Search

e12576 Background: From the results of ACOSOG Z0011, IBCSG23-01 and AMAROS trials, axilla surgery in node-positive breast cancer (BC) tends to be less invasive with sentinel node biopsy (SNB) followed by adjuvant therapy and regional node irradiation (RNI). However, optimized axilla treatment including SNB without RNI is still debated. The Japanese Society for Sentinel Node Navigation Surgery conducted a multi-institutional prospective cohort study to compare SNB with SNB followed by axillary lymph node dissection (ALND) in cases with positive-sentinel lymph nodes (SLN)(UMIN No. 000011782, Jpn J Clin Oncol, p.876-9, 2014). Methods: Female BC patients with cT1-3N0-1M0 were eligible. When 1 to 3 positive micrometastases or macrometastases in SLN were confirmed by histological or molecular diagnosis, SNB alone or additional ALND had been decided by physician’s discretion. Primary chemotherapy before or after SNB was acceptable for registration. Lymph node sampling was also allowed in the SNB group. Cases with bilateral BC, isolated tumor cells only in SLN, past history of invasive cancer within 5 years at the registration were ineligible. The primary endpoint was the 5-year recurrence rate of regional node (RN) in the SNB group. The secondary endpoint was overall survival (OS). We planned to collect 240 patients to reject that the 5-year recurrence rate of RN was more than 10% assuming the rate 5%. To compare the SNB group and ALND group, the propensity score matching (PSM) was performed. Matching variables were initial treatment, metastatic size and numbers of SLN, clinical stage, age, body mass index, menopausal status, family history, past history of invasive cancer, breast surgery. Results: Eight-hundred eighty cases had been registered between 2013 and 2016. In the 871 eligible cases, 308 cases were the SNB group. At the median follow-up of 6.3 years, 5-year recurrence rate of RN was 2.7% [95% confidence interval, 1.4% to 5.4%] and 5-year OS was 97.6% [94.9% to 98.8%]. After PSM, 209 cases were matched in the SNB and ALND group. Among them, 343 cases (82%) received operation at initial treatment. Partial and total mastectomy was performed in 225 (54%) and 193 cases (46%), respectively. One-positive SLN was recorded in 366 cases (88%), 2 in 48 (11%) and 3 in 4 (1%). Macrometastases and micrometastases in SLN were diagnosed in 271 (65%) and 147 cases (35%), respectively. Three-hundred seventy-six cases (90%) belonged to luminal-like subtype. RNI was underwent in 42 cases (20%) of the SNB group and 13 cases (6%) of the ALND group. Five-year recurrence rate of RN was 2.1% [0.8% to 5.5%] and 2.0% [0.8% to 5.3%] for the SNB and ALND group, respectively. Conclusions: Our series suggests that RNI is not necessary for regional control in cases with 1 to 3 positive SLN. In conclusion, SNB alone is acceptable in cases with fewer metastatic SLN. Clinical trial information: UMIN No. 000011782.

e13111Background: Breast cancer is the most frequent cancer among women both in US and Japan. Triple negative breast cancer (TNBC), a subset of breast cancers that are negative for estrogen recepto...

e23122 Background: Breast cancer is the leading cancer among women both in US and Japan. Triple negative breast cancer (TNBC) is the subset of breast cancers that are negative for estrogen receptor (ER), progesterone receptor, and HER2. While the selective ER modulator tamoxifen or anti-HER2 therapy for breast cancer patients with ER positive or HER2 protein overexpression are the most successful examples of targeted therapies, only limited therapies are available for patients with TNBCs, which are associated with a poor prognosis. Advance in next-generation sequencing enables us to identify actionable driver mutations that can be potentially treated by targeted therapies in each cancer patient. The aim of this study is to examine actionable driver mutations in TNBCs in Japan by comprehensive genomic sequencing (CGS) with 435 gene panel, and compare the driver events in Japan with TCGA database to validate the utility of CGS. Methods: We examined all exons of 435 known cancer genes in Japanese TNBC patients (N = 53) by CGS and evaluated for concordance among independent data obtained from The Cancer Genome Atlas-TNBC whole exome sequencing database (N = 123). Results: Oncogenic driver mutations were identified in 51 of 53 Japanese patients (96%) with TNBC and 36 of 53 patients (67%) harbored mutations in genes associated with FDA-approved targeted therapies, indicating the potential clinical utility of a large gene panel for evaluating patients with TNBC. Among 80 total genetic alterations, frequently mutated genes ( > 10% patients) were TP53, PIK3CA and PTEN. Overall, the mutation spectrum of the Japanese patients is similar to that of the TCGA population, except amplification of MYC. Conclusions: Use of a CGS panel of 435 genes can reliably identify all of the critical mutations in TNBC patients, which are similar as TCGA data. The information derived from CGS can be used to determine the optimal treatment for TNBC patients.

145 Background: Saliva is an easily accessible and informative biological fluid which has high potential for the early diagnosis of diseases. Saliva-based diagnostics, particularly those based on metabolomics technology, offer a promising clinical strategy by characterizing the association between salivary analysts and a particular disease. The aim of this study is to investigate potential biomarkers in human saliva to facilitate the early diagnosis of breast cancer. Methods: We conducted a comprehensive metabolite analysis of saliva samples obtained from 60 breast cancer patients and 20 healthy controls, using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). Statistical analyses were performed by using a nonparametric Mann-Whitney U test, multiple logistic regression and the receiver operating characteristics (ROC) to evaluate the predictive power of biomarkers. Results: Forty-nine patients (81.7 %) had invasive ductal carcinoma (IDC) and 9 patients (15.0 %) had ductal carcinoma in situ. In 33 patients receiving neoadjuvant treatment, saliva samples were obtained just before surgery. After removing the concomitantly observed peaks and noise peaks, an average of 205 peaks were derived from the metabolites. Among these peaks, five potential salivary biomarkers demonstrated significantly higher concentrations in breast cancer patients comparing with healthy individuals (p < 0.05). Especially, salivary biomarkers obtained from patients with IDC before neoadjuvant treatments tended to be higher concentrations than those obtained after treatment. In these metabolites, the area under the ROC curves (AUCs) were 0.765 for substance A, 0.716 for substance B, 0.809 for substance C, 0.819 for substance D and 0.850 for substance E. Conclusions: Salivary metabolites are promising biomarkers for the early diagnosis of breast cancer.

26 Background: HOXB9 is a member of class I HOX genes and is known to be overexpressed in human breast cancer and related to EMT, tumor angiogenesis, and radio-resistance (PNAS 2010), being significant prognostic factor in breast cancer patients. (Ann Surg Oncol 2012). Methods: We analyzed the upstream promoter region to determine the critical sequence for HOXB9 transcription. The multiple lengths of HOXB9 promoter region were cloned into pGL3 luciferase vector and each of them were checked by luciferase assay. Then made E2F1 overexpressed cell-lines: MCF7, MCF10A and MDA-MB231 to check HOXB9 gene expression. ChIP assay performed using E2F1 induced MCF7 cell-line model and EMSA made with T47D cells and E2F1 antibody. Results: We demonstrated luciferase activity of MDA-MB231 cells and overexpressed HOXB9 was determined at the region from -404 to -392 bps, showing that the mentioned region plays the critical role for the transcription of HOXB9. Computer prediction system for the binding ability to critical sequence raised several candidate: E2F1, PAX-5, P53, ETS-1, NFY-A, Sp-1, which are known to be related with cancer progression and we confirmed these genes could induce HOXB9. E2F1 overexpressed breast cancer cell-lines indicate high expression of HOXB9 show the correlation between E2F1 and HOXB9 expression. Luciferase assay method done by constructing pGL3 with HOXB9 critical sequence and analyzed using wild-type MCF7 and E2F1 induced MCF7 cells showed high luciferase activity of HOXB9 critical sequence plasmid in E2F1 induced MCF7 cell-line by compare to WT. ChIP assay and EMSA assay methods performed using E2F1 overexpressed MCF7 and T47D cell-lines model revealed high precipitation rate with E2F1 antibody and HOXB9 expression than negative control. Conclusions: HOXB9 is plays the role as the accelerator of breast cancer progression and discovering the properties of the gene and related factors will help to understand new signaling pathways of breast cancer development. E2F1 is a strong candidate direct binds to HOXB9 critical sequence and correlates with HOXB9 expression. Manipulating of E2F1 expression may regulate HOXB9 and reduce cancer progression and metastasis.

51 Background: For patients with clinically node-negative, early-stage breast cancer, sentinel lymph node biopsy (SLNB) has replaced axillary lymph node dissection (ALND) as a standard method for staging of regional lymph nodes. Regional recurrences after negative (SLNB) have generally been reported in the range of 0.5 to 2% and factors associated with regional recurrence in patients with negative SLNB are still to be elucidated. In this study, we evaluated regional recurrence rates and predictors of regional recurrences in patients with negative SLNB. Methods: Between January 2001 and December 2012, 1,322 patients with clinical node-negative invasive breast cancer less than 3cm underwent SLNB at Keio University Hospital. Of 1,322 patients with SLNB, 1,033 patients with negative SLNs were included in this study. Sentinel lymph nodes (SLNs) were detected using a combined method of blue dye and small-sized technetium-99m-labeled tin colloid. Intraoperative frozen examination was performed with hematoxylin and eosin (HE) staining. SLNs, fixed and embedded in paraffin, were additionally diagnosed with HE staining and immunohistochemical analysis. Results: Median age was 57.0 years (range, 25-89) and median tumor size was 1.9cm (range, 0.5-6.0cm). After a median follow-up of 54.8 months, there were 13 regional (1.3%) and 26 distant recurrences (2.5%). Median disease-free interval of regional and distant recurrences was 32.6 and 22.7 months, respectively (p=0.761). Higher nuclear grade (NG) was significantly correlated with regional and distant recurrences (p=0.001 and p=0.008). The rate of lymphovascular invasion (LVI) was significantly higher in patients with regional recurrences comparing with patients without recurrences (58.3% vs. 27.6%, p=0.026), however LVI was not a significant predictor of distant recurrences (p=0.072). Estrogen receptor negativity was significantly correlated with distant recurrences (p=0.013), whereas it was not associated with regional recurrences (p=0.626). Conclusions: Regional recurrences were rare in early-stage breast cancer patients with negative SLNB. LVI and NG can be used as predictive factors of regional recurrences after negative SLNB.

168 Background: Despite clinical usefulness of trastuzumab, intrinsic or acquired resistance to trastuzumab is a common clinical phenomenon. However, the mechanisms of resistance to trastuzumab have not been fully elucidated. The objective of this study was to determine the possible mechanisms of resistance to trastuzumab as neoadjuvant therapy in women with HER2-overexpressing operable breast cancer. Methods: Patients with operable breast cancer received 12 cycles of weekly paclitaxel plus weekly trastuzumab before surgery. All tumors were HER2-positive by immunohistochemistry (IHC) or fluorescence in situ hybridization. Expressions of ER, PgR, Ki67, PTEN, phosphorylated IGF-1R (pIGF-1R) and MUC4 were performed by IHC in core needle biopsy samples at baseline. PIK3CA mutation status was evaluated by sequencing of PIK3CA exons 9 and 20 using PCR amplification and direct sequencing. Results: Thirty-seven patients were enrolled and assessable for clinical and pathologic responses. The pCR rate was 48.6% (18/37). Negative, moderate and strong membranous expression of MUC4 was observed in 3 (8.1%), 18 (48.6%) and 12 (32.4%) patients, respectively. Membranous staining for pIGF1-R was negative in 24 (64.9%) patients. PTEN loss was observed in 33.3% (8/24) of the tumor examined. PIK3CA sequence analysis of the 13 tumors identified 2 mutations in exon 20 and 2 mutations in exon 9, corresponding to a PIK3CA mutation frequency of 30.8%. MUC4 status did not affect the pCR rate. Although membranous pIGF1-R expression did not affect pCR rate in hormone receptor-positive patients (66.7% vs. 50.0%), hormone receptor-positive patients with positive membranous pIGF1-R tended to show higher pCR rate compared with negative pIGF1-R (41.2% vs. 0%). PTEN loss and/or PIK3CA mutation were not significantly associated with pCR rate. Conclusions: These data indicate that aberrant downstream signaling caused by loss of PTEN and/or PIK3CA mutation, alternative signaling from IGF-1R and masking with MUC4 were not important mechanisms of resistance to trastuzumab.

97 Background: Hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-positive tumors generally cluster within the luminal/HER2 subset; whereas HR-negative/HER2-positive tumors reside in HER2-enriched subset. We investigated whether the efficacy of HER2-targeted therapy for HER2-positive tumors differs by HR status. Methods: Sixty-eight patients with operable breast cancer received trastuzumab plus taxane based therapy before surgery at Keio University Hospital from March 2009 to April 2012. All tumors were HER2-positive by immunohistochemistry (IHC) or fluorescence in situ hybridization. Expressions of ER, PgR, and Ki67 were performed by IHC in core needle biopsy samples at baseline. Pathological complete response (pCR) defined as no invasive residuals in breast. All patients with luminal/HER2 tumors received adjuvant endocrine therapy in addition to adjuvant trastuzumab monothereapy. Results: Sixty-eight patients with HER2-positive tumors were divided into 35 (51.5%) patients with luminal/HER2 tumors and 33(48.5%) patients with HER2-enriched tumors, respectively. There were no significant differences in tumor size, clinical nodal status, nuclear grade, and Ki67 status between the two groups. Clinical complete response rate and objective response rate were similar between the two groups. Patients with luminal/HER2 tumors were significantly younger than patients with HER2-enriched tumors (median age (range): 53 (35-78) vs. 61 (31-72), p=0.041). Compared to patients with luminal/HER2 tumors, patients with HER2-enriched tumors had significantly higher pCR rate (28.6% vs. 69.7%, p=0.002). With 24 months median follow-up, no significant differences were observed between the two groups with respect to disease-free survival. Estimated 2-year disease-free survival for luminal/HER2 and HER2-enriched was 94.3% and 97.0%, respectively (p=1.000). Conclusions: HER2-enriched breast cancer showed significantly higher pCR rate to HER2-targeted therapy compared with luminal/HER2 breast cancer. However, there was no significant difference in disease-free survival between the two groups.

e11598 Background: The growing number of anti-HER2 agents suggests the eventual need for defining the optimal choice of neoadjuvant therapy for HER2-positive breast cancer. Multiple-treatments meta-analysis synthesizes information from a network of trials and combines direct and indirect evidence on the relative effectiveness. An indirect estimate of the benefit of A over B can be obtained by comparing trials of A v C with trials of B v C. In this study, we assessed the efficacy and safety of neoadjuvant therapy for HER2-positive breast cancer by conducting the direct and indirect comparisons from multiple RCTs. Methods: The primary outcome of the study was the number of the patients who achieved pathological complete response (pCR) defined as no invasive residual in breast or node. Secondary objectives were the number of patients who completed the treatment as planned and adverse events including diarrhea, neutropenia, cardiac events and skin disorder. Results: We identified 1047 articles by database search and 10 studies met our criteria. A total of 2247 patients in 7 different treatment arms were assessed; chemotherapy (CT) alone, CT with single or dual anti-HER2 agents and dual anti-HER2 agents without CT. Anti-HER2 agents evaluated were trastuzumab (T-mab), lapatinib, pertuzumab (P-mab). There was no significant difference between dual targeting treatment arms (CT + T-mab + lapatinib v CT + T-mab + P-mab, OR; 1.11, [0.42-2.86], p=0.41), however, lapatinib reduced the treatment completion mainly due to adverse events. Patients in dual targeting arms had significantly higher incidence of pCR than in other treatment arms. (CT + T-mab + P-mab v CT + T-mab, OR; 2.29, [1.02-5.02], p=0.02) Surface under the cumulative ranking probability curve (SUCRA) also indicated that CT + T-mab + P-mab had the highest probability of being the best treatment arm for pCR followed by CT + T-mab + lapatinib and CT + T-mab. Conclusions: This study provides evidence that combining two anti-HER2 agents with chemotherapy are the most effective treatment arms. Considering the cost and limited medical resources, CT + T-mab showed a well-balanced profile for efficacy, completion and safety.

92 Background: Estrogen receptor (ER), progesterone receptor (PgR), and HER2 status are important biological markers for making decisions about breast cancer treatments. Although changes of hormone receptor (HR) and HER2 status with recurrence of breast cancer are clinically experienced, the frequency of discordance and clinical significance are still unknown. Thus, we investigated ER, PgR, and HER2 status with primary tumors and recurrent lesions, and assessed discordance rates and prognosis. Methods: We retrospectively identified recurrent breast cancer patients who had biopsies or resections of recurrent lesions between January 2007 to April 2012 at Keio University Hospital. HR status was assessed by immunohistochemistry (IHC) and determined using the Allred score. HR status was defined as positive when score was 3 and more. HER2 status was assessed by IHC and fluorescence in situ hybridization (FISH) analysis. We defined HER2 positivity as 3+ staining intensity by IHC or the presence of HER2 gene amplification by FISH. Results: Among 32 recurrences, 40% (13) were loco-regional recurrences (LLR) and 60% (19) were distant metastases (DM) (lung 14; liver 3; brain 1; pleura 1). Discordance rates in ER, PgR and HER2 status between the primary tumors and the recurrence lesions were 12.5%, 31.3%, and 13.8%, respectively. The most common change was loss of PgR. Changings from negative to positive in ER, PgR and HER2 status were found in 6.3%, 3.1%, and 3.4% of the patients, respectively. All gains were found in distant metastases. Discordance in ER and HER2 were more common in DM (15.8% and 17.6%) comparing with LRR (7.7% and 8.3%). Loss of HR status was not associated with a shorter time to progression (TTP) (ER: 19.3 vs. 8.5 months, p=0.185; PgR: 4.4 vs. 11.5 months, p=0.907). Patients with discordant HER2 status had significantly shorter TTP than with concordant status (0.9 vs. 11.5 months, p=0.012). Conclusions: Discordance of biological markers between primary and recurrent breast cancers were seen in 10-30%. Although HR discordance was not associated with prognosis, patients with HER2 discordance had poorer TTP. Tissue confirmation should be considered for making effective treatment decisions in recurrent breast cancer.

132 Background: S-1, an oral fluoropyrimidine formulation that combines tegafur, 5-chloro-2,4-dihydroxypryidine, and potassium oxonate in a molar ratio of 1:0.4:1 has been widely used against solid cancers including gastric, colorectal, pancreatic, lung and breast cancer. In a phase II study, the response rate (RR) was 41.7% and the median survival was 872 days among taxane-pretreated patients with metastatic breast cancer (MBC). However, the predictive factor of S-1 in patients with MBC has not been determined yet. The purpose of this study is to investigate the correlation between 5-FU-related enzyme and clinical efficacy of S-1 in patients with MBC. Methods: Forty-eight patients with MBC were treated with S-1 twice daily at a dose of 80 mg/m2 for 4 weeks, followed by a 2-week rest interval. Laser-captured microdissection was performed from the formalin-fixed, paraffin-embedded tumor sections at surgery and the expression of 5-FU-related enzyme including thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) was evaluated by RT-PCR. Results: The median age was 58 years. ER, PgR, HER2 was positive in 57, 28, and 14% of the entire patients, respectively. The median number of pretreated chemotherapy regimens was 2 (range 0-6). The sites of metastatic disease were the viceral in 25 patients, bone in 9 patients and soft tissue in 17 patients. The overall response rate (RR) was 22.9% (11/48) and clinical benefit rate was 41.7% (20/48). The median time to tumor progresssion (TTP) was 14.3 months (range 6.0 – 22.7). ER, PgR and HER2 status was not significantly correlated with RR. TS expression was significantly associated with clinical efficacy of S-1. Moreover, it is notable that triple negative breast cancer (TNBC) revealed lower TS expression than luminal type (TNBC vs luminal type = 80% (8/10) vs 36.4% (4/11), P= 0.054, OR 0.143, 95% CI 0.020-1.032). According to the correlation between metastatic sites and RR, better RR was noted for soft tissue metastases (soft tissue vs visceral = 25% (5/20) vs 84% (5/6), OR 15.0 (95%CI 1.40–161.05). Conclusions: This study demonstrated that TS is a significant predicitve factor of S-1 in patients with MBC.

124 Background: The sequential combination of anthracycline and docetaxel (TXT) is one of the standard regimen in the preoperative setting, however, the adverse events of TXT such as febrile neutropenia, fatigue, edema is not low at all. Several reports showed a synergistic effect of combination chemotherapy with 5-FU and TXT through down-regulation of dihydropyrimidine dehydrogenase (DPD) and thymidine synthetase (TS). (Suto et al., Oncol Rep 2006) Okazaki et al. reported the trial of pre-operative combination chemotherapy of TS-1 (80mg/m2) and TXT (40mg/m2) with 13% of pCR rates (HER2-negative), and less adverse events. This study's purpose was to determine the efficacy of TS-1 and TXT followed by FEC in preoperative setting. Methods: This was a single-arm, clinical study evaluating the patients with stage II/III and HER2 negative breast cancer received 4 cycles of TS-1 (TS-1 80 mg/m2 on days 1-14 and docetaxel 40 mg/m2 on day 1 every 3 weeks), followed by 4 cycles of FEC (fluorouracil 500 mg/m2, epirubicin 100 mg/m2, cyclophosphamide 500 mg/m2on day 1 every 3 weeks). The primary end point was the pCR rate. The secondary end points were the clinical response rate, adverse drug reactions. The sample size of this study (55 patients) was calculated based on pCR rate obtained in previous studies of the sequential combination of anthracycline and TXT in preoperative setting (HER2-negative). Results: From November 2009 to April 2010, 55 patients were enrolled and 50 patients were assessable for clinical and pathologic responses. The overall response rate (caliper) was 82.0%. The pCR rate was 12.0% (6/50). The breast-conserving rate in patients whose tumor size was larger than 3 cm was 53.3%. PgR status was significantly correlated with pCR. (p=0.024) Grade 4 neutropenia was observed in one patient (2.0%) at the first cycle. The most common grade 3/4 non-hematologic adverse event was hand-foot syndrome, observed in 12 patients (24.5%). One patient (2.0%) showed grade 4 diarrhea. Conclusions: These results indicate that the sequential combination of TS-1 and docetaxel followed by FEC is a well-tolerated, effective neoadjuvant treatment for stage II/III breast cancer.

166 Background: Nipple-sparing mastectomy (NSM) has been gathering increased recognition as an alternative to more traditional mastectomy approaches. However, the oncological safety of these procedures is still to be elucidated. The purpose of this study is to investigate technical feasibility of NSM without increasing the risk of local recurrence and complications. Methods: Ninety-eight stage 0-III breast cancer patients received NSM between January 2001 and April 2012 at Keio University Hospital. Eligible patients were without skin involvement and not indicated for breast-conserving therapy by preoperative imaging. The pathological analysis of the subareolar tissue was performed and nipple-areolar-complex (NAC) would be removed if positive. Four hundred seventy-four breast cancer patients who had mastectomy during the same period were used as a control group. Results: The median age was 46.0 years (range 29–68). The mean clinical tumor size was 2.8±1.2cm and the mean distance between the nipple and the tumor was 2.5±1.7cm. Ninety-one patients (92.9%) had clinically node-negative status. NAC involvement was observed in 3 patients (3.1%). Three patients (3.1%) presented with epidermal necrosis. After a median follow-up period of 37.3 months (range 1-136), there were 7 recurrences (7.1%), including 4 local recurrences (4.1%) and 3 distant metastases (3.1%). There was no statistically significant difference in local recurrence between the patients with NSM and mastectomy (4.1% vs. 3.2%; p=0.16). Immediate and delayed reconstruction was performed in 2 cases (2.0%) and 41 cases (41.8%), respectively. Conclusions: Our results suggested NSM may provide oncologic safety for strongly motivated and carefully selected patients.

10531 Background: Homeobox B9 (HOXB9) is known to be overexpressed in human breast cancer and profoundly related to tumorigenicity, lung metastasis and radio-resistance. (Hayashida, PNAS 2010, and Chiba, PNAS 2011). However, little is known about the relation between the expression of HOXB9 and angiogenesis in colorectal cancer (CRC). We aimed to clarify the impact of HOXB9 in CRC and evaluate the importance for bevacizumab treatment. Methods: The expression of HOXB9 in human CRC specimens was analyzed. Then, we introduced HOXB9 construct into human CRC cell lines and examined TGFβ signaling and angiogenic factors. Xenograft model was established by these cell lines either with or without the administration of bevacizumab (5mg/kg, weekly) intraperitoneally. Finally, we examined the mRNA levels of consecutive patients who were treated by chemotherapy with bevacizumab in our institute and calculated the Kaplan- Meier curve with log-rank test. Results: 47 of 69 surgical specimens (67%) showed positive expression of HOXB9 mRNA. The high HOXB9 mRNA levels significantly correlated with poor differentiation and liver metastasis. The HOXB9-overexpressed cell lines showed significantly higher expression of TGFβ signaling target genes and angiogenic factors. HOXB9 overexpression significantly increased tumor volume and burden with higher microvessel density in vivo, even though the cell proliferation decreased in vitro. Notably, HOXB9-overexpressed tumor was dramatically shrunk by administration of bevacizumab (tumor shrinkage rate; 93% vs. 42% in HT29, 83% vs. 27% in HCT116). Patients with high expression of HOXB9 in tumor showed significantly longer progression free and overall survival periods (n=39). Conclusions: Our results demonstrated that patients with high expression of HOXB9 in tumor had better prognosis with bevacizumab treatment but worse without. In vivo and in vitro experiments revealed that HOXB9 might orchestrate angiogenesis and establish positive feedback between cancer cells and microenvironment. Bevacizumab might inhibit the feedback to reduce tumor growth dramatically. Therefore, HOXB9 may work as a potential surrogate marker of bevacizumab treatment in CRC.

e11060 Background: Despite clinical usefulness of trastuzumab, intrinsic or acquired resistance is a common clinical phenomenon. Potential mechanisms of resistance include aberrant downstream signaling caused by loss of PTEN and/or PIK3CA mutation, alternative signaling from IGF-1R and masking with MUC4. Methods: Patients with operable breast cancer received 12 cycles of weekly paclitaxel (80 mg/m2 IV) plus weekly trastuzumab (4mg/kg loading dose followed by 2 mg/kg IV) before surgery. All tumors were HER2-positive by immunohistochemistry (IHC) or fluorescence in situ hybridization. Expressions of ER, PgR, Ki67, PTEN, phosphorylated IGF-1R (pIGF-1R) and MUC4 were performed by IHC in core needle biopsy samples at baseline. ER and PgR status was assessed using Allred score. For Ki67 labeling index, a total of 400 cells were counted from three consecutive high-power magnifications. PTEN, pIGF-1R and MUC4 expression level was scored semiquantitatively based on staining intensity. PIK3CA mutation status was evaluated by sequencing of PIK3CA exons 9 and 20 using PCR amplification and direct sequencing. Results: Thirty-seven patients were enrolled and assessable for clinical and pathologic responses. The pCR rate was 48.6% (18/37). ER and PgR were positive in 18 (48.6%) and 16 (43.2%) patients, respectively. Negative, moderate and strong membranous expression of MUC4 was observed in 3 (8.1%), 18 (48.6%) and 12 (32.4%) patients, respectively. Membranous staining for pIGF1-R was negative in 24 (64.9%) patients. PTEN loss was observed in 33.3% (8/24) of the tumor examined. PIK3CA sequence analysis of the 13 tumors identified 2 mutations in exon 20 and 2 mutations in exon 9, corresponding to a PIK3CA mutation frequency of 30.8%. MUC4 and pIGF1-R status did not affect the pCR rate. PTEN loss and/or PIK3CA mutation were not significantly associated with pCR rate. pCR rate was significantly correlated with PgR negativity (p=0.004) and higher Ki67 (p=0.01). Conclusions: These data indicate that aberrant downstream signaling caused by loss of PTEN and/or PIK3CA mutation, alternative signaling from IGF-1R and masking with MUC4 were not important mechanisms of resistance to trastuzumab.

63 Background: The expression analysis in ER positive human breast cancer already demonstrated the loss of nuclear expression of BTG2, the cell cycle regulator, in 46% of tumors and showed a significant inverse correlation with cyclinD1 expression. Our analysis of Oncomine database suggested that BTG2 expression might be related to the clinical outcome in patients with tamoxifen treatment. Methods: The examinations to evaluate whether BTG2 expression affects the sensitivity against tamoxifen were conducted both in vitro and in vivo. Results: In vitro cellular proliferation assay using tamoxifen against ER positive cell lines revealed that T47D expressing high level of endogenous BTG2 showed more drug sensitivity than MCF7 expressing low level of BTG2. (MCF7; IC50=4.48uM, T47D; =1.31uM) Tetracycline-inducible BTG2 expression model was developed in MCF7 and the cellular proliferation was strongly inhibited by the concomitant administration of tetracyclin and tamoxifen in vitro. Then, the cell line was injected into the mammary fat pad of immunodeficient mice. After the tumors grew more than 125mm3, these tumor bearing mice were administered the water including tetracyclin and/or tamoxifen pellet for 3 weeks. Tumor growth ratio was significantly suppressed in the concomitant administration of tetracyclin and tamoxifen in comparison with the animal treated with single agent. (p=0.044) Consistently, tumor weight and Ki67 expression were also significantly suppressed in the mice administered both tamoxifen and tetracyclin. (BW; p=0.034, Ki67; p=0.039) The cohort of 60 patients treated with adjuvant tamoxifen monotherapy was available online. Comparison of disease-free survival (DFS) of patients with positive BTG2 expression versus patients with loss of BTG2 expression revealed an univariate association in the dataset (log-rank probability, p=0.029). Multivariate analysis indicated that BTG2 expression resulted an independent prognostic factor for DFS (HR, 0.69; 95% CI, 0.495 to 0.963; p=0.029). Conclusions: Despite further validation studies need to be conducted, BTG2 expression may be useful biomarker to identify patients appropriate for tamoxifen treatment.

140 Background: Sentinel lymph node biopsy (SLNB) is a more sensitive and accurate nodal staging procedure than axillary lymph node dissection (ALND). Because of detailed pathologic evaluation in SLNs, more nodal micrometastases have been identified. However, it remains controversial whether to perform ALND for patients with micrometastases in SLNs and their prognostic significance is also a matter of debate. The purpose of this study is to determine the non-sentinel lymph node (NSLN) status and prognosis of the patients with micrometastatic SLNs. Methods: A prospective database of 1,012 clinically node-negative, T1-T2 breast cancer patients, who underwent SLNB from January 2002 to Dec 2010 at Keio University Hospital was analyzed. SLNs were detected using a combined method of isosulfun blue dye and small-sized technetium-99m-labeled tin colloid. Intraoperative frozen examination was performed with hematoxylin and eosin (HE) staining. SLNs, fixed and embedded in paraffin, were additionally diagnosed with HE staining and immunohistochemical (IHC) analysis. Results: Micrometastases in SLNs were found in 69 (6.8%) of 1,012 patients. Thirty eight (55.1%) of 69 patients with micrometastatic SLNs underwent immediate or delayed ALND and revealed no NSLN metastasis. Among 31 (44.9%) patients with micrometastatic SLNs who omitted ALND and axillary radiation therapy, no axillary lymph node recurrence has been observed after a median follow-up of 50 months, although 29 patients (93.5%) in these 31 patients received adjuvant systemic therapy. There is no significant difference in recurrence free survival between the patients with micrometastatic and negative SLNs (98.0% vs. 95.7%, respectively). Conclusions: These date suggested that it may not be necessary to perform ALND for the patients with micrometastatic SLNs and the presence of micrometastases in SLNs may not worsen prognosis with proper systemic therapy.

33 Background: We demonstrated that HOXB9, a member of homeobox genes, expression promoted tumor neovascularization and metastasis in vitro and in vivo assay. These findings imply that overexpression of HOXB9 contributes to tumor progression through activation of signaling pathways that alter both tumor-specific cell fates and tumor-stromal microenvironment, leading to increased invasion and metastasis. (Hayashida et al., PNAS 2010) We sought to determine whether these results could be extended to the clinical application. In this study, we evaluated the correlation between HOXB9 expression, clinical outcomes, and the clinicopathological variables in breast cancer patients, and the contribution of HOXB9 expression to tumor cell proliferation and angiogenesis. Methods: A consecutive series of 141 patients with invasive ductal carcinoma who underwent surgical treatment were examined. HOXB9 protein expression was analyzed immunohistochemically using the anti-human HOXB9 polyclonal antibody. Immunostaining of Ki-67, CD31, and CD34 were performed to evaluate the association of proliferation and tumor angiogenesis with HOXB9 expression. Results: Of 141 tumor specimens immunostained for HOXB9, 69 specimens (48.9%) were positive staining. Univariate logistic regression revealed ER and PgR negativity, HER2 positivity, high nuclear grade, and large pathological tumor size as significant variables associated with HOXB9 expression. Moreover, 12 (92.3%) out of 13 triple negative breast cancer showed HOXB9 expression. The disease-free survival (DFS) and the overall survival were significantly different between the HOXB9 positive and negative group; HR=20.714, p=0.001, HR 9.206, p=0.003, respectively. A Multivariate analysis indicated that HOXB9 expression was the only independent prognostic factor for DFS (HR=15.532, p=0.009). In subgroup analysis, HOXB9 positive tumors showed a significant increase in the number of vasculature and the Ki-67 ratio in comparison with HOXB9 negative. Conclusions: Our results suggest that HOXB9 expression promoting the tumor proliferation and the angiogenesis is a significant prognostic factor in breast cancer.

220 Background: The B-cell translocation gene-2 (BTG2) belongs to a class of proteins known as the Tob and BTG antiproliferative protein family. It was shown that estrogen and progesterone suppress BTG2 expression for the development of mammary gland. We demonstrated that proliferation rate of low level BTG2 expression in MCF7 was strongly inhibited by the administration of tamoxifen. In postmenopausal breast cancer patients, androgens can be converted to mitogenic estrogens by aromatase in breast cancer cells. Based on these results, we hypothesized that BTG2 expression affects the sensitivity against aromatase inhibitior. Methods: We used tetracycline-inducible BTG2 expression model in MCF7 stably transfected with the human aromatase gene (MCF7/tet/aro) as in vitro models of aromatase-driven breast cancer. The effects of BTG2 expression and administration of anastrozole in breast cancer cells were assessed by proliferation assays. Results: Administration of androstendion increased 79.1% of cellular proliferation, suggested that introduced aromatase gene worked well. Elevated level of BTG2 mRNA expression by tetracycline treatment was confirmed by Quantitative-RTPCR. Anastrozole treatment (100nM) reduced 37.8% of cellular proliferation ability, whereas the concomitant administration of tetracycline and anastorozole reduced 59.0% of cellular proliferation. These results suggested that the inhibitory effect of anastrozol for cellular proliferation was enhanced under the condition of BTG2 expression. Conclusions: Our results suggested loss of BTG2 expression may be affects the sensitivity against aromatase inhibitor.

e11503 Background: Sentinel lymph node biopsy (SLNB) is a more sensitive and accurate nodal staging procedure than axillary lymph node dissection (ALND). Because of detailed pathologic evaluation in SLNs, more nodal micrometastases have been identified. However, it remains controversial whether to perform ALND for patients with micrometastases in SLNs and their prognostic significance is also a matter of debate. The purpose of this study is to determine the non-sentinel lymph node (NSLN) status and prognosis of the patients with micrometastatic SLNs. Methods: A prospective database of 1,012 clinically node-negative, T1-T2 breast cancer patients, who underwent SLNB from January 2002 to Dec 2010 at Keio University Hospital was analyzed. SLNs were detected using a combined method of isosulfun blue dye and small-sized technetium-99m-labeled tin colloid. Intraoperative frozen examination was performed with hematoxylin and eosin (HE) staining. SLNs, fixed and embedded in paraffin, were additionally diagnosed wi...

10546 Background: Recently, we reported that HOXB9, a member of homeobox genes, expression promoted tumor neovascularization and metastasis in in vitro and in vivo asssay. These findings imply that...

662 Background: Clinical trials suggest that sensitivity to chemotherapy differs across breast cancer subtypes. This study's purpose was to evaluate the clinical utility of breast cancer subtypes in the prediction of pathologic complete response (pCR) in a cohort of breast cancer patients receiving neoadjuvant chemotherapy (NACT). Methods: Patients with stage II/III breast cancer received 4 cycles of XT (capecitabine 1650 mg/m2 on days 1-14 and docetaxel 60 mg/m2 on day 8 every 3 weeks), followed by 4 cycles of FEC (fluorouracil 500 mg/m2, epirubicin 90 mg/m2, cyclophosphamide 500 mg/m2 on day 1 every 3 weeks). Immunohistochemical analysis of estrogen receptor (ER), progesterone receptor (PgR), HER2, epidermal growth factor receptor (EGFR), cytokeratin (ck) 5/6, and Ki67 was performed in core needle biopsy samples at baseline. Tumors were classified as luminal A (ER+ and/or PgR+, and Ki67 20%), HER2 (ER- PgR-, and HER2+), or triple-negative (ER-, PgR-, and HER2-...