| Breast cancer is one of the most commonly occurring malignant tumors in women, and it is the second most common type of cancer in women. For patients with breast ductal carcinoma in situ, the presence of invasive carcinoma not only directly affects the treatment and prognosis, but also has strong impact on patients’ psychological and life status. However, how to assess intraductal carcinoma with or without the presence of invasive carcinoma is very difficult, especially early stage invasive carcinoma.Early stage invasive carcinoma includes microinvasive carcinoma which is not more than 1mm in diameter and the other early invasive carcinoma which is 1 to 2mm in diameter. Early stage invasive carcinoma is frequently found in background of high nuclear grade ductal carcinoma in situ(DCIS), less frequently with other types of DCIS or lobular carcinoma in situ. A more comprehensive definition is present in the World Health Organization Classification of Tumors of the Breast, where a microinvasive breast carcinoma is characterized ‘‘by one or more clearly separate microscopic foci of infiltration of tumor cells into the mammary stroma, each≤1 mm in size.’’ Because of the smallest of the foci, histopathological identification of microinvasive breast carcinoma in its earliest phases is very challenged for pathologists. On one hand, fibrosis, inflammatory reaction, and other stromal changes around in situ carcinoma may mask microinvasive foci on routine single-stain, on the other hand, breast carcinoma in its earliest phases is easy to be ignored. Other knds of 1-2 mm early invasive carcinoma of breast can also have the same problem and increase the difficulties of diagnosis.Histologically, the hallmark of invasion is the lack of myoepithelial cells(MECs).Immunohistochemistry(IHC) is one of the most important diagnostic methods, a combination of p63 and other MEC markers, such as smooth muscle myosin heavy chain and Calponin, are often suggested.However, IHC can only mark one kind of antibodies(myoepithelial cell layers),some cancer cells(express epithelial markers) and fibroblasts and macrophagocytes(cannot express epithelial markers) cannot be marked in breast stromal, IHC may also ignored the real cancer cells in breast stromal.Double immunostaining, which can simultaneously mark two different kinds of cell biomarkers in the same kind of tissue with different cell types, we can use this method to mark two kinds of different cells(MECs and epithelial cells) in the same time and the same tissue, may be a helpful method for the diagnostic of early stage breast cancer. In this study, I combined the myoepithelial cell markers(Calponin and p63 antibody) with epithelial cell markers(E-cadherin and CK7 antibody) to coaktail antibodies, using IHC and two antibodies coaktail immunostains and three antibodies immunostains in breast tissue,finding which combination and method is the best one for identify early stage invasive carcinoma.Then,use the new combination and method in some cases that are difficult to assure whether there is early stage invasive carcinoma and some other cases with high nuclear grade ductal carcinoma in situ,to determine whether the best combination and method can improve the diagnostic rate of early stage invasive breast carcinoma and applicable used in the department of pathology. ObjectiveAnalysis and compare the result of IHC(Calponin and p63),two antibodies cocktail double staining(Calponin+E-cadherin and p63+CK7),three antibodies cocktail double staining(Calponin/p63+E-cadherin and Calponin/P63+CK7) and three antibodies sequential double staining(Calponin/p63+E-cadherin and Calponin/P63 +CK7)in breast tissue,then, finding the optimal combination and method in this study.To investigate if the optimal combination and method can improve the diagnostic rate of early stage invasive breast cancer in some difficultly diagnostic cases.To investigate the occurrence of early stage invasive cancer in some cases with breast ductal carcinoma in situ. Methods1.Detect the expression of Calponin and p63 protein in breast tissue related to myepithelial cells with Maxvision 2-steps of IHC,detect the expression of Calponin+E-cadherin and p63+CK7 atibodies combination in normal breast tissue related to myepithelial cells and epithelial cells with two antibodies coaktail double staining, detect the expression of Calponin/p63+E-cadherin and Calponin/p63+CK7 atibody combination in normal breast tissue related to myepithelial cells and epithelial cells with three antibodies coaktail double staining and three antibodies sequential double staining.2.Choose 34 cases which is difficult to assure early stage invasive breast cancer(including doubt invasive cases and other cases need to group consultation),use the optimal combination and method to detect the tissue from that cases.3. Use the optimal combination and method to detect the tissue from 29 cases with diagnose of high nuclear grade ductal carcinoma in situ. Results1.The results of detection for normal breast tissue of staining level of myepithelial cell layer suggest that three antibodies coaktail double staining is better than three antibodies sequential double staining(p<0.05),and also better than IHC(p<0.05) and two antibodies coaktail double staining(p<0.05). The results of scoring system of myepithelial cell layer and epithelial cell layer suggest that, the difference between coaktail double staining and sequential double staining with the combination of Calponin/p63+E-cadherin was no statistically significant(P>0.05),but the difference between coaktail double staining and sequential double staining with the combination of Calponin/p63+CK7 was statistically significant(p<0.05). Judgement of scoring system and microscope image suggest that the optimal combination and method are Calponin/p63+E-cadherin combination and coaktail double staining.2. Detection of cases which is difficult to determine to microinvasive breast cancer with coaktail double staining and Calponin/p63+E-cadherin suggest that there are 21 cases with invasive carcinoma, including 2 cases(5.89%)are focal sheet invasives, 5 cases(14.71%)are diameter of tumor size>1mm,14 cases(41.18%) are microinvasive breast cancer(≤1mm),the diagnostic rate is increase than before.3. Detection of cases diagnosed of high nuclear grade ductal carcinoma in situ with coaktail double staining and Calponin/p63+E-cadherin suggests that there are 4 cases(13.79%) occurred with microinvasive carcinoma(≤1mm). Conclusion1.In breast tissue,coaktail double staining with the combination of Calponin/p63 + E-cadherin can improve the specificity and sensitivity of chermogenic of myepithelial cell layer and epithelial cell layer and make them easy to read. so, we concluded that it is the optimal method and combination in this study.2.Coaktail double staining with the combination of Calponin/p63+E-cadherin can improve the diagnostic of early invasive carcinoma of breast, has some clinical value in department of pathology. |
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