Mutsuko Yamamoto-Ibusuki

Targeted therapies for ER+/HER2- metastatic breast cancer

The majority of breast cancers present with estrogen receptor (ER)-positive and human epidermal growth factor receptor (HER2)-negative features and might benefit from endocrine therapy. Although endocrine therapy has notably evolved during the last decades, the invariable appearance of endocrine resistance, either primary or secondary, remains an important issue in this type of tumor. The improvement of our understanding of the cancer genome has identified some promising targets that might be responsible or linked to endocrine resistance, including alterations affecting main signaling pathways like PI3K/Akt/mTOR and CCND1/CDK4-6 as well as the identification of new ESR1 somatic mutations, leading to an array of new targeted therapies that might circumvent or prevent endocrine resistance. In this review, we have summarized the main targeted therapies that are currently being tested in ER+ breast cancer, the rationale behind them, and the new agents and combinational treatments to come.

Clinical significance of CYLD downregulation in breast cancer

Abstract Cylindromatosis (CYLD) is a tumor suppressor gene that is mutated in familial cylindromatosis, a rare autosomal dominant disorder associated with numerous benign skin adnexal tumors. CYLD is now known to regulate various signaling pathways, including transforming growth factor-β signaling, Wnt/β-catenin signaling, and NF-κB signaling by deubiquitinating upstream regulatory factors. Downregulation of CYLD has been reported in several malignancies; however, the clinical significance of CYLD expression in many malignancies, including breast cancer, remains to be elucidated. This study investigated the clinical significance of CYLD in breast cancer and its roles in tumor progression. We evaluated CYLD expression in matched normal breast tissue samples and tumor breast tissue samples from 26 patients with breast cancer and in a series of breast cancer cell lines. In addition, by means of immunohistochemistry, we investigated CYLD protein expression and its clinical significance in 244 breast cancer cases. We also analyzed the effects of CYLD repression or overexpression on breast cancer cell viability, cell migration, and NF-κB activity with or without receptor activator of NF-κB ligand (RANKL) stimulation. Breast cancer tissues demonstrated significantly reduced CYLD mRNA expression compared with normal breast tissues. Downregulation of CYLD promoted cell survival and migratory activities through NF-κB activation, whereas CYLD overexpression inhibited those activities in MDA-MB-231 cells. As an important finding, CYLD overexpression also inhibited RANKL-induced NF-κB activation. Our immunohistochemical analysis revealed that reduced CYLD protein expression was significantly correlated with estrogen receptor negativity, high Ki-67 index, high nuclear grade, decreased disease-free survival, and reduced breast cancer-specific survival in primary breast cancer. Moreover, reduced CYLD expression was an independent factor for poor prognosis in breast cancer. CYLD downregulation may promote breast cancer metastasis via NF-κB activation, including RANKL signaling.

Clinical significance of monitoring ESR1 mutations in circulating cell-free DNA in estrogen receptor positive breast cancer patients

Background The measurement of circulating cell-free DNA (cfDNA) may transform the management of breast cancer patients. We aimed to investigate the clinical significance of sequential measurements of ESR1 mutations in primary breast cancer (PBC) and metastatic breast cancer (MBC) patients. Results ESR1 mutations ratio in the PBC groups was used as the minimum cutoff for determining increases in cfDNA ESR1 mutation ratio. An increase in cfDNA ESR1 mutations was found in 13 samples of cfDNA from 12 (28.6%) out of 42 MBC patients. A total of 10 (83.3%) out of 12 MBC patients with increase cfDNA ESR1 mutations showed a poor response to treatment. In survival analysis, increase cfDNA ESR1 mutations may predict a shorter duration of post-endocrine-therapy effectiveness (P = 0.0033). Methods A total of 119 patients (253 plasma samples) with breast carcinoma were enrolled in this study. Cases were selected if archival plasma samples were available from PBC before and after treatment and from MBC gathered more than twice at the time of progression. cfDNA was isolated from the 77 PBC patients (154 plasma samples) and from the 42 MBC patients (99 plasma samples). To investigate any changes in each cfDNA ESR1 mutation before and after treatment, we analyzed the difference with cfDNA ESR1 mutations ratio in the first blood sample using droplet digital polymerase chain reaction (ddPCR). Conclusions We demonstrate that ddPCR monitoring of the recurrent ESR1 mutation in cfDNA of MBC patients is a feasible and useful method of providing relevant predictive information.

Droplet digital polymerase chain reaction assay for screening of ESR1 mutations in 325 breast cancer specimens

Droplet digital polymerase chain reaction (ddPCR), which could perform thousands of PCRs on a nanoliter scale simultaneously, would be an attractive method to massive parallel sequencing for identifying and studying the significance of low-frequency rare mutations. Recent evidence has shown that the key potential mechanisms of the failure of aromatase inhibitors-based therapy involve identifying activating mutations affecting the ligand-binding domain of the ESR1 gene. Therefore, the detection of ESR1 mutations may be useful as a biomarker predicting an effect of the treatment. We aimed to develop a ddPCR-based method for the sensitive detection of ESR1 mutations in 325 breast cancer specimens, in which 270 primary and 55 estrogen receptor-positive (ER+) metastatic breast cancer (MBC) specimens. Our ddPCR assay could detect the ESR1 mutant molecules with low concentration of 0.25 copies/μL. According to the selected cutoff, ESR1 mutations occurred in 7 (2.5%) of 270 primary breast cancer specimens and in 11 (20%) of 55 ER+ MBC specimens. Among the 11 MBC specimens, 5 specimens (45.5%) had the most common ESR1 mutation, Y537S, 4 specimens (36.3%) each had D538G, Y537N, and Y537C. Interestingly, 2 patients had 2 ESR1 mutations, Y537N/D538G and Y537S/Y537C, and 2 patients had 3 ESR1 mutations, Y537S/Y537N/D538G. Biopsy was performed in heterochrony in 8 women twice. In 8 women, 4 women had primary breast cancer and MBC specimens and 4 women had 2 specimens when treatment was failure. Four of these 8 women acquired ESR1 mutation, whereas no ESR1 mutation could be identified at first biopsy. ddPCR technique could be a promising tool for the next-generation sequencing-free precise detection of ESR1 mutations in endocrine therapy resistant cases and may assist in determining the treatment strategy.

High survivin mRNA expression is a predictor of poor prognosis in breast cancer: A comparative study at the mRNA and protein level

BackgroundSurvivin plays a key role in the initiation and progression of breast cancer. However, its prognostic relevance to breast cancer patients has long been a matter of debate. The purpose of this study was to examine the expression of survivin and its role in predicting clinical outcome in a series of human breast cancer cases both at the mRNA and protein level.MethodsFormalin-fixed paraffin-embedded tumor tissues from 245 female patients with invasive breast cancer and 13 patients with ductal carcinoma in situ were examined for survivin mRNA by quantitative real-time RT-PCR (RT-qPCR). In addition, 237 of these tumors with invasive breast cancer were available for immunohistochemistry (IHC). The relationship between survivin status and clinicopathological characteristics and prognosis was evaluated.ResultsRT-qPCR revealed that high levels of survivin mRNA were strongly associated with high nuclear grade, positive axillary lymph nodes, negative hormone receptor status, positive Her2 amplification, higher Ki67 labeling index, and presence of vascular invasion. In the Cox proportional regression model analysis, survivin mRNA was shown to be a significant univariate parameter for relapse-free survival (RFS), distant relapse-free survival (DRFS), and breast cancer-specific survival (BCSS) as well as a significant multivariate parameter for RFS, DRFS, and BCSS. In hormone receptor (HR)-positive/Her2-negative subtype cases, survivin mRNA expression was also an independent predictor in terms of DRFS. Immunohistochemically, positive staining was seen in the cytoplasm and/or nucleus of cancer cells, although this did not correlate with the mRNA level, and harbored no prognostic value.ConclusionsHigh mRNA expression of survivin was an independent marker of poor prognosis both in the entire cohort and in the HR-positive/Her2-negative subtype, whereas the protein expression of survivin was not. These findings suggest that RT-qPCR can provide more reliable data than IHC in validating the prognostic significance of survivin for breast cancer patients.

A comprehensive analysis of Aurora A; transcript levels are the most reliable in association with proliferation and prognosis in breast cancer

BackgroundAurora A kinase, a centrosomal serine/threonine kinase which plays an essential role in chromosome segregation during cell division, is commonly amplified and/or over expressed in human malignancies. Aurora A is suggested to be one of the proliferation parameters which is an independent prognostic factor for early invasive breast cancer patients; however the individual clinical or prognostic relevance of this gene has been a matter of debate.MethodsA comprehensive analysis of Aurora A at the levels of gene expression, gene copy number and protein expression was performed for 278 primary invasive breast cancer patients; and the correlation with clinical outcomes were investigated.ResultsAurora A gene expression level not only correlated with gene amplification, but was also significantly associated with several clinicopathological parameters and patient prognosis. Patients with higher nuclear grade, negative progesterone receptor status and higher Ki67 expressed higher levels of Aurora A mRNA, which was associated not only with poor relapse-free survival (RFS) but was also found to be a significant multivariate parameter for RFS. Aurora A protein expression was also significantly associated with clinicopathological characteristics; lymph node status, nuclear grade, estrogen receptor status and Ki67, but not with prognosis. By contrast, Aurora A gene amplification correlated with tumor size, nuclear grade and Ki67, and had no prognostic value.ConclusionOur data indicate that Aurora A gene expression is an effective tool, which defines both tumor proliferation potency and patient prognosis.

Prognostic role of PIK3CA mutations of cell-free DNA in early-stage triple negative breast cancer

PIK3CA is an oncogene that encodes the p110α component of phosphatidylinositol 3‐kinase (PI3K); it is the second most frequently mutated gene following the TP53 gene. In the clinical setting, PIK3CA mutations may have favorable prognostic value for hormone receptor‐positive breast cancer patients and, during the past few years, PIK3CA mutations of cell‐free DNA (cfDNA) have attracted attention as a potential noninvasive biomarker of cancer. However, there are few reports on the clinical implications of PIK3CA mutations for TNBC patients. We investigated the PIK3CA major mutation status of cfDNA as a noninvasive biomarker of cancer using droplet digital polymerase chain reaction (ddPCR), which has high level sensitivity and specificity for cancer mutation, in early‐stage 49 triple negative breast cancer (TNBC) patients. A total of 12 (24.4%) of 49 patients had PIK3CA mutations of cfDNA. In a median follow up of 54.4 months, the presence of PIK3CA mutations of cfDNA had significant impacts on relapse‐free survival (RFS; P = 0.0072) and breast cancer‐specific survival (BCSS; P = 0.016), according to the log‐lank test. In a Cox proportional hazards model, the presence of PIK3CA mutations of cfDNA had significant prognostic value in the univariate and multivariate analysis. Additionally, the presence of PIK3CA mutations of cfDNA was significantly correlated with positive androgen receptor phosphorylated form depending on PI3K signaling pathway (pAR) which is independent favorable prognostic factors of TNBC. We demonstrated that the presence of PIK3CA major mutations of cfDNA could be a discriminatory predictor of RFS and BCSS in early‐stage TNBC patients and it was associated with PI3K pathway‐dependent AR phosphorylation.

Comparison of prognostic values between combined immunohistochemical score of estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, Ki-67 and the corresponding gene expression score in breast cancer.

In the clinical diagnosis of breast cancer, immunohistochemistry panels with estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2) and Ki-67 are routinely used, and they have been proposed for the classification of breast tumors into distinct subtypes. Gene expression analysis with formalin-fixed paraffin-embedded material have also become widely available recently, but the prognostic values of corresponding gene panels compared with these four immunohistochemical panels had never tested. We independently evaluated the 5-year relapse risk-estimation scores using semiquantitative data of four immunohistochemical panels (Ku-IHC4 score) and compared these with the results of four-gene expression profiling of formalin-fixed paraffin-embedded specimens (Ku-FFPE4 score) in a consecutive series of 235 primary invasive breast cancer patients. Ku-IHC4 score was revealed to be an independent predictor of recurrence other than Ku-FFPE4 score in a multivariate model analyzed by classical clinical parameters (Ku-IHC4 score vs Ku-FFPE4 score; χ2: 14.2 vs 2.5, P: 0.0002 vs 0.11). When patients were trichotomized into high-, intermediate- and low-risk groups using the thresholds determined from the approximately calculated 5-year relapse rate, Kaplan–Meier analyses showed a significant difference among the three groups in Ku-IHC4 score (log-rank, P<0.0001), but not in Ku-FFPE4 score. The high-risk group according to Ku-FFPE4 score showed contradictory low recurrence rates (Ku-IHC4 score vs Ku-FFPE4 score, 53.1 vs 24.8%), which might be caused by risk-dependently extended error ranges. We show that the Ku-IHC4 score, consisted with semiquantitative measures of immunohistochemistry, provides better prognostic information than the corresponding quantitative RNA measurements. Prognostication tools such as the Ku-IHC4 score may be potentially useful in screening which patients had better be assessed by further testing using other genes rather than ER, PgR, HER2 and Ki-67 to determine critical aspects of therapeutic decision making.

Clinical significance of androgen receptor and its phosphorylated form in breast cancer

Attention to hormone receptor (HR) function in breast cancer has largely focused on estrogen receptor (ER), progesterone receptor (PgR), andhuman epidermal growth receptor 2 (HER2). The success of therapies targeted at these receptors has led to interest in androgen receptor (AR), a member of the steroid receptor (SR) subfamily, as another possible target in breast cancer therapy. Immunohistochemical studies have revealed an independent prognostic value of AR in ERa-positive breast cancer (Castellano et al. 2010), and the prognosis of AR-positive/ERa-negative tumors has been reported to be better than that of AR-negative one (Kuenen-Boumeester et al. 1996). These studies have revealed that AR plays an inherent role in breast cancer cell signaling. Much effort has been invested in elucidating the mechanism of action of AR in human epithelial cells. This includes research on posttranslational modifications, including phosphorylation, ubiquitination, acetylation, and sumoylation (Ward & Weigel 2009). Among these, AR phosphorylation has been shown to play a critical role in signal transduction (Lin et al. 2001), and a large number of phosphorylation sites have been identified, with the majority near the N-terminus. AR is phosphorylated at serine-213/210 (pAR) by the phosphatidylinositol 3-kinase/Akt signaling pathway (Wen et al. 2000), which modulates AR transcriptional activity (Lin et al. 2001, 2003). The objectives of the current study were to examine AR and pAR levels in a large series of breast cancers and to assess the impactofARandpAR expressiononrelapsefree survival (RFS) and breast cancer-specific survival (BCSS) in all breast cancers. A total of 379 consecutive patients (median age 58 years (range 21–93)) with invasive breast cancer were enrolled from a database that included patient and tumor characteristics, as well as the pathology findings at the initial and relapse diagnoses. All patients signed informed consent forms and were treated at the Department of Breast and Endocrine Surgery of our center in the period between 2001 and 2009. Patients were periodically examined at the Kumamoto University Hospital or affiliated hospitals. The median follow-up period was 66.1 months (range 0.23–145.1 months). AR was detected with mouse monoclonal AR-318 from Leica Biosystems (Newcastle, UK). Phospho-AR was detected with mouse monoclonal 156C135.2 from Abcam plc (Cambridge, UK). The levels of AR nuclear protein expression were determined in tissue samples of 379 consecutive female patients. In samples that showed detectable AR, the levels of pAR nuclear and cytoplasmic expression were determined because pAR may be strongly stained even if AR staining is weak (Fig. 1A). All samples were scored using a histoscore (HS), multiplying the percentage (0–100) of cells stained at a given intensity by the staining intensity score (IS) (0, none; 1, weak; 2, moderate; and 3, intense). AR and nuclear and cytoplasmic expression of pAR were observed in benign breast tissue. However, the patterns of both AR and phosphorylated AR expression were increased in breast cancers compared with benign controls (Ren et al. 2013). Compared with other studies in which AR expression was dichotomized as having positive cells or not, we used a HS (O10) for AR and a IS (R2) for pAR as a cutoff point, which we believe more accurately reflects the expression of AR and its phosphorylation status in breast cancer (Fig. 1A). The median HS was 126 in nuclear AR, 112 in nuclear pAR, and zero in cytoplasmic pAR (Fig. 1B). Nuclear staining of AR was observed in 76% of the samples. The correlation of AR expression and patient characteristics is summarized in Table 1. Positive ARs were found in the groups of patients with lower nuclear grade (PZ0.0008), positive ERa (P!0.0001), positive PgR E n d o cr in e -R e la te d C a n ce r Letter to the editor T Takeshita et al. AR and AR phosphorylation in breast cancer 20 :5 L15–L21

Fibroblast growth factor receptor-1 protein expression is associated with prognosis in estrogen receptor-positive/human epidermal growth factor receptor-2-negative primary breast cancer

Recently, research into the development of new targeted therapies has focused on specific genetic alterations to create advanced, more personalized treatment. One of the target genes, fibroblast growth factor receptor‐1 (FGFR1), has been reported to be amplified in estrogen receptor (ER)‐positive subtype breast cancer, and is considered one possible mechanism of endocrine resistance through cross‐talk between ER and growth factor receptor signaling. We performed a comprehensive analysis of FGFR1 at the levels of gene copy number, transcript and protein expression, and examined the relationships between FGFR1 status and clinicopathological parameters, including prognosis in 307 ER‐positive/HER2‐negative primary breast cancer patients treated with standard care at our institute. Most notably, a high level of FGFR1 protein expression was observed in 85 patients (27.7%), and was positively associated with invasive tumor size (P = 0.039). Furthermore, univariate analysis revealed that high FGFR1 protein expression was significantly correlated with poor relapse‐free survival rate (P = 0.0019, HR: 2.63, 95% confidence interval: 1.17–5.98), and showed a tendency towards an increase in recurrent events if the observation period extended beyond the 5 years of the standard endocrine treatment term. FGFR1 gain/amplification was found in 43 (14.0%) patients, which was only associated with higher nuclear grade (P = 0.010). No correlation was found between FGFR1 mRNA expression levels and any clinicopathological factors. Overall, the level of FGFR1 protein expression may be a biomarker of ER‐positive/HER2‐negative primary breast cancer with possible resistance to standard treatment, and may be a useful tool to identify more specific patients who would benefit from FGFR‐1 targeted therapy.