Abstract Introduction: Intraoperative evaluation of sentinel lymph nodes (SLN) in breast cancer patients are performed using Touch preparation (TP) and/or frozen section (FS). Touch preparation for intraoperative evaluation of SLN is quick and known to be a highly sensitive and specific method for detection of metastasis. Detecting metastases in SLN intraoperatively can be challenging in patients who receive neoadjuvant systemic therapy (NST). In our hospitals, we have been routinely evaluating SLN intraoperatively in patients who have undergone (NST), including those with known metastasis to an axillary lymph node (LN) prior to therapy. Objective: To compare the sensitivity and specificity of TP and frozen section (FS) in the intraoperative evaluation of SLN in the neoadjuvant setting. Material and Methods: This retrospective review study was approved by the institutional review board. Four hundred ninety-eight SLN from 142 patients were included in this study. The intraoperative results for TP and FS were compared with the final pathology results. Relevant clinical and pathological findings such as type of surgery, tumor grade, histologic subtype, and size of metastasis were reviewed. Results: Of the 498 SLN evaluated intraoperatively, 341 were by TP only, 57 by FS only and 100 by both. Of the 341 SLN examined by TP only, 313 (92%) were interpreted as negative and 28 (8%) as positive for carcinoma intraoperatively. Eighteen LN turned out to be false negative (FN) with no false positives (FP) (sensitivity=62%, specificity=100%). In the false negative cases, 12 LN had micrometastasis, 6 macrometastasis and 1 showed isolated tumor cells (ITC). The size of the macrometastatic ranged from 3 mm to 10 mm. Of the 57 LN examined by FS only, 48 were true negative and 9 were true positive (sensitivity=100%, specificity=100%). Of the 100 LN evaluated by both TP and FS, 59 were interpreted as negative and 41 as positive for carcinoma. There were 8 false negatives and 1 false positive (sensitivity=83%, specificity=98%). Of the 8 false negatives, 7 showed micrometastasis and 1 LN had ITC. Discussion: In neoadjuvant cases, both the primary tumor as well as lymph node metastases can show therapy effect such as fibrosis, necrosis and/or histiocytic aggregates. Evaluating SLN in NST cases can be challenging secondary to these effects. The TP slides are often paucicellular in SLN with treatment effect. Residual tumor cells are often trapped in a fibrotic scar and do not transfer onto the TP slide leading to low sensitivity. Therefore, for optimal intraoperative evaluation of SLN in NST cases, frozen section with or without touch preparation, is recommended. Citation Format: Sahoo S, Mir M, Sarode V, Fang Y, Peng Y, Gwin K, Hwang H. Intraoperative evaluation of sentinel lymph nodes after neoadjuvant systemic therapy in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-03-16.

Abstract Background: Accurate assessment of HER2 status is critical for selecting patients who will benefit from trastuzumab therapy. There is still no consensus regarding the optimal method to assess HER2 status. Computerized image analysis has been shown to provide a more accurate and objective way for quantification of HER2 expression by IHC than manual evaluation. It has been suggested that the use of image analysis may help to resolve some of the discrepancies between IHC and FISH assay. We compared the results of HER2 expression by IHC using automated image analysis with fluorescent in situ hybridization (FISH) assay. Design: Testing for HER2 expression by IHC and FISH was performed on 2853 specimens at UT Southwestern Medical Center between the years 2002 to 2011. Quantification of IHC HER2 expression was done by image analysis and scored as positive (>2.0), borderline (1.5 to 2.0) and negative. (<1.5). The PathVysion kit was used for FISH assay to evaluate HER2 amplification. Ratios >2.0, 1.8 to 2.0 and <1.8 were defined positive, borderline and negative amplification respectively. Results: IHC compared to FISH Conclusion: Despite improvements in IHC testing, the FISH assay may be a better method for determining HER2 status. Factors such as tissue fixation, scoring methods and choice of antibodies may contribute to the lower specificity of IHC. In the amplified group, the gene amplification ratio correlated with protein expression, being highest in the IHC positive cases and lowest in those that were negative. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-04.

To characterize prognostic values of androgen receptor (AR) in triple-negative (TN) breast cancers, we investigated AR expression status and levels, explored an association of AR expression with metastatic disease, and correlated AR expression with Ki-67 in TN invasive breast carcinomas. AR expression was analyzed with immunohistochemistry in 121 cases of TN tumors. Thirty-nine cases had distant metastatic disease and 82 had locoregional disease only. AR was positive in 38 (31.4%) of the 121 cases. Our results indicate that among the AR-positive TN tumors, distant metastases are significantly associated with lower expression of AR compared with cases with only locoregional disease, and that AR expression negatively correlates with Ki-67 expression. These findings suggest that decreased intratumoral AR expression may be predictive of distant metastatic disease and AR expression levels may have potential prognostic value in AR-expressing TN tumors.

Abstract Background: Molecular subtypes of breast cancer have been characterized by gene expression analysis. Luminal subtypes are hormone receptor (HR) positive and Her2/neu negative. Immunohistochemistry (IHC) has been used as a surrogate test for gene expression. Ki67 is a proliferation marker that identifies high-risk subtype of luminal breast cancer. Recently, the proposed Ki67 index (KI) of 14% was suggested as a cut-off to distinguish between luminal A and luminal B tumors (JNCI. 2009;101:736-750). The oncotype dx (ODx) is a 21- gene test that provides prognostic and predictive information in early stage HR-positive breast cancer patients. The test is reported as low (<18), intermediate (18-30) and high (>30) risk recurrence scores (RS) Design: We investigated the relationship between ODx RS and luminal subtypes of breast cancers using the KI of 14% as the cut-off for distinguishing luminal A and B tumors. Biomarker analysis (ER, PR, Her2/neu, Ki67 and p53) was performed as part of the diagnostic work-up using standard IHC procedures. Scoring was done by automated image analysis. Her2/neu FISH was performed on all IHC 2+ and 3 + results. Pathologic parameters such as, tumor size, grade, and presence of LVI were evaluated. Ploidy was performed by the Autocyte system (Tripath) on Feulgen stained paraffin sections. Results: We identified 106 patients with HR positive breast cancer who were tested for ODx from February 2006 to May 2010. 85/106 had Ki67 data available for analysis. 46/85 (54%) were luminal A and 39/85 (46%) luminal B tumors. The mean KI in luminal A was 6.93% versus 31.1% in luminal B (P<0.0001). The mean tumor size was 1.94 and 1.92 cm in luminal A and B respectively. Grade 1, 2 and 3 comprised 28/85 (32.9%), 49 (57.6%) and 8 (9.41%) of all tumors respectively. Of the grade1 tumors, 75% were luminal A, and grade 3 tumors were predominantly luminal B (p=0.013). LVI was present in 16 cases, 11 (68.8%) in luminal B and 5/16 (31.2%) in luminal A tumors (p=0.0416). Luminal A tumors were predominantly diploid 30/45 (66.6%) and luminal B were mostly aneuploid 21/32 (65.6%) (p=0.019). The overall mean RS in luminal A and B tumors was 14.67 and 20.15 respectively (P<0.0004). Luminal A tumors had low RS in 32/46 (66.6%). and luminal B tumors had predominantly intermediate/high RS in 23/39 (62.1%) (p=0.0082). ER Allred Scores were 7.1 and 7.3 and percent positivity was 88.1% and 91% respectively for luminal A and B subtypes. PR Allred scores were 5.6 and 5.8, and percent positivity was 62.1 and 52.9% for A and B tumors respectively. Information regarding treatment was available in 72 cases. 19 (26.3%) were treated by a combination of chemo and anti-hormonal therapy and 53 (73.6%) were treated by anti hormonal therapy alone, 8 of the 19 (42.1%) luminal A patients received combination therapy versus 11 (57.8%) in the luminal B category (p=0.277). Conclusion: Ki67 is a useful marker that showed significant correlation with RS by ODx. Luminal A tumors are more likely to be low grade, diploid with low RS compared to luminal B tumors. Ki67 in conjunction with other pathologic parameters may serve as a surrogate marker for the ODx RS. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-07.

Abstract Background: Oncotype Dx (ODx) is a 21-gene RT-PCR based assay that provides prognostic and predictive information in patients with hormone receptor (HR) positive breast cancer. The test predicts the likelihood of disease recurrence in the form of a recurrence score (RS), which is categorized into low, intermediate and high-risk types. We examined the relationship between ODx RS and histologic parameters, DNA ploidy, tumor markers (ER, PR, Her2, Ki67, p53) expression by immunohistochemistry (IHC). Design: We identified 106 patients with HR-positive invasive breast cancer who were tested for ODx from the department files. Pathologic variables such as, tumor size, grade, histologic type, lymphovascular invasion (LVI) were reviewed. Tumor markers ER, PR, Her2, p53 expression, Ki67 index (KI) and ploidy were performed as part of the patient's diagnostic work-up using standard procedures. Scoring was done by automated image analysis. Results of biomarker expression were compared to the ODx RS. All Her2neu 3+ and 2+ results by IHC were confirmed by FISH. Ploidy was performed by the Autocyte system (Tripath) on Feulgen stained sections. Oncotype RS was reported as low (<18), intermediate (18-30) and high risk (>30). Results: Mean tumor size was 1.96 cm. Of the 106 cases, 87 (82%), 14 (13.2%) and 2(1.8%) were ductal, lobular and mixed types respectively. Grade 1, 2 and 3 comprised 35 (33.0%), 62 (58.4%) and 9 (8.5%) respectively. Twenty-one (19.8%) had LVI. Oncotype RS was low in 56 (52.8%), intermediate in 45 (42.4%) and high in 5 (4.7%). Mean tumor size in low RS group was 2.2 cm versus 1.6 cm in the intermediate/high RS group (p <0.05). There was no significant association of grade and LVI with RS. ER Allred score was 7.2, 7.2 and 8.0 and percent positivity was 88.0%, 91.1% and 96.3% in low, intermediate and high RS respectively (p=0.6325). The PR Allred score was 6.0, 5.3 and 3.5 and percent positivity was 64.7%, 51.8% and 16.6% in low, intermediate and high RS respectively (p=0.0432). There was 100% concordance between ER by IHC and ODx. Concordance for PR was 71/75 (94.6%). The four discordant PR results by ODx had mean score of 20% by IHC. The mean KI was 14.4%, 21.8% and 25.6% in low, intermediate and high RS respectively (p=0.0248). Low KI (<14%) and high KI (>14%) showed significant correlation with RS (p=0.0055). Her2neu IHC was negative in 43/49 (87.7%) and borderline in 6/49 (12.2%) and all were negative by FISH. Her2 FISH showed 100% (44/44) concordance with Her2 results by ODx. There was no association between DNA ploidy and RS (p=0.7143). P53 expression showed no significant correlation with RS (p=0.2602). Patients in the intermediate/high RS group were more likely to be treated with a combination of chemo plus anti-hormonal therapy (P<0.0001) and radiation (P<0.05) compared to the low RS group. Conclusions: This study shows a complete concordance between ER IHC and Her2 FISH with RT-PCR by ODx. Low PR scores and high KI predict high RS by ODx. There is a significant association between KI and ODx RS. Ki67 index identifies low and high RS groups, which correspond to the luminal A, and B subtypes of breast cancer respectively. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-10.

Abstract Abstract #202 Introduction: Controversy remains over whether to perform sentinel node biopsy (SNB) before or after neoadjuvant chemotherapy (NAC). We examined the practice patterns, feasibility, and accuracy of this procedure in high risk breast cancer patients treated with NAC in a multi-institutional correlative science study. Methods: Patients with biopsy-proven breast cancer >3 cm enrolled into the I-SPY TRIAL to undergo 4 weeks of anthracycline-based NAC, 4 weeks of taxane treatment, then surgical intervention. Study protocol did not dictate axillary treatment. Timing of SNB was dictated by the surgeon. Practice patterns, outcome of SNB and axillary lymph node dissection (ALND), locoregional recurrence and distant metastases were recorded with a mean follow-up of 2.9 years. Results: 237 patients enrolled, 221 completed the trial, 210 had complete data at the time of analysis; Table 1 shows axillary practice patterns. Overall, 43% had a positive SNB and/or ALND after NAC. 129 (61% of 210) patients presented with clinically positive nodes, 39 of which had a post-NAC SNB. 5/39 had no ALND (all SNB negative). Table 2 shows results for those who had a post-NAC SNB and ALND. For this subset of patients, sentinel node ID, accuracy, and false negative (FN) rates were 80%, 91% and 15% respectively. If SNB was negative, 20% of patients still had a positive ALND. 81 (39% of 210) patients presented with clinically negative nodes, 22 of which had a post-NAC SNB. 8/22 post-NAC SNB patients had no ALND (6 negative, 2 positive for 1mm disease). Table 2 shows results for those who had a post-NAC SNB and ALND. For this subset of patients, sentinel node ID, accuracy, and FN rates were 100%, 100% and 0%. Overall, there were 26 deaths; 96% occurred in those who presented with clinically positive nodes, 77% had positive post-NAC nodes. A negative axilla post-NAC was predictive of longer DFS over those with axillary disease post-NAC (p<0.05). Conclusions: In clinically node negative patients, post-NAC SNB is feasible and accurate before or after NAC. Our data suggests that a post-NAC SNB is sufficient; this avoids an additional operation and allows us to gain information on post-NAC axillary status which is of prognostic significance. In clinically positive patients, SNB does not adequately reflect axillary disease; even when SNB was negative, 20% still had axillary disease. At this time, we recommend that ALND be performed on all clinically node positive patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 202.