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Background: Questions have been raised as to an increased risk of local recurrence with breast-conserving surgery (BCS) post NAC highlighting the uncertainty around optimal margin width in this patient population. We examined the association between margin status and local recurrence-free survival (LRFS) in patients who underwent BCS following NAC., Methods: We performed a retrospective cohort study of adult female patients with stage I-III breast cancer who underwent NAC followed by BCS between 2012 and 2021 at two cancer centers. Margins were categorized as "close" if they were < 1 mm., Results: The full cohort included 544 patients with a median age of 53 years (interquartile range [IQR] 44-64). Pathologic complete response (pCR) was achieved in 41.2% of the overall cohort (n = 224). Of the 320 with residual disease, 29.4% (n = 94) had at least one close margin, and 10.9% (n = 35) had ≥2 close margins. Median follow-up was 55 months (IQR 32-83); 4.8% had an ipsilateral breast recurrence (n = 26). Patients with pCR had a higher 5-year LRFS than those with residual disease (98.0% vs. 91.6%, p = 0.02). There was no difference in 5-year LRFS between the margin categories (clear vs. 1 close margin vs. ≥2 close margins) in those with residual disease (92.2% vs. 88.9% vs. 92.9%) (p = 0.78)., Conclusions: In patients undergoing BCS post-NAC, those who achieved pCR had a significantly higher LRFS compared with those with residual disease at the time of surgery, but LRFS was not associated with margin width nor the number of close margins., (© 2024. Society of Surgical Oncology.)

Importance: Between 5% and 10% of breast cancer cases are associated with an inherited germline pathogenic or likely pathogenic variant (GPV) in a breast cancer susceptibility gene (BCSG), which could alter local and systemic therapy recommendations. Traditional genetic testing criteria misses a proportion of these cases., Objective: To evaluate the prevalence and clinicopathological associations of GPVs in 2 groups of BCSGs among an ethnically diverse cohort of women with newly diagnosed breast cancer., Design, Setting, and Participants: This cross-sectional study, conducted at 3 Montreal hospitals between September 2019 and April 2022, offered universal genetic counseling and testing to all women with a first diagnosis of invasive breast cancer. Women were offered an obligatory primary panel of BRCA1, BRCA2, and PALB2 (B1B2P2) and an optional secondary panel of 14 additional BCSGs. Eligible participants were women 18 years of age or older who received a diagnosis of a first primary invasive breast cancer not more than 6 months before the time of referral to the study. Data were analyzed from November 2023 to June 2024., Results: Of 1017 referred patients, 805 were eligible and offered genetic counseling and testing, and 729 of those 805 (90.6%) consented to be tested. The median age at breast cancer diagnosis was 53 years (range, 23-91 years), and 65.4% were White and of European ancestry. Fifty-four GPVs were identified in 53 patients (7.3%), including 39 patients (5.3%) with B1B2P2 and 15 patients (2.1%) with 6 of the 14 secondary panel BCSGs (ATM, BARD1, BRIP1, CHEK2, RAD51D, and STK11). On multivariable analysis, clinical factors independently associated with B1B2P2-positive status included being younger than 40 years of age at diagnosis (odds ratio [OR], 6.83; 95% CI, 2.22-20.90), triple-negative breast cancer (OR, 3.19; 95% CI, 1.20-8.43), high grade disease (OR, 1.68; 95% CI, 1.05-2.70), and family history of ovarian cancer (OR, 9.75; 95% CI, 2.65-35.85). Of 39 B1B2P2-positive patients, 13 (33.3%) were eligible for poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors., Conclusions and Relevance: In this cross-sectional universal genetic testing study of women with newly diagnosed invasive breast cancer, the prevalence of GPVs was 7.3%, with 5.3% of patients testing positive for B1B2P2. Among B1B2P2-women women, one-third were eligible for PARP inhibitors.

Introduction: The impact of timing of genetic testing on uptake of risk reducing mastectomy (RRM) in affected female BRCA1/2 or PALB2 carriers remains an area of evolving interest, particularly with the introduction of mainstream genetic testing initiatives., Methods: Women with stage I-III breast cancer and a confirmed germline pathogenic variant in BRCA1/2 or PALB2 between 2000 and 2023 were identified from an institutional genetics database. Uptake of RRM was evaluated according to disclosure of genetic testing results before or after index surgery for a first diagnosis of breast cancer., Results: The cohort included 287 female BRCA1/2 or PALB2 carriers with a median age of 44 years (IQR, 36-52). Overall, 155 (54 %) carriers received genetic testing results before and 132 (46 %) after index breast surgery. Receipt of genetic testing results before surgery was associated with a higher rate of index bilateral mastectomy (58.7 % vs. 7.6 %, p < 0.001) and a commensurate decrease in adjuvant radiation (41.9 % vs. 74.2 %, p < 0.001). At a median follow up of 4.4 years after genetic testing, 219 (76.3 %) affected carriers had undergone bilateral RRM, including 83.9 % with preoperative knowledge and 67.4 % of patients with postoperative knowledge of their germline pathogenic variant (log rank, p < 0.001). On multivariate regression, disclosure of genetic testing results before index breast surgery was independently associated with long-term uptake of bilateral mastectomy (HR 1.69, 95 % CI 1.21-2.38)., Conclusion: Genetic testing results delivered prior to index breast surgery increase uptake of bilateral RRM in affected BRCA1/2 and PALB2 carriers. Efforts to mainstream genetic testing would help optimize surgical decision-making., Competing Interests: Declaration of competing interest Jean-Francois Boileau reports honoraria from Roche, Novartis, Genomic Health, Pfizer, Allergan, and Merck; personal fees from Roche, Genomic Health, NanoString Technologies, Pfizer, Eli Lilly, Novartis, and Merck; and travel support from Roche, GlaxoSmithKline, Novartis, Pfizer, and Lifecell outside the submitted work., (© 2024 Elsevier Ltd, BASO ∼ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Purpose: The goal of this study was to identify the preoperative predictors of pathologic nodal metastases (pN+) in cT1cN0 HER2+ breast cancer undergoing upfront surgery., Methods: We retrospectively reviewed data from women with cT1-T2N0 HER2+ breast cancer treated with neoadjuvant therapy (NAC) or upfront surgery at our institution between 2012 and 2023. Factors associated with management strategy were evaluated, and in those undergoing upfront surgery, univariate analyses were performed to identify the clinicopathologic factors associated with nodal metastases., Results: Overall, 255 women with cT1-T2N0 HER2+ breast cancer met inclusion criteria, including 170 (68.6%) upfront surgery patients and 85 (31.4%) who underwent NAC. The median age at diagnosis was 59 years (range, 27-90 years). Younger age, larger clinical tumor size, high-grade disease, ER-PR-HER2+ subtype, and year of diagnosis after 2019 were significantly associated with receipt of NAC (p < 0.05). In those undergoing upfront surgery, 25.3% were pN+ , including 32.5% of cT1cN0 tumors. Factors associated with nodal involvement included age under 50, larger clinical tumor size, lymphovascular invasion (LVI), multifocality/multicentricity, and abnormal lymph nodes on axillary ultrasound (p < 0.05). In subset analysis of cT1cN0 HER2+ cases, LVI remained the strongest predictor of pN + disease (73.3% vs. 22.6%, p < 0.001). Patients with cT1cN0 HER2+ breast cancer under 50 years had a 47.1% likelihood of pN+ disease., Conclusion: Patients with cT1cN0 breast cancer have a 32.5% likelihood of nodal metastases, with higher incidence with younger age, LVI, multifocality/multicentricity, and abnormal axillary ultrasound. The presence of these factors may identify the patients who would benefit from treatment with neoadjuvant chemotherapy., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: The recent ACOSOG Z11102 trial demonstrated low recurrence rates with breast conserving surgery (BCS) in women with multiple ipsilateral breast cancers (MIBC). Questions remain regarding the oncologic safety of BCS in women with MIBC receiving neoadjuvant chemotherapy (NAC)., Methods: We conducted a retrospective cohort study of adult patients who underwent BCS following NAC for stage I-III breast cancer from 2012 to 2021 at two academic centers. Descriptive statistics were used to summarize the data and the Kaplan-Meier method was used to provide estimates for recurrence and survival outcomes. MIBC was defined as ≥2 foci of malignancy., Results: A total of 544 patients were included; 29.4% (n = 160) ER+/HER2-, 17.7% (n = 96) ER+/HER2+, 18.2% (n = 99) ER-/HER2+, and 34.7% (n = 189) with ER-/HER2-disease. Overall, 80.5% (n = 438) had unifocal breast cancer while 19.5% (n = 106) had MIBC. Of patients with MIBC, 90.6% (n = 96) had multifocal and 9.4% (n = 10) had multicentric disease. Pathologic complete response was achieved in 41.1% of patients with MIBC versus 41.5% of patients with unifocal disease (p = 0.94). At a median follow-up of 55 months (IQR 32-83); 4.8% of patients in the unifocal group and 4.7% of patients in the MIBC group had had a local recurrence (p = 0.97). There was no difference in 5-year local recurrence-free survival (p = 0.92), recurrence-free survival (p = 0.06), or overall survival (p = 0.07) between the groups., Conclusion: In this large cohort of women undergoing BCS post-NAC, there was no significant difference in in breast tumor recurrence or survival outcomes between patients with unifocal disease and those with MIBC., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ipshita Prakash reports a relationship with F Hoffmann-La Roche Ltd that includes: speaking and lecture fees. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Published by Elsevier Ltd.)

Introduction: Primary prevention of breast cancer in women at elevated risk includes several strategies such as endocrine prevention and risk-reducing mastectomy (RRM). The objective of this study was to evaluate awareness of different preventive strategies across high-risk subgroups., Patients and Methods: Women referred for high risk evaluation between 2020 and 2023 completed an initial risk-assessment questionnaire that included questions around perceived lifetime risk and consideration of preventive strategies. One-way analysis of variance (ANOVA) and chi-squared tests were used to compare differences across different high-risk subgroups., Results: 482 women with a median age of 43 years (20-79 years) met inclusion criteria; 183 (38.0%) germline pathogenic variant carriers (GPV), 90 (18.7%) with high-risk lesions (HRL) on breast biopsy, and 209 (43.4%) with strong family history (FH) without a known genetic predisposition. Most high-risk women reported that they had considered increased screening and surveillance (83.7%) and lifestyle strategies (80.6%), while fewer patients had considered RRM (39.8%) and endocrine prevention (27.0%). Prior to initial consultation, RRM was more commonly considered in GPV carriers (59.4%) relative to those with HRL (33.3%) or strong FH (26.3%, p < 0.001). Based on current guidelines, 206 (43%) patients were deemed eligible for endocrine prevention, including 80.5% with HRL and 39.0% with strong FH. Prior consideration of endocrine prevention was highest in patients with HRL and significantly lower in those with strong FH (47.2% HRL versus 31.1% GPV versus 18.7% FH, p = 0.001)., Conclusions: Endocrine prevention is the least considered preventive option for high-risk women, despite eligibility in a significant proportion of those presenting with HRL or strong FH., (© 2023. Society of Surgical Oncology.)

Background: Risk-reducing mastectomy (RRM) helps prevent breast cancer in high-risk women but also carries a risk of unanticipated supplemental surgeries. We sought to determine the likelihood of supplemental surgeries following RRM., Methods: We performed a retrospective cohort study of female patients with a confirmed germline pathogenic variant (GPV) in a breast cancer susceptibility gene (BRCA1/2, PALB2 and others) who underwent bilateral or contralateral RRM at our institution between 2006 and 2022. Supplemental surgeries were defined as any operation requiring general or local anesthesia performed outside of the initially planned procedure(s). The Kaplan-Meier method was used to estimate the 5-years cumulative incidence of supplemental surgery., Results: Of 560 GPV carriers, RRMs were performed in 258 (46.1%) women. The median age of the cohort was 44 years (interquartile range 37-52 years), with 33 (12.8%) patients undergoing RRM without reconstruction and 225 (87.2%) undergoing RRM with reconstruction. Following surgery, 34 patients (13.2%) developed early (< 30 days) postoperative complications, including infection, hematoma, seroma, loss of the nipple areola complex, flap necrosis, implant exposure and/or prosthesis removal. At a median follow-up of 3.8 years, 94 (36.4%) GPV carriers underwent at least one reoperation. Participants who experienced an early postoperative complication had the highest rate of reoperation (85.3% vs. 29.0%; p < 0.001) and a significantly higher likelihood of multiple additional surgical interventions (41.2% vs. 10.7%; p < 0.001). The 5-years rate of supplemental surgery was 39.2% [95% confidence interval (CI) 32.7-46.5] in the overall cohort and 31.5% (95% CI 24.9-39.3) in patients without an early postoperative complication., Conclusions: Unanticipated supplemental surgeries occur in 40% of GPV carriers following RRM and in nearly one-third of patients without early postoperative complications., (© 2023. Society of Surgical Oncology.)

Background: The purpose of this study is to evaluate preoperative predictors of nodal metastases in patients with early-stage, HER2-positive (HER2+) breast cancer., Methods: The SEER Database was queried to identify women with a first diagnosis of stage I-II (T1-T2) HER2-positive breast cancer treated with upfront surgery in 2018. Multivariable logistic regression was used to identify clinical characteristics independently associated with nodal involvement., Results: Overall, 3333 women with stage I-II HER2+ breast cancer met inclusion criteria and were included in the study. The median age at diagnosis was 59 years (IQR, 51-69 years). Most patients underwent breast-conserving surgery (60.9%), with a median of 3 (IQR 2-4) axillary lymph nodes removed. On final pathology, 762 (22.9%) of T1-T2 HER2+ patients were node positive; 2.7% pN0[i+], 3.7% pN1mi, 15.1% pN1, and 1.4% pN2. Women less than 40 years and those between 40 and 49 years showed the highest proportion of axillary lymph node metastasis, in 33.7% and 30.7% respectively, and declining with age (p < 0.001). Patients with triple-positive breast cancer had the highest rates of nodal involvement (24.8%), compared to 20.7% ER+/PR-/HER2+ and 19.6% of HER2-enriched patients (p = 0.006). On adjusted analysis, age, biologic subtype, tumour size, and type of surgery remained independent predictors of nodal involvement. On subgroup analysis, women under age 50 with T1c HER2-enriched or triple-positive breast cancer had a 33% and 35% incidence of nodal involvement, which declined with age., Conclusions: The likelihood of pathologic nodal involvement in early-stage HER2+ breast cancer is contingent on age, ER/PR status, and tumour size., Competing Interests: Declaration of competing interest Jean-Francois Boileau reports honoraria from Roche, Novartis, Genomic Health, Pfizer, Allergan, and Merck; personal fees from Roche, Genomic Health, NanoString Technologies, Pfizer, Eli Lilly, Novartis, and Merck; and travel support from Roche, GlaxoSmithKline, Novartis, Pfizer, and Lifecell outside the submitted work. The remaining authors have no conflicts of interest to disclose., (Copyright © 2023. Published by Elsevier Ltd.)

Background: Approximately 80% of all breast cancers (BCs) are currently categorized as human epidermal growth factor receptor 2 (HER2)-negative [immunohistochemistry (IHC) 0, 1+, or 2+/in situ hybridization (ISH) negative]; approximately 60% of BCs traditionally categorized as HER2-negative express low levels of HER2. HER2-low (IHC 1+ or IHC 2+/ISH-) status became clinically actionable with approval of trastuzumab deruxtecan to treat unresectable/metastatic HER2-low BC. Greater understanding of patients with HER2-low disease is urgently needed., Patients and Methods: This global, multicenter, retrospective study (NCT04807595) included tissue samples from patients with confirmed HER2-negative unresectable/metastatic BC [any hormone receptor (HR) status] diagnosed from 2014 to 2017. Pathologists rescored HER2 IHC-stained slides as HER2-low (IHC 1+ or IHC 2+/ISH-) or HER2 IHC 0 after training on low-end expression scoring using Ventana 4B5 and other assays at local laboratories (13 sites; 10 countries) blinded to historical scores. HER2-low prevalence and concordance between historical scores and rescores were assessed. Demographics, clinicopathological characteristics, treatments, and outcomes were examined., Results: In rescored samples from 789 patients with HER2-negative unresectable/metastatic BC, the overall HER2-low prevalence was 67.2% (HR positive, 71.1%; HR negative, 52.8%). Concordance was moderate between historical and rescored HER2 statuses (81.3%; κ = 0.583); positive agreement was numerically higher for HER2-low (87.5%) than HER2 IHC 0 (69.9%). More than 30% of historical IHC 0 cases were rescored as HER2-low overall (all assays) and using Ventana 4B5. There were no notable differences between HER2-low and HER2 IHC 0 in patient characteristics, treatments received, or clinical outcomes., Conclusions: Approximately two-thirds of patients with historically HER2-negative unresectable/metastatic BC may benefit from HER2-low-directed treatments. Our data suggest that HER2 reassessment in patients with historical IHC 0 scores may be considered to help optimize selection of patients for treatment. Further, accurate identification of patients with HER2-low BC may be achieved with standardized pathologist training., Competing Interests: Disclosure All authors received nonfinancial support (assistance with manuscript preparation) from Articulate Science, LLC, and Helios Medical Communications Ltd funded by AstraZeneca. Additional disclosures are as follows: GV reports consulting or advisory fees from Agilent, AstraZeneca, Daiichi Sankyo, and Roche; participation in speakers bureaus and data safety monitoring advisory boards for Agilent, AstraZeneca, Daiichi Sankyo and MSD Oncology, and Roche; personal travel fees from AstraZeneca and Roche; and grants from Roche. MB reports consulting or advisory fees from AstraZeneca and Roche Canada; and contracted research funding from Pfizer Canada. NN reports grants from Chugai, Daiichi Sankyo, Eisai, Lilly, Mochida, Novartis, and Pfizer; and participation in speakers bureaus for AstraZeneca, Chugai, Daiichi Sankyo, Eisai, Lilly, Novartis, and Pfizer. ET reports consulting or advisory fees from Daiichi Sankyo and Lilly; and fees for non-CME services from AstraZeneca, Daiichi Sankyo, and Lilly. SB reports consulting or advisory fees from AstraZeneca, Lilly, MSD, Novartis, Pfizer, Roche, and Sanofi; and contracted research funding from AstraZeneca, Lilly, GSK, MSD, Novartis, Pfizer, and Roche. FPL reports consulting or advisory fees from Agendia, AstraZeneca, Daiichi Sankyo, Eisai, Exact Sciences, GSK, MammaPrint, MSD, Myriad Genetics, Novartis, Pfizer, Roche, Seagen, and Veracyte; contracted research funding from AstraZeneca, MammaPrint, MSD, Myriad Genetics, Roche, and Veracyte; and personal travel fees from AstraZeneca and Seagen. NH reports participation in speakers bureaus for AstraZeneca, Chugai, Genomic Health, Kirin, Novartis, and Pfizer. JS reports research funding from AstraZeneca, Boehringer Ingelheim, Daiichi Sankyo, GSK, Eli Lilly, MSD, Novartis, Pfizer, Roche, and Sanofi; and partnerships with Roche. RTS reports consulting and non-CME service fees from AstraZeneca, Gilead, Lilly, Novartis, and Pfizer. AMB reports consulting or advisory fees from AbbVie, Agendia, Bayer, Biotheranostics, Coherus BioSciences, Daiichi Sankyo, Lilly, Eisai, Genentech/Roche, General Electric, Gilead Sciences, Immunomedics, Merck, Michael J. Hennessy Associates, Myriad Genetics, Novartis, OncLive, Pfizer, Puma Biotechnology, Seagen, and Tyme; and contracted research funding from AstraZeneca, Daiichi Sankyo, Lilly, Genentech/Roche, Gilead Sciences, Merck, Novartis, and Puma Biotechnology. CSO reports consulting or advisory fees from Roche and Seagen; and participation in speakers bureaus for Lilly. FS reports no conflict of interests. GH reports being a shareholder in Rhythm Biosciences Limited. DV reports a consulting or advisory role with OPEN Health; employment with AstraZeneca and may own stock or stock options in that company. GDJ reports a consulting or advisory role with AstraZeneca/MedImmune. AM is an employee of Daiichi Sankyo and may own stock or stock options in that company. AL and VGM are employees of AstraZeneca and may own stock or stock options in that company. Data sharing Data underlying the findings described in this manuscript may be obtained in accordance with AstraZeneca’s data sharing policy described at https://astrazenecagrouptrials.pharmacm.com/ST/Submission/Disclosure. Data for studies directly listed on Vivli can be requested through Vivli at www.vivli.org. Data for studies not listed on Vivli could be requested through Vivli at https://vivli.org/members/enquiries-about-studies-not-listed-on-the-vivli-platform/. AstraZeneca Vivli member page is also available outlining further details: https://vivli.org/ourmember/astrazeneca/. Disclaimers Medical writing support was funded by AstraZeneca in accordance with Good Publication Practice (GPP) guidelines (http://www.ismpp.org/gpp-2022). The manuscript was reviewed for medical accuracy by AstraZeneca and Daiichi Sankyo; however, the authors retained full control of the content and made the final decisions for all aspects of this article., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Purpose: In KATHERINE, adjuvant T-DM1 reduced risk of disease recurrence or death by 50% compared with trastuzumab in patients with residual invasive breast cancer after neoadjuvant therapy (NAT) comprised of HER2-targeted therapy and chemotherapy. This analysis aimed to identify biomarkers of response and differences in biomarker expression before and after NAT., Experimental Design: Exploratory analyses investigated the relationship between invasive disease-free survival (IDFS) and HER2 protein expression/gene amplification, PIK3CA hotspot mutations, and gene expression of HER2, PD-L1, CD8, predefined immune signatures, and Prediction Analysis of Microarray 50 intrinsic molecular subtypes, classified by Absolute Intrinsic Molecular Subtyping. HER2 expression on paired pre- and post-NAT samples was examined., Results: T-DM1 appeared to improve IDFS versus trastuzumab across most biomarker subgroups, except the HER2 focal expression subgroup. High versus low HER2 gene expression in residual disease was associated with worse outcomes with trastuzumab [HR, 2.02; 95% confidence interval (CI), 1.32-3.11], but IDFS with T-DM1 was independent of HER2 expression level (HR, 1.01; 95% CI, 0.56-1.83). Low PD-L1 gene expression in residual disease was associated with worse outcomes with trastuzumab (HR, 0.66; 95% CI, 0.44-1.00), but not T-DM1 (HR, 1.05; 95% CI, 0.59-1.87). PIK3CA mutations were not prognostic. Increased variability in HER2 expression was observed in post-NAT versus paired pre-NAT samples., Conclusions: T-DM1 appears to overcome HER2 resistance. T-DM1 benefit does not appear dependent on immune activation, but these results do not rule out an influence of the tumor immune microenvironment on the degree of response., (©2023 The Authors; Published by the American Association for Cancer Research.)

Background: Phyllodes tumours of the breast are rare fibroepithelial neoplasms with a propensity for recurrence. While surgical excision remains the standard of care, the optimal margin width is an area of active investigation. Recent studies have questioned the necessity for wide, local excision., Methods: We conducted a retrospective, cohort study of patients with phyllodes tumours treated at our institution between 2003 and 2021. Demographic, histopathological, and recurrence data were captured; malignant phyllodes were excluded. Cox proportional hazard models were used to identify covariates associated with local recurrence., Results: Of 187 patients with phyllodes tumours, 82.9% (n = 155) were classified as benign while 17.1% (n = 32) were borderline. Initial surgical margins were positive in 26.2% (n = 49), < 2 mm in 50.8% (n = 95), and ≥ 2 mm in 23% (n = 43) patients. Among patients with positive margins, 61.2% (n = 30) underwent margin revision. At a median follow-up of 2.9 years, the recurrence rate was 3.7%. On univariate analysis, only a positive margin at the time of initial surgery and not margin width was significantly associated with a higher rate of disease recurrence (hazard ratio [HR] 9.52, 95% confidence interval [CI] 1.85-49.2), as was a size greater than 4 cm on preoperative imaging (HR 10.78, 95% CI 0.97-120.1). Revision of an initially positive margin was not significantly associated with decreased local recurrence (p = 1)., Conclusions: In this large cohort of benign and borderline phyllodes tumours, positive resection margins and not margin width at the initial surgery were associated with a increased recurrence. Individualization of decisions regarding margin reexcision is important., (© 2022. Society of Surgical Oncology.)

Background: Women with history of chest irradiation for Hodgkin lymphoma are at increased risk of developing bilateral breast cancer, although contralateral breast cancer risk estimates in this population remain undefined., Methods: We queried the SEER database for women treated with radiation therapy for Hodgkin lymphoma prior to age 30 years and were diagnosed with a subsequent breast cancer between 1990-2016. Trends in surgical management and the 5- and 10-year cumulative incidence of contralateral breast cancer were evaluated., Results: The cohort included 295 women with a median age of 22 years (range 8-30 years) at Hodgkin lymphoma diagnosis, and 42 years (range 22-65 years) at breast cancer diagnosis. Overall, 263 (89.2%) presented with unilateral breast cancer, while 32 (10.8%) presented with synchronous bilateral breast cancer. Breast-conserving surgery was performed in 17.3% of patients, while mastectomy was performed in 82.7%. In 263 patients presenting with unilateral breast cancer, 50 (19.0%) underwent breast-conserving surgery and 213 (81.0%) underwent mastectomy. Subgroup analysis of mastectomy patients demonstrated a 40.5% bilateral mastectomy rate. The 5-year incidence of contralateral breast cancer in women who underwent unilateral surgery was 9.4% [95% confidence interval (CI), 5.6-15.4%], increasing to 20.2% (95% CI, 13.7-29.2%) at 10-year and 29.9% (95% CI, 20.8-41.9%) at 15-year follow-up., Conclusions: Women with a history of prior chest radiation for Hodgkin lymphoma with a diagnosis of breast cancer have a 10-year contralateral breast cancer risk of 20%. These findings support consideration of contralateral prophylactic mastectomy during surgical decision-making for management of this high-risk patient population., (© 2022. Society of Surgical Oncology.)

Background: This study sought to determine the likelihood of occult malignancy during risk-reducing mastectomy in high-penetrance pathogenic variant carriers to help refine axillary staging recommendations., Methods: The authors performed a retrospective cohort study analyzing all female carriers of pathogenic variants in BRCA1/2, PALB2 or other genes who underwent prophylactic surgery at their institution between 2006 and 2021. Occult breast cancer was defined as the unanticipated presence of in situ or invasive malignancy on pathologic evaluation of prophylactic mastectomy specimens., Results: Of 523 women, 243 carriers met the inclusion criteria for the study including 124 BRCA1 (51.0%), 108 BRCA2 (44.4%), and 11 PALB2, TP53, CDH1, or PTEN (4.6%) carriers. The median age was 44 years (interquartile range, 37-52 years). Overall, 128 women (52.7%) underwent bilateral prophylactic mastectomies, and 115 (47.3%) underwent contralateral prophylactic mastectomy. In the 371 mastectomies performed, 16 (4.3%) occult malignancies were diagnosed. Most of the occult malignancies were ductal carcinoma in situ (13 mastectomies, 3.5%), whereas 3 mastectomies (0.8%) contained invasive breast cancer. If Breast Imaging Reporting and Data System (BIRADS) 1-2 or BIRADS 3 findings were reported on preoperative magnetic resonance imaging (MRI), the rate of occult malignancy decreased to 3.0 and 2.8%, respectively, per mastectomy. The patient-level factors associated with a likelihood of occult breast cancer greater than 10% included a history of prior breast cancer, age exceeding 60 years, and BIRADS 4 findings on preoperative imaging., Conclusions: Occult invasive malignancy was detected in less than 1% of the risk-reducing mastectomies performed for women with BRCA1/2 or PALB2 pathogenic variants. Sentinel lymph node biopsy can be safely avoided when BIRADS 1-3 findings are reported on preoperative MRI., (© 2022. Society of Surgical Oncology.)

Background: Childhood and young adult survivors of Hodgkin lymphoma (HL) are at elevated risk of developing breast cancer, yet little data exist on the tumor characteristics that develop in this high-risk patient population., Methods: The National Cancer Institute's Surveillance, Epidemiology, and End Results database was used to identify breast cancers diagnosed between 1990 and 2016 in women who had received prior radiation therapy for HL at age 30 years or younger. Clinicopathologic features of subsequent breast cancers (breast cancer after radiation therapy for HL [BC-HL]) were examined and compared with breast cancers diagnosed in women who had no prior malignancy (breast cancer with no prior malignancy [BC-NPM])., Results: In total, 321 breast cancers were identified in 257 women who had a history of radiation therapy for HL. The median age at HL diagnosis was 22 years (interquartile range, 18-26 years), and nearly all patients in the BC-HL group (97.9%) were diagnosed ≥8 years after radiation therapy. Overall, 56 patients in the BC-HL group (21.8%) developed bilateral breast cancer. Compared with women who had BC-NPM, those who had BC-HL were younger (43 vs 60 years; P < .001) and were less likely to present with ductal carcinoma in situ (8.4% vs 14.9%; P = .001). On multivariable analysis that included adjustment for age, invasive BC-HL was associated with smaller (≤2 cm) tumor size (odds ratio, 1.64; 95% CI, 1.25-2.15) and upper outer quadrant tumors (odds ratio, 1.37; 95% CI, 1.04-1.81) compared with BC-NPM. In a subset analysis of 102 women who had HER2/neu status available, the distribution of biologic subtype was not significantly different between BC-HL and BC-NPM (P = .16)., Conclusions: Breast cancers in women who previously received radiation therapy for HL are characterized by earlier onset disease, although most remain estrogen receptor-positive and have early stage disease at presentation., Lay Summary: Women who have had radiation therapy for Hodgkin lymphoma at a young age are at increased risk of developing early onset breast cancer; however, most of these breast cancers are sensitive to hormones (estrogen receptor-positive) and are diagnosed at early stages. Because these breast tumors are estrogen receptor-positive, medications that prevent breast cancer by blocking the effect of or lowering hormone levels (also termed endocrine prevention) may be useful in this group of high-risk women., (© 2021 American Cancer Society.)

Purpose: Distant metastases are present in 6% or more of patients with newly diagnosed breast cancer. In this context, locoregional therapy for the intact primary tumor has been hypothesized to improve overall survival (OS), but clinical trials have reported conflicting results., Methods: Women presenting with metastatic breast cancer and an intact primary tumor received systemic therapy for 4-8 months; if no disease progression occurred, they were randomly assigned to locoregional therapy for the primary site (surgery and radiotherapy per standards for nonmetastatic disease) or continuing sysmetic therapy. The primary end point was OS; locoregional control and quality of life were secondary end points. The trial design provided 85% power to detect a 19.3% absolute difference in the 3-year OS rate in randomly assigned patients. The stratified log-rank test and Cox proportional hazards model were used to compare OS between arms. Cumulative incidence of locoregional progression was compared using Gray's test. Quality-of-life assessment used standard instruments., Results: Of 390 participants enrolled, 256 were randomly assigned: 131 to continued systemic therapy and 125 to early locoregional therapy. The 3-year OS was 67.9% without and 68.4% with early locoregional therapy (hazard ratio = 1.11; 90% CI, 0.82 to 1.52; P = .57). The median OS was 53.1 months (95% CI, 47.9 to not estimable) in the systemic therapy arm and 54.9 months (95% CI, 46.7 to not estimable) in the locoregional therapy arm. Locoregional progression was less frequent in those randomly assigned to locoregional therapy (3-year rate: 16.3% v 39.8%; P < .001). Quality-of-life measures were largely similar between arms., Conclusion: Early locoregional therapy for the primary site did not improve survival in patients presenting with metastatic breast cancer. Although it was associated with improved locoregional control, this had no overall impact on quality of life., Competing Interests: Lori J. GoldsteinStock and Other Ownership Interests: CureVacHonoraria: Daiichi Sankyo, Roche/Genentech, Amgen, Mylan, Merck, Eisai, Immunomedics, Exact SciencesConsulting or Advisory Role: Genentech, Genomic Health, Merck, Mylan, Immunomedics, Amgen, Eisai, Exact SciencesResearch Funding: Merck (Inst), Genentech/Roche (Inst)Other Relationship: Daiichi Sankyo David CellaStock and Other Ownership Interests: FACIT.orgConsulting or Advisory Role: AbbVie, GlaxoSmithKline, Pfizer, Astellas Pharma, Novartis, Bristol Myers Squibb, Asahi Kasei, Ipsen, Mei PharmaResearch Funding: Novartis (Inst), Ipsen (Inst), Pfizer (Inst), PledPharma (Inst), Bristol Myers Squibb (Inst), AbbVie (Inst), Regeneron (Inst), Clovis Oncology (Inst) Mark BasikHonoraria: Roche CanadaResearch Funding: Pfizer, LabCorp Mehra GolshanConsulting or Advisory Role: AbbVie, BertisResearch Funding: Breast Cancer Research Foundation Christine A. LeeConsulting or Advisory Role: OlympusSpeakers' Bureau: AstraZeneca/Merck, OncoCyte Joseph A. SparanoStock and Other Ownership Interests: MetaStatConsulting or Advisory Role: Genentech/Roche, Novartis, AstraZeneca, Celgene, Lilly, Celldex, Pfizer, Prescient Therapeutics, Juno Therapeutics, Merrimack, Adgero Biopharmaceuticals, Cardinal Health, Pfizer, GlaxoSmithKline, CStone Pharmaceuticals, Epic Sciences, Daiichi Sankyo, BMSiSpeakers' Bureau: Eisai, CertaraResearch Funding: Prescient Therapeutics (Inst), Deciphera (Inst), Genentech/Roche (Inst), Merck (Inst), Novartis (Inst), Novartis (Inst), Merrimack (Inst), Radius Health (Inst), Olema Pharmaceuticals (Inst)Travel, Accommodations, Expenses: Menarini Silicon Biosystems, Roche/Genentech, Adgero Biopharmaceuticals, Myriad Genetics, Pfizer, AstraZeneca, Rhenium Medical Gildy V. BabieraStock and Other Ownership Interests: PolyPidHonoraria: Insightec, GleolanConsulting or Advisory Role: Insightec, Gleolan, Theracal, Polypid, NektarPatents, Royalties, Other Intellectual Property: Patent Holder for DNX 2401 and DNX2440 Owned by DNAtrixTravel, Accommodations, Expenses: Gleolan, Insightec Sarika JainEmployment: G1 TherapeuticsStock and Other Ownership Interests: G1 Therapeutics Carla S. FisherConsulting or Advisory Role: Biom'UpTravel, Accommodations, Expenses: Biom'Up Amye J. TevaarwerkOther Relationship: Epic Systems Lynne I. WagnerStock and Other Ownership Interests: Johnson & Johnson, Lilly, Gilead SciencesConsulting or Advisory Role: Celgene, AthenexTravel, Accommodations, Expenses: Celgene George W. SledgeLeadership: Syndax, Tessa TherapeuticsStock and Other Ownership Interests: Syndax, Tessa Therapeutics, PionyrConsulting or Advisory Role: Symphogen, Synaffix, Syndax, Verseau Therapeutics, GRAIL, AstraZeneca, G1 TherapeuticsResearch Funding: Genentech/Roche (Inst), Pfizer (Inst)Travel, Accommodations, Expenses: Verseau Therapeutics, Tessa TherapeuticsNo other potential conflicts of interest were reported.

Purpose: To examine the proportion of older women with ER + HER2- breast cancer receiving non-operative management versus surgery, and to evaluate the use of axillary staging and adjuvant radiation in this population., Methods: We queried the SEER database to identify all women aged 70 years or older with stage I-III ER + HER2- invasive breast cancer diagnosed between 2010 and 2016. We evaluated trends in non-operative management, breast surgery, axillary staging, and adjuvant radiation according to age at diagnosis., Results: We identified 57,351 older women with ER + HER2- disease. Overall, 3538 (6.2%) of the cohort underwent non-operative management, 38,452 (67.0%) underwent breast-conserving surgery (BCS), and 15,361 (26.8%) underwent mastectomy. The proportion of patients undergoing non-operative management increased from 2.8% among 70-74-year-old women to 30.1% in those ≥ 90 years old (p < 0.001). In 53,813 women who underwent surgery, 36,850 (68.5%) underwent sentinel lymph node biopsy, while 10,861 (20.2%) underwent axillary lymph node dissection. Subgroup analysis of 29,032 older women undergoing BCS for stage I ER + HER2- breast cancer revealed a 14.2% rate of omission of axillary staging, increasing from 5.3% in those 70-74 years to 67.6% in those ≥ 90 years old (p < 0.001). Receipt of adjuvant radiation occurred in 63.3% of older women following BCS and 18% post-mastectomy, with similar trends towards omission in older age groups., Conclusion: Primary breast surgery remains the dominant management strategy for the majority of older women with ER + HER2- breast cancer. Omission of axillary staging and adjuvant radiation are used in a minority of eligible women undergoing breast conservation for early-stage disease., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN is often down-regulated in cancer, and there are conflicting data on PTEN's role as breast cancer biomarker. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody crossreactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. Our iMALDI assay only requires a low-cost benchtop MALDI-TOF mass spectrometer, which simplifies clinical translation. We validated our assay's precision and accuracy, with simultaneous enrichment of both target proteins not significantly affecting the precision and accuracy of the quantitation when compared to the PTEN- and p110α-singleplex iMALDI assays (<15% difference). The multiplexed assay's linear range is from 0.6-20 fmol with accuracies of 90-112% for both target proteins, and the assay is free of matrix-related interferences. The inter-day reproducibility over 5-days was high, with an overall CV of 9%. PTEN and p110α protein concentrations can be quantified down to 1.4 fmol and 0.6 fmol per 10 μg of total tumor protein, respectively, in various tumor tissue samples, including fresh-frozen breast tumors and colorectal cancer liver metastases, and patient-derived xenograft (PDX) tumors.

The tumor suppressor PTEN is the main negative regulator of PI3K/AKT/mTOR signaling and is commonly found downregulated in breast cancer (BC). Conflicting data from conventional immunoassays such as immunohistochemistry (IHC) has sparked controversy about PTEN's role as a prognostic and predictive biomarker in BC, which can be largely attributed to the lack of specificity, sensitivity, and interlaboratory standardization. Here, we present a fully standardized, highly sensitive, robust microflow immuno-MRM (iMRM) assay that enables precise quantitation of PTEN concentrations in cells and fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissues, down to 0.1 fmol/10 μg of extracted protein, with high interday and intraday precision (CV 6.3%). PTEN protein levels in BC PDX samples that were determined by iMRM correlate well with semiquantitative IHC and WB data. iMRM, however, allowed the precise quantitation of PTEN-even in samples that were deemed to be PTEN negative by IHC or western blot (WB)-while requiring substantially less tumor tissue than WB. This is particularly relevant because the extent of PTEN downregulation in tumors has been shown to correlate with severity. Our standardized and robust workflow includes an 11 min microflow LC-MRM analysis on a triple-quadrupole MS and thus provides a much needed tool for the study of PTEN as a potential biomarker for BC.

Breast cancer diagnosed within 10 years following childbirth is defined as postpartum breast cancer (PPBC) and is highly metastatic. Interactions between immune cells and other stromal cells within the involuting mammary gland are fundamental in facilitating an aggressive tumor phenotype. The MNK1/2-eIF4E axis promotes translation of prometastatic mRNAs in tumor cells, but its role in modulating the function of nontumor cells in the PPBC microenvironment has not been explored. Here, we used a combination of in vivo PPBC models and in vitro assays to study the effects of inactivation of the MNK1/2-eIF4E axis on the protumor function of select cells of the tumor microenvironment. PPBC mice deficient for phospho-eIF4E (eIF4E S209A ) were protected against lung metastasis and exhibited differences in the tumor and lung immune microenvironment compared with wild-type mice. Moreover, the expression of fibroblast-derived IL33, an alarmin known to induce invasion, was repressed upon MNK1/2-eIF4E axis inhibition. Imaging mass cytometry on PPBC and non-PPBC patient samples indicated that human PPBC contains phospho-eIF4E high-expressing tumor cells and CD8 + T cells displaying markers of an activated dysfunctional phenotype. Finally, inhibition of MNK1/2 combined with anti-PD-1 therapy blocked lung metastasis of PPBC. These findings implicate the involvement of the MNK1/2-eIF4E axis during PPBC metastasis and suggest a promising immunomodulatory route to enhance the efficacy of immunotherapy by blocking phospho-eIF4E. SIGNIFICANCE: This study investigates the MNK1/2-eIF4E signaling axis in tumor and stromal cells in metastatic breast cancer and reveals that MNK1/2 inhibition suppresses metastasis and sensitizes tumors to anti-PD-1 immunotherapy., (©2021 American Association for Cancer Research.)

Bioenergetic perturbations driving neoplastic growth increase the production of reactive oxygen species (ROS), requiring a compensatory increase in ROS scavengers to limit oxidative stress. Intervention strategies that simultaneously induce energetic and oxidative stress therefore have therapeutic potential. Phenformin is a mitochondrial complex I inhibitor that induces bioenergetic stress. We now demonstrate that inflammatory mediators, including IFNγ and polyIC, potentiate the cytotoxicity of phenformin by inducing a parallel increase in oxidative stress through STAT1-dependent mechanisms. Indeed, STAT1 signaling downregulates NQO1, a key ROS scavenger, in many breast cancer models. Moreover, genetic ablation or pharmacological inhibition of NQO1 using β-lapachone (an NQO1 bioactivatable drug) increases oxidative stress to selectively sensitize breast cancer models, including patient derived xenografts of HER2+ and triple negative disease, to the tumoricidal effects of phenformin. We provide evidence that therapies targeting ROS scavengers increase the anti-neoplastic efficacy of mitochondrial complex I inhibitors in breast cancer.

Purpose: Accrual to clinical trials that challenge well-established treatment paradigms represents a unique challenge. Physician opinions on investigation of a novel approach to breast cancer treatment, in which patients with complete response to neoadjuvant chemotherapy are offered omission of lumpectomy, are unknown. NRG-CC006 sought to describe physician attitudes toward a novel approach to breast cancer treatment., Methods: We recruited 18 participants in the fields of surgery, medical oncology, and radiation oncology to participate in the semi-structured telephone interviews. Main outcomes are qualitative themes associated with omission of surgery., Results: Of 18 interview participants, specialty and gender were evenly represented across surgery, medical oncology, and radiation oncology. Qualitative themes included general attitudes toward treatment de-escalation, stakeholder considerations, and trial/protocol considerations. The vast majority of participants expressed interest in investigation of omission of surgery, with all participants endorsing need for further investigation into treatment de-escalation. Stakeholder considerations in opening such a trial emphasized need for multidisciplinary involvement and, particularly, the unique role of surgeons as gatekeepers in breast cancer treatment. Finally, participants endorsed a need for further foundational studies to develop ways to predict complete pathologic response to chemotherapy without surgical intervention., Conclusions: Physicians expressed interest in investigating a novel approach to breast cancer treatment that would omit surgery in complete responders to neoadjuvant chemotherapy. Multidisciplinary input, and specifically surgeon engagement, will be key to the success of future investigations. Ongoing work to develop approaches to predict pathologic complete response accurately is needed to achieve the promise of this idea. ClinTrials #: BR005: NCT03188393 June 13, 2017.

Background: The oncologic safety of sentinel lymph node biopsy (SLNB) alone for clinically node-positive (cN1-2) patients who convert to pathologic node-negativity (ypN0) after neoadjuvant chemotherapy (NAC) is not well established., Methods: This study retrospectively identified 244 consecutive patients with a diagnosis of cT1-3cN0-2 breast cancer who underwent NAC followed by SLNB at the authors' institution between 2013 and 2018. The patients were categorized as clinically node-negative (cN0) or cN1-2 before the onset of NAC, and the Kaplan-Meier method was used to compare locoregional and distant recurrence rates after SLNB alone for ypN0 patients., Results: Among 244 patients who underwent NAC followed by surgery with SLNB for axillary staging, 112 (45.9%) were cN0 at presentation, whereas 132 (54.5%) had biopsy-proven cN1-2 disease and converted to cN0 after treatment. Of the patients presenting with cN0 disease, 102 (91.1%) were ypN0 on SLNB pathology compared with 60 cN1/2 patients (45.5%; p < 0.001). Regional nodal irradiation was administered to 5% of the cN0/ypN0 patients compared with 70.7% of the cN1-2/ypN0 patients (p < 0.001). Overall, 211 patients were treated with SLNB alone and had a median follow-up period of 36 months (interquartile range [IQR], 24-53 months). For 101 cN0/ypN0 patients who underwent SLNB alone, the 5-year local and regional recurrence rates were respectively 5.7% (95% confidence interval [CI], 2.4-13.8) and 1% (95% CI 0.1-7.0). For 58 cN1-2/ypN0 patients who underwent SLNB alone, the 5-year local and regional recurrence rates were respectively 4.1% (95% CI 1.0-15.5) and 0%, with no axillary recurrences noted., Conclusion: For ypN0 patients, SLNB alone after NAC is associated with low and acceptable short-term axillary recurrence rates. Additional follow-up data from prospective clinical trials are needed to confirm long-term oncologic safety and define optimal local therapy recommendations.

Background: Cancer cells cooperate with cells that compose their environment to promote tumor growth and invasion. Among them, adipocytes provide lipids used as a source of energy by cancer cells and adipokines that contribute to tumor expansion. Mechanisms supporting the dynamic interactions between cancer cells and stromal adipocytes, however, remain unclear., Methods: We set-up a co-culture model with breast cancer cells grown in 3D as mammospheres and human adipocytes to accurately recapitulate intrinsic features of tumors, such as hypoxia and cancer cell-adipocytes interactions., Results: Herein, we observed that the lipid droplets' size was reduced in adipocytes adjacent to the mammospheres, mimicking adipocyte morphology on histological sections. We showed that the uncoupling protein UCP1 was expressed in adipocytes close to tumor cells on breast cancer histological sections as well as in adipocytes in contact with the mammospheres. Mammospheres produced adrenomedullin (ADM), a multifactorial hypoxia-inducible peptide while ADM receptors were detected in adipocytes. Stimulation of adipocytes with ADM promoted UCP1 expression and increased HSL phosphorylation, which activated lipolysis. Invalidation of ADM in breast cancer cells dramatically reduced UCP1 expression in adipocytes., Conclusions: Breast tumor cells secreted ADM that modified cancer-associated adipocytes through paracrine signaling, leading to metabolic changes and delipidation. Hence, ADM appears to be crucial in controlling the interactions between cancer cells and adipocytes and represents an excellent target to hinder them.

Background: Nipple-sparing mastectomy (NSM) remains controversial in patients with high-risk breast cancer. The objective of this study was to assess surgical and oncologic outcomes of NSM and to evaluate associations of outcomes with high-risk features., Methods: A retrospective review was conducted of all NSM cases performed for breast cancer at 2 academic cancer centers between January 2013 and August 2018., Results: Of the 175 patients who underwent NSM, 13 (7.4%) had locally advanced breast cancer (LABC), 52 (29.2%) had previous neoadjuvant chemotherapy, 21 (12.0%) had previous radiation therapy, 40 (22.8%) received postmastectomy radiation, 27 (15.4%) had de-epithelialized skin reduction, and 13 (7.4%) had free nipple grafting. The median duration of follow-up was 24 months. Nipple necrosis (4 cases; 2.2%) was associated with previous radiation (9.5%; P = .018), skin reduction (11.1%; P = .001), and nipple grafting (15.4%; P = .001). The nipple-areolar complex margin (NAC) was involved with invasive disease in 1 case. Local recurrence occurred in 8 cases (4.6%), with 1 in-NAC recurrence. Overall survival was 98.3%, and disease-free survival (DFS) was 88.6%. LABC was associated with worse DFS (hazard ratio, 4.28; P = .011), with all 4 recurrences being distant., Conclusions: Previous radiation, skin reduction, and nipple grafting are associated with an increased risk of NAC necrosis. None of these should be considered absolute contraindications, but patients should be counseled appropriately. Although LABC is associated with worse DFS, relapses are systemic. Longer follow-up is needed to establish oncologic safety in unselected breast cancer patients., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Subsets of breast tumors present major clinical challenges, including triple-negative, metastatic/recurrent disease and rare histologies. Here, we developed 37 patient-derived xenografts (PDX) from these difficult-to-treat cancers to interrogate their molecular composition and functional biology. Whole-genome and transcriptome sequencing and reverse-phase protein arrays revealed that PDXs conserve the molecular landscape of their corresponding patient tumors. Metastatic potential varied between PDXs, where low-penetrance lung micrometastases were most common, though a subset of models displayed high rates of dissemination in organotropic or diffuse patterns consistent with what was observed clinically. Chemosensitivity profiling was performed in vivo with standard-of-care agents, where multi-drug chemoresistance was retained upon xenotransplantation. Consolidating chemogenomic data identified actionable features in the majority of PDXs, and marked regressions were observed in a subset that was evaluated in vivo. Together, this clinically-annotated PDX library with comprehensive molecular and phenotypic profiling serves as a resource for preclinical studies on difficult-to-treat breast tumors.

Purpose: The microenvironment of metastatic breast cancer is incompletely characterized, despite prior evidence that it plays a key role in the biology of metastasis. A major component of the tumor stroma is the carcinoma-associated fibroblast (CAF), which has been shown to communicate with other stromal and cancer cells to create a protumorigenic milieu. Our study was designed to characterize human CAFs from different metastatic sites., Experimental Design: We collected eight carcinoma-associated fibroblasts (mCAFs) from different metastatic sites and compared them with CAFs from primary tumors (pCAFs) and with normal breast fibroblasts (NFs). Molecular profiles and effects on breast cancer cell growth, on response to doxorubicin and on T-cell proliferation were compared., Results: We observed marked differences in mCAFs compared with pCAFs and NFs with respect to in vitro proliferation and effects on breast cancer cell migration, spheroid growth, invasion, response to doxorubicin, and in vivo tumor growth. We found marked transcriptomic differences between mCAFs and pCAFs, including increased expression of IFN-related genes and IGF2 in the former. Cluster analysis revealed two groups of mCAFs, with the liver mCAFs clustering together, with increased PDGFA expression. Treatment with an antibody against insulin-like growth factors (BI836845) inhibited growth of mixed mCAF-tumor cell xenografts in vivo . Also, mCAFs had a suppressive effect on T-cell proliferation., Conclusions: This is the first comparative analysis of a set of CAFs from metastatic sites in breast cancer. It revealed a marked protumorigenic effect in these mCAFs, which occurs in part through increased expression of IGF2., (©2019 American Association for Cancer Research.)

Background: The sentinel node biopsy following neoadjuvant chemotherapy (SN FNAC) study has shown that in node-positive (N+) breast cancer, sentinel node biopsy (SNB) can be performed following neoadjuvant chemotherapy (NAC), with a low false negative rate (FNR = 8.4%). A secondary endpoint of the SN FNAC study was to determine whether axillary ultrasound (AxUS) could predict axillary pathological complete response (ypN0) and increase the accuracy of SNB., Methods: The SN FNAC trial is a study of patients with biopsy-proven N+ breast cancer who underwent SNB followed by completion node dissection. All patients had AxUS following NAC and the axillary nodes were classified as either positive (AxUS+) or negative (AxUS-). AxUS was compared with the final axillary pathology results., Results: There was no statistical difference in the baseline characteristics of patients with AxUS+ versus those with AxUS-. Overall, 82.5% (47/57) of AxUS+ patients had residual positive lymph nodes (ypN+) at surgery and 53.8% (42/78) of AxUS- patients had ypN+. Post NAC AxUS sensitivity was 52.8%, specificity 78.3%, and negative predictive value 46.2%. AxUS FNR was 47.2%, versus 8.4% for SNB. If post-NAC AxUS- was used to select patients for SNB, FNR would decrease from 8.4 to 2.7%. However, using post-NAC AxUS in addition to SNB as an indication for ALND would have led to unnecessary ALND in 7.8% of all patients., Conclusion: AxUS is not appropriate as a standalone staging procedure, and SNB itself is sufficient to assess the axilla post NAC in patients who present with N+ breast cancer.

Dysregulation of histone modifications promotes carcinogenesis by altering transcription. Breast cancers frequently overexpress the histone methyltransferase EZH2, the catalytic subunit of Polycomb Repressor Complex 2 (PRC2). However, the role of EZH2 in this setting is unclear due to the context-dependent functions of PRC2 and the heterogeneity of breast cancer. Moreover, the mechanisms underlying PRC2 overexpression in cancer are obscure. Here, using multiple models of breast cancer driven by the oncogene ErbB2, we show that the tyrosine kinase c-Src links energy sufficiency with PRC2 overexpression via control of mRNA translation. By stimulating mitochondrial ATP production, c-Src suppresses energy stress, permitting sustained activation of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which increases the translation of mRNAs encoding the PRC2 subunits Ezh2 and Suz12. We show that Ezh2 overexpression and activity are pivotal in ErbB2-mediated mammary tumourigenesis. These results reveal the hitherto unknown c-Src/mTORC1/PRC2 axis, which is essential for ErbB2-driven carcinogenesis.

Trastuzumab is integral to HER2+ cancer treatment, but its therapeutic index is narrowed by the development of resistance. Phosphorylation of the translation initiation factor eIF2α (eIF2α-P) is the nodal point of the integrated stress response, which promotes survival or death in a context-dependent manner. Here, we show an anti-tumor function of the protein kinase PKR and its substrate eIF2α in a mouse HER2+ breast cancer model. The anti-tumor function depends on the transcription factor ATF4, which upregulates the CDK inhibitor P21 CIP1 and activates JNK1/2. The PKR/eIF2α-P arm is induced by Trastuzumab in sensitive but not resistant HER2+ breast tumors. Also, eIF2α-P stimulation by the phosphatase inhibitor SAL003 substantially increases Trastuzumab potency in resistant HER2+ breast and gastric tumors. Increased eIF2α-P prognosticates a better response of HER2+ metastatic breast cancer patients to Trastuzumab therapy. Hence, the PKR/eIF2α-P arm antagonizes HER2 tumorigenesis whereas its pharmacological stimulation improves the efficacy of Trastuzumab therapy.

The mechanisms by which breast cancers progress from relatively indolent ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) are not well understood. However, this process is critical to the acquisition of metastatic potential. MAPK-interacting serine/threonine-protein kinase 1 (MNK1) signaling can promote cell invasion. NODAL, a morphogen essential for embryogenic patterning, is often reexpressed in breast cancer. Here we describe a MNK1/NODAL signaling axis that promotes DCIS progression to IDC. We generated MNK1 knockout (KO) or constitutively active MNK1 (caMNK1)-expressing human MCF-10A-derived DCIS cell lines, which were orthotopically injected into the mammary glands of mice. Loss of MNK1 repressed NODAL expression, inhibited DCIS to IDC conversion, and decreased tumor relapse and metastasis. Conversely, caMNK1 induced NODAL expression and promoted IDC. The MNK1/NODAL axis promoted cancer stem cell properties and invasion in vitro . The MNK1/2 inhibitor SEL201 blocked DCIS progression to invasive disease in vivo . In clinical samples, IDC and DCIS with microinvasion expressed higher levels of phospho-MNK1 and NODAL versus low-grade (invasion-free) DCIS. Cumulatively, our data support further development of MNK1 inhibitors as therapeutics for preventing invasive disease. SIGNIFICANCE: These findings provide new mechanistic insight into progression of ductal carcinoma and support clinical application of MNK1 inhibitors to delay progression of indolent ductal carcinoma in situ to invasive ductal carcinoma., (©2019 American Association for Cancer Research.)

Emerging evidence has illustrated the importance of epigenomic reprogramming in cancer, with altered post-translational modifications of histones contributing to pathogenesis. However, the contributions of histone modifiers to breast cancer progression are unclear, and how these processes vary between molecular subtypes has yet to be adequately addressed. Here we report that genetic or pharmacological targeting of the epigenetic modifier Ezh2 dramatically hinders metastatic behaviour in both a mouse model of breast cancer and patient-derived xenografts reflective of the Luminal B subtype. We further define a subtype-specific molecular mechanism whereby EZH2 maintains H3K27me3-mediated repression of the FOXC1 gene, thereby inactivating a FOXC1-driven, anti-invasive transcriptional program. We demonstrate that higher FOXC1 is predictive of favourable outcome specifically in Luminal B breast cancer patients and establish the use of EZH2 methyltransferase inhibitors as a viable strategy to block metastasis in Luminal B breast cancer, where options for targeted therapy are limited.

AXL is activated by its ligand GAS6 and is expressed in triple-negative breast cancer cells. In the current study, we report AXL expression in HER2-positive (HER2 + ) breast cancers where it correlates with poor patient survival. Using murine models of HER2 + breast cancer, Axl, but not its ligand Gas6, was found to be essential for metastasis. We determined that AXL is required for intravasation, extravasation, and growth at the metastatic site. We found that AXL is expressed in HER2 + cancers displaying epithelial-to-mesenchymal transition (EMT) signatures where it contributes to sustain EMT. Interfering with AXL in a patient-derived xenograft (PDX) impaired transforming growth factor β (TGF-β)-induced cell invasion. Last, pharmacological inhibition of AXL specifically decreased the metastatic burden of mice developing HER2 + breast cancer. Our data identify AXL as a potential anti-metastatic co-therapeutic target for the treatment of HER2 + breast cancers., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

With current advances in neoadjuvant systemic therapy (NST) and improved breast imaging, the potential of nonoperative therapy for invasive breast cancer has emerged as a viable option when utilizing meticulous image-guided percutaneous biopsy to document pathologic complete response. Feasibility clinical trials utilizing this approach are being performed by teams of investigators from single and multicenter/cooperative groups around the world. Imaging alone after NST lacks sufficient sensitivity and specificity in predicting pCR and therefore cannot be utilized for clinical selection of patients for omission of surgery. Imaging with adequate sampling after NST of the residual lesions (or around the remaining clip if a complete radiologic response occurs) appears to be essential in selecting patients with pCR to lower the false-negative rates based on initial reported feasibility studies to identify pCR without surgery that range from 5 to 49%. In this manuscript, recently completed, ongoing, and planned clinical feasibility trials and a new omission of surgery trial are described. Drastic rethinking of all diagnostic and therapeutic management strategies that are ordinarily utilized for patients who receive standard breast cancer surgery is required. A roadmap of essential questions and issues that will have to be resolved as the field of nonoperative breast cancer management advances is described in detail.

Background: The primary cilium is a microtubule-based and nonmotile organelle functioning as a cellular antenna that is involved in the regulation of cell proliferation, differentiation, and migration. In breast cancer cells, the primary cilium is a structure that decreases in incidence with increasing degrees of transformation and may be biologically more important in estrogen receptor (ERα)-negative breast cancer cells. Split ends (SPEN) is an ERα corepressor that we have identified as a tumor suppressor protein in ERα-positive breast cancer cells whose hormone-independent roles in breast cancer have never been explored., Methods: We determined the hormone-independent transcriptional program regulated by the ERα cofactor SPEN in breast cancer using DNA microarrays. The biological functions regulated by SPEN independently of hormones were studied in vitro in ERα-positive and ERα-negative breast cancer cells. Finally, we examined the clinical relevance of SPEN expression in cohorts of breast cancer samples with outcome data., Results: We found that SPEN is coexpressed with a number of genes involved in ciliary biology, including the ciliogenic transcription factor RFX3, in a hormone-independent manner. SPEN reexpression in T47D cells containing a nonsense mutation in SPEN restored the primary cilium, whereas its knockdown in MCF10A and Hs578T cells considerably decreased primary cilia levels. We also report that SPEN regulates migration in breast cells, but only in those harboring primary cilia, and that KIF3A silencing, a critical factor in primary cilia, partially reverses SPEN's effects, suggesting that SPEN may coordinate cellular movement through primary cilia-dependent mechanisms. Finally, we found that high SPEN RNA levels were predictive of early metastasis in two independent cohorts of 77 (HR 2.25, P = 0.03) and 170 (HR = 2.23, P = 0.004) patients with ERα-negative breast cancer., Conclusions: Together, our data demonstrate a role for SPEN in the regulation of primary cilia formation and cell migration in breast cancer cells, which may collectively explain why its expression is associated with time to metastasis in cohorts of patients with ERα-negative breast cancers.

Purpose: AZD8931 is an orally bioavailable, reversible tyrosine kinase inhibitor of EGFR, HER2, and HER3 signaling. The Phase II MINT study (ClinicalTrials.gov NCT01151215) investigated whether adding AZD8931 to endocrine therapy would delay development of endocrine resistance in patients with hormone-sensitive advanced breast cancer., Methods: Patients were randomized 1:1:1 to receive daily anastrozole (1 mg) in combination with AZD8931 20 mg twice daily (bid), AZD8931 40 mg bid, or placebo. The primary objective was evaluation of progression-free survival (PFS) in patients treated with combination AZD8931 and anastrozole versus anastrozole alone. Secondary objectives included assessment of safety and tolerability, objective response rate, and overall survival., Results: At the interim analysis, 359 patients were randomized and received anastrozole in combination with AZD8931 20 mg (n = 118), 40 mg (n = 120), or placebo (n = 121); 39 % of patients (n = 141) had a progression event. Median PFS (HR; 95 % CI vs placebo) in the AZD8931 20, 40 mg, and placebo arms was 10.9 (1.37; 0.91-2.06, P = 0.135), 13.8 (1.16; 0.77-1.75, P = 0.485), and 14.0 months, respectively. No indication of clinical benefit was observed following treatment with AZD8931 for the secondary endpoints. Safety findings showed a greater incidence of diarrhea (40, 51, and 12 % for AZD8931 20, 40 mg, and placebo, respectively), rash (32, 48, and 12 %), dry skin (19, 25, and 2 %), and acneiform dermatitis (16, 28, and 2 %) in patients treated with AZD8931 versus placebo., Conclusions: AZD8931, in combination with endocrine therapy, does not appear to enhance endocrine responsiveness and is associated with greater skin and gastrointestinal toxicity.

Approximately 70% of breast cancers express the estrogen receptor (ER)α and are treated with the ERα antagonist, tamoxifen. However, resistance to tamoxifen frequently develops in advanced breast cancer, in part due to a down-regulation of ERα corepressors. Nuclear receptor corepressors function by attenuating hormone responses and have been shown to potentiate tamoxifen action in various biological systems. Recent genomic data on breast cancers has revealed that genetic and/or genomic events target ERα corepressors in the majority of breast tumors, suggesting that the loss of nuclear receptor corepressor activity may represent an important mechanism that contributes to intrinsic and acquired tamoxifen resistance. Here, the biological functions of ERα corepressors are critically reviewed to elucidate their role in modifying endocrine sensitivity in breast cancer. We highlight a mechanism of gene repression common to corepressors previously shown to enhance the antitumorigenic effects of tamoxifen, which involves the recruitment of histone deacetylases (HDACs) to DNA. As an indicator of epigenetic disequilibrium, the loss of ERα corepressors may predispose cancer cells to the cytotoxic effects of HDAC inhibitors, a class of drug that has been shown to effectively reverse tamoxifen resistance in numerous studies. HDAC inhibition thus appears as a promising therapeutic approach that deserves to be further explored as an avenue to restore drug sensitivity in corepressor-deficient and tamoxifen-resistant breast cancers.

The treatment of breast cancer has benefitted tremendously from the generation of estrogen receptor-α (ERα)-targeted therapies, but disease relapse continues to pose a challenge due to intrinsic or acquired drug resistance. In an effort to delineate potential predictive biomarkers of therapy responsiveness, multiple groups have identified several uncharacterized cofactors and interacting partners of ERα, including Split Ends (SPEN), a transcriptional corepressor. Here, we demonstrate a role for SPEN in ERα-expressing breast cancers. SPEN nonsense mutations were detectable in the ERα-expressing breast cancer cell line T47D and corresponded to undetectable protein levels. Further analysis of 101 primary breast tumors revealed that 23% displayed loss of heterozygosity at the SPEN locus and that 3% to 4% harbored somatically acquired mutations. A combination of in vitro and in vivo functional assays with microarray-based pathway analyses showed that SPEN functions as a tumor suppressor to regulate cell proliferation, tumor growth, and survival. We also found that SPEN binds ERα in a ligand-independent manner and negatively regulates the transcription of ERα targets. Moreover, we demonstrate that SPEN overexpression sensitizes hormone receptor-positive breast cancer cells to the apoptotic effects of tamoxifen, but has no effect on responsiveness to fulvestrant. Consistent with these findings, two independent datasets revealed that high SPEN protein and RNA expression in ERα-positive breast tumors predicted favorable outcome in patients treated with tamoxifen alone. Together, our data suggest that SPEN is a novel tumor-suppressor gene that may be clinically useful as a predictive biomarker of tamoxifen response in ERα-positive breast cancers., (©2015 American Association for Cancer Research.)

Metabolic reprogramming is a hallmark of cellular transformation, yet little is known about metabolic changes that accompany tumor metastasis. Here we show that primary breast cancer cells display extensive metabolic heterogeneity and engage distinct metabolic programs depending on their site of metastasis. Liver-metastatic breast cancer cells exhibit a unique metabolic program compared to bone- or lung-metastatic cells, characterized by increased conversion of glucose-derived pyruvate into lactate and a concomitant reduction in mitochondrial metabolism. Liver-metastatic cells displayed increased HIF-1α activity and expression of the HIF-1α target Pyruvate dehydrogenase kinase-1 (PDK1). Silencing HIF-1α reversed the glycolytic phenotype of liver-metastatic cells, while PDK1 was specifically required for metabolic adaptation to nutrient limitation and hypoxia. Finally, we demonstrate that PDK1 is required for efficient liver metastasis, and its expression is elevated in liver metastases from breast cancer patients. Our data implicate PDK1 as a key regulator of metabolism and metastatic potential in breast cancer., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Cancer-associated fibroblasts (CAFs) play an important role in breast cancer pathogenesis by paracrine regulation of breast cancer cell biology. Several in vitro and mouse models have characterized the role of cell contact and cytokine molecules mediating this relationship, although few reports have used human CAFs from breast tumors., Methods: Primary breast CAF cultures were established and gene expression profiles analysed in order to guide subsequent co-culture models. We used a combination of colorimetric proliferation assays and gene expression profiling to determine the effect of CAFs on the MCF-7 breast cancer cell in an indirect co-culture system., Results: Using gene expression profiling, we found that a subgroup of breast CAFs are positive for a type one interferon response, confirming previous reports of an activated type one interferon response in whole tumor datasets. Interferon positive breast cancer patients show a poor prognostic outcome in an independent microarray dataset. In addition, CAFs positive for the type one interferon response promoted the growth of the MCF-7 breast cancer cell line in an indirect co-culture model. The addition of a neutralizing antibody against the ligand mediating the type one response in fibroblasts, interferon-β, reverted this co-culture phenotype. CAFs not expressing the interferon response genes also promoted the growth of the MCF-7 breast cancer cell line but this phenotype was independent of the type one fibroblast interferon ligand., Conclusions: Primary breast CAFs show inter-patient molecular heterogeneity as evidenced by interferon response gene elements activated in a subgroup of CAFs, which result in paracrine pro-proliferative effects in a breast cancer cell line co-culture model.

Purpose: An increasing proportion of patients (> 30%) with node-positive breast cancer will obtain an axillary pathologic complete response after neoadjuvant chemotherapy (NAC). If sentinel node (SN) biopsy (SNB) is accurate in this setting, completion node dissection (CND) morbidity could be avoided., Patients and Methods: In the prospective multicentric SN FNAC study, patients with biopsy-proven node-positive breast cancer (T0-3, N1-2) underwent both SNB and CND. Immunohistochemistry (IHC) use was mandatory, and SN metastases of any size, including isolated tumor cells (ypN0[i+], ≤ 0.2 mm), were considered positive. The optimal SNB identification rate (IR) ≥ 90% and false-negative rate (FNR) ≤ 10% were predetermined., Results: From March 2009 to December 2012, 153 patients were accrued to the study. The SNB IR was 87.6% (127 of 145; 95% CI, 82.2% to 93.0%), and the FNR was 8.4% (seven of 83; 95% CI, 2.4% to 14.4%). If SN ypN0(i+)s had been considered negative, the FNR would have increased to 13.3% (11 of 83; 95% CI, 6.0% to 20.6%). There was no correlation between size of SN metastases and rate of positive non-SNs. Using this method, 30.3% of patients could potentially avoid CND., Conclusion: In biopsy-proven node-positive breast cancer after NAC, a low SNB FNR (8.4%) can be achieved with mandatory use of IHC. SN metastases of any size should be considered positive. The SNB IR was 87.6%, and in the presence of a technical failure, axillary node dissection should be performed. We recommend that SN evaluation with IHC be further evaluated before being included in future guidelines on the use of SNB after NAC in this setting., (© 2014 by American Society of Clinical Oncology.)

Background: Chromosomal breakage followed by faulty DNA repair leads to gene amplifications and deletions in cancers. However, the mere assessment of the extent of genomic changes, amplifications and deletions may reduce the complexity of genomic data observed by array comparative genomic hybridization (array CGH). We present here a novel approach to array CGH data analysis, which focuses on putative breakpoints responsible for rearrangements within the genome., Results: We performed array comparative genomic hybridization in 29 primary tumors from high risk patients with breast cancer. The specimens were flow sorted according to ploidy to increase tumor cell purity prior to array CGH. We describe the number of chromosomal breaks as well as the patterns of breaks on individual chromosomes in each tumor. There were differences in chromosomal breakage patterns between the 3 clinical subtypes of breast cancers, although the highest density of breaks occurred at chromosome 17 in all subtypes, suggesting a particular proclivity of this chromosome for breaks. We also observed chromothripsis affecting various chromosomes in 41% of high risk breast cancers., Conclusions: Our results provide a new insight into the genomic complexity of breast cancer. Genomic instability dependent on chromosomal breakage events is not stochastic, targeting some chromosomes clearly more than others. We report a much higher percentage of chromothripsis than described previously in other cancers and this suggests that massive genomic rearrangements occurring in a single catastrophic event may shape many breast cancer genomes.

Most women with estrogen receptor expressing breast cancers receiving anti-estrogens such as tamoxifen may not need or benefit from them. Besides the estrogen receptor, there are no predictive biomarkers to help select breast cancer patients for tamoxifen treatment. CCND1 (cyclin D1) gene amplification is a putative candidate tamoxifen predictive biomarker. The RSF1 (remodeling and spacing factor 1) gene is frequently co-amplified with CCND1 on chromosome 11q. We validated the predictive value of these biomarkers in the MA.12 randomized study of adjuvant tamoxifen vs. placebo in high-risk premenopausal early breast cancer. Premenopausal women with node-positive/high-risk node-negative early breast cancer received standard adjuvant chemotherapy and then were randomized to tamoxifen (20 mg/day) or placebo for 5 yrs. Overall survival (OS) and relapse-free survival (RFS) were evaluated. Fluorescent in-situ hybridization was performed on a tissue microarray of 495 breast tumors (74% of patients) to measure CCND1 and RSF1 copy number. A multivariate Cox model to obtain hazard ratios (HR) adjusting for clinico-pathologic factors was used to assess the effect of these biomarkers on Os and RFS. 672 women were followed for a median of 8.4 years. We were able to measure the DNA copy number of CCND1 in 442 patients and RSF1 in 413 patients. CCND1 gene amplification was observed in 8.7% and RSF1 in 6.8% of these patients, preferentially in estrogen receptor-positive breast cancers. No statistically significant interaction with treatment was observed for either CCND1 or RSF1 amplification, although patients with high RSF1 copy number did not show benefit from adjuvant tamoxifen (HR = 1.11, interaction p = 0.09). Unlike CCND1 amplification, RSF1 amplification may predict for outcome in high-risk premenopausal breast cancer patients treated with adjuvant tamoxifen.

To examine the significance of intratumor genetic heterogeneity (ITGH) of the androgen receptor (AR) gene in breast cancer, patient-matched samples of laser capture microdissected breast tumor cells, adjacent normal breast epithelia cells, and peripheral blood leukocytes were sequenced using a novel next generation sequencing protocol. This protocol measured the frequency of distribution of a variable AR CAG repeat length, a functional polymorphism associated with breast cancer risk. All samples exhibited some degree of ITGH with up to 30 CAG repeat length variants identified. Each type of tissue exhibited a different distribution profile of CAG repeat lengths with substantial differences in the frequencies of zero and 18-25 CAG AR variants. Tissue differences in the frequency of ARs with each of these CAG repeat lengths were significant as measured by paired, twin t-tests. These results suggest that preferential selection of 18-25 CAG repeat length variants in breast tumors may be associated with breast cancer, and support the observation that shorter CAG repeats may protect against breast cancer. They also suggest that merely identifying variant genes will be insufficient to determine the critical mutational events of oncogenesis, which will require measuring the frequency of distribution of mutations within cancerous and matching normal tissues., (© 2013 Wiley Periodicals, Inc.)

Introduction: High prion protein (PrP) levels are associated with breast, colon and gastric cancer resistance to treatment and with a poor prognosis for the patients. However, little is known about the underlying molecular mechanism(s) regulating human PrP gene (PRNP) expression in cancers. Because endoplasmic reticulum (ER) stress is associated with solid tumors, we investigated a possible regulation of PRNP gene expression by ER stress., Methods: Published microarray databases of breast cancer tissues and breast carcinoma cell lines were analyzed for PrP mRNA and ER stress marker immunoglobulin heavy chain binding protein (BiP) levels. Breast cancer tissue microarrays (TMA) were immunostained for BiP and PrP. Breast carcinoma MCF-7, MDA-MB-231, HS578T and HCC1500 cells were treated with three different ER stressors - Brefeldin A, Tunicamycin, Thapsigargin - and levels of PrP mRNA or protein assessed by RT-PCR and Western blot analyses. A human PRNP promoter-luciferase reporter was used to assess transcriptional activation by ER stressors. Site-directed mutagenesis identified the ER stress response elements (ERSE). Chromatin immunoprecipitation (ChIP) analyses were done to identify the ER stress-mediated transcriptional regulators. The role of cleaved activating transcription factor 6α (ΔATF6α) and spliced X-box protein-1 (sXBP1) in PRNP gene expression was assessed with over-expression or silencing techniques. The role of PrP protection against ER stress was assessed with PrP siRNA and by using Prnp null cell lines., Results: We find that mRNA levels of BiP correlated with PrP transcript levels in breast cancer tissues and breast carcinoma cell lines. PrP mRNA levels were enriched in the basal subtype and were associated with poor prognosis in breast cancer patients. Higher PrP and BiP levels correlated with increasing tumor grade in TMA. ER stress was a positive regulator of PRNP gene transcription in MCF-7 cells and luciferase reporter assays identified one ER stress response element (ERSE) conserved among primates and rodents and three primate-specific ERSEs that regulated PRNP gene expression. Among the various transactivators of the ER stress-regulated unfolded protein response (UPR), ATF6α and XBP1 transactivated PRNP gene expression, but the ability of these varied in different cell types. Functionally, PrP delayed ER stress-induced cell death., Conclusions: These results establish PRNP as a novel ER stress-regulated gene that could increase survival in breast cancers.

Stroma in the tumor microenvironment plays a critical role in cancer progression, but how it promotes metastasis is poorly understood. Exosomes are small vesicles secreted by many cell types and enable a potent mode of intercellular communication. Here, we report that fibroblast-secreted exosomes promote breast cancer cell (BCC) protrusive activity and motility via Wnt-planar cell polarity (PCP) signaling. We show that exosome-stimulated BCC protrusions display mutually exclusive localization of the core PCP complexes, Fzd-Dvl and Vangl-Pk. In orthotopic mouse models of breast cancer, coinjection of BCCs with fibroblasts dramatically enhances metastasis that is dependent on PCP signaling in BCCs and the exosome component, Cd81 in fibroblasts. Moreover, we demonstrate that trafficking in BCCs promotes tethering of autocrine Wnt11 to fibroblast-derived exosomes. This work reveals an intercellular communication pathway whereby fibroblast exosomes mobilize autocrine Wnt-PCP signaling to drive BCC invasive behavior., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes.

Tumor-suppressor genes (TSGs) have been classically defined as genes whose loss of function in tumor cells contributes to the formation and/or maintenance of the tumor phenotype. TSGs containing nonsense mutations may not be expressed because of nonsense-mediated RNA decay (NMD). We combined inhibition of the NMD process, which clears transcripts that contain nonsense mutations, with the application of high-density single-nucleotide polymorphism arrays analysis to discriminate allelic content in order to identify candidate TSGs in five breast cancer cell lines. We identified ARID1A as a target of NMD in the T47D breast cancer cell line, likely as a consequence of a mutation in exon-9, which introduces a premature stop codon at position Q944. ARID1A encodes a human homolog of yeast SWI1, which is an integral member of the hSWI/SNF complex, an ATP-dependent, chromatin-remodeling, multiple-subunit enzyme. Although we did not find any somatic mutations in 11 breast tumors, which show DNA copy-number loss at the 1p36 locus adjacent to ARID1A, we show that low ARID1A RNA or nuclear protein expression is associated with more aggressive breast cancer phenotypes, such as high tumor grade, in two independent cohorts of over 200 human breast cancer cases each. We also found that low ARID1A nuclear expression becomes more prevalent during the later stages of breast tumor progression. Finally, we found that ARID1A re-expression in the T47D cell line results in significant inhibition of colony formation in soft agar. These results suggest that ARID1A may be a candidate TSG in breast cancer.

Background: Gene fusions arising from chromosomal translocations have been implicated in cancer. However, the role of gene fusions in BRCA1-related breast cancers is not well understood. Mutations in BRCA1 are associated with an increased risk for breast cancer (up to 80% lifetime risk) and ovarian cancer (up to 50%). We sought to identify putative gene fusions in the transcriptomes of these cancers using high-throughput RNA sequencing (RNA-Seq)., Methods: We used Illumina sequencing technology to sequence the transcriptomes of five BRCA1-mutated breast cancer cell lines, three BRCA1-mutated primary tumors, two secretory breast cancer primary tumors and one non-tumorigenic breast epithelial cell line. Using a bioinformatics approach, our initial attempt at discovering putative gene fusions relied on analyzing single-end reads and identifying reads that aligned across exons of two different genes. Subsequently, latter samples were sequenced with paired-end reads and at longer cycles (producing longer reads). We then refined our approach by identifying misaligned paired reads, which may flank a putative gene fusion junction., Results: As a proof of concept, we were able to identify two previously characterized gene fusions in our samples using both single-end and paired-end approaches. In addition, we identified three novel in-frame fusions, but none were recurrent. Two of the candidates, WWC1-ADRBK2 in HCC3153 cell line and ADNP-C20orf132 in a primary tumor, were confirmed by Sanger sequencing and RT-PCR. RNA-Seq expression profiling of these two fusions showed a distinct overexpression of the 3' partner genes, suggesting that its expression may be under the control of the 5' partner gene's regulatory elements., Conclusions: In this study, we used both single-end and paired-end sequencing strategies to discover gene fusions in breast cancer transcriptomes with BRCA1 mutations. We found that the use of paired-end reads is an effective tool for transcriptome profiling of gene fusions. Our findings suggest that while gene fusions are present in some BRCA1-mutated breast cancers, they are infrequent and not recurrent. However, private fusions may still be valuable as potential patient-specific biomarkers for diagnosis and treatment.

The liver represents the third most frequent site of metastasis in patients with breast cancer. We performed in vivo selection using 4T1 breast cancer cells to identify genes associated with the liver metastatic phenotype. Coincident with the loss of numerous tight-junctional proteins, we observe claudin-2 overexpression, specifically in liver-aggressive breast cancer cells. We further demonstrate that claudin-2 is both necessary and sufficient for the ability of 4T1 breast cancer cells to colonize and grow in the liver. The liver-aggressive breast cancer cells display a claudin-2-mediated increase in their ability to adhere to extracellular matrix (ECM) components, such as fibronectin and type IV collagen. Claudin-2 facilitates these cell/matrix interactions by increasing the cell surface expression of α(2)β(1)- and α(5)β(1)-integrin complexes in breast cancer cells. Indeed, claudin-2-mediated adhesion to fibronectin and type IV collagen can be blocked with neutralizing antibodies that target α(5)β(1) and α(2)β(1) complexes, respectively. Immunohistochemical analyses reveal that claudin-2, although weakly expressed in primary human breast cancers, is readily detected in all liver metastasis samples examined to date. Together, these results uncover novel roles for claudin-2 in promoting breast cancer adhesion to the ECM and define its importance during breast cancer metastasis to the liver.