| Purpose:Adenoid cystic carcinoma,ACC,is a rare subtype of invasive breast carcinoma,accounted for 0.1-1% of breast tumor.Previous studies showed ACC in breast are histopathologically similar to primary salivary ACC.The biologically characteristic fusion gene MYB-NFIB can also be found in breast ACC.Breast ACC usually does not metastasize to axillary lymph nodes or distant organs.With precise prognosis and even simple excision of the lesion,breast ACC don't undergo any further treatments.There are lots of studies on primary salivary ACC,but only case report or small sample studies on breast ACC.We collected 20 cases of breast ACC to analyze the histopathological features and expression of MYB and CD117 in breast ACC.Then we detected fusion gene MYB-NFIB in breast ACC to discuss the application of MYB and CD117 and MYB-NFIB in diagnosis of breast ACC.Materials and Methods:1.Cases and criteria: Based on the diagnostic criteria of breast ACC in 2012 WHO breast tumor classification and 2014 Rosen's breast pathology,we screened 20 cases of breast ACC diagnosed by the Department of Pathology inWest China Hospital from 2007 to 2016,confirmed by two breast pathology specialists.We included biopsy samples and excision samples.We get ER,PR expression and HER2 status from diagnosis reports.We set 20 cases of salivary ACC as control group and the only frozen breast ACC sample as positive control in q RT-PCR.2.Immunohistochemical staining: Detected the expression of MYB and CD117 in 20 breast ACC,20 primary salivary ACC and 10 collagenous spherulosis,10 adenomyoepithelioma cases by immunohistochemical staining.3.RNA extraction: Extracted total RNA in 20 breast ACC and 5 primary salivary ACC with the expression of MYB protein.Used Trizol to extract the total RNA in frozen breast ACC sample.4.q RT-PCR: Designed 9 pairs of fusion gene MYB-NFIB prime,including 9,10,12 and 14 exons in MYB gene and 8a,8c and 9 exons in NFIB gene,acquiring QRT-PCR products of each sample.5.Fusion gene MYB-NFIB electrophoresis: Detected the expression of 9 pairs of fusion gene MYB-NFIB for each sample by electrophoresis.6.Fusion gene MYB-NFIB sequence: Detected by Boke Co.Ltd.Results:1.Clinical data: All of the 20 patients with breast ACCs are female,age 33~73(mean age,51.5).There are 14 cases on left breast(14/20,70%),6 cases on right breast(6/20,30%).Nine cases underwent modified radical mastectomy(include axillary lymph nodes excision)with axillary lymph nodes metastasis in1 case.Eight cases underwent simple breast tumor resection.Three cases underwent fine needle aspiration and underwent no further therapy.2.Histopathological features : Six cases in 20 of breast ACC are grade 1without any solid pattern,8 cases showed solid pattern less than 30% are grade2,6 cases are grade 3(3 cases showed characteristic basaloid solid pattern ACC).Compared to primary salivary ACC,7 are grade 1,8 are grade 2,5 are grade 3.The frozen sample of breast ACC is grade 3.3.Immunohistochemical staining:(1).Tumor gland epithelial and myoepithelial of breast ACC both can be MYB protein positivity,which express in the nucleus.Sixteen cases(16/20,80%)of breast ACC showed MYB positivity.One of the 4 MYB negativity breast ACC is grade1(1/6,16.7%).Two cases are grade 2(2/8,25%).One case are grade 3(1/6,16.7%).(2).All breast ACC showed CD117 positivity,which express in cytomembrane and cytoplasm.Six cases of breast ACC showed focal positivity in CD117 staining,some cases also showed weak positivity in CD117 staining.There are 2 cases of breast ACC showed more than 90% areas are CD117 strong positivity,one of which is grade 1 without MYB expression.(3).Review the immunohistochemical of ER,PR and HER2: two breast ACCs showed focal ER positivity,3 breast ACC showed focal PR positivity,2 cases are HER2 1+,1case are HER2 2+.In all the 20 cases of primary salivary ACC,13 cases(13/20,65%)express MYB,3 in 7 MYB negativity cases are grade 1(3/7,42.9%),2cases are grade 2(2/8,25%),2 cases are grade 3.All the 20 primary salivaryACC cases showed CD117 positivity,only 2 cases showed scattered focally intermediate to weak positivity.4.q RT-PCR of fusion gene MYB-NFIB: One breast ACC didn't underwent q RT-PCR for low concentration of RNA.We found MYB-NFIB fusion gene in6 cases(6/19,31.6%).We detected 3 fusion gene in frozen sample,but no fusion gene was detected in FFPE sample of this case.All of the 6 cases with MYB-NFIB fusion gene showed MYB and CD117 strong positivity,but we didn't detected MYB-NFIB fusion gene in the 2 cases with CD117 expression more than 90%.Fusion gene MYB-NFIB are only found in 2 cases of primary salivary ACC.5.Sequencing of fusion gene MYB-NFIB: Sequencing comparing showed 2cases in 4 high MYB expression of primary salivary ACC(case 3 and case 6)and 5 cases of breast ACC(case 5,case 10,case 15,case 22 and case 26)got stable fusion gene MYB-NFIB on 1334F-1197 R.Sequencing comparing results also showed 4 cases of breast ACC(case 1,case 10,case 13 and case 15)got fusion gene MYB-NFIB on 1925F-1197 R.Fusion gene MYB-NFIB on1432F-1197 R was detected in one primary salivary ACC.Fusion gene MYB-NFIB was found in 6 cases of 19 breast ACC(31.6%,6/19).Conclusion:1.CD117 expression is one of the characteristic immunophenotyping of breast ACC,which can help diagnosis even not specifical.2.Fusion gene MYB-NFIB and high expression of MYB protein is specific inbreast ACC,detecting those protein or fusion gene is helpful in differential diagnosis breast ACC from collagenous spherulosis,adenomyoepithelioma,invasive cribriform carcinoma and neuroendocrine carcinoma.3.RNA is easily degraded in FFPE sample,frozen samples maybe more suitable for fusion gene MYB-NFIB detection.4.Gene sequencing can verify the reliability of electrophoresis of MYB-NFIB. |
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