Figure 5. Tissue sections stained for b rachyury expression, corresponding to a lung adenocarcinoma (A); a squamous carcinoma (B); and a bronchioalveolar carcinoma (BAC), mucinous type (C). Lung tumor cells invading a blood vessel, positive for brac hyury expression (D). IHC staining for brachyury in a bone metastasis of breast cancer (E). Figure 6B. Expression of brachyury mRNA in breast tumor tissues. Brachyury mRNA expression, as determined by real - time PCR, is shown for breast primary tumor tissues grouped by histological tumor grade (Nottingham grading). E 16 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential 1.2.5 Expression of Brachyury Protein in Breast Tumor Samples The expression of brachyury was further analyzed in breast tissues by IHC with a murine anti- brachyury monoclonal Ab . Twenty- seven of 30 primary breast tumors (90%) were positive for brachyury expression . Sixteen benign breast tissues were negative for brachyury, with the exception of 2 cases of fibroadenoma where focal expression of brachyury protein was detected. Expression of brachyury was further evaluated in matched primary carcinoma and metastatic lymph nodes from 2 patients. As shown in Table 2, brachyury was expressed at higher levels in tumor-positive lymph nodes compared to the primary tumor, while the tumor-negative lymph node was negative for brachyury express ion in both cases. Additional metastatic lesions (pleura, bone, and brain) also exhibited high levels of brachyury expression (Table 2 , Figure 5E). Table 2. Expression of brachyury protein in primary and metastatic breast carcinoma lesions by immunohist ochemistry utilizing a murine monoclonal anti -brachyury Ab Pt Tissue Brachyury % Positivity Intensity 6 Breast primary tumor 30 + 6 Met+ lymph node (a) 90 + 6 Met+ lymph node (b) 90 + 6 Non-met lymph node (c) Neg Neg 9 Breast primary tumor Focal + 9 Met+ lymph node (a) 60 ++ 9 Met+ lymph node (b) 60 ++ 9 Non-met lymph node (c) Neg Neg 31 Pleura 90 + 32 Bone 90 ++ 33 Bone 90 + 34 Brain 70 ++ Matched breast primary tumor and metastatic lymph nodes from 2 patients and metastatic lesions from additional patients were analyzed for brachyury expression by IHC with a murine monoclonal anti -brachyury antibody. Two lymph nodes positive for metastasis from each patient (a, b) and one lymph node negative for metastasis from the same patient (c) were assayed. 17 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential 1.2.6 Brachyury Expression and Prognosis Breast tumor gene expression data (n = 4010) derived from 23 datasets from the Gene Expression Omnibus ( GEO) at the National Center for Biotechnology Information ( NCBI) was compiled . Each sample was assigned into low (first quartile, lowest 25%), i ntermediate ( second quartile , intermediate 50%) and high (third quartile, highest 25%) subgroups according to brachyury mRNA levels . A Kaplan -Meier estimate of survival was used to evaluate differences in prognosis among the 3 subgroups. Among 357 breast cancer patients treated with tamoxifen monotherapy as adjuvant therapy for 5 years post-diagnosis, we found that high brachyury expression significantly correlated with higher risk of recurrence ( p = 0.0283, n = 357, Figure 7A) and distant metastasis ( p = 0.0150, n = 332, Figure 7B). To minimize the effect of clinical confounding factors such as tumor size, grade, nodal status, age, HER2, ER and PR status that might cause false positive association between gene expression and poor prognosis, a Cox Proportional-Hazards Regression (COXPH) survival analysis was conducted (n = 270). When mRNA expression signal was used as a continuous variable, increased expression of brachyury was significantly associated with higher risk of recurrence ( p = 2.77x10 -5, n = 270) and distant metastasis ( p = 0.0001, n = 270). 18 Abbreviated Title: Phase I MVA -Brachyury -TRICOM Version Date: September 14, 2016 Confidential 1.2.7 Brachyury Confers Chemotherapy Resistance The LTIB has developed human lung carcinoma cell lines that have been either stably knocked down for expression of brachyury (sh/b rachyury) or transfected to stably overexpress brachyury (ph/brachyury). Brachyury downregulation significantly enhances sensitivity to chemotherapy, while stable overexpression of brachyury in human lung cancer cells is associated with a significant decrease in cell death mediated by various chemotherapy agents in vitro (Fig ure 8). Figure 7.