Breast Cancer Research and Treatment

Anti-tumor and anti-metastasis efficacy of E6201, a MEK1 inhibitor, in preclinical models of triple-negative breast cancer Abstract Purpose Triple-negative breast cancer (TNBC) lacks the receptor targets estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, and thus, it does not respond to receptor-targeted treatments. TNBC has higher recurrence, metastasis, and mortality rates than other subtypes of breast cancer. Mounting data suggest that the MAPK (also known as RAS-RAF-MEK-ERK) pathway is an important therapeutic target in TNBC. Methods To evaluate anti-tumor and anti-metastasis efficacy of E6201, we used cell proliferation assay, soft agar assay, cell cycle assay, Annexin V staining assay, immunoblotting analysis, immunohistochemistry, migration assay, invasion assay, mammary fat pad xenograft, and experimental and spontaneous metastasis xenograft models. We also evaluated the anti-tumor efficacy of E6201 plus CDK4/6 inhibitor, mTOR inhibitor, or ATR inhibitor. Results E6201 inhibited TNBC cell colony formation, migration, and invasion in a dose-dependent manner. E6201 induced G1 cell cycle arrest and apoptosis. E6201 inhibited TNBC xenograft growth and inhibited TNBC lung metastasis and improved mouse survival in experimental metastasis and spontaneous metastasis assays. Immunohistochemical staining demonstrated that E6201 decreased the metastatic burden in the lung and decreased phosphorylated ERK expression in a dose-dependent manner. Combination of E6201 with CDK4/6 inhibitor or mTOR inhibitor enhanced E6201's in vitro anti-tumor efficacy. Conclusion These results indicate that E6201 exhibits anti-tumor efficacy against TNBC in vitro and anti-metastasis efficacy against TNBC in vivo. These results provide a rationale for further clinical development of E6201 as a MAPK-pathway-targeted therapy for TNBC.

4-Hydroxytamoxifen enhances sensitivity of estrogen receptor α-positive breast cancer to docetaxel in an estrogen and ZNF423 SNP-dependent fashion Abstract Purpose In early stage, ERα-positive breast cancer, concurrent use of endocrine therapy and chemotherapy has not been shown to be superior to sequential use. We hypothesized that genetic biomarkers can aid in selecting patients who would benefit from chemo-endocrine therapy. Our previous studies revealed that ZNF423 is a transcription factor for BRCA1 and an intronic single nucleotide polymorphism (SNP) in ZNF423, rs9940645, determines tamoxifen response. Here, we identified mitosis-related genes that are regulated by ZNF423 which led us to investigate taxane response in a rs9940645 SNP- and tamoxifen-dependent fashion. Methods The Cancer Genome Atlas (TCGA) breast cancer dataset was used to identify genes correlated with ZNF423. Quantitative reverse transcription PCR, chromatin immunoprecipitation, and luciferase reporter assays were used to validate the gene regulation. We used CRISPR/Cas9 to engineer paired ZR-75-1 cells which differ only in ZNF423 rs9940645 SNP genotype to test SNP-dependent phenotypes including cell cycle and cell viability. We validated our findings in an additional two breast cancer cell lines, Hs578T-ERα and HCC1500. Results Mitosis-related genes VRK1 and PBK, which encode histone H3 kinases, were experimentally validated to be regulated by ZNF423. ZNF423 knockdown decreased VRK1 and PBK expression and activity. Additionally, ZNF423 knockdown enhanced docetaxel-induced G2/M arrest and cytotoxicity through VRK1 or PBK regulation. Lastly, cells carrying the rs9940645 variant genotype had increased G2/M arrest and decreased cell viability when treated with docetaxel in combination with estradiol and 4-OH-TAM. Conclusions We identified ZNF423 regulated genes involved in the G2/M phase of the cell cycle. 4-OH-TAM sensitized ERα-positive breast cancer cells to docetaxel in a ZNF423 SNP-dependent manner. Our findings suggest that patients with rs9940645 variant genotype may benefit from concurrent tamoxifen and docetaxel. This would impact a substantial proportion of patients because this SNP has a minor allele frequency of 0.47.

Influence of adherence to adjuvant endocrine therapy on disease-free and overall survival: a population-based study in Catalonia, Spain Abstract Objective To assess adherence to endocrine therapy and its relation to recurrence and mortality in women with early breast cancer. Methods This is a retrospective cohort study in population-based cancer registries in two Catalonian provinces of Spain. We included all cases of invasive stage I–III breast cancer diagnosed from 2007 to 2011 and with follow-up to 2017. Adherence to endocrine therapy was measured by means of prescription refills. Patients were considered non-adherent if they filled less than 80% of their prescriptions. After collecting data from patients' medical records, we analysed clinical variables and their relation with adherence by means of logistic and Cox regression models. Results The study included 2413 women. Five-year adherence was 84.5%; the greatest risk for non-adherence was in women under 50 years of age, diagnosed with stage III cancer, treated with neoadjuvant therapy, or receiving tamoxifen or sequential treatment. Adverse effects were associated with greater adherence. Non-adherence was significantly and independently associated with recurrence (hazard ratio [HR] 1.71, 95% confidence interval [CI] 1.16–2.51) and all-cause mortality (HR 2.11, 95% CI 1.62–2.74), after adjusting for age and tumour stage. Conclusions Although non-adherence was relatively infrequent in this population-based study, its impact on the risk of recurrence and mortality was considerable. Clinicians should make efforts to ensure therapeutic adherence during clinical follow-up of women with breast cancer.

Association of antidiabetic medication and statins with breast cancer incidence in women with type 2 diabetes Abstract Purpose To address the possible association between the use of metformin, other forms of antidiabetic medication (ADM) and statins with the incidence of breast cancer in women with type 2 diabetes (T2D). Methods Data were collected from a Finnish nationwide diabetes database (FinDM). The study cohort consisted of women diagnosed with T2D in 1996–2011 in Finland. In full-cohort analysis, Poisson regression was used to estimate hazard ratios (HRs) in relation to use of metformin, insulin, other forms of oral ADM and statins. In nested case–control analysis, up to 20 controls were matched for age and duration of diabetes to each case of breast cancer. Conditional logistic regression was used to estimate HRs in relation to medication use and cumulative use of different forms of ADM, and statins. Results 2300 women were diagnosed with breast cancer during follow-up. No difference in breast cancer incidence was observed between metformin users [HR 1.02, 95% confidence interval (CI) 0.93–1.11] or statin users (HR 0.97, 95% CI 0.89–1.05) compared with non-users. In nested case–control analysis the results were similar. Use of insulin (HR 1.18, 95% CI 1.03–1.36) was associated with a slightly increased incidence of breast cancer. Conclusions No evidence of an association between the use of metformin or statins and the incidence of breast cancer in women with T2D was found. Among insulin users, a slightly higher incidence of breast cancer was observed.

Risk of ipsilateral breast tumor recurrence in primary invasive breast cancer following breast-conserving surgery with BRCA1 and BRCA2 mutation in China Abstract Purpose BRCA1/2 germline mutations are associated with a high risk of breast cancer, which may preclude mutation carriers from breast-conserving surgery (BCS). This study retrospectively examined whether mutation status influenced the rate of ipsilateral breast tumor recurrence (IBTR) after BCS  in Chinese women. Methods Patients who underwent BCS were enrolled in carriers group and non-carriers group according to their BRCA1/2 mutation status in the study. The correlations were analyzed between IBTR incidence and BRCA1/2mutation. The IBTR cases were further separated into new primary tumor (NP) and true local recurrences (TR). The risk factors of NP were studied in multivariate analysis. Results 1947 consecutive Chinese women with primary invasive breast cancer were selected. 103 patients were identified as BRCA1/2 mutation carriers and 1844 were non-carriers. BRCA1/2 mutation carriers were younger (P < 0.001) with more often negative HER-2 expression (P = 0.01) and tumor size over 2 cm (P = 0.04) than non-carriers. The median follow-up for all patients was 80 months. The rate of IBTR was 3.9% in mutated carriers and 2.0% in non-carriers, respectively (P = 0.16). In IBTR cases, NP incidence was 3.9% in carrier group and 0.6% in non-carrier group, respectively (P < 0.01). After adjustment of all clinical-pathological factors, BRCA1/2mutation was the only statistical risk factor of NP incidence (HR = 6.29, P = 0.002), while positive lymph node was nearly statistically significant (HR = 2.70, P = 0.06). Conclusions BCS may be a rational option for Chinese BRCA1/2 mutation carriers. High NP incidence in mutation carriers should be paid close attention in the future.

Incidence of peripheral edema in patients receiving PI3K/mTOR/CDK4/6 inhibitors for metastatic breast cancer Abstract Purpose This study evaluated development of edema in patients receiving PI3K/mTOR/CDK4/6 targeted therapy for metastatic breast cancer (MBC). Methods We reviewed medical records of 160 patients receiving targeted therapy with PI3K/mTOR/CDK4/6 inhibitors to treat MBC (n = 160; 185 treatment occurrences). Clinicopathologic data, treatment details, and edema incidence were recorded. Results Edema incidence was 43.1% (69/160) overall and 25.6% (41/160) in the upper extremity ipsilateral to the treated breast. In 185 therapy regimens administered, 6.8% of patients on a PI3K inhibitor, 8.8% of patients on an mTOR inhibitor, and 9.2% of patients on a CDK4/6 inhibitor experienced new onset or worsened preexisting upper extremity edema. Further, 9.1% of patients on a PI3K inhibitor, 18.8% of patients on an mTOR inhibitor, and 10.5% of patients on a CDK4/6 inhibitor experienced new onset or worsened preexisting edema elsewhere in the body. Multivariate logistic regression showed that, beyond the established breast cancer-related lymphedema (BCRL) risk factors [axillary lymph node dissection (Odds Ratio (OR) 2.69, p = 0.020), regional lymph node irradiation (OR 6.47, p < 0.001), and body-mass index ≥ 30 kg/m2 (OR 3.46, p = 0.006)], a relative decrease in serum albumin after 3 months of treatment increased risk of developing edema (OR 2.07, p = 0.062). Neither duration nor type of therapy were significant risk factors for edema. Conclusion PI3K/mTOR/CDK4/6 inhibitors may influence the development of edema, which may cause or exacerbate progression of BCRL in patients with MBC. The varied incidence of edema between therapeutic regimens warrants vigilant monitoring of patients treated with these therapies, especially those at high risk of developing BCRL.

Loss of HER2 after HER2-targeted treatment Abstract Purpose HER2 expression has been reported to be discordant between primary tumor and metastatic tissue. Patients and methods HER2 discordance and relation to HER2-targeted treatment was investigated in 227 patients with primary breast cancer. Results HER2 discordance between primary biopsy and second biopsy after neoadjuvant or adjuvant treatment was observed in 20.7%. This discordance was related only to the use of HER2-targeted treatment: 30 of 33 (90.9%) women with downgraded HER2 expression underwent a HER2-targeted therapy, whereas in the group of patients with concordant HER2 expression, only 32 of 180 (17.8%) received HER2-targeted treatment (p < 0.0001). HER2 discordance was associated with reduced disease-free survival but not overall survival. In a second cohort, including patients with HER2 overexpressing tumors, trastuzumab treatment was associated with change of HER2 expression from positive to negative in 47.3% of cases. Addition of pertuzumab increased the rate of HER2 loss up to 63.2%. Notably, the interval between last HER2-targeted treatment and the time of surgical excision of the tumor after neoadjuvant chemotherapy (NACT) or the biopsy of the metachronous metastasis was associated with a significant change in HER2 expression. The median time between NACT and the time of surgical excision was 23 days (range 5–81 days) for tumors with decreased HER2 expression and 51 days (range 10–179 days) for tumors with concordant HER2 expression. Furthermore, median time between the end of adjuvant treatment and second histology of the metachronous metastases accounted for 15 days (range 2–165 days) and 478 days (range 7–2739 days) was observed in the group of patients with decreased or unchanged HER2 expression, respectively. Conclusion The interval between anti-HER2 treatment and the determination of HER2 in second histology is strongly associated with HER2 expression.

SNAIL is induced by tamoxifen and leads to growth inhibition in invasive lobular breast carcinoma Abstract Purpose Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer that is predominantly estrogen receptor alpha (ER)-positive (+) and is thus treated with endocrine therapies. Herein, we sought to understand the molecular underpinnings of the 4-hydroxytamoxifen (4OHT) resistance in ILC by assessing the potential role of the epithelial-to-mesenchymal transition transcription factor (EMT-TF) SNAIL (SNAI1). Methods Using a series of breast cancer cell lines, we measured the basal, estrogen and 4OHT-induced expression of SNAIL and other EMT-TF family members by quantitative reverse transcription-polymerase chain reaction and immunoblotting. Chromatin immunoprecipitation experiments were performed to assess ER binding to the SNAIL promoter. Cell proliferation, cell cycle and apoptosis were assessed in 2D cultures. 3D growth was assessed in Matrigel and Collagen I cultures. Results Estrogen and 4OHT induced SNAIL expression, but not that of the other EMT-TF family members SLUG (SNAI2) and SMUC (SNAI3), with the 4OHT effect being specific to the lobular but not the ductal subtype. We observed estrogen and 4OHT-induced ER recruitment to the SNAI1 promoter and high endogenous basal levels of SNAIL and several EMT-TFs in ILC cell lines. While SNAIL knockdown had a minor impact on the 4OHT partial agonism in estrogen-depleted conditions, it led to a surprising increase in cell proliferation in full serum. In complementary experiments, inducible SNAI1 overexpression caused decreased proliferation, associated with a cell cycle arrest in G0/G1. Additionally, apoptosis was observed in BCK4 cells. Conclusion These data suggest a previously unrecognized role for SNAIL in ILC, substantiating a context-dependent behavior for this EMT-TF.

The effect of scalp cooling on CIA-related quality of life in breast cancer patients: a systematic review Abstract Purpose Chemotherapy-induced alopecia (CIA) remains a distressing adverse event of cancer treatment but may be prevented by scalp cooling. The effectiveness of scalp cooling, however, is dependent on the chemotherapy regimen with successful hair preservation (i.e., < 50% hair loss) in 41–59% of women on taxane-based therapies in comparison to 16–36% on anthracycline-based therapies. Despite the potential utility, use of scalp cooling has shown a more equivocal impact on quality of life (QoL). In this review, we aim to evaluate the use of scalp cooling for CIA and quantitative QoL measures. Methods A systematic review of PubMed, Embase, Web of Science, and Cochrane databases for clinical studies on scalp cooling to prevent CIA published before October 29, 2018 was performed. Clinical studies with 5 or more patients that reported on a quantitative QoL measure were included and graded according to a modified five-point scale from the Oxford Centre for Evidence-Based Medicine. Results Studies meeting inclusion criteria included 4 randomized clinical trials (RCT), 8 cohort studies, and 1 cross-sectional study with 1282 unique patients. The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (QLQ-C30: 46%) and Breast Cancer Module (QLQ-BR23: 46%) represented the most commonly used QoL assessments. Overall, 4 (31%) of the 13 studies concluded that scalp cooling was associated with significant improvements in QoL measures; 8 (62%) determined that there was either non-significant or no improvements; and 1 (7.7%) provided a mixed conclusion. Although 2 (50%) RCT demonstrated that scalp cooling can effectively prevent CIA depending on the chemotherapy regimen, these studies did not show that successful hair preservation was associated with improved QoL measures. Conclusions This review demonstrates that scalp cooling is not consistently associated with significant QoL improvements as assessed by EORTC QLQ-C30 and -BR23. Representing a critical limitation, more than one-third of the studies did not subcategorize QoL outcomes for successfully or unsuccessfully scalp-cooled patients but rather reported on QoL measures for all scalp-cooled patients in general. Failure to prevent hair loss in patients undergoing an expensive and potentially uncomfortable treatment likely contributes to decreased well-being, impacting the overall distribution of QoL measures in scalp cooling patients compared to controls. Future studies should incorporate validated QoL instruments specific to hair disease and classify QoL outcomes for scalp-cooled patients based on the degree of hair preservation.

Quality of life in pre- and postmenopausal patients with early breast cancer: a comprehensive analysis from the prospective MaLife project Abstract Purpose Quality of life (QoL) plays an important role in recovery—especially after an incisive diagnosis such as breast cancer. Here, we present a comprehensive assessment of QoL for pre- and postmenopausal patients, starting from initial systemic treatment of early breast cancer until 3 years later, in patients from a so-called "real-world" setting. Methods 251 premenopausal and 478 postmenopausal patients with early breast cancer have been recruited into the longitudinal MaLife project within the prospective, multicentre, German Tumour Registry Breast Cancer between 2011 and 2015. The questionnaires FACT-G, FACT-Taxane, FACT-ES, EORTC QLQ-BR23, BFI and HADS were filled in at start of treatment (T0), 6, 12, 24 and 36 months later. The proportion of patients with clinically meaningful changes at 36 months was determined. Results This first interim analysis shows that the FACT-G global QoL improved over time regardless of the menopausal status. However, clinically meaningful decrease of social/family well-being (48–51%), arm symptoms (44–49%) and symptoms of neurotoxicity (55–56%) was frequently reported 3 years after start of treatment. Many premenopausal patients also reported a clinically meaningful worsening of endocrine symptoms (64%), emotional well-being (36%) and fatigue intensity (37%). Additionally, 3 years after start of treatment, 15% of the patients were classified as doubtful cases and 18% as definite cases of anxiety. Conclusions Despite improvements in global QoL, breast cancer survivors report worsened ailments 3 years after start of therapy. Follow-up care should distinguish between premenopausal patients needing special attention for emotional/menopausal issues, and postmenopausal patients needing particular care regarding physical concerns.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com