SOX10 Is a Novel Sensitive Diagnostic Marker for Adenoid Cystic Carcinoma of the Breast: Immunohistochemical Analysis of Adenoid Cystic Carcinoma Chen Yang M.D., Lingxin Zhang M.D., Souzan Sanati M.D. Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, Missouri, United States Background Adenoid cystic carcinoma (AdCC) of the breast is a rare triple-negative breast cancer with favorable prognosis. Given the low prevalence of this tumor, its defining features are not well established. The immunophenotypic features of AdCC of the breast remains unclear except for the fact that it is commonly triple negative (ER, PR, and Her-2 negative). SRY-related HMG-box 10 (SOX10) protein is a transcription factor known to be crucial in the specification of the neural crest and maintenance of Schwann cells and melanocytes. Expression of SOX10 as a diagnostic marker has been previously established in salivary gland AdCC and basal-like breast carcinoma; however, SOX-10 has not been studied in primary breast AdCC, especially in comparison with salivary AdCCs. Here, we examined SOX10 expression in AdCCs from breast, salivary gland, and soft tissue metastasis, and correlated its expression with other markers to investigate the histogenesis of this tumor. Our goal was to identify innovative markers that could serve as novel diagnostic or prognostic biomarkers. Insight into the immunophenotype of these tumors would also help us in understanding their cell of origin and potentially explain the difference in prognosis in tumors arising in different anatomical sites despite similar histopathologic features. Results All cases of breast (100%) and metastatic (100%) AdCCs and all but one case of salivary AdCC showed diffuse nuclear positivity (>50% of cells) for SOX10 (Figure 1). Thus, SOX10 is proven to be a sensitive diagnostic marker for AdCCs of the breast, salivary origin, and metastatic sites. Except one case of salivary gland AdCC which showed loss of beta-catenin expression and developed subsequent metastasis to the T10 vertebra, all cases of AdCC showed strong and diffuse membrane beta-catenin expression. Whether the loss of beta-catenin expression has any prognostic significance cannot be determined from this study due to our small sample size. Future studies with larger sample size is recommended. EMA is an epithelial marker which highlights the luminal cells in AdCCs. Interestingly, EMA expression was different in breast, salivary gland, and metastatic AdCCs; the lowest expression (both percentage and intensity) was seen in breast AdCCs and the highest expression in metastatic AdCCs (p<0.01, Figure 2). This difference in expression might be related to different histogenesis of these tumors which ultimately may result in different outcomes despite similar morphologies. Further studies are needed to better characterize these differences. There were no significant differences in expression of CK7, p63, CK5/6, C-kit, and Ki-67 (p>0.05). Table 2 and Figure 2 summarize the results. Similar to AdCC of salivary gland, SOX10 is a sensitive marker for diagnosis of AdCCs of the breast. This marker provides important insight into histogenesis of AdCC of breast and its cell origin. Additional studies are needed to investigate genetic and microenvironmental differences that contribute to different outcomes of these tumors. H&E Sox-10 EMA Ki-67 β-catenin Breast Salivary gland Metastasis Figure 1. Sox-10, EMA, Ki-67, and beta-catenin microphotographs of the AdCC tumor cells. Note the diffuse nuclear staining for Sox-10 and diffuse membranous staining for beta catenin in all three cases. EMA is more commonly expressed in salivary and metastatic AdCCs. The Ki-67 shows some variation amongst individual cases but are similar in regard to each category. TABLE 1. Breast AdCC Salivary AdCC Metastatic AdCC Age (year) 46-76 36-77 51-67 Sex 1M/10F 5M/12F 2M/3F Size (cm) 0.7-6.0 1.3-6.1 1.9-3.9 Margin Stat 1pos/10neg 9pos/5neg* 2pos/3neg DFS (year) No recur 0-7 (4.5) 0-5 (2.5) OS NDRTD** NDRTD Methods and Design A total of 11 consecutive cases of breast AdCC (from 1992 to 2014) with formalin-fixed paraffin-embedded (FFPE) blocks were obtained from archives of the Washington University School of Medicine. 17 age-matched salivary gland AdCCs and 5 metastatic AdCCs from salivary primary were also retrieved. Clinical data were collected and summarized in Table 1. These cases were centrally reviewed by three pathologists to confirm the diagnosis. Tissue microarrays of 3 mm cores of each case were made in triplicates. The expression of SOX10, Ki-67, c-kit, beta-catenin, EMA, p63, CK7, CK5/6 were assessed by immunohistochemistry (IHC), with adequate positive and negative controls. The IHC results were interpreted by three pathologists with the following criteria: nuclear staining for SOX10, Ki-67, p63, membranous staining for EMA, membranous/cytoplasmic staining with beta-catenin, C-kit, and cytoplasmic staining for CK7, and CK5/6 were considered positive. The percentage of cells that were positive and the intensity of reactions were evaluated. The intensity was scored as 1 (weak), 2 (moderate), or 3 (strong). For Ki-67, any reactivity was considered positive regardless of intensity. The immunohistochemistry results are analyzed and summarized in Table 2. * Three biopsy cases were not included ** No death related to disease Figure 2. Staining percentage of tumor cells Reference 1. Ho AS, Kannan K, Roy DM, et al. The mutational landscape of adenoid cystic carcinoma. Nat Genet. 2013; 45(7):791-8. 2. Ivanov SV, Panaccione A, Nonaka D, Prasad ML, Boyd KL, Brown B, Guo Y, Sewell A, Yarbrough WG. Diagnostic SOX10 gene signatures in salivary adenoid cystic and breast basal-like carcinomas. Br J Cancer. 2013;109(2):444-51. 3. Frierson HF Jr, El-Naggar AK, Welsh JB, Sapinoso LM, Su AI, Cheng J, Saku T, Moskaluk CA, Hampton GM. Large scale molecular analysis identifies genes with altered expression in salivary adenoid cystic carcinoma. Am J Pathol. 2002;161(4):1315-23. TABLE 2. Breast AdCC Salivary AdCC Metastatic AdCC Percentage (%) Mean Intensity (Range) SOX10 93 (63-100) 2.23 (1-3) 83 (19-100) 2.14 (1-3) 96 (75-100) 2.50 (2-3) Beta-catenin 100 (100) 2.72 (2-3) 94 (30-100) 2.41 (1-3) CK7 73 (15-100) 2.45 (1-3) 82 (6-100) 2.67 (2-3) 81 (25-100) 3.00 (3) CK5/6 66 (0-100) 2.29 (1-3) 79 (50-100) 2.43 (1-3) 50 (5-65) 2.25 (1-3) P63 93 (2-88) 2.55 (2-3) 64 (1-97) 50 (2-77) 1.83 (1-3) EMA 3 (0-17) 0.72 (1-3) 12 (1-52) 1.49 (1-3) 34 (1-93) 2.00 (1-3) C-kit 68 (25-100) 2.19 (1-3) 61 (13-100) 1.94 (1-3) 50 (15-87) 1.67 (1-2) Ki-67 7 (1-20) N/A 6 (1-27) 12 (5-25) Disclosure The authors have no relevant funding sources or conflicts of interest to disclose for this work