A prediction model for early systemic recurrence in breast cancer using a molecular diagnostic analysis of sentinel lymph nodes: A large-scale, multicenter cohort study.

BACKGROUND: The one-step nucleic acid amplification (OSNA) assay can quantify the cytokeratin 19 messenger RNA copy number as a proxy for sentinel lymph node (SN) metastasis in breast cancer. A large-scale, multicenter cohort study was performed to determine the prognostic value of the SN tumor burden based on a molecular readout and to establish a model for the prediction of early systemic recurrence in patients using the OSNA assay. METHODS: SN biopsies from 4757 patients with breast cancer were analyzed with the OSNA assay. The patients were randomly assigned to the training or validation cohort at a ratio of 2:1. On the basis of the training cohort, the threshold SN tumor burden value for stratifying distant recurrence was determined with Youden's index; predictors of distant recurrence were investigated via multivariable analyses. Based on the selected predictors, a model for estimating 5-year distant recurrence-free survival was constructed, and predictive performance was measured with the validation cohort. RESULTS: The prognostic cutoff value for the SN tumor burden was 1100 copies/µL. The following variables were significantly associated with distant recurrence and were used to construct the prediction model: SN tumor burden, age, pT classification, grade, progesterone receptor, adjuvant cytotoxic chemotherapy, and adjuvant anti-human epidermal growth factor receptor 2 therapy. The values for the area under the curve, sensitivity, specificity, and accuracy of the prediction model were 0.83, 63.4%, 81.7%, and 81.1%, respectively. CONCLUSIONS: Using the OSNA assay, the molecular readout-based SN tumor burden is an independent prognostic factor for early breast cancer. This model accurately predicts early systemic recurrence and may facilitate decision-making related to treatment.

The multigene classifiers 95GC/42GC/155GC for precision medicine in ER-positive HER2-negative early breast cancer.

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.

A 95-gene signature stratifies recurrence risk of invasive disease in ER-positive, HER2-negative, node-negative breast cancer with intermediate 21-gene signature recurrence scores.

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: 206 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.

Development of Prediction Model Including MicroRNA Expression for Sentinel Lymph Node Metastasis in ER-Positive and HER2-Negative Breast Cancer.

BACKGROUND: 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. PATIENTS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Vasculogenic mimicry is associated with trastuzumab resistance of HER2-positive breast cancer.

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.

Prognostic value of tumor cell DNA content determined by flow cytometry using formalin-fixed paraffin-embedded breast cancer tissues.

PURPOSE: The use of formalin-fixed paraffin-embedded (FFPE) tumor tissues in flow cytometry (FCM)-based determination of tumor cell DNA content is more complicated than the use of fresh-frozen tissues. This study aimed to accurately measure tumor cell DNA content from FFPE tissues by separating tumor cells from stromal cells through FCM and investigating its prognostic impact. METHODS: We separately measured the DNA contents of tumor cells and stromal cells by gating with pan-cytokeratin and vimentin (FCMC/V). We evaluated tumor cell DNA contents [DNA index (DI)] of 290 FFPE tumor tissues and classified them into low and high DI groups, using a cutoff DI value determined through an unbiased computational method. RESULTS: The distribution of DI was bimodal, and a cutoff value was determined at a DI of 1.26. The high-DI tumors were associated with aggressive phenotypes and had significantly worse distant recurrence-free intervals (DRFI) than low-DI tumors. Multivariate analysis revealed that lymph node metastasis, Ki67, and DI were independent factors affecting DRFI. Accordingly, patients with low-DI/low-Ki67 tumors had excellent outcomes compared with other tumor types. Multiploid tumors were associated with increased lymphocytic infiltration and aggressive phenotypes. CONCLUSIONS: The DI of FFPE tumors could be precisely determined through FCMC/V. A combination of DI and Ki67 analyses may be able to predict the prognoses of breast cancer patients with greater accuracy.

PAM50 for prediction of response to neoadjuvant chemotherapy for ER-positive breast cancer.

PURPOSE: 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. PATIENTS AND METHODS: 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). RESULTS: 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%). CONCLUSIONS: 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.

Clinicopathological analysis of homologous recombination-deficient breast cancers with special reference to response to neoadjuvant paclitaxel followed by FEC.

PURPOSE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Mutational Analysis of AKT1 and PIK3CA in Intraductal Papillomas of the Breast with Special Reference to Cellular Components.

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.

Tetramethylrhodamine is an essential scaffold of azide probe in detecting cellular acrolein.

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.

Construction of a novel multi-gene assay (42-gene classifier) for prediction of late recurrence in ER-positive breast cancer patients.

PURPOSE: Prediction models for late (> 5 years) recurrence in ER-positive breast cancer need to be developed for the accurate selection of patients for extended hormonal therapy. We attempted to develop such a prediction model focusing on the differences in gene expression between breast cancers with early and late recurrence. METHODS: For the training set, 779 ER-positive breast cancers treated with tamoxifen alone for 5 years were selected from the databases (GSE6532, GSE12093, GSE17705, and GSE26971). For the validation set, 221 ER-positive breast cancers treated with adjuvant hormonal therapy for 5 years with or without chemotherapy at our hospital were included. Gene expression was assayed by DNA microarray analysis (Affymetrix U133 plus 2.0). RESULTS: With the 42 genes differentially expressed in early and late recurrence breast cancers in the training set, a prediction model (42GC) for late recurrence was constructed. The patients classified by 42GC into the late recurrence-like group showed a significantly (P = 0.006) higher late recurrence rate as expected but a significantly (P = 1.62 × E-13) lower rate for early recurrence than non-late recurrence-like group. These observations were confirmed for the validation set, i.e., P = 0.020 for late recurrence and P = 5.70 × E-5 for early recurrence. CONCLUSION: We developed a unique prediction model (42GC) for late recurrence by focusing on the biological differences between breast cancers with early and late recurrence. Interestingly, patients in the late recurrence-like group by 42GC were at low risk for early recurrence.

Highly sensitive detection of ESR1 mutations in cell-free DNA from patients with metastatic breast cancer using molecular barcode sequencing.

PURPOSE: 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). METHODS: 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). RESULTS: 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 < 0.01). Moreover, all the patients without these mutations by MB-NGS were found to have no mutations by ddPCR. CONCLUSION: In conclusion, MB-NGS could successfully detect ESR1 mutations in cfDNA with a higher sensitivity of 0.1% than conventional NGS and was considered as clinically useful as ddPCR.

Intraoperative Nomograms, Based on One-Step Nucleic Acid Amplification, for Prediction of Non-sentinel Node Metastasis and Four or More Axillary Node Metastases in Breast Cancer Patients with Sentinel Node Metastasis.

BACKGROUND: One-step nucleic acid amplification (OSNA) for cytokeratin 19 messenger RNA is an intraoperative diagnostic procedure for the detection of lymph node metastasis. OBJECTIVE: This study aimed to construct intraoperative nomograms using OSNA for the prediction of non-sentinel lymph node (NSLN) metastasis and four or more axillary lymph node (ALN) metastases. METHODS: Of the 4736 breast cancer patients (T1-3, N0) who underwent sentinel lymph node (SLN) biopsy and had SLNs examined intraoperatively with OSNA, 623 with SLN metastasis treated with completion ALN dissection (cALND) were retrospectively analyzed, and were randomly divided into training (n = 312) and validation (n = 311) sets. RESULTS: Of the clinicopathological parameters available preoperatively and intraoperatively, the multivariate analysis of the training set revealed that clinical tumor size and total tumor load (TTL) determined by OSNA were significantly associated with NSLN metastasis, and that clinical tumor size, number of macrometastatic SLNs, and TTL were significantly associated with four or more ALN metastases. Nomograms for NSLN metastasis and four or more ALN metastases were constructed using these parameters, and their area under the receiver operating characteristic curve (AUC) of the validation set were both 0.70, with a diagnostic accuracy similar to that of previously reported postoperative nomograms. CONCLUSIONS: We constructed intraoperative nomograms using OSNA for the prediction of NSLN metastasis and four or more ALN metastases. These nomograms are as accurate as the conventional postoperative nomograms and might be helpful for decision making regarding the indication for cALND or the choice of adjuvant chemotherapeutic regimens and radiation field.

Comparison of quality of life between 2-year and 3-or-more-year administration of leuprorelin acetate every-3-months depot in combination with tamoxifen as adjuvant endocrine treatment in premenopausal patients with endocrine-responsive breast cancer: a randomized controlled trial.

BACKGROUND: We conducted an open-label, randomized controlled trial evaluating the appropriate treatment duration of leuprorelin acetate 3-month depot, TAP-144-SR (3M), administered postsurgically every 3 months for 2 years versus 3 or more (up to 5) years, in combination with tamoxifen, for 5 years in premenopausal endocrine-responsive breast cancer patients and reported similar survival benefit in the two treatment groups. We hereby present patient-reported quality of life (QOL) data obtained from this trial. METHODS: Three self-administered QOL questionnaires (QOL-ACD, QOL-ACD-B, FACT-ES subscale) were used, and the difference in QOL score changes between the two groups was analyzed using a mixed-effects model for repeated measures. RESULTS: Eligible patients (N = 222) were randomly assigned to a 2-year (2YG, N = 112) or 3-or-more-year treatment group (3YG, N = 110). The time courses of the three QOL scores during the trial period were similar in the two groups. The mean changes in the QOL scores from week 96 were largely stable through week 240 in the 3YG, but showed significantly greater improvement in the score changes from week 96 in the 2YG than the 3YG. Symptoms associated with menopause such as hot flashes and sweating contributed to these results. Menstruation recovery was associated with significantly greater improvement of these symptoms in the 2YG than the 3YG. CONCLUSIONS: Patient-reported menopause-associated symptoms and QOL improved after discontinuation of the LH-RH agonist administration and menstruation recovery. QOL information should be a consideration in long-term treatment.

Endocrine sensitivity of estrogen receptor-positive breast cancer is negatively correlated with aspartate-ß-hydroxylase expression.

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-ß-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.

Fulvestrant 500 mg versus anastrozole 1 mg for hormone receptor-positive advanced breast cancer (FALCON): an international, randomised, double-blind, phase 3 trial.

BACKGROUND: Aromatase inhibitors are a standard of care for hormone receptor-positive locally advanced or metastatic breast cancer. We investigated whether the selective oestrogen receptor degrader fulvestrant could improve progression-free survival compared with anastrozole in postmenopausal patients who had not received previous endocrine therapy. METHODS: In this phase 3, randomised, double-blind trial, we recruited eligible patients with histologically confirmed oestrogen receptor-positive or progesterone receptor-positive, or both, locally advanced or metastatic breast cancer from 113 academic hospitals and community centres in 20 countries. Eligible patients were endocrine therapy-naive, with WHO performance status 0-2, and at least one measurable or non-measurable lesion. Patients were randomly assigned (1:1) to fulvestrant (500 mg intramuscular injection; on days 0, 14, 28, then every 28 days thereafter) or anastrozole (1 mg orally daily) using a computer-generated randomisation scheme. The primary endpoint was progression-free survival, determined by Response Evaluation Criteria in Solid Tumors version 1·1, intervention by surgery or radiotherapy because of disease deterioration, or death from any cause, assessed in the intention-to-treat population. Safety outcomes were assessed in all patients who received at least one dose of randomised treatment (including placebo). This trial is registered with ClinicalTrials.gov, number NCT01602380. FINDINGS: Between Oct 17, 2012, and July 11, 2014, 524 patients were enrolled to this study. Of these, 462 patients were randomised (230 to receive fulvestrant and 232 to receive anastrozole). Progression-free survival was significantly longer in the fulvestrant group than in the anastrozole group (hazard ratio [HR] 0·797, 95% CI 0·637-0·999, p=0·0486). Median progression-free survival was 16·6 months (95% CI 13·83-20·99) in the fulvestrant group versus 13·8 months (11·99-16·59) in the anastrozole group. The most common adverse events were arthralgia (38 [17%] in the fulvestrant group vs 24 [10%] in the anastrozole group) and hot flushes (26 [11%] in the fulvestrant group vs 24 [10%] in the anastrozole group). 16 (7%) of 228 patients in in the fulvestrant group and 11 (5%) of 232 patients in the anastrozole group discontinued because of adverse events. INTERPRETATION: Fulvestrant has superior efficacy and is a preferred treatment option for patients with hormone receptor-positive locally advanced or metastatic breast cancer who have not received previous endocrine therapy compared with a third-generation aromatase inhibitor, a standard of care for first-line treatment of these patients. FUNDING: AstraZeneca.

Detection of ESR1 mutations in plasma and tumors from metastatic breast cancer patients using next-generation sequencing.

PURPOSE: Liquid biopsy using digital PCR (dPCR) has been widely used for the screening of ESR1 mutations, since they are frequently identified in the hotspot. However, dPCR is limited to the known mutations. Therefore, we aimed to analyze the utility of next-generation sequencing (NGS) to discover novel ESR1 mutations. METHODS: Whole exon sequencing of the ESR1 gene using NGS was performed in 16 primary and 47 recurrent tumor samples and 38 plasma samples from hormone receptor-positive metastatic breast cancer patients. Functional analyses were then performed for the novel mutations we detected. RESULTS: We identified no mutations in primary tumors and six mutations in five recurrent tumors, including three types of known mutations (Y537C, Y537N, and D538G) and two novel mutations (E279V and G557R). We also identified seven mutations in five plasma samples, including three types of known mutations (S463P, Y537S, and D538G) and one mutation not reported in COSMIC database (L536H). All nine patients with ESR1 mutations were treated with aromatase inhibitors (AIs) prior to sampling, and the mutations were frequently detected in patients who received AI treatments in the metastatic setting. Among the three novel mutations (E279V, L536H, and G557R), L536H, but not E279V and G557R, showed ligand-independent activity. All three mutant proteins showed nuclear localization and had no relation with non-genomic ER pathways. CONCLUSIONS: Although the molecular mechanisms of the E279V and G557R mutations remain unclear, our data suggest the utility of NGS as a liquid biopsy for metastatic breast cancer patients and the potential to identify novel ESR1 mutations.

A small-molecule inhibitor of SMAD3 attenuates resistance to anti-HER2 drugs in HER2-positive breast cancer cells.

PURPOSE: Resistance against anti-HER2 drugs in HER2-positive breast cancer is a major obstacle to the improving prognosis. Transforming growth factor ß (TGFß) is a cytokine involved in the acquisition of more malignant phenotypes through epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties. The aim of this study was to investigate the effects of TGFß and its downstream SMAD pathway on resistance to anti-HER2 drugs. METHODS: HER2-positive breast cancer cell lines were stimulated with TGFß for 14 days. Then, the sensitivity to trastuzumab and lapatinib and the expression levels of various EMT and CSC markers were examined. The correlation of nuclear SMAD3 expression in untreated breast tumor tissues with trastuzumab efficacy in neoadjuvant settings was examined. The effect of a small-molecule inhibitor of SMAD3 (SIS3) on resistance to anti-HER2 drugs was explored. RESULTS: We found that continuous activation of the TGFß-SMAD3 pathway induced resistance to anti-HER2 drugs and CSC traits in HER2-positive breast cancer cells. The induction of drug resistance by TGFß required strong activation of SMAD3. In fact, activated SMAD3 regulated multiple genes that harbor SMAD-binding elements and are involved in trastuzumab resistance. Nuclear SMAD3 expression in tumor tissue was inversely correlated with sensitivity to neoadjuvant treatment with trastuzumab. SIS3 not only prevented the acquisition of resistance to anti-HER2 drugs but also restored trastuzumab sensitivity in trastuzumab-resistant cells. CONCLUSIONS: This study indicates that the TGFß-SMAD3 pathway plays an important role in the induction and maintenance of resistance to anti-HER2 drugs. Thus, SMAD3 is a potential therapeutic target that can inhibit resistance and restore sensitivity to anti-HER2 drugs.

Relationship between Pulmonary Adverse Events and Everolimus Exposure in Japanese and Non-Japanese Patients: A Meta-Analysis of Oncology Trials.

AIMS: This meta-analysis explores the relationship between the everolimus minimum (Cmin) and maximum (Cmax) exposure and the risk for pulmonary adverse events (AEs) in Japanese versus non-Japanese patients. METHODS: Patient-level data from patients treated with daily everolimus in advanced solid tumor trials were evaluated using a Cox regression model, stratified by cancer type or treatment arm, with log-transformed time-averaged Cmin or Cmax as a time-varying covariate. Kaplan-Meier analysis was used to evaluate the relationship between pulmonary AEs and pharmacokinetic parameters. RESULTS: Thirty studies were identified. In the Cmin population (n = 1,962), all-grade pulmonary AE incidence was significantly higher in Japanese versus non-Japanese patients (19.9 vs. 9.4%). Pharmacokinetic parameters were similar between Japanese and non-Japanese patients. A 2-fold increase in everolimus Cmin significantly increased the risk for the first any-grade pulmonary AE in Japanese (risk ratio: 1.824; 95% CI: 1.141-2.918) and non-Japanese patients (risk ratio: 1.406; 95% CI: 1.156-1.710). CONCLUSIONS: The risk for pulmonary AEs is related to everolimus exposure. Local monitoring and reporting differences might account for the significantly higher reported incidence of low-grade everolimus-associated pulmonary AEs in Japanese versus non-Japanese patients. Patients should be carefully monitored for early signs of pulmonary AEs, and appropriate medical management should be implemented.