Terumasa Sawada

The β-catenin signaling pathway induces aggressive potential in breast cancer by up-regulating the chemokine CCL5

β-Catenin signaling plays a pivotal role in the genesis of a variety of malignant tumors, but its role in breast cancer has not been fully elucidated. Here, we examined whether deregulation of β-catenin signaling is related to the aggressive characteristics of certain types of breast cancers. Analysis of cytokine levels in MDA-MB-231 cells overexpressing a constitutively active form of β-catenin (CAβ-catenin) revealed a higher level of CCL5 expression. Cells transfected with CAβ-catenin or stimulated with recombinant CCL5 exhibited increased cell invasion activity and spheroid formation in vitro. Furthermore, CAβ-catenin-transfected MDA-MB-231 cells formed larger tumor masses that contained more Ki-67-positive cells and infiltrating lymphocytes than did the control cells. An inhibitor of CCR5 and a pan-CXCR neutralizing antibody dramatically reduced CAβ-catenin-promoted activities. In addition to CCL5, 6-BIO, a chemical activator of β-catenin, induced cell invasion and spheroid formation in MDA-MB-231 cells. Furthermore, high levels of nuclear β-catenin accumulation were detected in breast cancer in patients with metastasis but not in those without metastasis. Nuclear β-catenin localization is related to increased CCL5 production in breast cancer. These findings suggest that β-catenin expression enhances tumor progression via chemokine production in breast cancers and that β-catenin signaling is a critical regulator of the aggressive traits of breast cancers.

Digital volumetric measurement of mammographic density and the risk of overlooking cancer in Japanese women

BackgroundBreast density often affects cancer detection via mammography (MMG). Because of this, additional tests are recommended for women with dense breasts. This study aimed to reveal trends in breast density among Japanese women and determine whether differences in breast density differentially affected the detection of abnormalities via MMG.MethodsWe retrospectively analyzed 397 control women who underwent MMG screening as well as 269 patients who underwent surgery for breast cancer for whom preoperative MMG data were available. VolparaDensity™ (Volpara), a three-dimensional image analysis software with high reproducibility, was used to calculate breast density. Breasts were categorized according to the volumetric density grade (VDG), a measure of the percentage of dense tissue. The associations between age, VDG, and MMG density categories were analyzed.ResultsIn the control group, 78% of women had dense breasts, while in the breast cancer group, 87% of patients had dense breasts. One of 36 patients with non-dense breasts (2.7%) was classified as category 1 or 2 (C-1 or C-2), indicating that abnormal findings could not be detected by MMG. The proportion of patients with breast cancer who had dense breasts and were classified as C-1 or C-2 was as high as 22.3%.ConclusionsThe proportions of Japanese women with dense breasts were high. In addition, the false-negative rate for women with dense breasts was also high. Owing to this, Japanese women with dense breasts may need to commonly undergo additional tests to ensure detection of breast cancer in the screening MMG.

Abstract 4687: Gene expression of breast cancer with CD44+CD24-/low genotype

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose: Breast cancer cells with a CD44+CD24-/low phenotype have been suggested to have tumor-initiating properties with stem cell-like. The purpose of this study is to clarify the gene expression profiling of cells with different CD44/CD24 genotypes within breast cancer. Methods: Laser-captured microdissection was used to select the isolation of cancer cells in 36 frozen tissues of breast cancer, and RNA extracted from these cells was examined by real-time RT-PCR to quantify CD44 and CD24 expressions. Human stem cell RT2 profiler PCR array was used for gene expression analysis in the groups of different CD44/CD24 genotype. Associations between different CD44/CD24 genotypes and clinical variables were assessed by Fishers exact test, and the Mann-Whitney U test was used for gene expression profiling. P<0.05 was considered significant. Results: Thirty-six tumors were divided into 3 groups. Group A was composed of CD44+CD24-/low genotype, in which the ratio of CD44/CD24 was >1.0. Group B was composed of CD44+CD24+ genotype, in which that was 0.1 < ratio ≤ 1.0. In group C of CD44-/lowCD24+ genotype, that was < 0.1. The number of tumors in group A, B and C was 5, 29 and 2, respectively. Regarding the correlation of CD44/CD24 status with tumor characteristics, tumors of group A were significantly associated with lymph node metastasis compared with those of group B (P=0.029). The number of tumor with HER 2 score 3 within group A and B was 0 and 11, respectively. There was no significantly difference in ER status and tumor size. As far as the signaling pathways, the number of expression genes for Notch pathway in group A was significantly greater than in group B (P=0.028), and that for Wnt pathway was not significantly different between group A and B. Overexpressed gene in group A was NUMB, which is related to the programmed cell death. MYST1 and SOX2 tended to show higher levels of group A than of group B. Conclusion: This study suggests that Notch pathway may be more important than Wnt pathway on breast cancer with CD44+CD24-/low genotype, and the CD44+CD24-/low status would be associated with low/negative HER2 expression. Moreover, the gene of self-renewal markers, such as MYST1, SOX2 and NUMB might be a target for therapy against breast cancer with low/negative HER2 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4687.

QOL Evaluation of Nab-Paclitaxel and Docetaxel for Early Breast Cancer

Objective A previous randomized phase II study showed that neoadjuvant nab-paclitaxel (nab-PTX) 100 mg/m2) was effective and well-tolerated in patients with HER2-negative early-stage breast cancer, compared with docetaxel (DTX). We evaluated patient outcomes in terms of the Functional Assessment of Cancer Therapy-Breast (FACT-B), as a measure of health-related quality of life (HRQoL). Materials and Methods Stage I-III HER2-negative breast cancer patients from the previous study were included. They received either four cycles of nab-PTX (100 mg/m2 days 1/8/15) every 4 weeks, or DTX (75 mg/m2 day 1) every 3 weeks, both followed by four cycles of 5-fluorouracil/epirubicin/cyclophosphamide (FEC). Patients completed a health-related quality-of-life questionnaire at baseline, after one and four cycles of taxanes, before administration of FEC, and after administration of one and four cycles of FEC. Results Thirty-six eligible patients were enrolled. The baseline characteristics of the two groups were well balanced. FACT-B scores at baseline and after four cycles of taxanes were 115/108 (DTX/nab-PTX) and 99/92, respectively. There were no significant differences between DTX and nab-PTX for FACT-B, FACT-B-Trial Outcome Index (FACT-B-TOI) and FACT-General. FACT-B and FACT-B TOI scores tended to decrease after one cycle and after four cycles of chemotherapy which did not recover to the baseline scores through the end of chemotherapy in each group. Conclusion There were no significant safety differences between nab-PTX and DTX. HRQoL tended to decrease during taxane-based anticancer treatment, with no significant differences between the treatments. We suggest that the HRQoL questionnaire has limited ability to evaluate different chemotherapy schedules. Trial registration UMIN000009855. Nov 20, 2012 registered.

Abstract P4-01-09: γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) is promising fluorescence probe for rapid diagnosis of breast cancer; - The feasibility study of real time imaging for breast cancer examination -

【Background and Aim】 To date, fluorescence imaging has been used gradually for real time diagnosis in various clinical situations. Evaluation of margin on surgical specimens is essential to decide whether additional resection should be performed for breast cancer surgery. In the same context, rapid assessment of biopsy specimen is crucial because when they do not contain any part of the lesions, re-examination should be need. A fluorescence probe named γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) was rapidly activated by an enzyme, γ-glutamyltransferase (GGT). It was overexpressed in variety of cancers so promising for clinical use. The aim of this study is to examine the usefulness of the probe for breast cancer detection. 【Material and Methods】 We investigated the patients of breast cancer or benign disease who received examination consecutively from March 2015 to February 2016 in our hospital. The samples were obtained by core needle biopsy (CNB) or vacuum-assisted breast biopsy (VAB).We sprayed the probe on these samples immediately after examination and shoot images by CCD camera. To evaluate the fluorescence intensity along the time, we used a filtered CCD camera that could detect specifically gGlu-HMRG color (Discovery® INDEC Medical Systems Inc.). The images were automatically obtained every 30 seconds for 10 minutes after adding the probe on the specimens. The average value of the image in each region of interest (ROI) was analyzed using image analysis program Image J (https://imagej.nih.gov/ij/). We investigated the change of fluorescence intensity with the passage of time. We also compared the fluorescence intensity of malignant lesions with benign ones, and analyzed whether the fluorescence intensity could distinguish the malignant lesions from benign ones. 【Result】 We obtained 362 samples from96 tumors. Fifty-six tumors with 215 samples were benign, while 40 tumors with 147samples are malignant histologically. The fluorescence was immediately observed after sprayed the probe. The intensity had been increasing in proportion to time. The malignant specimens were rapidly increasing; in contrast, the benign ones were slowly. For example, when it took 60 seconds after spraying the probe that the intensity increase up to some level in malignant specimens, while benign one took 240 seconds up to the same level on average. Comparing the malignant lesions with benign ones after sprayed 120 seconds, the fluorescence intensity was higher in malignant specimens than benign ones (average fluorescence intensity; benign 0.9, malignant 2.3 p=0.0138). By ROC analysis whether the fluorescence intensity could distinguish the malignant lesions from benign, AUC, sensitivity and specificity was 0.63, 70% and 57%, respectively (cut off 0.2). 【Conclusion】 The probe was contributory to distinguish malignant and benign lesions and may be useful for the rapid diagnosis of CNB in practice. We are now trying to seek a more accurate probe to differentiate benign and malignant lesion as a next step. Citation Format: Takamaru T, Akashi ST, Kuwayama T, Sawada T, Hirota Y, Urano Y, Nakamura S. γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) is promising fluorescence probe for rapid diagnosis of breast cancer; - The feasibility study of real time imaging for breast cancer examination - [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-01-09.

Abstract P4-01-03: Digital measurement of mammographic density and the risk of overlooking cancer in Japanese women

Introduction: mammographic density is a strong risk factor for breast cancer and an essential determinant of screening sensitivity, but the breast density of Japanese women has yet to be objectively examined. Single mammographic examination fails to detect approximately half of breast cancers in women with dense breasts. We objectively evaluated mammographic density to clarify the relation between breast density and incidence of breast cancer in Japanese women. Method: We enrolled 269 patients diagnosed with breast cancer and 397 healthy controls, who participated in screening mammography at Showa University Hospital. Breast density was measured using Volpara (a software application which analyses breast composition volumetrically). Results: The average age of both control and breast cancer groups was 55. Of the control group, 308 (78%) were characterized as having dense breasts, being defined as volumetric density grade (VDG) 3 or 4. More than 50% of the total population (all ages) had dense breasts (VDG 3 or 4) and more than 20% were extremely dense (VDG:4). Of the breast cancer patients, 232 (87%) were VDG 3 or 4. 52 (23%) of those with dense breasts showed negative results in single mammography examination. In primary breast cancer patients with dense breasts, 30% of those under age 70, but only 13% of those over age 70, showed no abnormal findings from mammographic examination. Conclusion and Discussion: In comparison of our present results and other papers, In the Japanese control group, 72% of patients had dense breasts (VDG 3 or 4) at age 50 or greater, but in Holland, for example, only 36% of women had breasts classed as dense. 87% of Japanese women with breast cancer had dense breasts, approximately the same ratio (88%) as found in Korea. For Japanese and other Asians, women under 70 years old with dense breasts may warrant additional screening. Citation Format: Sawada T, Ikeda M, Kuwayama T, Akashi ST, Nakamura S. Digital measurement of mammographic density and the risk of overlooking cancer in Japanese women. [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 P4-01-03.

Abstract P1-01-03: Interim analysis of the validation study on the clinical usefulness of the ICG fluorescence method for detecting sentinel lymph nodes in early breast cancer compared with the RI method (fICG-BR02)

Background: Sentinel lymph node (SLN) biopsy guided by radioisotope (RI), blue dye or in combination methods is common. A high identification rate is reported for the RI-guided method. On the other hand, it has the demerits of radiation exposure, being expense, and it can only be used in a radiation-controlled area. The blue dye method, however, is safe and inexpensive, but the identification rate is lower compared with the RI method and requires training. The indocyanine green (ICG) fluorescent method involves the application of the fluorescing property. Lymph flow can be traced from outside the body with a photodynamic eye (PDE) camera simultaneously with an operation procedure. The ICG method is safe, inexpensive and requires little training, therefore its use will be widely permitted in any general hospitals. Recently, based on several retrospective clinical trials, the identification rate with the ICG fluorescent method has been reported to be equal to or greater than the RI method. The purpose of this multicenter study is to prospectively assess the diagnostic performance of SLN biopsy using the ICG fluorescence technique compared with RI. Materials and methods: In this validation study, the patients aged from 20 to 75 years-old with operable primary invasive breast cancer (cT1c-2N0M0) have been nominated and required the written informed consent. All candidates underwent SLN biopsy using the combined methods with RI and ICG fluorescence. The target sample size was 840 patients to evaluate the sensitivity of ICG method as a primary endpoint, and after 200 patients were enrolled we analyzed the identification rate and the SLN-positive rate of the RI and ICG methods respectively as the interim analysis planned beforehand. Results: Two hundred eligible patients were enrolled in this study from May 2011 to February 2012. Their median age was 53.0 years (range: 27-74 years). The number of patients with cT1c was 106 and that with cT2 was 94. The identification rate of the RI and the ICG method was 97% (194/200) and 96% (192/200) respectively. Of the 194 patients that were identified with the RI method, 186 (95.9%) were also identified with the ICG method. ICG identified 6 patients that were not identified by RI. On the other hand, RI identified 8 patients that were not identified by ICG. The SLN-positive rate was 25.5% (51/200). This rate was higher than we expected. Of the 51 patients with positive metastatic lymph nodes, 23 patients (21.7%) had cT1c breast cancer and 28 (29.8%) had cT2 breast cancer. The positive rate of the first SLN was 23.5% (47/200) and tumor cells skipped to the second or further tier in four cases (2%). Conclusions: The ICG-guided SLN biopsy procedure achieved a high identification rate almost equal to that with the RI method. Using this combination method, the identification rate was 100%. We will assess the sensitivity and the additive effect of combining the ICG fluorescence method with the RI method, etc., when all 840 patients have been enrolled (UMIN000005167). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-01-03.

Validation study on the clinical usefulness of the ICG fluorescence method for detecting sentinel lymph node in early-stage breast cancer in comparison with the dye method.

1122 Background: Sentinel lymph node (SLN) biopsy using indocyanine green (ICG) fluorescence method is a technique that utilizes the fluorescing property of ICG reagent that is used in the dye method. Flow of lymph can be confirmed as real time image from outside the body, and this method is the best for intra-operative sentinel lymph node biopsy. To assess the diagnostic performance of SLN biopsy using ICG fluorescence technique, prospective multicenter study was performed compared with blue dye method. METHODS Patients with T1-3 primary breast cancer without lymph node metastasis were assigned in this study. A combination of ICG as a fluorescence emitting source and indigocarmine as a blue dye were injected in the subreolar area and lymphatic flows were traced with PDE camera (a charge coupled device, Hamamatsu Photonics Co, Japan) and a real time image guided surgery enabled to identify the florescence signal of SLN after meticulous dissection. Extracted lymph nodes were examined for the first SLN or the second and further SLNs and classified in terms of ICG fluorescence, blue dye. Primary endpoint in this study is the difference in the number of lymph nodes identified by the fluorescence and the dye method. Secondary endpoints are identification rate, metastatic rate to SLNs and metastatic rate by order of SLNs. RESULTS Ninety-nine eligible patients were included in this study. The number of lymph nodes identified by fluorescence method was significantly larger than dye method (p<0.001). The SLN identification rate of fluorescence and dye method was 99% and 78%, respectively (p<0.001). Twenty of 99 patients had a positive SLN and ICG fluorescence method identified all these patients. The first SLN identified had both ICG fluorescence and blue dye (77%) or ICG fluorescence alone (23%). Of the 20 patients with lymph node metastasis, 100% was positive in the first SLN. CONCLUSIONS SLN biopsy using ICG fluorescence achieves a high identification rate and is superior to dye method. The first SLN identified by ICG fluorescence method represents the exact axillary status. Direct comparison between ICG fluorescence and radio isotope method is currently on the way.