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Background: Neoadjuvant chemotherapy (NAC) with taxanes, followed by fluorouracil, epirubicin, and cyclophosphamide (FEC), with concurrent trastuzumab is known to achieve a high pCR rate of more than 60% for HER2-positive breast cancer (BC) as well as good prognoses in those obtaining pCR. On the other hand, the prognostic significance of tumor-infiltrating lymphocytes (TILs) has recently been described in triple-negative BC. However, the prognostic and predictive values of TILs in HER2-positive BC remain unclear. In the present study, we examined the grades of TILs in pre-treatment cancer tissues and residual tumors after NAC with trastuzumab, and also investigated its predictive utility for pCR and prognostic power for HER2-positive BC. Patients and Methods: A total of 128 Japanese women with HER2-positive BC received either paclitaxel or docetaxel followed by FEC, with concomitant trastuzumab. The proportional grades of stromal (Str)-TILs in pre-treatment biopsy specimens and residual tumors after NAC with trastuzumab were determined as follows: low grade (0-10%), intermediate grade (10-40%), and high grade (40-90%), using the criteria of the International Working Group for TILs in BC. Analysis 1: The relationship between the grades of Str-TILs in pre-treatment tumors and pCR rates was investigated. Relapse-free survival (RFS) and cancer-specific survival (CSS) were analyzed for a correlation with pre-treatment Str-TILs. Analysis 2: Alterations in the grade of Str-TILs were examined in the residual tumors of non-pCR patients, and RFS and CSS were analyzed for a correlation with residual Str-TILs. Results: pCR was achieved in 83 out of the 128 patients (pCR rate, 64.8%) who received NAC with trastuzumab, and RFS was significantly better in the pCR group than in the non-pCR group (p = 0.0071). Analysis 1: The patient distribution of the Str-TILs grade in pre-treatment tumors was as follows: high: 24 (18.8%); intermediate: 38 (29.7%); and low: 66 (51.6%). pCR rates correlated with the Str-TILs grade in pre-treatment tumors: 83.3% in the high group, 71.1% in the intermediate group, and 54.5% in the low group (p = 0.026); however, the Str-TILs grade in pre-treatment tumors did not correlate with survival. Analysis 2: In 45 non-pCR patients, the distribution of the Str-TILs grade in residual tumors was as follows: high: 9 (20.0%); intermediate: 8 (17.8%); and low: 28 (62.2%), respectively. In non-pCR patients, the rate of a high Str-TILs grade was greater in residual tumors than in pre-treatment tumors (residual, 20.0%, pre-treatment, 8.9%). RFS was significantly better with a high grade than with a low grade of residual Str-TILs (p = 0.033). Conclusions: The status of TILs in pre-treatment tumors predicted responses to NAC concomitant with trastuzumab in HER2-positive BC. The grade of TILs was higher in residual tumors than in pre-treatment tumors, and, among non-pCR patients, the prognosis of patients with a high residual-TILs grade was better prognosis than that of patients with a low residual-TILs grade. We speculate that an examination of TILs in residual tumors after NAC with trastuzumab may be necessary for selecting patients with a good prognosis from non-pCR patients. Citation Format: Kurozumi S, Inoue K, Matsumoto H, Hayashi Y, Tozuka K, Kubo K, Komatsu K, Takai K, Nagai SE, Oba H, Horiguchi J, Takeyoshi I, Kurosumi M. Prognostic value of tumor-infiltrating lymphocytes in residual tumors after neoadjuvant chemotherapy concomitant with trastuzumab for HER2-positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-14-17.

BACKGROUND: Addition of trastuzumab to neoadjuvant chemotherapy (NAC) improved pathologic complete response (pCR) rate in HER2-positive breast cancer. Although recent trials have shown favorable prognosis with NAC plus trastuzumab, clinicopathological factors to predict the outcome have not been fully understood. The aim of this study was to investigate the survival after NAC with trastuzumab and to explore the predictive factors. PATIENTS AND METHODS: This is a multicenter retrospective observational study. Patients with HER2-positive primary breast cancer treated with NAC plus trastuzumab from 2001 to 2010 were identified from the institutional database. Primary end point was disease-free survival (DFS). pCR was defined as ypT0/is+ypN0. Kaplan-Meier method was used to estimate DFS. Logistic regression and proportional hazard analysis were used to identify clinicopathological factors to predict pCR and DFS, respectively. RESULTS: 733 patients were included in the analysis (whole dataset). 425 were ER/PgR-negative (HR- dataset) and 306 were ER/PgR-positive (HR+ dataset). Radiation therapy was performed in 90% of lumpectomy and 31% of mastectomy. Hormonal therapy was performed in 84% of HR+ dataset. pCR rate was 45% in whole dataset, 60% in HR- dataset, and 34% in HR+ dataset. Table 1 showed the result of multivariate analysis for pCR in whole dataset. When HR+ and HR- dataset were analyzed separately, no definitive predictors for pCR were identified in multivariate analysis. Although the patients with pCR showed a significantly favorable prognosis than those without pCR at 3 years DFS, in whole dataset (93% vs 83%, p<0.0001) and HR- dataset (94% vs 80%, p<0.0001), there was no significant difference in HR+ dataset (89% vs 86%, p = 0.10). Different predictors were selected for DFS when multivariate analysis was conducted separately between HR- and HR+ dataset (Table 2). CONCLUSIONS: In this observational study, we clarified predictors for pCR and DFS in HER2-positive patients treated with neoadjuvant trastuzumab containing therapy based on tumor subtype. Our results may help us to predict the prognosis more precisely and to simulate the disease course. Table 1) Multivariate logistic regression analysis for pCR in whole datasetFactorsOR95%CIp-valuePost- vs Pre-menopause1.50(1.05-2.15)0.026*cT1-2 vs cT3-41.72(1.16-2.59)0.008*ER/PgR-negative vs ER/PgR-positive3.32(2.30-4.82)<0.0001*Grade 3 vs 1-21.28(0.89-1.84)0.183 Table 2) Multivariate proportional hazard analysis for DFSFactors†HR95%CIp-valueWhole dataset   Pre- vs Post-menopause1.61(1.04-2.52)0.033*cN2-3 vs cN03.06(1.58-6.24)0.001*cN1 vs cN02.26(1.23-4.41)0.007*Grade 3 vs 1-21.87(1.20-2.97)0.006*non-pCR vs pCR1.90(1.18-3.13)0.008*HR- dataset   Pre- vs Post-menopause1.70(1.01-2.85)0.046*cT3-4 vs cT1-21.86(1.09-3.17)0.024*non-pCR vs pCR3.28(1.90-5.87)<0.0001*HR+ dataset   cN2-3 vs cN05.01(1.79-16.19)0.002*cN1 vs cN03.50(1.40-10.61)0.006*Grade 3 vs 1-22.95(1.52-5.87)0.001*†Only factors with statistical significance Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-20.

Background : The detection of circulating tumor cells (CTCs) in peripheral blood is an independent predictor of the efficacy of systemic therapy and a prognostic marker for patients with metastatic breast cancer. One of the leading techniques to detect CTCs uses immune-magnetic separation followed by immunocytochemistry. A microdevice can capture and enumerate CTCs using distinctive physiological difference (size and deformability) between cancer cells and blood cells. This microdevice thus obtains a larger CTC yield than that of affinity based separation which enriches the samples from a particular subgroup of cells based on biomarker (EpCAM) used. In this study, we investigated CTCs in peripheral blood from metastatic breast cancer patients using this microdevice. Patients and methods: We examined blood samples of 9 patients with heavily treated locally recurrent or metastatic breast cancer. Informed consent from these patients was obtained before blood extraction. Blood samples were taken into sodium EDTA tubes after discarding the first 1ml of blood samples. Two ml whole blood were subjected to the microdevice (Clear cell system), and CTCs were trapped in the microwells: Trapped cells were analyzed by immunocytochemistry with monoclonal antibodies specific for leukocytes (CD45) and epithelial cells (CK8/18), along with 4,2-diamidino-2-phenylndole dihydrochloride (DAPI) for nuclei. CK8/18- positive, DAPI-positive and CD45-negative cells were defined as CTCs. Three patients were examined using both this microdevice and affinity-based separation with EpCAM, to compare the yield of CTCs. Results: Of the 9 patients: 7 had ER-positive primary tumors, and 6 had PgR-positive ones, HER2 overexpression was detected in 2 primary tumors. CTCs were detected in 8 patients. The single patient in whom CTCs were not detected suffered from local recurrence (axillary lymph node metastasis) only, with no distant metastases. We were also unable to detect CTCs using EpCAM affinity method for this patient. The number of detected CTCs in the other patients ranged from 19/2ml to 156/2ml (mean 90/2ml), and the sizes of CTCs varied from 5 to 16μm. CK8/18-negative and DAPI positive were detected in most patients, and these cells tended to be larger than CK8/18-positive cells, suggesting that epithelial–mesenchymal transition (EMT) might occur in CTCs. The total number of CTCs detected by the microdevice from 2 patients was larger than that of CTCs detected by EpCAM affinity method (107/2ml vs 1/7.5ml, and 19/2ml vs 39/7.5ml). Conclusion: CTCs detected by this microdevice varied in regard to the size of trapped cells and characteristics examined by immunochemistry, suggesting the heterogeneity of CTCs. Further research on this heterogeneity is vital in order to develop personalized treatment for patients with metastatic breast cancer. Citation Format: Tozuka K, Nagai SE, Inoue K, Komatsu K, Matsumoto H, Hayashi Y, Kurozumi S, Suganuma M. Enumeration of heterogeneous circulating tumor cells (CTCs) in metastatic breast cancer patients based on size and deformability. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-20.

Background The detection of circulating tumor cells (CTCs) in peripheral blood is an independent predictor of the efficacy of systemic therapy, and also a prognostic marker for patients with metastatic breast cancer. One of the main methods to detect CTCs is CellSearch system, which uses immune-magnetic separation followed by immunocytochemistry. A microdevice (CTChip from ClearCell system) can capture and enumerate CTCs based on distinctive physiological differences (size and deformability) between cancer cells and blood cells. CTChip thus obtains a larger CTC yield than affinity-based separation, which enriches a particular subgroup of cells expressing EpCAM. In this study, we enumerate CTCs in peripheral blood from early and metastatic breast cancer patients using a size-based method. Patients and methods We examined blood samples from a total of 18 early and metastatic breast cancer patients, after obtaining written informed consent. Blood samples were taken in sodium EDTA tubes after discarding the first 1ml of blood from the syringe. Two ml blood samples were applied to CTChip (ClearCell system), and CTCs were eventually trapped in the microwells of the CTChip. Trapped cells were analyzed by immunocytochemistry with monoclonal antibodies specific for leukocytes (CD45) and epithelial cells (CK8/18), along with 4',6-diamidino-2-phenylindole (DAPI) for nuclei: CK8/18-positive, DAPI-positive and CD45-negative cells more than 10 μm in diameter were defined as CTCs. Eight patients were examined using both the CTChip and CellSearch system to compare the yield of CTCs. Results Of 18 patients, 6 were de novo stage IV, 6 were recurrent and 6 were early stage breast cancer patients. Of primary tumors, 8 were HER2- and ER and/or PR +, 6 were HER2-and ER- and PR-, 3 were HER2+ and ER and/or PR +, and one was HER2+ and ER- and PR-. Using CTChip, detected CTCs ranged from 3 - 107 cells/2 ml in all cases: 3 - 83 for early stage, 19 - 156 for stage IV and 21 - 146 for recurrent. The number of CTCs found in recurrent patients tended to be higher than in early stage patients. Size-based method using CTChip clearly showed high sensitivity compared with the CellSearch system, which detected CTCs in only 2 cases out of 8. In analysis by immunochemistry, we found CK-negative, CD45-negative and DAPI positive cells with larger diameter (>16 μm) than CK-positive CTCs in most patients, and the numbers were higher in stage IV (8.5 cells of median value) and recurrent (13 cells) patients than in early stage patients (1.5 cells). Our study suggested that CK-negative large cells might be CTCs with epithelial–mesenchymal transition (EMT). Conclusion This size-based technology enables us to capture CTCs regardless of EpCAM expression. Enumerated CTCs varied in size and positivity of CK8/18, suggesting the heterogeneity of CTCs. Further research, especially focusing on EMT will be crucial to understand the key mechanism of metastasis and drug resistance. Citation Format: Tozuka K, Nagai SE, Kubo K, Komatsu K, Takai K, Inoue K, Matsumoto H, Hayashi Y, Tsuboi M, Yamada Y, Wang X, Suganuma M. Enumeration of heterogeneous circulating tumor cells (CTCs) using size-based method in early, and metastatic, breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-08.