Genomic alterations associated with resistance and circulating tumor DNA dynamics for early detection of progression on CDK4/6 inhibitor in advanced breast cancer.

Combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy significantly improves outcome for patients with estrogen receptor-positive (ER+) metastatic breast cancer, but drug resistance and thus disease progression inevitably occur. Herein, we aimed to identify genomic alterations associated with combined CDK4/6i and endocrine therapy resistance, and follow the levels of specific mutations in longitudinal circulating tumor DNA (ctDNA) for early detection of progression. From a cohort of 86 patients with ER+ metastatic breast cancer we performed whole exome sequencing or targeted sequencing of paired tumor (N = 8) or blood samples (N = 5) obtained before initiation of combined CDK4/6i and endocrine therapy and at disease progression. Mutations in oncogenic genes at progression were rare, while amplifications of growth-regulating genes were more frequent. The most frequently acquired alterations observed were PIK3CA and TP53 mutations and PDK1 amplification. Longitudinal ctDNA dynamics of mutant PIK3CA or private mutations revealed increased mutation levels at progression in 8 of 10 patients (80%). Impressively, rising levels of PIK3CA-mutated ctDNA were detected 4-17 months before imaging. Our data add to the growing evidence supporting longitudinal ctDNA analysis for real-time monitoring of CDK4/6i response and early detection of progression in advanced breast cancer. Further, our analysis suggests that amplification of growth-related genes may contribute to combined CDK4/6i and endocrine therapy resistance.

ESF1 and MIPEP proteins promote estrogen receptor-positive breast cancer proliferation and are associated with patient prognosis.

BACKGROUND: Estrogen receptor-positive (ER+) breast cancer accounts for two-thirds of all breast cancers, and its early and late recurrences still threaten patients' long-term survival and quality of life. Finding candidate tumor antigens and potential therapeutic targets is critical to addressing these unmet needs. METHOD: The isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis was employed to identify the differentially expressed proteins (DEPs) between ER + breast cancer and corresponding adjacent normal tissue. Candidate DEPs were screened by bioinformatic analyses, and their expression was confirmed by immunohistochemical (IHC) staining and western blot. A series of in vitro experiments, including wound healing assay, colony formation, and cell cycle assay, were performed to reveal the functions of selected DEPs. Additionally, their clinical significances were further analyzed. RESULT: A total of 369 DEPs (fold change ≥ 2.0 or ≤ 0.66, P < 0.05) were discovered. Compared with normal tissue, 358 proteins were up-regulated and 11 proteins were down-regulated in ER + breast cancer. GO and KEGG enrichment analysis showed that DEPs were closely associated with RNA regulation and metabolic pathways. STRING analysis found ESF1 and MIPEP were the hub genes in breast cancer, whose increased expressions were verified by the IHC staining and western blot. Knocking down ESF1 and MIPEP inhibited colony formation and increased cell apoptosis. Besides, knocking down ESF1 inhibited wound healing but not MIPEP. In addition, ESF1 and MIPEP expression were negatively associated with patient prognosis. CONCLUSION: The upregulation of ESF1 and MIPEP promoted ER + breast cancer proliferation, which might provide novel targets for the development of new therapies.

Modeling of Mouse Experiments Suggests that Optimal Anti-Hormonal Treatment for Breast Cancer is Diet-Dependent.

Estrogen receptor positive breast cancer is frequently treated with anti-hormonal treatment such as aromatase inhibitors (AI). Interestingly, a high body mass index has been shown to have a negative impact on AI efficacy, most likely due to disturbances in steroid metabolism and adipokine production. Here, we propose a mathematical model based on a system of ordinary differential equations to investigate the effect of high-fat diet on tumor growth. We inform the model with data from mouse experiments, where the animals are fed with high-fat or control (normal) diet. By incorporating AI treatment with drug resistance into the model and by solving optimal control problems we found differential responses for control and high-fat diet. To the best of our knowledge, this is the first attempt to model optimal anti-hormonal treatment for breast cancer in the presence of drug resistance. Our results underline the importance of considering high-fat diet and obesity as factors influencing clinical outcomes during anti-hormonal therapies in breast cancer patients.

Structure-Guided Identification of Novel Aromatase Inhibitors Targeting Breast Carcinoma.

Aromatase inhibitors play a critical therapeutic role in treating ER+ breast cancer, especially in postmenopausal women. However, their efficacy is often limited by resistance and severe side effects. Identifying new compounds that can disrupt aromatase enzyme function is essential. In this study, structural anomalies in the aromatase enzyme were corrected through energy minimization, and the structure was validated via Ramachandran plot. We screened 170,269 natural compounds from the ASINEX Biodesign library using high-throughput screening algorithms to target the aromatase enzyme. Molecular docking identified three compounds: BDD30170158, BDE33872639, and BDE30177677, all showing stable binding interactions with the aromatase enzyme. Molecular dynamics simulations over 100 ns confirmed the conformational stability of these compounds. Although all three compounds exhibited the desired pharmacokinetic and drug metabolism properties, only one compound (BDE33872639) was identified as a non-blocker, demonstrating a reduced risk of adverse cardiac effects. This compound exhibits significant potential as a novel aromatase inhibitor, warranting further experimental research to develop it as a therapeutic option for ER+ breast cancer.

Drugging the PI3K/AKT/mTOR Pathway in ER+ Breast Cancer.

The frequent activation of the PI3K/AKT/mTOR pathway and its crucial role in estrogen receptor-positive (ER+) breast cancer tumorigenesis and drug resistance has made it a highly attractive therapeutic target in this breast cancer subtype. Consequently, the number of new inhibitors in clinical development targeting this pathway has drastically increased. Among these, the PIK3CA isoform-specific inhibitor alpelisib and the pan-AKT inhibitor capivasertib were recently approved in combination with the estrogen receptor degrader fulvestrant for the treatment of ER+ advanced breast cancer after progression on an aromatase inhibitor. Nevertheless, the clinical development of multiple inhibitors of the PI3K/AKT/mTOR pathway, in parallel with the incorporation of CDK4/6 inhibitors into the standard of care treatment in ER+ advanced breast cancer, has led to a multitude of available therapeutic agents and many possible combined strategies which complicate personalizing treatment. Here, we review the role of the PI3K/AKT/mTOR pathway in ER+ advanced breast cancer, highlighting the genomic contexts in which the various inhibitors of this pathway may have superior activity. We also discuss selected trials with agents targeting the PI3K/AKT/mTOR and related pathways as well as the rationale supporting the clinical development of triple combination therapy targeting ER, CDK4/6 and PI3K/AKT/mTOR in ER+ advanced breast cancer.

Racial disparities in neutrophil counts among patients with metastatic breast cancer during treatment with CDK4/6 inhibitors.

PURPOSE: The three CDK4/6 inhibitors (CDK4/6i) approved for use in HR-positive/HER2-negative metastatic breast cancer (MBC), palbociclib, ribociclib, and abemaciclib, are generally well tolerated; however, neutropenia is a common toxicity. Within the general population, neutropenia has been shown to be more common in individuals of African descent. The landmark CDK4/6i trials in MBC lacked racial diversity in their patient populations. We aimed to assess the toxicity profiles of CDK4/6is in a racially diverse population. METHODS: We conducted a retrospective study at Montefiore Medical Center in patients with HR-positive/HER2-negative MBC prescribed CDK4/6i as first or subsequent line therapy between January 2015 and April 2020. Baseline characteristics and laboratory data at various treatment timepoints were collected. RESULTS: The final analysis included 182 patients, of whom 46% were Black. Baseline absolute neutrophil count (ANC) was lower in the Black vs. Non-Black cohort (p = 0.001) but the change in ANC from baseline (delta-ANC) was smaller in the Black cohort, and the ANC at different treatment timepoints was similar between groups. There was no difference in the rate of infection or number of dose delays/reductions between racial groups. We did not find any difference in PFS between Black and Non-Black groups, regardless of the presence of CDK4/6i-induced neutropenia. CONCLUSION: We analyzed toxicity profiles of 182 patients with HR-positive/HER2-negative MBC treated with CDK4/6i. Despite the lower baseline ANC seen in our Black cohort, treatment toxicities were similar between racial groups. Long-term outcomes with CDK4/6i therapy, measured by PFS, were similar between Black vs. Non-Black patients.

Carbidopa suppresses estrogen receptor-positive breast cancer via AhR-mediated proteasomal degradation of ERα.

Estrogen receptor-α (ERα) promotes breast cancer, and ER-positive cancer accounts for ~ 80% of breast cancers. This subtype responds positively to hormone/endocrine therapies involving either inhibition of estrogen synthesis or blockade of estrogen action. Carbidopa, a drug used to potentiate the therapeutic efficacy of L-DOPA in Parkinson's disease, is an agonist for aryl hydrocarbon receptor (AhR). Pharmacotherapy in Parkinson's disease decreases the risk for cancers, including breast cancer. The effects of carbidopa on ER-positive breast cancer were evaluated in cell culture and in mouse xenografts. The assays included cell proliferation, apoptosis, cell migration/invasion, subcellular localization of AhR, proteasomal degradation, and tumor growth in xenografts. Carbidopa decreased proliferation and migration of ER-positive human breast cancer cells in vitro with no significant effect on ER-negative breast cancer cells. Treatment of ER-positive cells with carbidopa promoted nuclear localization of AhR and expression of AhR target genes; it also decreased cellular levels of ERα via proteasomal degradation in an AhR-dependent manner. In vivo, carbidopa suppressed the growth of ER-positive breast cancer cells in mouse xenografts; this was associated with increased apoptosis and decreased cell proliferation. Carbidopa has therapeutic potential for ER-positive breast cancer either as a single agent or in combination with other standard chemotherapies.

Estrogen Receptor Gene Polymorphism as a Possible Genetic Risk Factor for Treatment Response in ER-Positive Breast Cancer Patients.

Estrogen receptor-α (ESR1) single nucleotide polymorphisms (SNPs) have been related to breast cancer (BC) susceptibility. In this retrospective study we investigated ESR1 SNPs in association with survival and treatment response in BC patients. Seven ESR1 SNPs were genotyped using TaqMan probe assay in 100 formalin-fixed paraffin embedded blocks of Egyptian ER+BC patients. Log-binomial regression was used to assess the association of 5 ESR1 SNPs with relative risk of non-response to adjuvant-hormonal treatment. We compared the performance of five machine learning classification models for prediction of treatment response. Predictive models were developed using rs1801132, rs2228480, and rs9322354 that were significantly associated with increased risk for non-response along with the relevant clinical features. Survival analysis was performed to detect prognostic significance of ESR1 SNPs in ESR+BC patients. rs1801132 (C), rs2228480 (A), and rs9322354 (G) minor alleles significantly increased the risk of non-response to tamoxifen by more than 81, 84, and 117%, respectively, in ER+BC patients on anthracycline/anthracycline-taxanes-based chemotherapy. Multivariate Cox regression survival analysis revealed that rs1801132 (C) and large tumor size were independent predictors for poor survival outcome in ER+BC. The best response predictive model was a combination random forest, K-nearest neighbor, and decision tree having an area under the curve of 0.94 and an accuracy of 90.8%. Our proposed predictive model based on ESR1 rs1801132, rs2228480, and rs9322354 SNPs represents a promising genetic risk stratification for selection patients who could benefit from tamoxifen therapy in such a way that might facilitate personalized medicine required to improve ER+BC patients' outcome.

Gene expression comparison between primary estrogen receptor-positive and triple-negative breast cancer with paired axillary lymph node metastasis.

The aim of this study is to characterize and compare changes in gene expression patterns of paired axillary lymph node (ALN) metastases from estrogen receptor (ER)-positive and triple-negative (TNBC) primary breast cancer (PBC). Patients with stage 2-3 PBC with macrometastasis to an ALN were selected. Gene expression of 2567 cancer-associated genes was analyzed with the HTG EdgeSeq system coupled with the Illumina Next Generation Sequencing (NGS) platform. Changes in gene expression between ER/PR-positive, HER2-negative PBC, and their paired ALN metastases were compared with TNBC and their paired ALN metastases. Fourteen pairs of ER-positive and paired ALN metastasis were analyzed. Compared with the PBC, ALN metastasis had 673 significant differentially expressed genes, including 348 upregulated genes and 325 downregulated genes. Seventeen pairs of TNBC and paired ALN metastasis were analyzed. ALN metastasis had 257 significant differentially expressed genes, including 123 upregulated genes and 134 downregulated genes. When gene expression of the ALN for ER-positive PBC was compared to that of TNBC, 97 genes were upregulated in both, and 115 genes were similarly downregulated. Common upregulated genes were associated with cell death, necrosis, and homeostasis. Common downregulated genes were those of migration, degradation of extracellular matrix, and invasion. Although ER-positive PBC and TNBC have a distinct gene expression profiles and distinct changes from PBC to ALN metastases, a significant number of genes are similarly up- or downregulated. Understanding the role of these common genomic changes may provide clues to understanding the metastatic process itself.

Quercetin exerts bidirectional regulation effects on the efficacy of tamoxifen in estrogen receptor-positive breast cancer therapy: An in vitro study.

Tamoxifen was widely applied in the therapy of estrogen receptor (ER)-positive breast cancer. With the purpose of determining the potential impacts of quercetin on its effectiveness, MCF-7 cells were selected as the in vitro model and several cellular biological behaviors (ie, cell proliferation, migration, invasion, cycle, apoptosis, and oxidative stress) were investigated. As results, quercetin showed contrasting dose-response to cellular behaviors dependent on the ROS-regulated p53 signaling pathways. In detail, quercetin promoted cell proliferation and inhibited cell apoptosis at low concentrations, whereas high-concentration resulted in apoptosis induction. Moreover, quercetin at a low concentration significantly inhibited tamoxifen-induced antiproliferation in MCF-7 cells, whereas high concentrations enhanced cell apoptosis in a synergetic manner. The real-time quantitative polymerase chain reaction analysis further implied that quercetin exerted its dual roles in tamoxifen-induced antiproliferative effects by regulated the gene expression involved in cell metastasis, cycle, and apoptosis through the ER pathways. Our present study provides a considerable support to the combination of quercetin and tamoxifen on human ER-positive breast carcinoma management.

Molecular mechanisms of resistance to CDK4/6 inhibitors in breast cancer: A review.

Deregulation of the cyclin D-CDK4/6-INK4-RB pathway leading to uncontrolled cell proliferation, is frequently observed in breast cancer. Currently, three selective CDK4/6 inhibitors have been FDA approved: palbociclib, ribociclib and abemaciclib. Despite promising clinical outcomes, intrinsic or acquired resistance to CDK4/6 inhibitors has limited the success of these treatments; therefore, the development of various strategies to overcome this resistance is of great importance. We highlight the various mechanisms that are directly or indirectly responsible for resistance to CDK4/6 inhibitors, categorizing them into two broad groups; cell cycle-specific mechanisms and cell cycle-nonspecific mechanisms. Elucidation of the diverse mechanisms through which resistance to CDK4/6 inhibitors occurs, may aid in the design of novel therapeutic strategies to improve patient outcomes. This review summarizes the currently available knowledge regarding mechanisms of resistance to CDK4/6 inhibitors, and possible therapeutic strategies that may overcome this resistance as well.

Integrative molecular profiling identifies a novel cluster of estrogen receptor-positive breast cancer in very young women.

Very young breast cancer patients are more common in Asian countries than Western countries and are thought to have worse prognosis than older patients. The aim of the current study was to identify molecular characteristics of young patients with estrogen receptor (ER)-positive breast cancer by analyzing mutations and copy number variants (CNV), and by applying expression profiling. The whole exome and transcriptome of 47 Korean young breast cancer (KYBR) patients (age <35) were analyzed. Genomic profiles were constructed using mutations, CNV and differential gene expression from sequencing data. Pathway analyses were also performed using gene sets to identify biological processes. Our data were compared with young ER+ breast cancer patients in The Cancer Genome Atlas (TCGA) dataset. TP53, PIK3CA and GATA3 were highly recurrent somatic mutation genes. APOBEC-associated mutation signature was more frequent in KYBR compared with young TCGA patients. Integrative profiling was used to classify our patients into 3 subgroups based on molecular characteristics. Group A showed luminal A-like subtype and IGF1R signal dysregulation. Luminal B patients were classified into groups B and C, which showed chromosomal instability and enrichment for APOBEC3A/B deletions, respectively. Group B was characterized by 11q13 (CCND1) amplification and activation of the ubiquitin-mediated proteolysis pathway. Group C showed 17q12 (ERBB2) amplification and lower ER and progesterone receptor expression. Group C was also distinguished by immune activation and lower epithelial-mesenchyme transition (EMT) degree compared with group B. This study showed that integrative genomic profiling could classify very young patients with breast cancer into molecular subgroups that are potentially linked to different clinical characteristics.

Interaction between dietary acrylamide intake and genetic variants for estrogen receptor-positive breast cancer risk.

PURPOSE: The association between dietary acrylamide intake and estrogen receptor-positive (ER+) breast cancer risk in epidemiological studies is inconsistent. By analyzing gene-acrylamide interactions for ER+ breast cancer risk, we aimed to clarify the role of acrylamide intake in ER+ breast cancer etiology. METHODS: The prospective Netherlands Cohort Study on diet and cancer includes 62,573 women, aged 55-69 years. At baseline, a random subcohort of 2589 women was sampled from the total cohort for a case-cohort analysis approach. Dietary acrylamide intake of subcohort members (n = 1449) and ER+ breast cancer cases (n = 844) was assessed with a food frequency questionnaire. We genotyped single nucleotide polymorphisms (SNPs) in genes in acrylamide metabolism, sex steroid systems, oxidative stress and DNA repair. Multiplicative interaction between acrylamide intake and SNPs was assessed with Cox proportional hazards analysis, based on 20.3 years of follow-up. RESULTS: Unexpectedly, there was a statistically non-significant inverse association between acrylamide and ER+ breast cancer risk among all women but with no clear dose-response relationship, and no association among never smokers. Among the results for 57 SNPs and 2 gene deletions, rs1056827 in CYP1B1, rs2959008 and rs7173655 in CYP11A1, the GSTT1 gene deletion, and rs1052133 in hOGG1 showed a statistically significant interaction with acrylamide intake for ER+ breast cancer risk. CONCLUSIONS: This study did not provide evidence for a positive association between acrylamide intake and ER+ breast cancer risk. If anything, acrylamide was associated with a decreased ER+ breast cancer risk. The interaction with SNPs in CYP1B1 and CYP11A1 suggests that acrylamide may influence ER+ breast cancer risk through sex hormone pathways.

Comparing protein and mRNA expressions of the human epidermal growth factor receptor family in estrogen receptor-positive breast cancer.

The human epidermal growth factor receptor (HER) family plays a vital role in the development of resistance to treatments in estrogen receptor (ER)-positive breast cancer. This study investigated the correlation between protein and mRNA expressions of the HER family in ER-positive breast cancer. We dissected regions of invasive cancer from the frozen tissues of 34 patients with ER-positive breast cancer using laser-capture microdissection, followed by evaluation of the mRNA levels of the ER and HER family (EGFR, HER2, HER3, and HER4) using the quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. In addition, we assessed the protein expressions of the ER and HER family using an immunohistochemical (IHC) assay. A significant correlation was observed between the ER protein and mRNA expressions. For HER2, HER3, and HER4, protein expressions significantly correlated with mRNA levels. We established significant correlations of the mRNA level between EGFR versus HER2, as well as EGFR versus HER3. Furthermore, a significant correlation of the mRNA level between HER2 and HER3 was illustrated. In conclusion, IHC evaluation may be reliable and representable for mRNA. Hence, this study established a marked correlation between the mRNA expressions of HER family members in patients with ER-positive breast cancer.

HER4 expression in estrogen receptor-positive breast cancer is associated with decreased sensitivity to tamoxifen treatment and reduced overall survival of postmenopausal women.

BACKGROUND: The sensitivity of estrogen receptor-positive breast cancers to tamoxifen treatment varies considerably, and the molecular mechanisms affecting the response rates are manifold. The human epidermal growth factor receptor-related receptor HER2 is known to trigger intracellular signaling cascades that modulate the activity of coregulators of the estrogen receptor which, in turn, reduces the cell sensitivity to tamoxifen treatment. However, the impact of HER2-related receptor tyrosine kinases HER1, HER3, and, in particular, HER4 on endocrine treatment is largely unknown. METHODS: Here, we retrospectively evaluated the importance of HER4 expression on the outcome of tamoxifen- and aromatase inhibitor-treated estrogen receptor-positive breast cancer patients (n = 258). In addition, we experimentally analyzed the efficiency of tamoxifen treatment as a function of HER4 co-expression in vitro. RESULTS: We found a significantly improved survival in tamoxifen-treated postmenopausal breast cancer patients in the absence of HER4 compared with those with pronounced HER4 expression. In accordance with this finding, the sensitivity to tamoxifen treatment of estrogen and HER4 receptor-positive ZR-75-1 breast cancer cells can be significantly enhanced by HER4 knockdown. CONCLUSION: We suggest an HER4/estrogen receptor interaction that impedes tamoxifen binding to the estrogen receptor and reduces treatment efficiency. Whether the sensitivity to tamoxifen treatment can be enhanced by anti-HER4 targeting needs to be prospectively evaluated.

SNAI2 upregulation is associated with an aggressive phenotype in fulvestrant-resistant breast cancer cells and is an indicator of poor response to endocrine therapy in estrogen receptor-positive metastatic breast cancer.

BACKGROUND: Endocrine resistance in estrogen receptor-positive (ER+) breast cancer is a major clinical problem and is associated with accelerated cancer cell growth, increased motility and acquisition of mesenchymal characteristics. However, the specific molecules and pathways involved in these altered features remain to be detailed, and may be promising therapeutic targets to overcome endocrine resistance. METHODS: In the present study, we evaluated altered expression of epithelial-mesenchymal transition (EMT) regulators in ER+ breast cancer cell models of tamoxifen or fulvestrant resistance, by gene expression profiling. We investigated the specific role of increased SNAI2 expression in fulvestrant-resistant cells by gene knockdown and treatment with a SNAIL-p53 binding inhibitor, and evaluated the effect on cell growth, migration and expression of EMT markers. Furthermore, we evaluated SNAI2 expression by immunohistochemical analysis in metastatic samples from two cohorts of patients with breast cancer treated with endocrine therapy in the advanced setting. RESULTS: SNAI2 was found to be significantly upregulated in all endocrine-resistant cells compared to parental cell lines, while no changes were observed in the expression of other EMT-associated transcription factors. SNAI2 knockdown with specific small interfering RNA (siRNA) converted the mesenchymal-like fulvestrant-resistant cells into an epithelial-like phenotype and reduced cell motility. Furthermore, inhibition of SNAI2 with specific siRNA or a SNAIL-p53 binding inhibitor reduced growth of cells resistant to fulvestrant treatment. Clinical evaluation of SNAI2 expression in two independent cohorts of patients with ER+ metastatic breast cancer treated with endocrine therapy in the advanced setting (N = 86 and N = 67) showed that high SNAI2 expression in the metastasis correlated significantly with shorter progression-free survival on endocrine treatment (p = 0.0003 and p = 0.004). CONCLUSIONS: Our results suggest that SNAI2 is a key regulator of the aggressive phenotype observed in endocrine-resistant breast cancer cells, an independent prognostic biomarker in ER+ advanced breast cancer treated with endocrine therapy, and may be a promising therapeutic target in combination with endocrine therapies in ER+ metastatic breast cancer exhibiting high SNAI2 levels.

Clinical significance of plasma cell-free DNA mutations in PIK3CA, AKT1, and ESR1 gene according to treatment lines in ER-positive breast cancer.

The somatic activation of PI3K/AKT pathway mutations, PIK3CA and AKT1, and ESR1 mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive procedure to quickly assess and monitor disease progression or therapeutic effect in breast cancer (BC) patients, but the clinical significance of these mutations in late treatment lines (TLs) remains unclear. The subjects of this study were a total of 251 plasma samples from 128 estrogen receptor-positive (ER+) BC patients. Of these plasma samples, 133 were from 73 primary BC (PBC) patients, and 118 plasma samples were from 68 metastatic BC (MBC) patients. We developed droplet digital PCR (ddPCR) assays to verify the clinical significance of PIK3CA, AKT1, and ESR1 mutations in these patients. cfDNA PIK3CA mutations were observed in 15.1% of the PBC patients, while a cfDNA AKT1 mutation was observed in 1.4% of patients, and cfDNA ESR1 mutations were observed in 2.7% of patients. Patients with detectable cfDNA PIK3CA mutations were not associated with clinical outcomes. According to the TL, the prevalence of the PIK3CA and ESR1 mutations in cfDNA were lower in early TLs compared with late TLs. In the early TL group, patients with cfDNA PIK3CA mutations had a shorter time to treatment failure (TTF) than patients without mutations (P = 0.035). However, there was no statistically significant difference between patients with or without cfDNA ESR1 mutations. However, in the late TL group, patients with cfDNA ESR1 mutations had a shorter TTF than patients without mutations (P = 0.048). However, there was no statistically significant difference between patients with or without cfDNA PIK3CA mutations. Since the prevalence of cfDNA AKT1 mutation is low in both PBC and MBC patients, the impact of AKT1 mutations on the prognosis remains unclear. We have demonstrated the difference in the clinical significance of the hotspot PIK3CA, AKT1, and ESR1 mutations in cfDNA for each TL in ER+ BC patients.

Therapeutic predictors of neoadjuvant endocrine therapy response in estrogen receptor-positive breast cancer with reference to optimal gene expression profiling.

PURPOSE: Neoadjuvant endocrine therapy (NAET) for estrogen receptor-positive primary breast cancer causes adequate tumor shrinkage, and is expected to be helpful for breast-conserving surgery, but the adaptation criteria, especially in regard to treatment duration, have never been elucidated. Re-visiting past gene expression profiles, we explored the data for specialized pre-therapeutic predictors and validated the results using our in-house clinical cohorts. METHODS: We sorted the genes related to a > 30% tumor volume reduction through NAET from a cDNA microarray data-set of GSE20181, then selected the top 40 genes. We validated these gene expression levels using pre-therapeutic biopsy samples obtained from patients treated with long-term NAET (over 4 months; N = 40). A short-term (2-8 weeks; N = 37) NAET cohort was also validated to clarify whether expression of these genes is also related to a rapid response of Ki67 and PEPI score. RESULTS: In the long-term group, higher expression of KRAS, CUL2, FAM13A, ADCK2, and LILRA2 was significantly associated with tumor shrinkage, and KRAS, MMS19, and IVD were related to lower PEPI score (≤ 3). Meanwhile in the short-term group, none of these genes except CUL2 showed a direct correlation with Ki67 reduction or PEPI score. This suggested that tumor shrinkage by NAET might be induced by response to the hypoxic environment (CUL2, FAM13A, KRAS) and activation of tumor immune system (LILRA2), without involving inhibition of proliferation. CONCLUSION: Expression of specific genes may allow selection of the most responsive patients for maximum tumor shrinkage with NAET.

Local estrogen axis in the human bone microenvironment regulates estrogen receptor-positive breast cancer cells.

BACKGROUND: Approximately 70% of all breast cancers express the estrogen receptor, and are regulated by estrogen. While the ovaries are the primary source of estrogen in premenopausal women, most breast cancer is diagnosed following menopause, when systemic levels of this hormone decline. Estrogen production from androgen precursors is catalyzed by the aromatase enzyme. Although aromatase expression and local estrogen production in breast adipose tissue have been implicated in the development of primary breast cancer, the source of estrogen involved in the regulation of estrogen receptor-positive (ER+) metastatic breast cancer progression is less clear. METHODS: Bone is the most common distant site of breast cancer metastasis, particularly for ER+ breast cancers. We employed a co-culture model using trabecular  bone tissues obtained from total hip replacement (THR) surgery specimens to study ER+ and estrogen receptor-negative (ER-) breast cancer cells within the human bone microenvironment. Luciferase-expressing ER+ (MCF-7, T-47D, ZR-75) and ER- (SK-BR-3, MDA-MB-231, MCF-10A) breast cancer cells were cultured directly on bone tissue fragments or in bone tissue-conditioned media, and monitored over time with bioluminescence imaging (BLI). Bone tissue-conditioned media were generated in the presence vs. absence of aromatase inhibitors, and testosterone. Bone tissue fragments were analyzed for aromatase expression by immunohistochemistry. RESULTS: ER+ breast cancer cells were preferentially sustained in co-cultures with bone tissues and bone tissue-conditioned media relative to ER- cells. Bone fragments analyzed by immunohistochemistry revealed expression of the aromatase enzyme. Bone tissue-conditioned media generated in the presence of testosterone had increased estrogen levels and heightened capacity to stimulate ER+ breast cancer cell proliferation. Pretreatment of cultured bone tissues with aromatase inhibitors, which inhibited estrogen production, reduced the capacity of conditioned media to stimulate ER+ cell proliferation. CONCLUSIONS: These results suggest that a local estrogen signaling axis regulates ER+ breast cancer cell viability and proliferation within the bone metastatic niche, and that aromatase inhibitors modulate this axis. Although endocrine therapies are highly effective in the treatment of ER+ breast cancer, resistance to these treatments reduces their efficacy. Characterization of estrogen signaling networks within the bone microenvironment will identify new strategies for combating metastatic progression and endocrine resistance.

Neoadjuvant Therapy with Weekly Nanoparticle Albumin-Bound Paclitaxel for Luminal Early Breast Cancer Patients: Results from the NABRAX Study (GEICAM/2011-02), a Multicenter, Non-Randomized, Phase II Trial, with a Companion Biomarker Analysis.

BACKGROUND: Nanoparticle albumin-bound paclitaxel (nab-Paclitaxel) is an alternative to standard taxanes for breast cancer (BC) treatment. We evaluated nab-Paclitaxel efficacy as neoadjuvant treatment for early estrogen receptor-positive (ER+), human epidermal growth factor receptor 2-negative (HER2-) disease. MATERIALS AND METHODS: Women with ER+, HER2-, stage II-III BC were treated preoperatively with four cycles of weekly nab-Paclitaxel (150 mg/m2), 3 weeks on and 1 week off. We hypothesized that poor pathological response rate (residual cancer burden [RCB] III; Symmans criteria) would be ≤16%. RESULTS: Eighty-one patients with a median age of 47 years were treated; 64.2% were premenopausal, and 69% of tumors were stage II. Residual cancer burden III rate was 28.4% (95% confidence interval [CI]: 18.6%-38.2%), RCB 0+I (good response) rate was 24.7% (95% CI: 15.3%-34.1%) and RCB 0 (complete response) rate was 7.4% (95% CI: 1.7%-13.1%). Objective response rate by magnetic resonance imaging was 76.5% and rate of conversion to breast conserving surgery was 40.0%. The most frequent grade 3 and 4 toxicity was neutropenia (12.3% and 3.7% of patients, respectively), without any febrile neutropenia. Sensory neuropathy grade 2 and 3 were seen in 25.9% and 2.5% of patients, respectively. Tumor secreted protein, acidic, cysteine-rich (SPARC) overexpression was significantly associated with RCB 0 (odds ratio: 0.079; 95% CI: 0.009-0.689; p = .0216). CONCLUSION: Despite failing to confirm an RCB III rate ≤16% in nab-Paclitaxel-treated patients, the RCB 0+I rate indicates a significant drug antitumor activity with low rates of grade 3-4 toxicity. Our exploratory biomarker analysis suggests a potential predictive role of complete response for SPARC. Confirmatory analyses are warranted, adapting dose and schedule to decrease peripheral neurotoxicity. (Trial registration: European Clinical Trials Database study number: 2011-004476-10; ClinicalTrials.gov: NCT01565499). IMPLICATIONS FOR PRACTICE: The pathological response rate (residual cancer burden [RCB]; Symmans criteria) of nanoparticle albumin-bound paclitaxel administered as neoadjuvant treatment for early estrogen receptor-positive, human epidermal growth factor receptor 2-negative disease was evaluated. Whereas poor response (RCB III) was 24.7%, similar to that for docetaxel, good response (RCB 0+I) reached 23.0%, far superior to the 13% for docetaxel, while keeping toxicity low. Exploratory biomarker analysis suggests secreted protein, acidic, cysteine-rich overexpression in tumor cells as a potential predictor of complete response (RCB 0). Findings point to an encouraging single-agent neoadjuvant treatment with low toxicity, which warrants future research and development.