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Purpose: Breast cancer stem cells have been shown to be associated with resistance to chemotherapy in vitro, but their clinical significance remains to be clarified. The aim of this study was to investigate whether cancer stem cells were clinically significant for resistance to chemotherapy in human breast cancers.Experimental Design: Primary breast cancer patients (n = 108) treated with neoadjuvant chemotherapy consisting of sequential paclitaxel and epirubicin-based chemotherapy were included in the study. Breast cancer stem cells were identified by immunohistochemical staining of CD44/CD24 and aldehyde dehydrogenase 1 (ALDH1) in tumor tissues obtained before and after neoadjuvant chemotherapy. CD44+/CD24− tumor cells or ALDH1-positive tumor cells were considered stem cells.Results: Thirty (27.8%) patients achieved pathologic complete response (pCR). ALDH1-positive tumors were significantly associated with a low pCR rate (9.5% versus 32.2%; P = 0.037), but there was no significant association between CD44+/CD24− tumor cell proportions and pCR rates. Changes in the proportion of CD44+/CD24− or ALDH1-positive tumor cells before and after neoadjuvant chemotherapy were studied in 78 patients who did not achieve pCR. The proportion of ALDH1-positive tumor cells increased significantly (P < 0.001) after neoadjuvant chemotherapy, but that of CD44+/CD24− tumor cells did not.Conclusions: Our findings suggest that breast cancer stem cells identified as ALDH1-positive, but not CD44+/CD24−, play a significant role in resistance to chemotherapy. ALDH1-positive thus seems to be a more significantly predictive marker than CD44+/CD24− for the identification of breast cancer stem cells in terms of resistance to chemotherapy.

Supplementary Data from Association of Breast Cancer Stem Cells Identified by Aldehyde Dehydrogenase 1 Expression with Resistance to Sequential Paclitaxel and Epirubicin-Based Chemotherapy for Breast Cancers

In clinical decision‐making, to decide the indication for adjuvant chemotherapy for estrogen receptor‐positive (ER+), human epidermal growth factor receptor‐2‐negative (HER2−), and node‐negative (n0) breast cancer patients, the accurate estimation of recurrence risk is essential. Unfortunately, conventional prognostic factors, such as tumor size, histological grade and ER, progesterone receptor (PR), and HER2 status as well as Ki67 index, are not sufficiently accurate for such estimation. Therefore, several multigene assays (MGAs) based on the mRNA expression analysis of multiple genes in tumor tissue have been developed to better predict patient prognosis. These assays include Oncotype DX, MammaPrint, PAM50, GGI, EndoPredict, and BCI. We developed Curebest™ 95‐Gene Classifier Breast (95GC) classifier, which is unique in that mRNA expression data of all 20 000 human genes are secondarily obtainable, as the 95GC assay is performed using Affymetrix microarray. This can capture mRNA expression of not only 95 genes but also every gene at once, and such gene expression data can be utilized by the other MGAs that we have developed, such as the 155GC, which is used for the prognostic prediction of ER+/HER2− breast cancer patients treated with neoadjuvant chemotherapy. We also developed the 42GC for predicting late recurrence in ER+/HER2− breast cancer patients. In this mini‐review, our recent attempt at the development of various MGAs, which is expected to facilitate the implementation of precision medicine in ER+/HER2− breast cancer patients, is presented with a special emphasis on 95GC., In this mini‐review, we discuss our progress since 2007 in developing multigene assays (MGAs) 95GC/42GC/155GC that may be used to implement precision medicine in ER+/HER2− patients. Although future studies still need to be performed in a larger number of patients, substantial progress has been achieved in developing multiple MGAs that can guide treatment‐related decision‐making in clinical settings.

HER2 amplification is seen in 20–25% of primary breast cancer cases, and HER2 detection is performed routinely in primary operable, as well as metastatic breast cancer patients. Currently, HER2 is the only gene of which amplification is routinely assayed by fluorescent in situ hybridization (FISH) and/or immunohistochemistry (IHC). However, histochemical assay (FISH/IHC) of multiple target genes is laborious and time-consuming, and simultaneous amplification by microarray is preferred. OncoScan™ is a microarray-based assay capable of whole-genome copy number analysis using DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues. In the current study, we aimed to investigate the impact of tumor cellularity on the accuracy of OncoScan™ in the determination of HER2 amplification. Our results demonstrated that HER2 amplification by OncoScan™ is accurate, and has a high concordance rate of 93.3% with FISH. However, the concordance rate is poor (66.7%) in cases with a tumor cellularity

Ten stage I/II breast cancer patients with one or two suspicious lymph nodes (LNs) detected on ultrasonography underwent sentinel LN biopsy (SLNB) after the introduction of Twirl® breast markers into each suspicious LN revealed that each Twirl®-marked LN was SLN and was likely the first SLN. Three patients had less than three SLN metastases and were candidates for sparing completion axillary lymph node dissection (cALND). Indeed, of them, one underwent breast-conserving surgery without cALND and the other two underwent total mastectomy with cALND. These results suggest that, as all suspicious LNs are SLNs, patients can be treated with SLNB alone if they fulfill the ACOSOG Z-0011 criteria, despite suspicious LNs yielding positive results on preoperative fine-needle aspiration cytology.

Humoral immunity plays a substantial role in the suppression of breast cancer. We have revealed that a high serum concentration of anti‐HER2 autoantibody (HER2‐AAb) is associated with favorable outcomes in patients with invasive breast cancer. Thus, we aimed to clarify the association between high serum concentration of HER2‐AAb and humoral immune response in the tumor microenvironment. Out of 500 consecutive patients with invasive breast cancer, we selected those whose HER2‐AAb values were high (n = 33) or low (n = 20) based on the distribution of HER2‐AAb values of 100 healthy individuals. Tumor and regional lymph node formalin‐fixed paraffin‐embedded samples prepared from the surgical specimens were subjected to immunohistochemistry. We confirmed that the recurrence‐free survival of the high HER2‐AAb group was significantly longer than that of the low HER2‐AAb group (p = 0.015). The numbers of tumor‐infiltrating CD20+ immune cells (ICs) (p, Anti‐HER2 autoantibody in breast cancer patients is positively associated with their outcomes. Enhanced humoral immunity in the tumor microenvironment is associated with autoantibody, providing a rationale for favorable outcomes in such patients.

The aim of our study is to find microRNAs (miRNAs) associated with sentinel lymph node metastasis (SLNM) and to develop a prediction model for SLNM in ER-positive and HER2-negative (ER+/HER2-) breast cancer.In the present study, only ER+/HER2- primary breast cancer was considered. The discovery set for SLNM-associated miRNAs included 10 tumors with and 10 tumors without SLNM. The training and validation sets both included 100 tumors. miRNA expression in tumors was examined comprehensively by miRNA microarray in the discovery set and by droplet digital PCR in the training and validation sets.In the discovery set, miR-98, miR-22, and miR-223 were found to be significantly (P 0.001, fold-change 2.5) associated with SLNM. In the training set, we constructed the prediction model for SLNM using miR-98, tumor size, and lymphovascular invasion (LVI) with high accuracy (AUC, 0.877). The accuracy of this prediction model was confirmed in the validation set (AUC, 0.883), and it outperformed the conventional Memorial Sloan Kettering Cancer Center nomogram. In situ hybridization revealed the localization of miR-98 expression in tumor cells.We developed a prediction model consisting of miR-98, tumor size, and LVI for SLNM with high accuracy in ER+/HER2- breast cancer. This model might help decide the indication for SLN biopsy in this subtype.

The diagnostic accuracy of the multigene panel test (MPT) and OncoScan™ in the determination of HER2 amplification in breast tumors remains controversial. In the present study, HER2 copy number was analyzed using both MPT and OncoScan™ in 45 breast tumors and was compared with that in fluorescent in situ hybridization (FISH) analysis. Tumors with low cellularity were examined using tumor cell enrichment and fluorescence‑activated cell sorting. Both MPT and OncoScan™ exhibited significant correlations with FISH with respect to the determination of HER2 amplification in breast tumors. However, the correlation coefficient was significantly higher for the comparison of MPT and FISH (r=0.770) compared with that between OncoScan™ and FISH (r=0.564). The accuracy of MPT (93.3%) was slightly higher compared with that in OncoScan™ (84.4%) in determining the HER2 status, which was mostly explained by the higher sensitivity of MPT in tumors with low cellularity (83.3 vs. 33.3%), but not in those with high cellularity (81.8 vs. 72.7%). The specificity was 100% for both tests. The MPT exhibited higher sensitivity in the determination of the amplification of other genes, including MYC, fibroblast growth factor receptor 1 and GATA binding protein 3 in tumors with low cellularity compared with that in tumors with high cellularity. OncoScan™ exhibited low sensitivity without tumor cell enrichment. The results suggested that MPT could be a promising method to determine HER2 status in breast tumors and that it could exhibit improved accuracy compared with that in OncoScan™ in tumors with low cellularity.

AIM To investigate the feasibility of hookwire-guided sentinel lymph node biopsy (SLNB) using contrast-enhanced ultrasonography (CEUS) followed by a one-step nucleic acid amplification (OSNA) assay. PATIENTS AND METHODS Clinical T1-2N0M0 breast cancer patients scheduled to undergo SLNB participated in this study. Both Sonazoid® and dye were used as tracers, and the most upstream sentinel lymph node (SLN) at each lymphatic flow detected by CEUS (First-SLN) was sampled under hookwire guidance, a procedure called "Sona-Hook". RESULTS In each of the 50 cases, at least one First-SLN was extracted by "Sona-Hook". All contrast-enhanced SLNs (CE-SLNs) were dye-positive, and the mean number of CE-SLNs sampled per patient was lower than that of dye-positive SLNs (1.48 vs. 1.88, p

Contrast-enhanced ultrasonography with Sonazoid (SNZ) enables real-time visualization, resulting in more precise identification of lymphatic flow to sentinel lymph nodes (SLNs). This study aimed to classify lymphatic drainage patterns to SLNs. Patients (n = 75) with T1–2 N0 M0 breast cancer received a periareolar injection of SNZ to identify SNZ-enhanced SLNs (SNZ-SLNs), followed by SLN biopsy with blue dye. The lymphatic drainage patterns were classified into four types: type A, single lymphatic route/single SLN; type B, multiple lymphatic routes/single SLN; type C, single lymphatic route/multiple SLNs; and type D, multiple lymphatic routes/multiple SLNs. SLNs were successfully identified in all patients using both blue dye and SNZ. The drainage lymphatic pathways identified were as follows: type A in 53 cases (70.7%), type B in seven (9.3%), type C in eight cases (10.7%) and type D in seven (9.3%). SNZ-SLN biopsy is a technically simple method with a 100% identification rate, enabling the real-time visualization of lymphatic flow to SNZ-SLNs.

Background Trastuzumab is a drug that targets the receptor tyrosine kinase HER2 and is essential for the treatment of HER2-positive breast cancer. Resistance to the drug leads to severe consequences, including disease recurrence, tumor enlargement, and metastasis. We hypothesized that trastuzumab treatment might be associated with phenotypic switching in HER2-positive breast cancer cells (BCCs), enabling them to escape and survive the effect of trastuzumab. Methods We conducted comprehensive immunophenotyping to detect phenotypic changes in HER2-positive BCCs treated with trastuzumab, based on criteria determined a priori. Based on immunophenotyping results, we characterized the vascular phenotypes of HER2-positive BCCs by western blotting, real-time RT-PCR, and tube formation assay. The vascular phenotype of tumor cells from clinical samples was evaluated by staining with periodic acid-Schiff and an anti-CD31 antibody. We explored small molecule inhibitors that suppress tube formation and determined the inhibitory mechanism. Results Out of 242 cell surface antigens, 9 antigens were significantly upregulated and 3 were significantly downregulated by trastuzumab treatment. All upregulated antigens were related to endothelial and stem cell phenotypes, suggesting that trastuzumab treatment might be correlated to switching to a vascular phenotype, namely, vasculogenic mimicry (VM). Several VM markers were upregulated in trastuzumab-treated cells, but these cells did not form tubes on Matrigel, a functional hallmark of VM. Upon analysis of three trastuzumab-resistant HER2-positive cell lines, we found that all three cell lines showed tube formation on Matrigel in the presence of angiogenic growth factors including EGF, FGF2, IGF1, or VEGF. Clinically, VM channels significantly increased in surviving cancer cell clusters of surgically removed tumors pretreated with trastuzumab and chemotherapy compared to both surgically removed tumors without prior systemic treatment and tumors biopsied before presurgical treatment with trastuzumab. Finally, we found that salinomycin completely suppressed VM in all three trastuzumab-resistant cell lines through disruption of actin cytoskeletal integrity. Conclusions VM promotes metastasis and worsens patient outcomes. The present study indicates that HER2-positive BCCs can exhibit VM in an angiogenic microenvironment after eventually acquiring trastuzumab resistance. The clinical finding supports this in vitro observation. Thus, targeting VM might provide a therapeutic benefit to patients with HER2-positive breast cancer. Electronic supplementary material The online version of this article (10.1186/s13058-019-1167-3) contains supplementary material, which is available to authorized users.

Background/aim One-step nucleic acid amplification (OSNA) is a newly developed procedure for detection of node metastasis by targeting CK19 mRNA. This study aimed to compare the prognosis of ER-positive/HER2-negative (ER+/HER2-) breast cancer patients with negative sentinel lymph node (SLN), as determined by OSNA with that determined by pathology. Patients and methods A total of 508 patients who underwent breast surgery and SLN biopsy were enrolled. Of 263 patients with negative SLN by OSNA (osN0), 239 were treated with endocrine therapy alone (osN0-ET), and of 107 with negative SLN by pathology (pN0), 103 were treated with endocrine therapy alone (pN0-ET). Results Distant relapse-free survival (DRFS) of osN0-ET group (99.5% at 6 years) was significantly better (p=0.044) than that of pN0-ET group. Multivariate analysis revealed that osN0 was significantly associated (p=0.019) with favorable DRFS. Conclusion ER+/HER2- breast cancer patients with negative SLN by OSNA show an excellent prognosis with endocrine therapy alone.

[Purpose]Homologous Recombination Deficiency (HRD) score has been developed to evaluate the DNA double-strand break repair function. BRCA1/2 germline mutation-associated breast cancers are known to show a high HRD score and a high sensitivity to platinum-based chemotherapy as well as PARP inhibitor. HRD can be theoretically induced by not only the dysfunction of BRA1/2 but also the dysfunction of the other molecules involved in homologous recombination, and, actually, high HRD score is reported to be seen in a significant proportion of sporadic breast tumors without BRCA1/2 mutation. The aim of the present study was to elucidate the clinicopathological characteristics of sporadic breast tumors with high HRD score as well as their sensitivity to sequential paclitaxel and FEC chemotherapy (P-FEC). [Methods]Tumor tissues obtained by Mammotome from 132 sporadic breast cancer patients (stage II-III) before neoadjuvant chemotherapy with P-FEC were subjected to assay for HRD score using Oncoscan CNV kit®. HRD score was a simple sum of NtAI, LOH, and LST (cutoff, 42), and were also subjected to the gene expression analysis using the Affymetrix microarray (U133 plus 2.0). BRCA1 promoter methylation was assayed by methylation specific real-time PCR. [Result]Of the 132 breast tumors, 32% showed a high HRD score which were significantly associated with high histological grade (P=0.001), negative progesterone receptor (P=0.023) and high Ki67 index (P=0.004). Triple negative breast cancer (TNBC)(n=19) showed a significantly (P [Conclusion]Approximately one third of sporadic breast tumors show a high HRD score, indicating the presence of homologous recombination dysfunction, and they are most often seen in TNBC with BRCA1 promoter methylation and never in LAR subtype. Interestingly, breast tumors with a high HRD score were less sensitive to P-FEC. Citation Format: Imanishi S, Naoi Y, Shimazu K, Shimoda M, Kagara N, Tanei T, Miyake T, Kim SJ, Noguchi S. HRD (homologous recombination deficiency) score and its clinicopathological features in neoadjuvant chemotherapy treated sporadic breast cancers [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 P3-11-06.

Background/aim To clarify the diagnostic utility of third-look, contrast-enhanced ultrasonography (CEUS) for MRI-detected breast lesions undetectable by unenhanced, second-look ultrasonography (MRI+/US- lesions). Patients and methods Clinical stage 0-IIA breast cancer patients who underwent CEUS for incidental MRI+/US- lesions (n=27; cohort 1) and patients with breast lesions detected only by MRI, to be examined by CEUS (n=15; cohort 2), were retrospectively analyzed. Results Of the 42 MRI+/US- lesions, 23 (55%) were detected by CEUS and then examined by needle biopsy. Pathological examination showed that 10 of these 23 lesions were malignant. None of the 19 lesions undetected by CEUS were found to be histologically malignant or developed malignancy with a median follow-up of 18.5 months. The accuracy, sensitivity, and specificity of CEUS plus needle biopsy were 98%, 91%, and 100%, respectively. Conclusion Third-look CEUS followed by needle biopsy is useful in the initial diagnosis of MRI+/US- lesions.

This study aimed to elucidate the clinicopathological characteristics of breast tumors with homologous recombination deficiency (HRD) and the sensitivity to neoadjuvant paclitaxel followed by fluorouracil, epirubicin, and cyclophosphamide (P-FEC). Tumor biopsy samples obtained before P-FEC from 141 patients with stages II–III breast cancer including the luminal (n = 76), luminal-HER2 (n = 13), HER2 (n = 17), and triple-negative (TNBC, n = 35) subtypes were subjected to assay for HRD score using the OncoScan CNV FFPE Assay Kit. HRD score was a simple sum of NtAI, LOH, and LST (cutoff, 42). TNBCs were also subjected to the gene expression assay using the Affymetrix microarray (U133 plus 2.0) and to the BRCA1 promoter methylation assay using the methylation-specific real-time PCR. Of the 141 breast tumors, 45 samples (32%) had high HRD scores and were associated with high histological grade (P = 0.001), negative progesterone receptor (P = 0.018), high Ki67 index (P = 0.032), and BRCA1 promoter methylation (P = 3.6e−07). The proportion of tumors with high HRD scores was significantly higher in the TNBC subtype than the others (P = 0.006). In the TNBC subtype, but not the others, high HRD scores were significantly (P = 0.001) associated with a low pathological complete response rate to P-FEC. Among the molecular TNBC subtypes, a majority of tumors belonging to the basal-like 1, immunomodulatory, mesenchymal, mesenchymal stem-like, but not luminal androgen receptor (LAR), subtypes had high HRD scores. Approximately one-third of sporadic breast tumors show a high HRD score, indicating the presence of homologous recombination dysfunction, and they are characterized by biologically aggressive phenotypes, most commonly in the TNBC subtype, and less sensitive to P-FEC.

Background: Endocrine therapy is clinically administered in hormone-responsive breast cancer. Combinations of fluoropyrimidine S-1 and an aromatase inhibitor or anti-estrogen are considered beneficial in Japan. Herein we assessed new combinations of S-1 and fulvestrant. Patients and Methods: Cytotoxicity of fulvestrant and 5-fluorouracil (5-FU) was assessed in hormone-responsive (MCF-7) and non-responsive (MDA-MB-231) breast cancer cell cultures. Fulvestrant and S-1 were evaluated for antitumor activity in mice and their effects on estrogen receptor (ER)-α and progesterone receptor (PgR) levels in MCF-7 xenografts using immunohistochemical methods. Results: Fulvestrant inhibited growth of MCF-7, but not of MDA-MB-231 xenografts. Combinations of 5-FU and fulvestrant were superior to monotherapy in vitro. In vivo antitumor activity of S-1/fulvestrant combination therapy was significantly (p

Purpose A subset of patients with intermediate 21-gene signature assay recurrence score may benefit from adjuvant chemoendocrine therapy, but a predictive strategy is needed to identify such patients. The 95-gene signature assay was tested to stratify patients with intermediate RS into high (95GC-H) and low (95GC-L) groups that were associated with invasive recurrence risk.Methods Patients with ER-positive, HER2-negative, node-negative breast cancer and RS 11-25 who underwent definitive surgery and adjuvant endocrine therapy without any cytotoxic agents were included. RNA was extracted from archived formalin-fixed, paraffin-embedded samples, and 95-gene signature was calculated. Results Two hundred six patients had RS of 11-25 (95GC-L, N = 163; 95GC-H, N = 43). In Cox proportional hazards model, 95GC-H was significantly associated with shorter time to recurrence than was 95GC-L (HR 5.94; 95%CI 1.81-19.53; P = 0.005). The correlation between 95-gene signature and 21-gene signature assay scores was not strong (correlation coefficient r = 0.27), which might suggest that 95-gene signature reflects biological characteristics differing from what 21-gene signature shows.Conclusions The 95-gene signature stratifies patients with ER-positive, HER2-negative, node-negative invasive breast cancer and intermediate RS of 11-25 into high and low groups that are associated with recurrence risk of invasive disease. Further retrospective analysis in the prospectively-accrued TAILORx population is warranted to confirm that 95-gene signature can identify patients who would benefit from adjuvant chemoendocrine therapy.

Background/aim Magnetic resonance imaging (MRI)-guided breast biopsy is a complex and time-consuming procedure. This study aimed to clarify the factors that affect the duration of the procedure. Patients and methods Twenty-eight examinations performed at our institute for 27 lesions detected solely on MRI were analyzed. The correlations between the clinicopathological factors and duration of the procedure were estimated. Results The needle guidance method was the only factor that significantly affected the duration of the MRI-guided vacuum-assisted breast biopsy (VAB) (p=0.012). The use of a computer-aided detection (CAD) system with grid breast compression plates had significantly shorter durations (62±12 min) than the manual calculation of coordinates with pillar-type compression plates (76±13 min). Conclusion This preliminary study showed that the use of a CAD system might shorten the duration of MRI-guided VAB.

Summary We aimed to validate the cosmetic utility of addition of nipple-areola recentralization (NAR) to rotation flap according to nipple tumor distance (NTD) as a volume displacement technique after breast conserving surgery (BCS) for lower-outer and upper-inner breast cancers. Twenty breast cancer patients who had been treated with rotation flap with (Group 1; n = 6) or without (Group 2; n = 14) NAR after BCS for lower-outer or upper-inner located tumors, and those who had undergone BCS without oncoplastic surgical technique for tumors in the same area (Control group; n = 43), were retrospectively investigated. Cosmetic outcome was evaluated using Harvard scale and/or BCCT.core. As a result, the ratio of patients categorized as excellent/good was 83% in Group 1 and 93% in Group 2, respectively, and there was no significant difference between them (P = 0.521). In addition, Group 1 + 2 showed a significantly higher ratio of patients classified as excellent/good than the control group (90% vs. 56%; P = 0.009). After adjustment of clinical background parameters using propensity score matching analysis between Group 1 + 2 and the control group, 12 pairs with similar background factors were matched. Among them, Group 1 + 2 showed a higher ratio of patients categorized as excellent/good than the control group (92% vs. 42%; P = 0.034). In conclusion, addition of NAR to rotation technique according to NTD may enable us to perform a volume displacement after BCS for lower-outer or upper-inner located tumors irrespective of NTD without sacrificing postoperative breast appearance.

We attempted to detect circulating tumor DNA (ctDNA), taking advantage of molecular barcode next-generation sequencing (MB-NGS), which can be more easily customized to detect a variety of mutations with a high sensitivity than PCR-based methods. Sequencing with a gene panel consisting of the 13 most frequently mutated genes in breast tumors from stage I or II patients revealed 95 somatic mutations in the 12 genes in 62% (62/100) of tumors. Then, plasma DNA from each patient (n = 62) before surgery was analyzed via MB-NGS customized to each somatic mutation, resulting in the detection of ctDNA in 16.1% (10/62) of patients. ctDNA was significantly associated with biologically aggressive phenotypes, including large tumor size (P = .004), positive lymph node (P = .009), high histological grade (P < .001), negative ER (P = .018), negative PR (P = .017), and positive HER2 (P = .046). Furthermore, distant disease-free survival was significantly worse in patients with ctDNA (n = 10) than those without ctDNA (n = 52) (P < .001). Our results demonstrate that MB-NGS personalized to each mutation can detect ctDNA with a high sensitivity in early breast cancer patients at diagnosis, and it seems to have a potential to serve as a clinically useful tumor marker for predicting their prognosis.

There is an urgent need for the development of a predictor of response to chemotherapy for ER-positive breast cancer which is less chemosensitive than for ER-negative breast cancer in order to avoid unnecessary chemotherapy. In the present study, intrinsic subtyping by PAM50 was evaluated for its ability to predict a response to chemotherapy. For this study, 124 patients with ER-positive breast cancer treated with neoadjuvant sequential paclitaxel and FEC (NAC) were evaluated. Tumor biopsy specimens obtained before NAC were subjected to intrinsic subtyping (IS) by gene expression (GE) using PAM50 (PAM50-IS) or immunohistochemistry (IHC-IS). Of the PAM50-ISs (Luminal A, Luminal B, HER2-enriched, and Basal-like), GE-Luminal A showed the lowest pCR rate (1.9%), and multivariate analysis revealed that GE-Luminal A was a significant (P = 0.031) predictor of non-pCR independently of other clinicopathological parameters, including Ki67, and tumor-infiltrating lymphocytes. Of the IHC-ISs, on the other hand, IHC-Luminal A was not significantly associated with pCR. We also found that breast tumors with low ER levels (1–9%), like ER-negative tumors, were mostly GE-HER2-enriched and GE-Basal-like, and more sensitive to NAC than those with high ER levels (≥ 10%). GE-Luminal A intrinsically subtyped by PAM50 was the least sensitive to NAC and very unlikely to attain pCR. IHC-Luminal A identified by IHC, on the other hand, was not significantly predictive of pCR. In addition, PAM50 revealed that tumors with low ER (1–9%) were more like ER-negative tumors than ER-positive tumors, and most such cases should therefore would better be treated with chemotherapy.

Background One-step nucleic acid amplification (OSNA) for cytokeratin 19 messenger RNA is an intraoperative diagnostic procedure for the detection of lymph node metastasis.

The pathologic feature of intraductal papillomas is defined as a papillary structure composed of a fibrovascular stromal core lined by luminal epithelial cells and myoepithelial cells. We used droplet digital PCR for the mutational analysis of AKT1 (E17K) and PIK3CA (H1047R, E542K, and E545K) in 60 papillomas. AKT1 and PIK3CA mutations were detected in 12 (20%) and 17 (28%) of the papillomas, respectively. In five tumors harboring mutations, mutational analysis of AKT1 or PIK3CA was performed separately using luminal epithelial cells and myoepithelial cells sorted using anti–cytokeratin 19 antibody and anti–α smooth muscle actin antibody. The two types of cells from a given papilloma had the identical mutation. Three patients with the PIK3CA mutation–positive papilloma developed breast cancers at the resection site of the papilloma, but none of these subsequent breast cancers had the PIK3CA mutation. These results indicate that a papilloma stems from a bipotent progenitor cell that contains the AKT1 or PIK3CA mutation and proliferates and differentiates to form the papilloma. Papilloma can be a risk factor for developing breast cancer but is unlikely to be its obligate precursor.

The international, phase III FALCON study (NCT01602380) in postmenopausal patients with hormone receptor-positive, locally advanced/metastatic breast cancer (LA/MBC) who had not received prior endocrine therapy, demonstrated statistically significant improvement in progression-free survival (PFS) for patients who received fulvestrant 500 mg vs anastrozole 1 mg. This subgroup analysis evaluated PFS in Asian (randomized in China, Japan, or Taiwan) and non-Asian patients from the FALCON study. Eligible patients (estrogen receptor- and/or progesterone receptor-positive LA/MBC; World Health Organization performance status 0–2; ≥ 1 measurable/non-measurable lesion[s]) were randomized. PFS was assessed via Response Evaluation Criteria in Solid Tumours version 1.1, surgery/radiotherapy for disease worsening, or death (any cause). Secondary endpoints included: objective response rate, clinical benefit rate, duration of response, and duration of clinical benefit. Consistency of effect across subgroups was assessed via hazard ratios and 95% confidence intervals (CIs) using a log-rank test. Adverse events (AEs) were evaluated. Of the 462 randomized patients, the Asian and non-Asian subgroups comprised 67 and 395 patients, respectively. In the Asian subgroup, median PFS was 16.6 and 15.9 months with fulvestrant and anastrozole, respectively (hazard ratio 0.81; 95% CI 0.44–1.50). In the non-Asian subgroup, median PFS was 16.5 and 13.8 months, respectively (hazard ratio 0.79; 95% CI 0.62–1.01). Secondary outcomes were numerically improved with fulvestrant vs anastrozole in both subgroups. AE profiles were generally consistent between Asian and non-Asian subgroups. Results of this subgroup analysis suggest that treatment effects in the Asian patient subgroup are broadly consistent with the non-Asian population.

Purpose This phase II study evaluated the efficacy and safety of anastrozole concurrent with tegafur/uracil (UFT) as neoadjuvant therapy for ER-positive postmenopausal breast cancer. Methods Postmenopausal Japanese women with ER-positive, HER2-negative, T2,N0-1,M0 breast cancer seen at tertiary hospitals were eligible for this open-label, randomized, multicenter study. Patients were randomized 1:1 by minimization to orally receive either anastrozole (1 mg once daily) plus UFT (tegafur/uracil combination in 1:4 molar ratio; 270 mg/m2/day in two divided doses) or anastrozole (as above) alone for 24 weeks. Tumor response was assessed by investigator and central review as per RECIST v1.1. The primary endpoint was the proportion of patients with best overall response of CR or PR [clinical response rate (RR)] determined by central radiologic review. Results The study was prematurely terminated due to Grade ≥ 3 liver dysfunction reported in 3 patients receiving anastrozole/UFT. Of 57 patients randomized before termination (29 anastrozole/UFT, 28 anastrozole), all were analyzed for safety and 56 (28 each group) for tumor response. Compared with anastrozole alone, anastrozole/UFT did not achieve significantly higher RR [39.3% (90% CI 23.8–56.5%) vs 14.3% (90% CI 5.0–29.8%); p = 0.0683, Fisher’s exact test], but produced significantly greater tumor shrinkage (mean tumor reduction rate 31.0 vs. 14.2%; p = 0.0181, unpaired t-test). Grade ≥ 3 adverse events were more common with anastrozole/UFT than with anastrozole (17.2 vs. 0%). Conclusion Although the study was terminated owing to the altered liver function, it showed that there was a trend to greater shrinkage of tumor in the combination group for ER-positive, HER2-negative postmenopausal breast cancer.

Although prognostic markers for early estrogen receptor (ER)‐positive breast cancer have been extensively developed, predictive markers for adjuvant endocrine therapy are still lacking. Focusing on the mechanisms underlying endocrine resistance, we investigated whether the endocrine sensitivity of ER‐positive breast cancer cells was correlated with the expression of aspartate‐β‐hydroxylase (ASPH), which is involved in the development of hepatocellular carcinoma. ASPH expression in ER‐positive and tamoxifen‐resistant breast cancer cells was upregulated by the MAPK and phosphoinositide‐3 kinase (PI3K) pathways, which both play pivotal roles in endocrine resistance. In the clinical setting, ASPH expression was negatively correlated with recurrence‐free survival of luminal B breast cancer patients that received adjuvant endocrine therapy, but not in patients that did not receive adjuvant endocrine therapy. Luminal B breast cancer is one of the intrinsic molecular subtypes identified by the Prediction Analysis of Microarray 50 (PAM50) multiple gene classifier, and because of its poor response to endocrine therapy, chemotherapy in addition to endocrine therapy is generally required after surgical resection. Our results suggest that the endocrine sensitivity of luminal B breast cancer can be assessed by examining ASPH expression, which promotes the consideration of a prospective study on the association between ASPH expression at the mRNA and protein levels in luminal B breast cancer and subsequent response to endocrine therapy.

We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB–NGS) targeting the hotspot segment (c.1600–1713). The sensitivity of MB–NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB–NGS. The results of MB–NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR). MB–NGS showed a higher sensitivity (0.1%) than NGS without barcode (1%) by reducing background errors. Of the cfDNA samples from 34 patients with metastatic breast cancer, NGS without barcode revealed seven mutations in six patients (17.6%) and MB–NGS revealed six additional mutations including three mutations not reported in the COSMIC database of breast cancer, resulting in total 13 ESR1 mutations in ten patients (29.4%). Regarding the three hotspot mutations, all the patients with mutations detected by MB–NGS had identical mutations detected by droplet digital PCR (ddPCR), and mutant allele frequency correlated very well between both (r = 0.850, p

The aim of this prospective study was to evaluate the feasibility of periareolar injection of the contrast agent Sonazoid (SNZ) followed by ultrasonography (US) for the identification of sentinel lymph node (SLN) in breast cancer patients with clinically negative node. Patients (n = 100) with T1‐2N0M0 breast cancer received a periareolar injection of SNZ followed by US to identify contrast‐enhanced SLN. Each contrast‐enhanced SLN underwent fine needle aspiration cytology (FNAC) followed by SLN biopsy with a conventional method using blue dye and/or radiocolloid (B/R). In almost all cases, contrast‐enhanced lymphatic vessels were clearly visualized by US soon after the periareolar injection of SNZ and the SLNs were easily identified with an identification rate of 98% (98/100) for SNZ and 100% (100/100) for B/R. The number of SLNs identified by SNZ (SNZ‐SLN) (mean per patient, 1.52) was significantly lower than that identified by B/R (B/R‐SLN) (2.19) (P

BACKGROUND Fulvestrant is a selective estrogen receptor degrader (SERD) with no known agonist effects. In the open-label Phase 2 FIRST study, fulvestrant 500 mg suggested improved efficacy as first-line treatment vs anastrozole in patients with hormone receptor-positive locally advanced or metastatic breast cancer (LA/MBC). The Phase 3, randomized, double-blind, multicenter FALCON trial (NCT01602380) compared fulvestrant 500 mg with anastrozole 1 mg in patients with hormone receptor-positive LA/MBC who had not received prior hormonal therapy. The primary endpoint of the study, progression-free survival (PFS) assessed via RECIST 1.1, surgery/radiotherapy for disease worsening, or death (any cause), was met, as shown by a statistically significant improvement in PFS for fulvestrant 500 mg vs anastrozole. This analysis evaluated PFS in the Asian patient subgroup, which included all randomized patients from centers in China, Japan, and Taiwan. METHODS Eligible patients had ER and/or progesterone receptor-positive breast cancer, WHO performance status 0–2, and ≥1 measurable/non-measurable lesion(s). Patients were randomized (1:1) to receive fulvestrant 500 mg (IM on Days 0, 14, 28, and each 28 days thereafter) or anastrozole 1 mg daily, and were stratified according to LA or MBC; prior or no prior treatment with chemotherapy for LA/MBC; and measurable or non-measurable disease. The consistency of effect across patient subgroups was assessed via hazard ratios and 95% confidence intervals using a log-rank test. RESULTS In total, 462 patients were randomized (n=230 fulvestrant 500 mg; n=232 anastrozole). The Asian subgroup comprised 67 patients (n=34 fulvestrant 500 mg; n=33 anastrozole). PFS outcomes for the Asian and non-Asian subgroups are presented (Table). The most commonly reported adverse event (AE) was arthralgia (18.2% vs 12.1% of patients with fulvestrant 500 mg and anastrozole, respectively). The rate of AEs leading to discontinuation of treatment was 3.0% and 3.0%, respectively. CONCLUSIONS Based on a preliminary assessment of 67 patients, the treatment effect in the Asian patient subgroup from the FALCON trial appears to be broadly consistent with the non-Asian population. PFS in Asian and non-Asian patient subgroupsGeographic regionNumber of patients (%) with event Hazard ratio (95% CI) Fulvestrant 500 mg n=230Anastrozole n=232 Asia19/34 (55.9%)22/33 (66.7%)0.81 (0.44, 1.50)Non-Asia124/196 (63.3%)144/199 (72.4%)0.79 (0.62, 1.01) Citation Format: Shao Z, Ellis MJ, Robertson JFR, Grinsted LM, Fazal M, Noguchi S. Progression-free survival results in postmenopausal Asian women: Subgroup analysis from a phase 3 randomized trial of fulvestrant 500 mg vs anastrozole for hormone receptor-positive advanced breast cancer (FALCON) [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 P2-08-09.

BACKGROUND Improved efficacy was suggested for fulvestrant 500 mg, a selective estrogen receptor degrader (SERD), vs anastrozole as first-line treatment for hormone receptor-positive locally advanced or metastatic breast cancer (LA/MBC) in the open-label Phase 2 FIRST trial. The Phase 3, randomized, double-blind, multicenter FALCON trial (NCT01602380) compared fulvestrant 500 mg with anastrozole 1 mg in patients with hormone receptor-positive LA/MBC who had not received prior hormonal therapy. The primary endpoint of the study was met, such that there was a statistically significant improvement in progression-free survival (PFS) for fulvestrant 500 mg vs anastrozole. We present an analysis of PFS for pre-specified patient subgroups in the FALCON trial. METHODS Eligible patients had ER and/or progesterone receptor (PgR)-positive breast cancer, WHO performance status 0–2 and ≥1 measurable/non-measurable lesion(s). Patients were randomized (1:1) to receive fulvestrant 500 mg (IM on Days 0, 14, 28, and each 28 days thereafter) or anastrozole 1 mg daily. The primary endpoint was PFS, assessed via RECIST 1.1, surgery/radiotherapy for disease worsening, or death (any cause). PFS was evaluated in patient subgroups defined by pre-specified baseline covariates. The consistency of effect across patient subgroups was assessed via hazard ratios and 95% confidence intervals using a log-rank test. RESULTS Overall, 462 patients were randomized to treatment: 230 received fulvestrant 500 mg and 232 received anastrozole. PFS outcomes in each patient subgroup are presented in the Table. CONCLUSIONS This analysis of patient subgroups from the FALCON trial suggests that treatment effects were largely consistent across the subgroups analyzed with some possible exceptions (e.g. patients with visceral vs non-visceral disease). Further work is ongoing to understand the possible treatment effect in these subgroups. PFS in pre-specified patient subgroupsSubgroup Number of patients (%) with event Hazard ratio (95% CI) Fulvestrant 500 mg n=230 Anastrozole n=232 Breast cancer type Locally advanced11/28 (39.3%)14/32 (43.8%)0.79 (0.36, 1.73)Metastatic132/202 (65.3%)152/200 (76.0%)0.78 (0.62, 0.99)Prior chemotherapy for LA/MBC Yes31/36 (86.1%)33/43 (76.7%)1.08 (0.66, 1.77)No112/194 (57.7%)133/189 (70.4%)0.75 (0.59, 0.97)Geographic regiona US/Canada16/25 (64.0%)19/24 (79.2%)0.66 (0.34, 1.30)Non-US/Canada127/205 (62.0%)147/208 (70.7%)0.81 (0.64, 1.03)Measurable disease Yes124/193 (64.2%)143/196 (73.0%)0.76 (0.60, 0.97)No19/37 (51.4%)23/36 (63.9%)0.99 (0.53, 1.82)ER-positive and PgR-positive Yes103/175 (58.9%)127/179 (70.9%)0.73 (0.56, 0.94)No40/55 (72.7%)39/53 (73.6%)1.04 (0.67, 1.62)Bisphosphonate use at baseline Yes44/61 (72.1%)53/62 (85.5%)0.69 (0.46, 1.03)No99/169 (58.6%)113/170 (66.5%)0.82 (0.63, 1.07)Visceral disease Yes92/135 (68.1%)87/119 (73.1%)0.99 (0.74, 1.33)No51/95 (53.7%)79/113 (69.9%)0.59 (0.42, 0.84)ªData for Asia/non-Asia subgroups are presented in a separate abstract Citation Format: Robertson JFR, Noguchi S, Shao Z, Grinsted LM, Fazal M, Ellis MJ. Progression-free survival results in patient subgroups from a Phase 3 randomized trial of fulvestrant 500 mg vs anastrozole for hormone receptor-positive advanced breast cancer (FALCON) [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 P2-08-02.

Rapidly growing cancer cells have increased levels of intracellular polyamines compared to normal, healthy tissues. Based on the selective reactivity of glycine propargyl esters, probes were synthesized that show evidence for selective polyamine reactivity, which was then applied for selective cancer cell imaging studies.

One-step nucleic acid amplification (OSNA) targeting cytokeratin 19 (CK19) mRNA expression and pathological examination are widely used for the intraoperative diagnosis of sentinel node (SN) metastasis. The aim of the present study was to develop a novel assay for detecting SN metastasis by targeting Ras association domain-containing protein 1 (RASSF1A) methylation in tumor cells, and to compare its performance with OSNA. Using digital PCR with methylation-specific restriction enzymes (RE-dMSP), our assay was able to detect ≥3 copies of methylated DNA per well, and was ≥10 times more sensitive than real-time PCR with bisulfite modification. OSNA lysates were examined using RE-dMSP and digital PCR for PIK3CA mutation, in the event that primary tumors were PIK3CA mutation-positive. RE-dMSP revealed a high concordance of 95.0% (153/161) with OSNA, and 100% (59/59) with PIK3CA mutation for detecting SN metastasis. In 11 breast cancer cell lines, the variation in methylated RASSF1A copy number was significantly lower than that of CK19 mRNA (2.8 vs. 10.5-fold; P

We previously developed a 95‑gene classifier (95GC) to classify ER‑positive/HER2‑negative/node‑negative (ER+/HER2‑/N0) breast cancer as high‑ and low‑risk. The present study aimed to devise a 95GC recurrence score (95GCRS) to estimate recurrence risk more precisely and, although the 95GC was originally developed using fresh‑frozen (FF) tissues, this was applied to formalin‑fixed paraffin‑embedded (FFPE) tissues. 95GCRS was calculated using between‑group analysis and denominated as a value from 0 to 100. Correlation of 95GCRS with distant recurrence rate and response to neoadjuvant chemotherapy (NAC) was evaluated in 257 patients with ER+/HER2-/N0 breast cancer treated with adjuvant hormonal therapy at Osaka University Hospital and in 425 patients with ER+ breast cancer treated with NAC at Osaka University Hospital and the University of Texas MD Anderson Cancer Center (GSE25066 dataset). Correlation of 95GCRS between FF and FFPE tissues was evaluated in paired tissues from 56 ER+/HER2‑/N0 breast cancer types obtained from patients without NAC treatment. Distant recurrence rates were remarkably low in patients with 95GCRS ≤50 and increased proportionally in patients with 95GCRS >50. Pathological complete response (pCR) rates to NAC were increased in proportion to 95GCRS, indicating a greater sensitivity of breast cancers with high 95GCRS to chemotherapy. 95GCRS was highly correlated (R=0.92) between FF and FFPE tissues, and the concordance rate (94.6%) of high‑ and low‑risk groups was also considerably high. Overall, the present study developed a 95GCRS that correlated with distant recurrence rate and pCR rate to NAC. The 95GC was applicable to FFPE tissues with a high concordance rate in FF tissues.

ESR1 mutations in breast cancer are known as one of the mechanisms of resistance to aromatase inhibitors. These mutations often occur in the hotspot regions in the ligand binding domain (LBD), but comprehensive mutational analysis has shown that mutations are observed throughout the whole LBD. We previously developed a molecular barcode sequencing (MB-NGS) technique to detect ESR1 hotspot mutations in plasma with high sensitivity. In this study, we have developed a multiplex MB-NGS assay that covers the whole LBD of ESR1. The assay demonstrated that the background errors in the plasma DNA of 10 healthy controls were below 0.1%; thus, the limit of detection was set at 0.1%. We analyzed the plasma DNA of 54 patients with estrogen receptor–positive metastatic breast cancer. Seventeen mutations were detected in 13 patients (24%), with variant allele frequencies ranging from 0.13% to 10.67%, including six rare mutations with a variant allele frequency

Fusion genes are involved in cancer, and their detection using RNA-Seq is insufficient given the relatively short reading length. Therefore, we proposed a shifted short-read clustering (SSC) method, which focuses on overlapping reads from the same loci and extends them as a representative sequence. To verify their usefulness, we applied the SSC method to RNA-Seq data from four types of cell lines (BT-474, MCF-7, SKBR-3, and T-47D). As the slide width of the SSC method increased to one, two, five, or ten bases, the read length was extended from 201 bases to 217 (108%), 234 (116%), 282 (140%), or 317 (158%) bases, respectively. Furthermore, fusion genes were investigated using STAR-Fusion, a fusion gene detection tool, with and without the SSC method. When one base was shifted by the SSC method, the reads mapped to multiple loci decreased from 9.7% to 4.6%, and the sensitivity of the fusion gene was improved from 47% to 54% on average (BT-474: from 48% to 57%, MCF-7: 49% to 53%, SKBR-3: 50% to 57%, and T-47D: 43% to 50%) compared with original data. When the reads are shifted more, the positive predictive value was also improved. The SSC method could be an effective method for fusion gene detection.

Tetramethylrhodamine (TAMRA)-phenyl azide is a chemical probe used to detect intracellular acrolein directly in live cells. Herein, we demonstrated that TAMRA is the optimum fluorophore for the probe. TAMRA-phenyl azide was used to reveal that high levels of acrolein are generated in a variety of breast cancer cells, regardless of the tumor subtype. These findings corroborate the analysis presented in our previous report, in which TAMRA-phenyl azide was used to label breast cancer tissues resected from breast cancer patients. Because high levels of acrolein were generated in all cancer cell types, we believe that acrolein detection may be useful as a general method for labeling cancerous tissues.