Calcification Breast Cancer Research-based Proteomics Technology Serum Diagnosis Model And The Calcification Mechanism

The morbidity and mortality of breast carcer are in the forefront of the overall tumors among women. Mammography is a main clinical procedure used worldwidely in screening breast cancer in women. Mammary calcifications were often identified in mammography screening. Specific calcifications were generally presented in patients with breast cancer. It's necessary that the characters of different type calcification be identified.It's concerned that the formation of breast carcer is a multistep, multistage and multifactor process like most other tumors. The conventional strategy of research on single gene has been challenged by the rapidly progress of proteomics, which can be utilized to observe the dynamical changes of nature and quantity of total protein during progression of disease. Proteomics can be used to find new protein markers and key molecules and therefore is an effective and high-throughout method for exploring mechanism of disease and for searching targets of diagnosis or therapy. High throughput genomics and proteomics techniques have facilitated a better understanding of diseases such as cancer by deciphering the unique molecular signature that predicts clinical outcomes and therapeutics. The nascent field of proteomics-the complete description of all the proteins encoded by the genome-promises to rapidly expand our understanding of cancer. An important goal of clinical proteomics is to develop robust, sensitive, and specific methodologies for the simultaneous analysis of all the proteins expressed by the human genome, and to establish "bio-signature" profiles that discriminate between disease states.One of these technological advances in proteomics is the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Applications of this technology have suggested great potential for the early detection of various cancers. Two dimensional gel electrophoresis (2-DE) is the standard of Mass spectrometry-based proteomics. Peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is now a routine tool for the identification of proteins separated by 2D electrophoresis. MALDI-TOFMS calculates the time of flight of peptides subjected to analysis, which is correlated to their M/Z. These peptide masses are used to search sequence databases and match with a known protein.It is common knowledge that approximately 50% of proteins in malignant tumors are glycosylated and that glycans play crucial roles in various biological events. There is still no any systematic glycomic method to screen aberrantly glycosylated proteins.The objective of this study is to find the rules of calcification in mammography. To identify the protein biomarkers indicative of calcification in serum derived from human breast cancer serum subjects with SELDI-based proteomics procedures. Then a clinical diagnostic and proteomic pattern is established with validation and evaluation. By using the technical platform of two-dimensional electrophoresis (2-DE) and MALDI-TOF-MS technique, in present study we observed the differentially expressed proteins in serum of human breast cancer with or without calcification, We got a group of significant differential proteins which provides the information of calcification. Then we validated some potential biomarkers. At the same time, we used MP technique, followed by lectin blot and precipitation to build up serum glycoprotein profiles of breast cancer with/without calcification, benign lesion with/without calcification, and to find some aberrantlyα1-6 fucosylated glycoproteins with different express which related to the development of malignant calcification in breast. All in all, we detected the change on hp-βprotein expression levels, and oligosaccharide structures in these different sera samples to discover whether these alterations played important roles in evolving process of special calcification in breast cancer. Objective:The aim of this study is to explore the features of breast calcification lesion on mammogram and to evaluate the significance of the calcification in the diagnosis of breast cancer.Methods:A total of 378 patients with breast lesion were detected with mammography before operation. The size and distribution and relation to pathology of calcification in the breast lesions were observed carefully. ER, PR and C-erbB-2 of the cancer feature were determined by immunohistochemistry (IHC) in 64 cases with breast ductal carcinoma.Results:Calcifications were detected in both malignant and benign breast lesions groups and showed statistically significant difference in the size and the distribution between the groups (P<0.05). Regarding the clustered miscrocalcif ication as the diagnostic criterion of imaging, the sensitivity and specificity were 43.9% and 84.2%. The positive expression rate of ER PR, C-erbB-2 had no statistical significance between the groups, but presented different features of calcification in breast ductal cancer.Conclusions:The clustered microcalcification inside breast lesion is an important feature in the diagnosis of breast cancer.Part Two:To Screen Serum Biomarkers with Relation to the Calcification on the Patients with Breast Cancer and Establish the Serum Diagnostic Proteomic Pattern Using SELDI-TOF-MSObjective:The aim of this study was to establish the platform of SELDI-TOF-MS protein chip and to screen serum proteomic tumor markers with the establishment and validation of the diagnostic models in patients with breast cancer, especially in those with calcification. Methods:Four groups of breast cancer with/without calcification, benign disease with/without calcification in breast were built up. Each group was composed of 40,32,33,21 patients separately. The serum samples were prepared from peripheral venous blood before operations. Special serum protein or peptide fingerprint was determined by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) measurement after treating the sample onto weak cation exchange (CM10) protein chip for each case. Detector sensitivity was set at 7 and laser intensity at 270. Spectra were analyzed with Ciphergen ProteinChip software (version 3.1) and normalized. Then intensity values for each peak were input into Biomarker Patterns Software for classification tree analysis and the best performing tree was chosen for testing. Second, the randomly selected samples were categorized with the decision tree being tested to ensure that the decision tree was valid.Results:1. Most of the proteins detected by protein chip ranged from 1500 to 50000 at the M/Z value. The coefficient variation (CV) of protein intensity and M/Z value was 0.161 and 0.0004 within the same protein chip. The CV of protein intensity and M/Z value was 0.16 and 1.6×10-8 among different protein chips.2. Diagnostic model established with special peaks related to malignant lesion(1) Comparing breast cancer group with benign group, a total of 54 protein peaks were identified according to serum protein fingerprints,19 significant differential proteins were found between two groups (P<0.05) Among 19 significant differential proteins,7 proteins were up regulated with the M/Z value of 8099,16204,16287,16465,16672,33123 and 40650, 12 proteins were down regulated with the M/Z value of 1746,1765,1771,1794,1842,1867,1888,1913,1934,1973,2025 and 9085 respect ively in cancer group. Serum protein profiles were generated using classification trees by Biomarker Patterns Software, which combined with 5 candidate proteins with the M/Z value of 16465,15633,7890,9561,4396 with a sensitivity of 94.3% and specificity of 97.6%。The validity of the decision tree was then challenged with a test set of 31 samples. As a result of blind assessment, a sensitivity of 86.8% and specificity of 85.7% were obtained.(2)Comparing breast cancer with calcification group to benign with calcification group, a total of 61 protein peaks were identified according to serum protein fingerprints,16 significant differential proteins were found between two groups (P<0.05). Among 16 significant differential proteins,6 proteins were up regulated with the M/Z value of 5987.9450. 15633,16287,16465 and 33068.10 proteins were down regulated with the M/Z value of 1842,1867,1890,1899,1913,1934,1954,1980,2025 and 3000 respectively in cancer group. Serum protein profiles were generated using classification trees by Biomarker Patterns Software, which combined with 3 candidate proteins with the M/Z value of 16465,9561,1792 with a sensitivity of 89.3% and specificity of 100.0%。The validity of the decision tree was then challenged with a test set of 24 samples. As a result of blind assessment, a sensitivity of 100.0% and specificity of 75.0% were obtained.3. Special peaks related to calcification lesion(1)Comparing breast cancer with calcification group to breast cancer without calcification group, a total of 54 protein peaks were identified according to serum protein fingerprints,14 significant differential proteins were found between two groups (P<0.05). Among 14 significant differential proteins,1 proteins were up regulated with the M/Z value of 3358,13 proteins were down regulated with the M/Z value of 1746,1765,1771,1794,10448,14320,14427,15445,15633,16204,24142,28811 and 29008 respectively in cancer with calcification group.(2)Comparing two benign groups with or without calcification, a total of 60 protein peaks were identified according to serum protein fingerprints,14 significant differential proteins were found between two groups (P<0.05). Among 14 significant differential proteins,3 proteins were up regulated with the M/Z value of 3140,3358 and 6938,11 proteins were down regulated with the M/Z value of 4704,5992,9452,10448,11943,15446,15658,24105,28811,29009 and 38365 respectively in calcification group. Conclusion:The diagnostic model established according to the different serum proteome fingerprints of caicification is the most valuable.The results of this study had significance for the diagnosis as well as understanding of breast cancer with calcification.Part Three:Proteomic Analysis on Serum Proteins of Human Breast Cancer Accompanied with Clustered MicrocalcificationObjective:The aim of this study was to search the critical serum protein molecules responsible for calcification of human breast ductal cancer and to explore the mechanism of the formation of calcification, this part compared and analyzed serum protein profiles of human breast ductal cancers with calcification(1a), breast ductal cancers with no calcification(1b), breast benign lesions with calcification (2a) and without calcifi cation (2b), by 2-DE and MALDI-TOF MS proteomic techniques.Methods:Four categories of serum samples were collected from human sera,10 samples in each category. The mixed protein from samples in each category, from which high abundance proteins(albumin and immunoglobulins) has been removed, was assayed repeatedly 3 times with 2-DE method. IPG stripe (18cm, pH3-10 NL) was used in the first dimensional electrophoresis, and in the second dimensional electrophoresis 12% SDS-PAG was used. Both analytical gels and preparative gels were stained.The analytical gels were then scanned using ImageScanner and analyzed with ImageMaster 2D Platinum 5.0 software. The protein points showing 3 or more than 3 fold difference and appearing in fifty percent or more than fifty percent gels were screened out. The spots with marked different expression were excised from gels and digested into peptides. These peptides were analyzed by MALDI-TOF MS and the peptide mass f ingerprintings (PMF) were got. Data were then searched with the search engine MASCOT (Matrix Science) against a NCBI nonredundant protein sequence database. All spots were identified by MALDI-TOF MS.Results:The mean detected numbers of protein spot in each category were as follows: 345±129 in group la,329±40 in group lb,296±27 in group 2a,308±43 in group 2b. Thirty-one protein points showing difference were screened out. All spots were identified by MALDI-TOF MS.Comparing breast cancer with calcification group to benign with calcification group:In malignant calcification group,2 proteins were special. The expressions of 8 proteins were up-regulated; on the other hand 7 proteins were down-regulated.Comparing cancer with calcification group to cancer with no calcification group:In malignant calcification group,3 proteins were special.The expressions of 5 proteins were up-regulated; on the other hand 3 proteins were down-regulated.Comparing benign disease with and without calcification groups:In benign calcification group, the expressions of 1 protein was up-regulated; on the other hand 4 proteins were down-regulated.Conclusion:To our knowledge, some identified proteins may be relative with carcinogenesis and progression of tumors and may play a role in metabolism of calcification. The results implied that various distinct different proteins may join together in breast calcification.Part Four:Verification on Differential Expression of haptoglobin and Its N-polysaccharide characteristic analysisObjective: To further validate haptoglobin expression level and approach the oligosaccharide structures of HP. Method:Sera from four groups of breast ductal cancers with calcification (1a), breast ductal cancers with no calcification (1b), breast benign lesions with calcification (2a) and benign without calcification (2b)were investigated for validation study (each group 10 sera). We examined sera total haptoglobin in the same four groups by immunoturbidimetry and western blot. Multiplexed proteomics technique (Glycoprotein stain Emerald 488 and Total protein stain SYPRO Ruby) coupled with two-dimensional electrophoresis (2-DE), followed by lectin blot with several different kinds of lectin and precipitation was used to inference glycoproteins with different structures which related to the formation of malignant calcification.Result:The results of immunoturbidimetry and western blot of the members in haptoglobin family (HP, Hp2-alpha and preprohaptoglobin), have shown the expressions of haptoglobin family in four groups of human sera, confirming the 2-DE result. And we built up serum glycoprotein profiles of cancers with calcification(la), breast ductal cancers with no calcification(lb), breast benign lesions with calcification(2a) and benign without calcification(2b) through the Image master 6.0 software to calculate vol% of HP, we found the extents of glycosylation of HP were the same as the protein express levels of HP except the preprohaptoglobin; AAL, LCA, DSA, Con A, PHA-E and RCA-I were employed to survey the alternations in their N-glycan structures. Compared to benign calcification group, AAL and LCA had a higher affinity to HP in malignant groups,especially in cancer with calcification group, PHA-E and RCA-I had obvious affinity to HP only in cancer with calcification group, while DSA and Con A almost had no affinity in four groups.Conclusions:The change on HP-βprotein expression levels and the alternations in their N-glycan structures in different serum samples have shown that these al terations played important roles in evolving process of mal ignant specific calcification of breast cancer. Haptoglobin was proved to have core-fucosylated type high mannose-core, triantennary type and terminal GalNAc type sugar chain structure. Fucosylated haptoglobin was increased in the serum in the process of malignant calcification.Conclusion1. The clustered microcalcification inside breast lesion is an important feature in the diagnosis of breast cancer.2. The diagnostic model established according to the different serum proteome fingerprints of caicification is the most valuable. The results of this study had significance for the diagnosis as well as understanding of breast cancer with calcification.3. To our knowledge, some identified proteins may be relative with carcinogenesis and progression of tumors and may play a role in metabolism of calcification. The results implied that various distinct different proteins may join together in breast calcification.4. The change on HP-βprotein expression levels and the alternations in their N-glycan structures in different serum samples have shown that these alterations played important roles in evolving process of malignant specific calcification of breast cancer. Haptoglobin was proved to have core-fucosylated type high mannose-core, triantennary type and terminal GalNAc type sugar chain structure. Fucosylated haptoglobin was increased in the serum in the process of malignant calcification.NOVELTY1. Establishment of preoperational serum protein fingerprints in patients with breast cancer accompanied with calcification.2. Establishment of clinical serum diagnostic proteomic pattern for screening breast cancer patients with calcification.3. The discriminated protein that we have analyzed and identified would supply us with more sensitive and specific index to identify the malignant calcification from benign ones and well understand the mechanism of clustered microcalcif ication in breast cancer. Among these proteins, some new molecular biomarkers would be found for malignant calcification and provide a therapeutic target in future.4. The influence of haptoglobin structure of sugar chain that may affect the occurrence and development process of malignant calcification was investigated firstly in our work.