者比例， 并且随着***转 移数量的增加， ER-a36阳性的患者比例增加。 实施例 5
乳腺癌患者手术后生存状况和 ER-a36表达的关系
ER-a36表达为阳性和阴性的乳腺癌患者经过乳腺癌手术后还有其他辅助治 疗的， 其术后生存情况如图 1所示。
从图 1A可见， 在手术结束后， ER-a66阳性的患者中， 随着手术后时间的增 力口， ER-a36、 ER-a66均为阳性患者的总体生存率小于 ER-a66阳性、 ER-a36阴性 患者的生存率。
从图 1B可见， 在手术结束后， ER-a66阳性的患者中， 随着手术后时间的增 力口， ER-a36阳性患者的无病生存率小于 ER-a36阴性患者的无病生存率。
从图 1C可见， 在手术结束后， ER-a66阴性的患者中， 其中 ER-a36阳性患者 的总体生存率小于 ER-a36阴性的患者的总体生存率。
从图 1D可见， 在手术结束后， ER-a66阴性的患者中， 其中 ER-a36阳性患者 的无病生存率小于 ER-a36阴性的患者的无病生存率。
因此， 可见无论 ER-a66的表达为阳性或者阴性， 只要患者的 ER-a36表达为 阳性， 患者的总体生存率和无病生存率均低于 ER-a36阴性的患者。 通常乳腺癌 患者在接受手术治疗后会出现癌症的原位复发或转移复发从而导致乳腺癌患者 死亡， 进而使乳腺癌患者总体生存率降低。 实施例 6
他莫西芬对高表达 ER-a36的乳腺癌细胞增殖的促进作用
选取 MCF-7乳腺癌细胞， 该细胞从美国菌种保藏中心 （ATCC) 获得。 通过 基因稳转技术， 转入外源性 ER-a36基因， 使 MCF-7乳腺癌细胞高表达 ER-a36。 将他莫西芬添加于该细胞培养基， 经过 6天培养， 通过图 2A可见， 他莫西芬 促进了 MCF-7乳腺癌细胞的增殖，在他莫西芬的促进下， MCF-7乳腺癌细胞的相 对生长率远大于空白对照组的 MCF-7乳腺癌细胞的相对生长率。
他莫西芬对低表达 ER-a36的乳腺癌细胞增殖的促进作用
选取未转染 ER-a36的 MCF-7乳腺癌细胞， 其低表达 ER-a36。
将他莫西芬添加于该细胞培养基， 经过 6天培养， 通过图 2B可见， 他莫西芬 并没有促进 MCF-7乳腺癌细胞的增殖。在他莫西芬的作用下， MCF-7乳腺癌细胞 的相对生长率小于空白对照组 MCF-7乳腺癌细胞的相对生长率。
因此可见，他莫西芬对于 ER-a36高表达的乳腺癌细胞并不能起到抑制作用， 反而促进了 ER-a36乳腺癌细胞的增殖。 但是， 他莫西芬对于 ER-a36低表达的乳 腺癌细胞不能起到促进增殖的作用。 所以， 他莫西芬如果用于 ER-a36高表达的 乳腺癌治疗中， 会增加乳腺癌细胞增殖和转移的风险。

Claims

权 利 要 求 书
1.一种乳腺癌预后诊断的方法， 该方法包括检测乳腺癌患者肿瘤标本组织 中 ER-a36的表达量， 当 ER-a36高表达时， 表示乳腺癌患者预后不良。
2.根据权利要求 1所述的方法， 其中所述的乳腺癌患者预后不良表示乳腺癌 患者存在乳腺癌复发和死亡的风险。
3.根据权利要求 2所述的方法， 其中所述的乳腺癌复发风险包括乳腺癌的原 位复发和转移复发。
4.根据权利要求 1所述的方法， 其中通过免疫组化的方法检测乳腺癌患者肿 瘤标本组织中 ER-a36的表达量。
5.根据权利要求 1所述的方法， 其中所述的 ER-a36高表达为通过免疫组化评 分标准得到 ER-a36的表达值在 5以上。
6.根据权利要求 5所述的方法， 其中 ER-a36的表达值为通过以下免疫组化的 评分标准得出： 细胞膜染色强度分值乘以细胞膜染色的分值 +细胞质染色强度分 值乘以细胞质染色的分值；
所述的染色强度分值为未染色 =0; 弱染色 =1 ; 中等染色 =2; 强染色 =3， 并 且所述的细胞膜染色强度分值为在细胞膜上染色强度的分值， 细胞质染色强度 分值为在细胞质上染色强度的分值；
所述的细胞膜染色分值为细胞膜染色细胞比例 0%对应的分值为 0; 细胞膜染 色细胞比例 1-24%对应的分值为 1， 细胞膜染色细胞比例 25-49%对应的分值为 2; 细胞膜染色细胞比例 50-74%对应的分值为 3 ; 细胞膜染色细胞比例 75-100%对应 的分值为 4;
所述的细胞质染色分值为细胞质染色细胞比例 0%对应的分值为 0; 细胞质染 色细胞比例 1-24%对应的分值为 1， 细胞质染色细胞比例 25-49%对应的分值为 2; 细胞质染色细胞比例 50-74%对应的分值为 3 ; 细胞质染色细胞比例 75-100%对应 的分值为 4。
7.根据权利要求 1所述的方法， 其中所述的 ER-a36高表达表示患者肿瘤标本 组织中的 ER-a36的表达量超过肿瘤组织中***或肿瘤周围癌旁正常组织的 表达量。
8.根据权利要求 1所述的方法， 其中所述的肿瘤标本组织为手术或穿剌的乳 腺肿瘤组织经处理后得到。
9.根据权利要求 1所述的方法， 其中通过特异性识别 ER-a36的抗体与肿瘤标 本组织相互作用， 检测肿瘤标本组织中 ER-a36的表达量。
10.根据权利要求 9所述的方法，其中所述的特异性识别 ER-a36抗体为鼠源或 兔源抗体。
11.根据权利要求 1所述的方法， 其中所述的肿瘤标本组织中含有恶性肿瘤细 胞。
12.根据权利要求 1所述的方法， 其中所述的乳腺癌患者包括经过药物治疗的 乳腺癌患者。
13.根据权利要求 12所述的方法， 其中所述的经过药物治疗的乳腺癌患者包 括经过他莫西芬治疗的乳腺癌患者。
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