Showing papers by "Naoto T. Ueno published in 2017"

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo

Abstract: Somatic cells acquire mutations throughout the course of an individual's life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.

Androgen receptor function and androgen receptor–targeted therapies in breast cancer: A review

Abstract: Importance The androgen receptor (AR) pathway is emerging as a potential therapeutic target in breast cancer. To date, AR-targeted drugs have been approved only for treatment of prostate cancer; however, AR-targeted treatment for breast cancer is an area of active investigation. Through review of preclinical studies, retrospective clinical studies, and clinical trials, we examined the biology of AR and AR-related pathways, the potential for AR-targeted therapies in breast cancer, and potential biomarkers for AR-targeted treatments. Observations The rate of AR positivity in breast cancer is about 60% to 80%. Biologically, the AR pathway has cross-talk with several other key signaling pathways, including the PI3K/Akt/mTOR and MAPK pathways, and with other receptors, including estrogen receptor and human epidermal growth factor receptor-2. The value of AR positivity as a prognostic marker has not yet been defined. Androgen receptor–targeted therapies, including AR agonists, AR antagonists, and PI3K inhibitors, have shown promising results in clinical trials in patients with breast cancer, and combinations of AR-targeted therapies with other agents have been investigated for overcoming resistance to AR-targeted therapies. Biomarkers to stratify patients according to the likelihood of response to AR-targeted drugs are yet to be established. Potential biomarkers of response to AR inhibitors include AR phosphorylation andARgene expression. Conclusions and Relevance Androgen receptor–targeted treatments for breast cancer are in development and have shown promising preliminary results. In-depth understanding of AR and AR-related signaling pathways would improve the treatment strategies for AR-positive breast cancer. Further preclinical and clinical studies of AR-targeted drugs alone and in combination with other drugs are justified and warranted to clarify the biology of AR and inform the development of AR-targeted therapies to improve survival outcome in patients with breast cancer.

Inflammatory breast cancer: a proposed conceptual shift in the UICC–AJCC TNM staging system

Abstract: Summary In the absence of histological criteria that distinguish between inflammatory and non-inflammatory breast cancer, diagnosis of inflammatory breast cancer relies entirely on the existence of clinical criteria as outlined by the TNM classification. This classification restricts patients presenting with clinical criteria characteristic of inflammatory breast cancer to subcategory T4d, which immediately relegates all patients with non-metastatic inflammatory breast cancer to stage 3, regardless of tumour size or nodal spread. Patients who present with metastatic disease are consigned to stage 4, and the TNM classification does not distinguish patients on the basis of the presence of inflammatory criteria. Evidence by our group and others suggests that patients with inflammatory breast cancer have significantly reduced overall survival among those who present with distant metastasis at diagnosis (stage 4). In light of these results, this Personal View addresses whether the current TNM staging classification accurately represents a distinction between patients with inflammatory and those with non-inflammatory breast cancer.

c-Jun N-terminal kinase promotes stem cell phenotype in triple-negative breast cancer through upregulation of Notch1 via activation of c-Jun

Abstract: c-Jun N-terminal kinase (JNK) plays a vital role in malignant transformation of different cancers, and JNK is highly activated in basal-like triple-negative breast cancer (TNBC). However, the roles of JNK in regulating cancer stem-like cell (CSC) phenotype and tumorigenesis in TNBC are not well defined. JNK is known to mediate many cellular events via activating c-Jun. Here, we found that JNK regulated c-Jun activation in TNBC cells and that JNK activation correlated with c-Jun activation in TNBC tumors. Furthermore, the expression level of c-Jun was significantly higher in TNBC tumors than in non-TNBC tumors, and high c-Jun mRNA level was associated with shorter disease-free survival of patients with TNBC. Thus, we hypothesized that the JNK/c-Jun signaling pathway contributes to TNBC tumorigenesis. We found that knockdown of JNK1 or JNK2 or treatment with JNK-IN-8, an adenosine triphosphate-competitive irreversible pan-JNK inhibitor, significantly reduced cell proliferation, the ALDH1+ and CD44+/CD24- CSC subpopulations, and mammosphere formation, indicating that JNK promotes CSC self-renewal and maintenance in TNBC. We further demonstrated that both JNK1 and JNK2 regulated Notch1 transcription via activation of c-Jun and that the JNK/c-Jun signaling pathway promoted CSC phenotype through Notch1 signaling in TNBC. In a TNBC xenograft mouse model, JNK-IN-8 significantly suppressed tumor growth in a dose-dependent manner by inhibiting acquisition of the CSC phenotype. Taken together, our data demonstrate that JNK regulates TNBC tumorigenesis by promoting CSC phenotype through Notch1 signaling via activation of c-Jun and indicate that JNK/c-Jun/Notch1 signaling is a potential therapeutic target for TNBC.

Novel therapeutic strategies in the treatment of triple-negative breast cancer.

Abstract: Triple-negative breast cancer (TNBC) is a heterogeneous subtype of breast cancer that is defined by negative estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status. Treating patients with TNBC remains clinically challenging, as patients are not candidates for endocrine or HER2-directed therapy. As a result, chemotherapy with traditional agents such as anthracyclines and taxanes remains the only available option with moderate success. Recent discoveries have revealed that TNBC is a heterogeneous disease at the clinical, histological and molecular levels. The use of biomarkers to identify distinct subsets of TNBC that derive the greatest benefit from presently approved as well as novel therapeutics has become the main focus of current research. The aim of this review is to explore the clinical and biological complexity of TNBC as well as identify novel therapeutic options that target the various molecular subsets of TNBC.

Selinexor (KPT-330) demonstrates anti-tumor efficacy in preclinical models of triple-negative breast cancer

Abstract: Selinexor (KPT-330) is an oral agent that has been shown to inhibit the nuclear exporter XPO1. Given the pressing need for novel therapies for triple-negative breast cancer (TNBC), we sought to determine the antitumor effects of selinexor in vitro and in vivo. Twenty-six breast cancer cell lines of different breast cancer subtypes were treated with selinexor in vitro. Cell proliferation assays were used to measure the half-maximal inhibitory concentration (IC50) and to test the effects in combination with chemotherapy. In vivo efficacy was tested both as a single agent and in combination therapy in TNBC patient-derived xenografts (PDXs). Selinexor demonstrated growth inhibition in all 14 TNBC cell lines tested; TNBC cell lines were more sensitive to selinexor (median IC50 44 nM, range 11 to 550 nM) than were estrogen receptor (ER)-positive breast cancer cell lines (median IC50 > 1000 nM, range 40 to >1000 nM; P = 0.017). In multiple TNBC cell lines, selinexor was synergistic with paclitaxel, carboplatin, eribulin, and doxorubicin in vitro. Selinexor as a single agent reduced tumor growth in vivo in four of five different TNBC PDX models, with a median tumor growth inhibition ratio (T/C: treatment/control) of 42% (range 31 to 73%) and demonstrated greater antitumor efficacy in combination with paclitaxel or eribulin (average T/C ratios of 27% and 12%, respectively). Collectively, these findings strongly suggest that selinexor is a promising therapeutic agent for TNBC as a single agent and in combination with standard chemotherapy.

Circulating tumor cells (CTCs) are associated with abnormalities in peripheral blood dendritic cells in patients with inflammatory breast cancer.

Abstract: CTCs are involved in tumor dissemination and are an independent prognostic factor in primary and metastatic breast cancer patients. Dendritic cells (DCs) are the most efficient antigen presenting cells and are comprised of plasmacytoid-(pDC) and myeloid-(mDC) derived DC subsets. This study aimed to correlate CTC counts with the peripheral blood DC immunophenotypes and functions of inflammatory breast cancer (IBC) patients. This study included 65 IBC patients. Peripheral blood (PB) was obtained from patients prior to starting a new line of chemotherapy for CTCs enumeration by CellSearch® and DC phenotype and function by flow cytometry; the characteristics of DCs were then correlated with CTC counts and clinical outcome. Twenty-one (32.3%) patients with CTCs ≥5 had a significantly inferior overall survival (OS) compared to patients with <5 CTCs (p=0.045). In addition, patients with ≥5 CTCs had a lower percentage of mDCs capable of producing TNF-α before or after activation through the toll-like receptor (TLR), as well as a lower percentage of mDCs producing IL-12 after TLR-activation. There was a positive correlation between CTCs counts and expression of the activation (CCR7) and costimulatory (CD86) receptors on TLR-activated mDCs and pDCs, respectively. Moreover, presence of high percentage of mDC capable to produce increased levels of TNF-α was independently associated with inferior OS (p = 0.0006). An increase in the percentage of mDC producing TNF-α might induce a pro-inflammatory environment that could play a role in determining the poor clinical outcome in IBC patients and could add further prognostic value to CTCs.

Cyclin E overexpression as a biomarker for combination treatment strategies in inflammatory breast cancer

Abstract: Inflammatory breast cancer (IBC) is a virulent form of breast cancer, and novel treatment strategies are urgently needed. Immunohistochemical analysis of tumors from women with a clinical diagnosis of IBC (n = 147) and those with non-IBC breast cancer (n = 2510) revealed that, whereas in non-IBC cases cytoplasmic cyclin E was highly correlated with poor prognosis (P < 0.001), in IBC cases both nuclear and cytoplasmic cyclin E were indicative of poor prognosis. These results underscored the utility of the cyclin E/CDK2 complex as a novel target for treatment. Because IBC cell lines were highly sensitive to the CDK2 inhibitors dinaciclib and meriolin 5, we developed a high-throughput survival assay (HTSA) to design novel sequential combination strategies based on the presence of cyclin E and CDK2. Using a 14-cell-line panel, we found that dinaciclib potentiated the activity of DNA-damaging chemotherapies treated in a sequence of dinaciclib followed by chemotherapy, whereas this was not true for paclitaxel. We also identified a signature of DNA repair–related genes that are downregulated by dinaciclib, suggesting that global DNA repair is inhibited and that prolonged DNA damage leads to apoptosis. Taken together, our findings argue that CDK2-targeted combinations may be viable strategies in IBC worthy of future clinical investigation.

EGFR signaling promotes inflammation and cancer stem-like activity in inflammatory breast cancer

Abstract: // Xiaoping Wang 1, 2, 3, * , Monica E. Reyes 1, 2, 3, * , Dongwei Zhang 1, 2, 3, * , Yohei Funakoshi 1, 2, 3 , Adriana P. Trape 1, 2, 3 , Yun Gong 1, 4 , Takahiro Kogawa 1, 2, 3 , Bedrich L. Eckhardt 1, 2, 3 , Hiroko Masuda 1, 2, 3 , David A. Pirman Jr 5 , Peiying Yang 6 , James M. Reuben 1, 7 , Wendy A. Woodward 1, 8 , Chandra Bartholomeusz 1, 2, 3 , Gabriel N. Hortobagyi 3 , Debu Tripathy 3 and Naoto T. Ueno 1, 2, 3 1 Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 2 Section of Translational Breast Cancer Research, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 3 Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 4 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 5 Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 6 Department of General Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 7 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 8 Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA * These authors contributed equally to this work Correspondence to: Naoto T. Ueno, email: nueno@mdanderson.org Xiaoping Wang, email: xiwang@mdanderson.org Keywords: inflammatory breast cancer, EGFR, COX-2, nodal, cancer stem-like cells Received: October 19, 2016 Accepted: June 17, 2017 Published: July 04, 2017 ABSTRACT Inflammatory breast cancer (IBC) is the most lethal and aggressive type of breast cancer, with a strong proclivity to metastasize, and IBC-specific targeted therapies have not yet been developed. Epidermal growth factor receptor (EGFR) has emerged as an important therapeutic target in IBC. However, the mechanism behind the therapeutic effect of EGFR targeted therapy is not well defined. Here, we report that EGFR regulates the IBC cell population that expresses cancer stem-like cell (CSC) markers through COX-2, a key mediator of inflammation whose expression correlates with worse outcome in IBC. The COX-2 pathway promoted IBC cell migration and invasion and the CSC marker-bearing population in vitro , and the inhibition of this pathway reduced IBC tumor growth in vivo . Mechanistically, we identified Nodal, a member of the TGFβ superfamily, as a potential driver of COX-2-regulated invasive capacity and the CSC phenotype of IBC cells. Our data indicate that the EGFR pathway regulates the expression of COX-2, which in turn regulates the expression of Nodal and the activation of Nodal signaling. Together, our findings demonstrate a novel connection between the EGFR/COX-2/Nodal signaling axis and CSC regulation in IBC, which has potential implications for new combination approaches with EGFR targeted therapy for patients with IBC.

Early clinical development of epidermal growth factor receptor targeted therapy in breast cancer

Abstract: Introduction: Epidermal growth factor receptor (EGFR) targeted treatment has been evaluated but has not shown a clear clinical benefit for breast cancer. This review article aims to consider the knowledge of the biological background of EGFR pathways in dissecting clinical studies of EGFR targeted treatment in breast cancer.Areas covered: This review focuses on the role of the EGFR pathway and the investigational drugs that target EGFR for breast cancer.Expert opinion: Recent studies have indicated that EGFR targeted therapy for breast cancer has some promising effects for patients with triple-negative breast cancer, basal-like breast cancer, and inflammatory breast cancer. However, predictive and prognostic biomarkers for EGFR targeted therapy have not been identified. The overexpression or amplification of EGFR itself may not be the true factor of induction of the canonical pathway as an oncogenic driver of breast cancer. Instead, downstream, non-canonical pathways related to EGFR may contribute...

Histone Deacetylase Inhibitor Enhances the Efficacy of MEK Inhibitor through NOXA-Mediated MCL1 Degradation in Triple-Negative and Inflammatory Breast Cancer.

Abstract: Purpose: Inflammatory breast cancer (IBC), diagnosed clinically, and triple-negative breast cancer (TNBC), diagnosed by molecular receptor status, are the two most aggressive forms of breast cancer, and both lack effective targeted therapies. We previously demonstrated involvement of histone deacetylase (HDAC) inhibitor entinostat in regulating apoptosis in IBC and TNBC cells; here, we aimed to identify novel combination therapy candidates.Experimental Design: Potential therapeutic targets were identified by mRNA expression profiling of TNBC and IBC cells treated with entinostat. Drug action and synergism were assessed by in vitro proliferation assays, tumor growth in vivo, and proteomic analyses. Gain/loss-of-expression studies were utilized to functionally validate the role of identified targets in sensitivity of TNBC and IBC cells to combination therapy.Results: Entinostat induced activity of the oncogenic ERK pathway and expression of proapoptotic NOXA. These are known to stabilize and degrade, respectively, MCL1, an antiapoptotic Bcl-2 protein. In breast cancer patients, high-MCL1/low-NOXA tumor expression correlated significantly with poor survival outcomes. Combination treatment of entinostat with MEK inhibitor pimasertib reduced the growth of TNBC and IBC cells in vitro and inhibited tumor growth in vivo The synergistic action of combination therapy was observed in TNBC and IBC cell lines in which NOXA expression was induced following entinostat treatment. The therapeutic activity depended on induction of mitochondrial cell death pathways initiated by NOXA-mediated MCL1 degradation.Conclusions: Our preclinical findings provide a rationale for the clinical testing of combination HDAC and MEK pathway inhibition for TNBC and IBC that exhibit elevated baseline tumor MCL1 expression. Clin Cancer Res; 23(16); 4780-92. ©2017 AACR.

Poor prognosis of patients with triple-negative breast cancer can be stratified by RANK and RANKL dual expression

Abstract: Purpose As clinical studies have correlated RANK expression levels with survival in breast cancer, and that RANK signaling is dependent on its cognate ligand RANKL, we hypothesized that dual protein expression further stratifies the poor outcome in TNBC.

Association between weight gain during adjuvant chemotherapy for early-stage breast cancer and survival outcomes.

Abstract: Obese and overweight women have an increased risk of breast cancer and worse outcomes at the time of diagnosis. Women tend to gain weight after breast cancer diagnosis and during chemotherapy for early-stage disease, which may in turn increase risk for worse outcomes. We examined if weight gained during adjuvant chemotherapy was associated with worse survival outcomes. We queried our database for data on patients who received adjuvant third-generation chemotherapy for early-stage breast cancer. Univariate and multivariate analyses by Cox regression were performed for survival outcomes across three categories according to BMI variation from start to end of chemotherapy: >0.5 kg/m2 loss or gain and stable BMI (±0.5 kg/m2 ). We included 1998 patients in this study. Women over 50 years old and postmenopausal were more likely to lose weight during adjuvant chemotherapy, whereas women under 30 years old gained more weight (P 0.5 kg/m2 compared to maintaining BMI was marginally associated with increased locoregional recurrence risk (HR: 2.53; 95% CI, 1.18-5.45; P = 0.017), adjusting for grade, stage, and radiation delivery. Weight variation during adjuvant chemotherapy for early-stage breast cancer may occur as both weight gain and weight loss in a balanced manner. Furthermore, this variation seems to be transient in nature and does not appear to significantly influence recurrence rates and overall survival.

Improved Locoregional Control in a Contemporary Cohort of Nonmetastatic Inflammatory Breast Cancer Patients Undergoing Surgery.

Abstract: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer characterized by rapid progression and early metastatic dissemination. The purpose of this study was to assess contemporary rates of local regional recurrence (LRR) in the era of trimodality therapy for nonmetastatic IBC and identify risk factors leading to local failure. A total of 114 patients with nonmetastatic IBC receiving trimodality therapy (neoadjuvant chemotherapy, surgery, and radiation therapy) were identified from a prospectively collected database from 2007 to 2015 and outcomes analyzed. Median age at diagnosis was 52 years, and the median follow-up was 3.6 years. Sixty-three (55%) patients presented with N2 IBC, and 52 patients (45%) presented with N3 IBC. Local regional recurrence was observed during follow-up for four patients; 25 died, and 85 were censored at last follow-up. Surgical margins were negative in 99% of patients (n = 113). The 2-year probability of LRR was 3.19% (95% confidence interval 1.03–9.90%). Five-year overall survival for this cohort was 69.14%. Improvement in disease-free survival was seen among patients with HER2+ subtype, clinical stage IIIB, complete or partial radiologic response to neoadjuvant therapy, pathologic complete response, and lower nodal burden on presentation. Locoregional recurrences were rare at a median of 3.6 years follow-up in a contemporary cohort of IBC patients treated with trimodality therapy. Although longer follow-up is needed, aggressive surgical resection to negative margins in the frame of trimodality therapy with curative intent can lead to LRR rates that mirror non-IBC rates.

Identification of frequent somatic mutations in inflammatory breast cancer

Abstract: Inflammatory breast cancer is an aggressive form of breast cancer that shows distinct clinical features from non-inflammatory breast cancer. Genomic understanding of inflammatory breast cancer will shed light on biological targets for this disease. Our objective was to identify targeted hotspot mutations using multiplex genome sequencing in inflammatory breast cancer and compare the findings with those for patients with non-inflammatory breast cancer to further recognize novel targets. We studied 400 patients with metastatic breast cancer who had somatic hotspot mutation testing using a 46- or 50-gene multiplex platform from March 2012 to December 2014. Among this population, 24 patients had inflammatory breast cancer and 376 patients had non-inflammatory breast cancer. We tested a total of 26 samples from 24 patients with inflammatory breast cancer. The average number of mutations per patient was higher in inflammatory breast cancer than in non-inflammatory breast cancer (1.23 vs. 0.65, respectively). Identified somatic mutations in inflammatory breast cancer were TP53 (n = 18, 75%), PIK3CA (n = 10, 41.7%), and ERBB2 (n = 4, 16.7%). TP53 and ERBB2 mutations were significantly more prevalent in inflammatory breast cancer than in non-inflammatory breast cancer (P < 0.01). All patients with ERBB2 mutations had hormone receptor (HR)+ primary tumors. TP53, PIK3CA, and ERBB2 were detected as three major somatic mutations in metastatic inflammatory breast cancer patients. While the inflammatory breast cancer TP53 and PIK3CA mutations mirrored previously reported data for metastatic non-inflammatory breast cancer, this is the first report of higher frequency of ERBB2 mutation in inflammatory breast cancer, especially in the HR+ subtype. Once validated in a larger cohort of inflammatory breast cancer patients, this novel finding could lead to development of treatments for HR+ inflammatory breast cancer.

Outcomes in patients with early-stage breast cancer who underwent a 21-gene expression assay.

Abstract: BACKGROUND Invasive disease-free survival (IDFS) rates are excellent in patients with breast cancer (BC) with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-), axillary lymph node-negative (LN-) tumors with a 21-gene expression assay recurrence score (RS) of 0 to 10. However, to the authors' knowledge, the outcomes among patients with an RS of 11 to 25 who are treated with endocrine therapy alone are unknown. METHODS In this retrospective single-institution study, the authors described the characteristics of patients with HR+, HER2-, LN- BC who underwent a 21-gene expression assay. In addition, among those individuals diagnosed between 2005 and 2011, we measured IDFS, recurrence-free survival, distant recurrence-free survival, and overall survival rates, focusing on patients with an RS of 11 to 25 by receipt of chemotherapy. The Kaplan-Meier method was used to estimate survival rates and multivariable Cox proportional hazards models were used to calculate hazard ratios and 95% confidence intervals (95% CIs). RESULTS Among 1424 patients, the RS distribution was 0 to 10 in 297 patients (21%), 11 to 25 in 894 patients (63%), and >25 in 233 patients (16%); of these, 1.7%, 15%, and 73.4% of patients, respectively, received chemotherapy. With a median follow-up of 58 months, those patients with an RS of 11 to 25 had an IDFS rate at 5 years of 92.6% (95% CI, 89.6%-94.7%), which was comparable between those who received chemotherapy and those who did not. The hazard ratios of the effect of chemotherapy were 1.64 for IDFS (95% CI, 0.73-3.71), 1.46 for recurrence-free survival (95% CI, 0.41-5.23), 1.25 for distant recurrence-free survival (95% CI, 0.32-4.92), and 2.19 for overall survival (95% CI, 0.44-11.0). CONCLUSIONS The results of the current study demonstrate similar outcomes with or without chemotherapy in patients with HR+, HER2-, LN- BC who have an RS of 11 to 25, but a benefit from chemotherapy in this group cannot be ruled out. Cancer 2017;123:2422–31. © 2017 American Cancer Society.

Androgen receptor expression on circulating tumor cells in metastatic breast cancer

Abstract: Purpose Androgen receptor (AR) is frequently detected in breast cancers, and AR-targeted therapies are showing activity in AR-positive (AR+) breast cancer However, the role of AR in breast cancers is still not fully elucidated and the biology of AR in breast cancer remains incompletely understood Circulating tumor cells (CTCs) can serve as prognostic and diagnostic tools, prompting us to measure AR protein expression and conduct genomic analyses on CTCs in patients with metastatic breast cancer Methods Blood samples from patients with metastatic breast cancer were deposited on glass slides, subjected to nuclear staining with DAPI, and reacted with fluorescent-labeled antibodies to detect CD45, cytokeratin (CK), and biomarkers of interest (AR, estrogen receptor [ER], and HER2) on all nucleated cells The stained slides were scanned and enumerated by non-enrichment-based non-biased approach independent of cell surface epithelial cell adhesion molecule (EpCAM) using the Epic Sciences CTC platform Data were analyzed using established digital pathology algorithms Results Of 68 patients, 51 (75%) had at least 1 CTC, and 49 of these 51 (96%) had hormone-receptor-positive (HR+)/HER2-negative primary tumors AR was expressed in CK+ CTCs in 10 patients Of these 10 patients, 3 also had ER expression in CK+ CTCs Single cell genomic analysis of 78 CTCs from 1 of these 3 patients identified three distinct copy number patterns AR+ cells had a lower frequency of chromosomal changes than ER+ and HER2+ cells Conclusions CTC enumeration and analysis using no enrichment or selection provides a non-biased approach to detect AR expression and chromosomal aberrations in CTCs in patients with metastatic breast cancer The heterogeneity of intrapatient AR expression in CTCs leads to the new hypothesis that patients with AR+ CTCs have heterogeneous disease with multiple drivers Further studies are warranted to investigate the clinical applicability of AR+ CTCs and their heterogeneity

Impact of statin use on outcomes in triple negative breast cancer

Abstract: Purpose: We sought to investigate if the use of HMG Co-A reductase inhibitors (statins) has an impact on outcomes among patients with triple negative breast cancer (TNBC). Methods: We reviewed the cases of women with invasive, non-metastatic TNBC, diagnosed 1997-2012. Clinical outcomes were compared based on statin use (defined as ever use during treatment vs. never use). We identified a subset of women for whom a 5-value lipid panel (5VLP) was available, including total cholesterol, low density lipoprotein, high density lipoprotein, very low density lipoprotein, and triglycerides. The Kaplan-Meier method was used to estimate median overall survival (OS), distant metastases-free survival (DMFS), and local-regional recurrence-free survival (LRRFS). A Cox proportional hazards regression model was used to test the statistical significance of prognostic factors. Results: 869 women were identified who met inclusion criteria, with a median follow-up time of 75.1 months (range 2.4-228.9 months). 293 (33.7%) patients used statins and 368 (42.3%) had a 5VLP. OS, DMFS, and LRRFS were not significant based on statin use or type. Controlling for the 5VLP values, on multivariable analysis, statin use was significantly associated with OS (HR 0.10, 95% CI 0.01-0.76), but not with DMFS (HR 0.14, 95% CI 0.01-1.40) nor LRRFS (HR 0.10 95% CI 0.00-3.51). Conclusions: Statin use among patients with TNBC is not associated with improved OS, although it may have a benefit for a subset of patients. Prospective assessment would be valuable to better assess the potential complex correlation between clinical outcome, lipid levels, and statin use.

Nomogram to predict pathologic complete response in HER2-positive breast cancer treated with neoadjuvant systemic therapy.

Abstract: Pathologic complete response (pCR) is associated with improved survival outcomes in patients with HER2-positive primary breast cancer. We developed a nomogram to predict the probability of pCR rates by using oestrogen receptor (ER) expression, progesterone receptor (PR) expression and HER2/CEP17 ratio as continuous variables. We retrospectively reviewed patients with stages I–III HER2-positive invasive breast cancer who had definitive surgery in 1999–2015 and received neoadjuvant systemic therapy (NST). Multivariate logistic regression models were applied to assess the effect of variables on pCR. A nomogram was built to estimate the probability of pCR. The discriminative ability was estimated by the concordance index (C-index). The accuracy was assessed graphically with a calibration curve. A total of 793 patients were included in the analysis. Low ER expression (P<0.001), high HER2/CEP12 ratio (P=0.03), and non-inflammatory breast cancer subtype (P=0.003) were associated with increased pCR rates. Regimens containing trastuzumab or trastuzumab and pertuzumab were associated with higher pCR rates than cytotoxic agents alone (P<0.001 and P<0.001, respectively). The C-index was 0.69. The calibration curve showed good agreement. Our nomogram predicted the pCR rate after NST among patients with HER2-positive primary breast cancer using clinicopathologic factors.

Thrombocytosis as a prognostic factor in inflammatory breast cancer.

Abstract: Platelets are essential components of hemostasis and also play an important role in the tumor microenvironment. The purposes of our research were to examine the role of thrombocytosis in inflammatory breast cancer (IBC) and to know which cytokine drives thrombocytosis. We reviewed the medical records of 3654 patients with stage I–III breast cancer treated between 1998 and 2013, including 230 patients (6%) with IBC. We used Chi-squared test or Fisher’s exact test to compare the variables between patients with and without thrombocytosis. Multivariate Cox regression models were used to determine the association of thrombocytosis with overall survival. We also examined baseline serum cytokine levels in 81 patients with primary IBC to determine the association of inflammatory cytokines with thrombocytosis. We found that thrombocytosis was the only variable that predicted prognosis. Fifty-five patients (1.5%) had thrombocytosis. Thrombocytosis was more prevalent in patients with IBC than in those with non-IBC (3.4% vs. 1.4%, p = 0.015). In patients with IBC, thrombocytosis was associated with worse overall survival [hazard ratio 2.38, 95% confidence interval (CI) 1.05–5.4, p = 0.0378]. Circulating levels of growth-regulated oncogene (GRO) (odds ratio 1.003, 95% CI 1.001–1.005, p = 0.0019) and transforming growth factor β (TGF-β) (odds ratio 1.3, 95% CI 1.128–1.499, p = 0.0003) were associated with thrombocytosis. Thrombocytosis was more prevalent in patients with IBC than in those with non-IBC and it was associated with poor prognosis. GRO and TGF-β were associated with thrombocytosis in IBC.

Rapid breast cancer disease progression following Cyclin Dependent Kinase 4 and 6 inhibitor discontinuation

Abstract: Background: CDK 4 and 6 inhibitors (CDK4/6i), which arrest unregulated cancer cell proliferation, show clinical efficacy in breast cancer. Unexpectedly, a patient treated on a CDK4/6i-based trial, as first-line therapy in metastatic breast cancer, developed rapid disease progression following discontinuation of study drug while receiving standard second-line therapy off trial. We thus sought to expand this observation within a population of patients treated similarly at The University of Texas MD Anderson Cancer Center. Methods: Using an IRB-approved protocol, 4 patients previously enrolled on CDK4/6i trials were analyzed for outcomes after discontinuing study drug. These patients were treated on a randomized trial of first-line endocrine therapy +/- a CDK4/6i. Rapid disease progression was defined as progression occurring within 4 months of CDK4/6i discontinuation. Results: In total, 4 patients developed rapid disease progression and died; 2 of whom died within 6 months of CDK4/6i discontinuation. Conclusion: This case series suggests a potential for rapid disease progression following CDK4/6i discontinuation. However, the clinical course following progression must be validated in large CDK4/6i clinical trials and standard-of-care cohorts. If confirmed, such observations may alter the algorithm for subsequent therapy in patients with disease progression on CDK4/6i. Nevertheless, the need remains to define a mechanistic basis for this rapid progression and formulate alternative therapeutic strategies.

Reverse phase protein array identification of triple-negative breast cancer subtypes and comparison with mRNA molecular subtypes

Abstract: // Hiroko Masuda 1, 2, 4 , Yuan Qi 3 , Shuying Liu 1 , Naoki Hayashi 1 , Takahiro Kogawa 1, 2 , Gabriel N. Hortobagyi 1 , Debu Tripathy 1 and Naoto T. Ueno 1, 2 1 Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 4 Department of Breast Surgical Oncology, Showa University Hospital, Tokyo, Japan Correspondence to: Naoto T. Ueno, email: nueno@mdanderson.org Keywords: triple-negative breast cancer, molecular subtype, functional, proteomics, mRNA microarray Received: October 19, 2016 Accepted: June 27, 2017 Published: July 31, 2017 ABSTRACT Background: Reverse phase protein array (RPPA) analysis, allows investigation of potential targets at the functional protein level,. We identified TNBC subtypes at the protein level using RPPA and compared them with mRNA molecular subtypes (TNBCtype, TNBCtype-4, and PAM50) that is unique in its availability of both RPPA and mRNA analyses. Methods: We classified the samples from 80 TNBC patients using both k-means and hierarchical consensus clustering analysis and performed Ingenuity Pathway Analysis. We also investigated whether we could reproduce the mRNA molecular subtypes using the RPPA dataset. Results: Both clustering methods divided all samples into 2 clusters that were biologically the same. The top canonical pathways included inflammation, hormonal receptors, and MAPK signaling pathways for the first cluster [“inflammation and hormonal-related (I/H) subtype”] and the GADD45, DNA damage, and p53 signaling pathways for the second cluster [“DNA damage (DD)-related subtype”]. Further k-means cluster analysis identified 5 TNBC clusters. Comparison between sample classification using the 5 RPPA-based k-means cluster subtypes and 6 gene-expression-based TNBCtype molecular subtypes showed significant association between the 2 classifications (p = 0.017). Conclusions: The I/H and DD subtypes identified by RPPA advance our understanding of TNBC’s heterogeneity from the functional proteomic perspective.

Scientific Summary from the Morgan Welch MD Anderson Cancer Center Inflammatory Breast Cancer (IBC) Program 10th Anniversary Conference.

Abstract: In 2006, a remarkable collaboration between University of Texas MD Anderson Cancer Center clinicians and Texas and New Mexico State legislators led to the formation of a dedicated IBC Research Program and Clinic at MD Anderson. This initiative provided funding and infrastructure to foster coordination of an IBC World Consortium of national and international experts, and launch the first ever IBC international conference in 2008, which brought together experts from around the world to facilitate collaborations and accelerate progress. Indeed great progress has been made since then. National and international experts in IBC convened at the 10th Anniversary Conference of the MD Anderson IBC Clinic and Research Program and presented the most extensive sequencing analysis to date comparing IBC to non-IBC, gene- and protein-based immunoprofiling of IBC versus non-IBC patients, and converging lines of evidence on the specific role of the microenvironment in IBC. Novel models, unique metabolic mechanisms, and prominent survival pathways have been identified and were presented. Multiple clinical trials based on the work of the last decade are in progress or in development. The important challenges ahead were discussed. This progress and a coordinated summary of these works are presented herein.

Using the National Cancer Data Base for quality evaluation to assess adherence to treatment guidelines for nonmetastatic inflammatory breast cancer.

Abstract: BACKGROUND Guidelines for the treatment of nonmetastatic inflammatory breast cancer (IBC) using trimodality therapy (TT) (chemotherapy, surgery, and radiotherapy) have remained largely unchanged since 2000. However, many patients with nonmetastatic IBC do not receive TT. It is unknown how patient-level (PL) and facility-level (FL) factors contribute to TT use. METHODS Using the National Cancer Data Base, patients with nonmetastatic IBC who underwent locoregional treatment from 2003 through 2011 were identified. The authors correlated PL factors, including demographic and tumor characteristics, with TT use. An observed-to-expected ratio for the number of patients treated with TT was calculated for each hospital by adjusting for significant PL factors. Hierarchical mixed effects models were used to assess the percentage of variation in TT use attributable to PL and FL factors, respectively. RESULTS Of the 542 hospitals examined, 55 (10.1%) and 24 (4.4%), respectively, were identified as significantly low and high outliers for TT use (P<.05). The percentage of the total variance in the use of TT attributable to the facility (11%) was nearly triple the variance attributable to the measured PL factors (3.4%). The nomogram generated from multivariate logistic regression of PL factors only allows a facility to assess TT use given their PL data. CONCLUSIONS FL factors rather than PL factors appear to contribute disproportionately to the underuse of TT in patients with nonmetastatic IBC. To improve treatment guideline adherence for patients with nonmetastatic IBC, it is critical to identify the specific FL factors associated with TT underuse. More organized FL intervention is required to train physicians and to build multidisciplinary teams. Cancer 2017. © 2017 American Cancer Society.

Bone metastasis-related signaling pathways in breast cancers stratified by estrogen receptor status.

Abstract: Background: Breast cancer bone metastasis (BCBM)-specific genes have been reported without considering biological differences based on estrogen receptor (ER) status. The aims of this study were to identify BCBM-specific genes using our patient dataset and validate previously reported BCBM-specific genes, and to determine whether ER-status-related biological differences matter in identification of BCBM-specific genes. Methods: We used Affymetrix GeneChips to analyze 365 primary human epidermal growth factor receptor 2 (HER2)-negative invasive breast cancer specimens. Genes that were differentially expressed between patients who developed bone metastasis and those who developed non-bone metastasis were identified using Cox proportional hazards model, and differential expression of gene sets was assessed using gene set analysis. We performed gene set analysis to determine whether biological function associated with bone metastasis were different by ER status using 2,246 functionally annotated gene sets assembled from Gene Ontology data base. Results: Among 16,712 probe sets, 592 were overexpressed in the bone metastasis cohort compared to the non-bone-metastasis cohort (false discovery rate ≤ 0.05). However, no BCBM-specific genes met our significance tests when the cancers were stratified by ER status. In ER-positive and ER-negative breast cancers, 151 and 125 gene sets, respectively, were overexpressed for BCBM and the majority of BCBM-related pathways were different. Of significant gene sets, only 13 gene sets were overlapped between ER-positive and -negative cohorts. Conclusion: ER-positive and ER-negative breast cancers have different biological pathways in BCBM development. We have yet to explore BCBM-related biomarkers and targets considering the biological features associated with BCBM depending on the ER status.