In the United States, more than 203,500 new cases of breast cancer were diagnosed in 2002, and 39,600 women died from breast cancer. Women in the United States have an approximately 11% chance of developing breast cancer in their lifetime. Currently, more than 1,000,000 women in the United States have stage II, III, or IV breast cancer. Although the current state of medicine cannot prevent and completely cure breast cancer, as the level of medical diagnosis and treatment advances, more and more patients are able to effectively manage their disease. In addition, early diagnosis of breast cancer (including self-examination of patient’s breast, mammogram, and clinical examination) can significantly reduce the mortality rate of patients. Although tumor markers are not a substitute for imaging, they can provide useful information for the clinical management of patients. Tumor markers are used clinically to select treatment options, predict disease progression and recurrence, and monitor the effectiveness of treatment. The following tumor markers are in use or recommended for use: Estrogen and progesterone receptors CA15-3 BR27.29 (CA27.29) CEA HER-2/neu (c-erb B2)
Estrogen and progesterone receptors
Estrogen and progesterone receptors are major tissue tumor markers for breast cancer, including p53,c-erb B2(HER-2/neu) and other steroid receptors. Steroid receptors are the only tissue markers currently included in the inclusion criteria specifications (14-17). Although it was too late for the NACB or EGTM to consider other markers when drafting the guidelines, the results of HER-2/neu testing are now used to determine whether patients with metastatic breast cancer should be treated with the monoclonal antibody Herceptin (trastuzumab).
Clinical applications of estrogen and progesterone receptors
In addition to the general characteristics of breast cancer, such as tumor size, pathological type, and axillary lymph node status, protein biomarkers are valuable in evaluating differences in tumor growth rate, invasiveness, and malignancy potential. Estrogen and progesterone receptors have been used as hormonal [e.g., tamoxifen, toremifene, droloxifene (“anti-estrogen”), medroxyprogesterone acetate, and megestrol acetate] treatment Predictors of response. Levels of estrogen and progesterone receptors are also used as predictors to evaluate the effects and side effects of hormone therapy. In addition, sex hormone receptors are also used as prognostic factors in the diagnostic evaluation of breast cancer or similar cases.
Overall, patients with estrogen receptor-positive tumors have a good prognosis, at least in the short term, while patients with estrogen receptor-negative tumors usually have malignant tumors. Approximately 30% of breast cancer patients with tumors that are estrogen receptor negative or greater than 75% of breast cancer patients with tumors that are estrogen receptor positive will develop metastases and die within 10 years. Estrogen and progesterone receptors have been used as prognostic indicators along with other factors to differentiate between tumor types in patients with high risk of recurrence (poor prognosis) and low risk of recurrence (good prognosis) breast cancer. The American Society of Clinical Oncology (ASCO) clinical practice guidelines agree that estrogen and progesterone receptor levels should be used to determine whether tumors have metastasized in pre- and post-menopausal breast cancer patients and to guide clinical management.
Reference Value Ranges
The range of reference values is uncertain because estrogen and progesterone receptors are rarely tested in women who do not have breast tumors. Results of ligand binding and enzyme immunoassay techniques have shown that sex hormone receptors are low (<15 fmol/mg protein) or undetectable in breast tissue without tumors, as confirmed by many studies using immunohistochemical assays. Usually 10 fmol/mg of extracted protein (by ligand binding) or 15 fmol/mg of protein (by enzyme immunoassay) is used as Cut-off value. The presence of estrogen and progesterone receptors in tumors with metastases of unknown pathological origin suggests a primary hormone-responsive breast cancer or other sex hormone-responsive tumors. The distribution of estrogen and progesterone receptors in biopsy tissue is influenced by the patient's age and menopausal status. In general, premenopausal women with breast cancer have lower receptor levels than postmenopausal women. Since the hormonal treatment regimen for breast cancer patients is determined by their hormonal response, which in turn is related to the estrogen and progesterone receptor levels in their biopsied tumor tissue, patients' receptor levels need to be tested.
Variations in estrogen beta receptors and other sex hormone receptors
Investigations have shown that regulatory proteins exhibit polymorphisms. For example, a new estrogen receptor gene, estrogen beta receptor (ER-β), has been identified and the estrogen alpha receptor (ER-α), which has been extensively studied, shows some degree of homology in the DNA and ligand binding regions (96% and 58% homology, respectively), but the two heterodimers have different ligand bonding ratios and biological activities. PR) co-expression is accompanied by some indicators of low bioinvasive breast cancer tumors suggesting that ER-β positive tumors tend to respond to anti-estrogen therapy, but further independent ER-β predictive values need to be established. Approximately 20 sex hormone receptor variants identified in the last decade have been shown to be defective due to breaks, exon deletions and point mutations, and further research into new reagents and reference samples is necessary to investigate the clinical significance of these naturally occurring isoforms and variants.
Pre-analytical considerations and specimen preservation
Regardless of the analytical technique chosen, proper selection and handling of biopsy specimens from breast tissue is essential for reproducibility of test results. First, after surgical excision, the tissue should be frozen at -20°C for the shortest possible time and transported to the laboratory in a frozen state. The optimal sectioning temperature (OCT) complex used to make frozen sections must be completely removed prior to tissue extraction; if dyes (such as commercially available Davidson dye cartridges) are used, the dyes should be removed from the tissue; and bleeding tissue and biopsies with a large amount of normal tissue attached should be avoided. Sex hormone receptors in tissue extracts are unstable at room temperature and are inactivated within 2 to 4 hours at 4°C. Tissue extracts should be frozen at -70°C immediately after extraction. The stability of receptors in frozen tissue extracts depends on the type of tumor, the blood and normal tissue contained in the tissue, and a number of other factors. If the extract is not used within 48 hours, the remaining tissue should be re-extracted for tissue extraction.
Considerations in Analysis
The precision (CV% of 4-15%) of existing radioligand binding assays and enzyme immunoassay (using standardized reagents and stable reference samples) methods is higher than required for clinical diagnosis. The total imprecision required for clinical diagnostic treatment is 10 to 20% at estrogen and progesterone levels of 30 to 50 fmol/mg of extracted protein and 30 to 40% at estrogen and progesterone levels >100 fmol/mg of extracted protein.
Considerations for reporting results after analysis
Data from collaborative clinical trials suggest that estrogen and progesterone receptor levels in human breast cancer biopsies are not clinically meaningful at levels less than 10-15 fmol/mg of extracted protein. -910 -9M to 1-910 -10M indicates a high affinity progesterone receptor. The ligand binding properties of estrogen β receptors and other sex hormone receptor isoforms have also been well studied. Cut-off values should be reported according to the analytical method. Some institutions also recommend reporting the age-specific receptor distribution status, since receptor levels are age-dependent.
CA15-3
CA15-3 is a high molecular weight mucin (glycoprotein) called MUC-1, which has been characterized by monoclonal antibodies (clone DF3) and anti-human fat globule antibodies (clone 115D8) to purified extracts of tumor membranes isolated from breast cancer metastases to the liver. The mucin-based tumor markers for breast cancer include BR27.29, CA-549, MCA, CA-M26, and CA-M29. these mucins have similar sensitivity and specificity, and the use of more than one mucin tumor marker does not provide more information. the FDA has approved only CA15-3 and BR27.29 for clinical use in patients with breast cancer and breast cancer metastases. diagnosis.
Clinical Applications
Although carcinoembryonic antigen (CEA) has been used for the detection of breast cancer metastasis, the results of a large number of recent studies have established the relative advantage of CA15-3. In short, CA15-3 levels are related to the patient’s clinical status and response to tumor therapy and are now used in the evaluation of 60-80% of patients with metastatic breast cancer. The findings suggest that CA15-3 tumor markers can be used to monitor the progression and regression of breast cancer patients during treatment. The EGTM guidelines recommend the use of both CA15-3 and CEA to improve the sensitivity of clinical testing. Several research groups have reported that the use of both CA15-3 and CEA in the clinical setting can detect more patients with early recurrent tumors than CA15-3 alone. Early detection of liver and bone metastases in patients with tumors has higher sensitivity using serial tumor markers, and the early diagnosis of tumors is extremely important for both patient treatment and survival. Currently CA15-3 is mainly used to monitor the activity of breast cancer. Future work is to develop guidelines for the clinical significance of CA15-3 for treatment and prognostic follow-up monitoring.
Analytical test prep and sample storage
CA15-3 is stable at 4°C for 24 hours. It is recommended that sera be stored at -20°C (short-term) or -70°C (long-term) for retesting. For long term storage, denaturing gel should not be used (CA15-3 exhibits significant instability in the presence of denaturing gel).
Precautions in analysis
Each laboratory should validate the analytical method, analytical precision and reference value range for CA15-3 to determine the cut-off value for clinical diagnosis.
Considerations for reporting results after analysis
Further studies should be performed to confirm the effects of gender, race, age and menopausal status on CA-153 expression in normal subjects and breast cancer patients, and to develop literature on reference value ranges for tumor markers.
BR27.29 (CA27.29)
BR27.29 is a member of the newly investigated family of mucin-based breast cancer tumor markers (including CA15-3), which are different types of antibodies produced from mucin antigen (MUC-1).The reaction sequences of the BR27.29 monoclonal antibody and the DF3 antibody used for CA15-3 analysis overlap in the antigenic determinant cluster mapping.
Clinical Applications
There have been numerous studies on the use of BR27.29 in the detection and diagnosis of breast cancer. Like CA15-3, BR27.29 can be used for routine screening of breast cancer, but it also lacks sensitivity and specificity. Some reports suggest that BR27.29 is more sensitive but less specific than CA15-3. In conclusion, BR27.29 levels have been used by many institutions to evaluate the recurrence of disease in breast cancer patients at high risk of recurrence and to monitor the progression of disease in patients with advanced disease. These applied studies suggest that BR27.29 levels reflect tumor activity and provide clinical evidence for the screening of patients with advanced tumor metastases. The use of BR27.29 to predict recurrence of disease in patients with stage II or III breast cancer has been reported. Recent studies have also suggested that BR27.29 levels can predict tumor bone metastasis in breast cancer patients with skeletal disease.
Pre-analytical considerations and preservation of specimens
Because BR27.29 is a member of the MUC-1 family of glycogen-containing antigens, it can be measured in the same way as other tumor-associated antigens using treated serum or plasma, provided that enzymatic cleavage of the antibodies during the analysis is primarily considered. Freshly isolated serum samples are used for the determination of BR27.29. Samples are stable for 24 hours at 4°C and are recommended to be stored at -20°C (short-term) and -70°C (long-term) for retesting to ensure stability. The effect of freeze-thaw on the stability of BR27.29 needs to be further investigated.
Precautions in Analysis
Each laboratory should validate the analytical method, precision, reference value range and clinical diagnostic cut-off values for BR27.29. The sensitivity and specificity of BR27.29 analytical tests should meet the requirements of clinical specifications. No effects of commonly used analytical matrices on BR27.29 analytical tests have been reported, but further studies on this need to be conducted. Studies on the sensitivity of analytical methods should be focused on quality control. It is recommended that a reference sample of BR27.29 be established and used as an in-house quality control to evaluate the test results.
Post-analytical results reporting considerations
It is recommended that the results report state that the clinical application of BR27.29 is limited to the follow-up of breast cancer patients with further lesions.
NACB and EGTM recommendations for the clinical application of breast cancer tumor markers
1. the status of estrogen and progesterone receptors is used to determine whether a breast cancer patient is responding to hormone therapy (e.g., tamoxifen). the ASCO (American Society of Clinical Oncology) clinical practice guidelines agree that the levels of these receptors should be quantified at the initial visit for premenopausal and postmenopausal breast cancer patients. Immunohistochemical analysis is performed only if the sample size of the biopsy tissue does not allow for receptor quantification. When fresh tissue is not available, paraffin-embedded tissue can also be used for immunochemical analysis.
2. When the estrogen and progesterone receptor levels are 30-35 fmol/mg of extracted protein, the total imprecision requirement for the determination of the clinical treatment plan is 10-20%; while when the estrogen and progesterone receptor levels are >100 fmol/mg of extracted protein, the total imprecision requirement for the determination of the results is 30-40%.
3. ASCO clinical practice guidelines suggest that quantitative estrogen and progesterone receptor assays should be performed in premenopausal and postmenopausal patients with metastatic breast cancer to aid in the development of clinical treatment plans. Immunohistochemical analysis should be performed only when quantitative receptor testing is not possible on biopsy tissue.
4. The determination of CA15-3 or BR27.29 is important for the early diagnosis of recurrence of disease in asymptomatic stage II and III breast cancer patients after treatment. High CA15-3 levels in breast cancer patients indicate the presence of tumor metastasis.
5. A decrease in CA15-3 concentration in the body circulation indicates that the patient is responding well to treatment, while a persistent or elevated CA15-3 concentration indicates that the patient is progressing or responding poorly to treatment. Therefore, it is recommended that CA15-3 be used to follow the clinical status of breast cancer patients. In addition, EGTM also recommends concomitant testing of CEA for early diagnosis of tumor metastasis.
6. Since elevated CA15-3 levels are also seen in other malignant and non-malignant diseases, the ASCO clinical practice guidelines state that CA15-3 alone should not be used for diagnosis and disease determination of breast cancer.
7. The clinical application of BR27.29 is limited to the follow-up of breast cancer patients with further lesions. 8. CA15-3 testing should be performed immediately after serum is isolated. Serum samples are stable for 24 hours when stored at 4°C. It is recommended that sera be stored at -20°C (short-term) or -70°C (long-term) for retesting. If long-term storage is required, isolate should not be used (CA15-3 exhibits significant instability in the presence of isolate).
Newly discovered breast cancer markers
Epidermal growth factor receptor
Epidermal growth factor (EGF) is a single-chain polypeptide with a molecular weight of 6000 and three disulfide bridge structures that make it highly thermally stable.The EGF receptor protein is a complex molecule with an extracellular region that attaches to EGF, a transmembrane portion that anchors the receptor to the cytoplasmic membrane, and an internal region that contains an ATP binding site and presents tyrosine transaminase kinase activity. The EGF receptor is present in certain breast, uterine and ovarian tumors and can be detected by binding to radiolabeled EGF. Because radiolabeled EGF is expensive, many laboratories prefer to use ligand competition assays that detect both specific binding capacity and EGF affinity. Overexpression (i.e., increased number) of EGF receptors in breast cancer biopsy specimens is strongly associated with shorter asymptomatic periods and decreased overall patient survival. Unlike steroidal hormone receptors, high levels of EGF receptors in breast cancer biopsy specimens are indicative of poor prognosis. Although the application of EGF receptors is not included in the ASCO clinical practice guidelines, EGF receptors show increasing value as a prognostic factor and predictor of trial response to new drugs for the treatment of EGF receptor-positive breast cancer.
HER-2/neu (c-erb B2) tumor protein
The neu oncogene, originally isolated from murine neuroblastoma, encodes a 185-kDa surface glycoprotein named “p185neu” that exhibits tyrosine aminotransferase kinase activity and is structurally similar to the EGF receptor. Studies on the properties and chromosomal localization of various molecules have revealed differences between the EGF receptor and p185neu. p185neu natural ligand is becoming a hot topic of research in many laboratories. The gene named c-erb B2, a homologue of the human c-neu gene, has been reported to be amplified (i.e., increased copy number) from human breast tumors, and it is associated with shorter asymptomatic periods and decreased overall patient survival. c-erb B2 is also known as HER-2/neu. HER-2/neu tumor protein levels are associated with prognosis in breast cancer patients, indicating In 1997, a panel of experts from ASCO concluded that the available data were not sufficient to recommend c-erbB2 (HER-2/neu) gene amplification and overexpression for the management of breast cancer patients. The theoretical difficulties in evaluating the clinical application of c-erbB2(HER-2/neu) assay test results are due to the high variability of the assay methods and the lack of internationally recognized reference products. In the latest edition of ASCO2000, it is recommended that testing for HER2/neu and amplification of the HER2/neu gene in patients with primary and recurrent breast cancer is valuable. The new edition of the ASCO 2000 errata also includes information on the use of tumor protein assays and test results to guide trastuzumabR therapy, selection of chemotherapy, and evaluation of treatment response. HER2/neu tumor protein assays can be performed with tumor extracts or circulating extracellular regions, but are not recommended for prognostic purposes.
Histone D
Histone D is an acidic lysosomal protease that is present in all cells. Histone D is catabolized in a manner similar to that of breast cancer and normal breast, and is a 52 KDa precursor substance secreted in cultured breast cancer cells mediated by estrogen. Pre-histone D is a phosphoglycoprotein that breaks down into three molecules with molecular weights of 48-kDa, 34-kDa, and 14-kDa, respectively, and monoclonal antibodies to them have been developed and used in the production of radioimmunoassay kits. Enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) kits have also been developed and used for the analysis of histone D in breast cancer extracts. There is evidence that overexpression of histone D in patients with node-negative breast cancer is associated with shorter asymptomatic periods and decreased overall survival. However, the ASCO panel concluded that the available data are not sufficient to recommend histone D for use in the diagnosis and treatment of patients with breast cancer.
Urokinase-type fibrinogen activators and their receptors and inhibitors
Urokinase-type fibrinogen activator (uPA) and tissue-type fibrinogen activator (tPA) are serine proteases whose function is to convert fibrinogen to fibrinolytic enzymes. During cell migration and tissue reconstruction in physiological and pathological situations, tPA is mainly involved in intravascular thrombolysis, while uPA mediates pericyte proteolysis. The enzyme precursor of uPA (pro-uPA inactive) is secreted by the cell and binds to its receptor (uPAR) at the cell surface. Specific proteases (fibrinolytic enzymes, trypsin, kinase-releasing enzymes, histones) convert pro-uPA to its active form. pro-uPA active form has a large molecular weight (MW=52,000) and consists of two polypeptide chains linked by disulfide bonds [A chain (158 amino acids, MW=32,000), B chain (253 amino acids, MW=20, 000)] Activated uPA converts fibrinogen to fibrinolytic enzymes. Fibrinolytic enzymes are broad-spectrum proteases that catalyze the degradation of multiple proteins (either invasive or translocated proteins) in the extracellular matrix. Fibrinogen activator inhibitors (PAI-1 and PAI-2), produced by normal and tumor cells, bind to their receptors to inhibit uPA activity. In cancer, uPA mediates the invasion of tumor cells. Assessment of uPA, PAI-1 and PAI-2 levels in tissue extracts from many tumors (including breast, ovarian, uterine, cervical, prostate, and intestinal cancers) has been reported. If uPA, PAI-1 and PAI-2 are all overexpressed in the same biopsy specimen, it may accurately predict disease recurrence and reduced overall patient survival. uPA and PAI-1 as biological diagnostic indicators for clinical use require further development of standardized ELISA assay kits and stable reference products.