Publications by authors named "Steffi Oesterreich"

137 Publications

Prognostic Utility of Breast Cancer Index to Stratify Distant Recurrence Risk in Invasive Lobular Carcinoma.

Clin Cancer Res 2021 Aug 10. Epub 2021 Aug 10.

Dana-Farber Cancer Institute, Boston, Massachusetts.

Purpose: The prognostic utility of Breast Cancer Index (BCI) for risk assessment of overall (0-10 years), early (0-5 years), and late (5-10 years) distant recurrence (DR) in hormone receptor-positive (HR+) invasive lobular carcinoma (ILC) was evaluated.

Experimental Design: BCI gene expression analysis was performed blinded to clinical outcome utilizing tumor specimens from patients with HR+ ILC from a multi-institutional cohort. The primary endpoint was time to DR. Kaplan-Meier analyses of overall, early, and late DR risk were performed, and statistical significance was evaluated by log-rank test and Cox proportional hazards regression. The prognostic contribution of BCI in addition to clinicopathologic factors was evaluated by likelihood ratio analysis.

Results: Analysis of 307 patients (99% ER+, 53% T1, 42% N+, 70% grade II) showed significant differences in DR over 10 years based on BCI risk categories. BCI low- and intermediate-risk patients demonstrated similar DR rates of 7.6% and 8.0%, respectively, compared with 27.0% for BCI high-risk patients. BCI was a significant independent prognostic factor for overall 10-year DR [HR = 4.09; 95% confidence interval (CI), 2.00-8.34; = 0.0001] as well as for both early (HR = 8.19; 95% CI, 1.85-36.30; = 0.0042) and late (HR = 3.04; 95% CI, 1.32-7.00; = 0.0224) DR. In multivariate analysis, BCI remained the only statistically significant prognostic factor for DR (HR = 3.49; 95% CI, 1.28-9.54; = 0.0150).

Conclusions: BCI is an independent prognostic factor for ILC and significantly stratified patients for cumulative risk of 10-year, early, and late DR. BCI added prognostic value beyond clinicopathologic characteristics in this distinct subtype of breast cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-21-0733DOI Listing
August 2021

How Researchers, Clinicians and Patient Advocates Can Accelerate Lobular Breast Cancer Research.

Cancers (Basel) 2021 Jun 22;13(13). Epub 2021 Jun 22.

Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15261, USA.

Breast cancer research and therapies have significantly advanced in recent years. However, Invasive Lobular Carcinoma (ILC), the second most common histological type of breast cancer and the sixth most frequently diagnosed cancer of women, has not always benefited from critical analysis, missing opportunities to better understand this important subtype. Recent progress understanding the biological and behavioral differences of ILC demonstrates that it is a unique subtype of breast cancer which can respond differently to common therapies. These new insights have increased interest in researching lobular breast disease. Concurrently, the formation of motivated patient-led advocacy organizations working in partnership with basic, translational and clinical researchers creates new opportunities, including connecting a dispersed patient population to research, encouraging new research funding and connecting patient advocates to researchers to advance common goals. This commentary will explore the unprecedented opportunity to drive multidisciplinary, multicenter and international collaborative research into lobular breast cancer that builds on recent research progress. Collaborative research partnerships that include advocates can result in a better understanding of ILC, identify targeted therapies and refine standard of care therapies that are currently equally applied to all breast cancers, resulting in improvements in the diagnosis, treatment and follow-up care for patients with ILC.
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http://dx.doi.org/10.3390/cancers13133094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8268486PMC
June 2021

Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised.

Elife 2021 06 28;10. Epub 2021 Jun 28.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, United States.

Molecular chaperones, such as Hsp70, prevent proteotoxicity and maintain homeostasis. This is perhaps most evident in cancer cells, which overexpress Hsp70 and thrive even when harboring high levels of misfolded proteins. To define the response to proteotoxic challenges, we examined adaptive responses in breast cancer cells in the presence of an Hsp70 inhibitor. We discovered that the cells bin into distinct classes based on inhibitor sensitivity. Strikingly, the most resistant cells have higher autophagy levels, and autophagy was maximally activated only in resistant cells upon Hsp70 inhibition. In turn, resistance to compromised Hsp70 function required the integrated stress response transducer, GCN2, which is commonly associated with amino acid starvation. In contrast, sensitive cells succumbed to Hsp70 inhibition by activating PERK. These data reveal an unexpected route through which breast cancer cells adapt to proteotoxic insults and position GCN2 and autophagy as complementary mechanisms to ensure survival when proteostasis is compromised.
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http://dx.doi.org/10.7554/eLife.64977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8275131PMC
June 2021

Prognostic factors and survival of patients undergoing surgical intervention for breast cancer bone metastases.

J Bone Oncol 2021 Aug 3;29:100363. Epub 2021 May 3.

Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

Introduction: Bone is the most common distant site of breast cancer metastasis. Skeletal lesions can cause significant morbidity due to pain, pathologic fracture, and electrolyte abnormalities. Current treatment for patients with bone metastases (BoM) from breast cancer is highly personalized and often involves a multidisciplinary approach with chemotherapy, hormone therapy, bone-targeted antiresorptive agents, radiation therapy, and surgery. We have retrospectively collected clinical data from a series of patients with bone metastases to evaluate the clinical characteristics, prognostic factors, and survival patterns of patients with breast cancer BoM receiving standard multimodal therapy.

Methods: A consecutive series of 167 patients with breast cancer BoM treated at a single institution between August 2013 and March 2020 were identified. Clinical information was obtained from the medical record and survival analyses were performed to evaluate patient outcomes and identify prognostic factors.

Results: Thirty-seven patients (22%) presented with BoM - bone metastases at the time of breast cancer diagnosis - and were 2.6 times more likely to die within the study period than those with asynchronous BoM (HR = 2.62, p = <0.0001). Patients who received bone-targeted medical therapy were 61% less likely to die after BoM diagnosis than those who did not (HR = 0.39, p = 0.001). Operative stabilization of BoM was more frequently employed in patients with lytic (p = 0.02) or mixed (p = 0.02) tumors than it was for those with blastic lesions. Patients treated with surgery had a lower overall bone metastasis survival than those treated without (p < 0.03).

Discussion: These findings reflect the current patterns in metastatic breast cancer treatment and associated outcomes. In a series of 167 consecutive patients, we demonstrate the natural history of breast cancer with BoM being treated with modern multimodal therapy. Understanding these treatment patterns and prognostic factors enhances the provider's ability to counsel patients and direct appropriate treatments.
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http://dx.doi.org/10.1016/j.jbo.2021.100363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8143999PMC
August 2021

A Novel Mouse Model for SNP in Steroid Receptor Co-Activator-1 Reveals Role in Bone Density and Breast Cancer Metastasis.

Endocrinology 2021 Aug;162(8)

Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.

The steroid receptor coactivator-1 (SRC-1) is a nuclear receptor co-activator, known to play key roles in both estrogen response in bone and in breast cancer metastases. We previously demonstrated that the P1272S single nucleotide polymorphism (SNP; P1272S; rs1804645) in SRC-1 decreases the activity of estrogen receptor in the presence of selective estrogen receptor modulators (SERMs) and that it is associated with a decrease in bone mineral density (BMD) after tamoxifen therapy, suggesting it may disrupt the agonist action of tamoxifen. Given such dual roles of SRC-1 in the bone microenvironment and in tumor cell-intrinsic phenotypes, we hypothesized that SRC-1 and a naturally occurring genetic variant, P1272S, may promote breast cancer bone metastases. We developed a syngeneic, knock-in mouse model to study if the SRC-1 SNP is critical for normal bone homeostasis and bone metastasis. Our data surprisingly reveal that the homozygous SRC-1 SNP knock-in increases tamoxifen-induced bone protection after ovariectomy. The presence of the SRC-1 SNP in mammary glands resulted in decreased expression levels of SRC-1 and reduced tumor burden after orthotopic injection of breast cancer cells not bearing the SRC-1 SNP, but increased metastases to the lungs in our syngeneic mouse model. Interestingly, the P1272S SNP identified in a small, exploratory cohort of bone metastases from breast cancer patients was significantly associated with earlier development of bone metastasis. This study demonstrates the importance of the P1272S SNP in both the effect of SERMs on BMD and the development of tumor in the bone.
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http://dx.doi.org/10.1210/endocr/bqab094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8248588PMC
August 2021

Outcomes After Sentinel Lymph Node Biopsy and Radiotherapy in Older Women With Early-Stage, Estrogen Receptor-Positive Breast Cancer.

JAMA Netw Open 2021 04 1;4(4):e216322. Epub 2021 Apr 1.

Women's Cancer Research Center, UPMC Hillman Cancer Center, Pittsburgh, Pennsylvania.

Importance: Overtreatment of early-stage breast cancer with favorable tumor biology in older patients may be harmful without affecting recurrence and survival. Guidelines that recommend deimplementation of sentinel lymph node biopsy (SLNB) (Choosing Wisely) and radiotherapy (RT) (National Comprehensive Cancer Network) have been published.

Objective: To describe the use rates and association with disease recurrence of SLNB and RT in older women with breast cancer.

Design, Setting, And Participants: This cohort study obtained patient and clinical data from an integrated cancer registry and electronic health record of a single health care system in Pennsylvania. The cohort was composed of consecutive female patients 70 years or older who were diagnosed with early-stage, estrogen receptor-positive, ERBB2 (formerly HER2)-negative, clinically node-negative breast cancer from January 1, 2010, to December 31, 2018, who were treated at 15 community and academic hospitals within the health system.

Exposures: Sentinel lymph node biopsy and adjuvant RT.

Main Outcomes And Measures: Primary outcomes were 5-year locoregional recurrence-free survival (LRFS) rate and disease-free survival (DFS) rate after SLNB and after RT. Secondary outcomes included recurrence rate, subgroups that may benefit from SLNB or RT, and use rate of SLNB and RT over time. Propensity scores were used to create 2 cohorts to separately evaluate the association of SLNB and RT with recurrence outcomes. Cox proportional hazards regression model was used to estimate hazard ratios (HRs).

Results: From 2010 to 2018, a total of 3361 women 70 years or older (median [interquartile range {IQR}] age, 77.0 [73.0-82.0] years) with estrogen receptor-positive, ERBB2-negative, clinically node-negative breast cancer were included in the study. Of these women, 2195 (65.3%) received SLNB and 1828 (54.4%) received adjuvant RT. Rates of SLNB steadily increased (1.0% per year), a trend that persisted after the 2016 adoption of the Choosing Wisely guideline. Rates of RT decreased slightly (3.4% per year). To examine patient outcomes and maximize follow-up time, the analysis was limited to cases from 2010 to 2014, identifying 2109 patients with a median (IQR) follow-up time of 4.1 (2.5-5.7) years. In the propensity score-matched cohorts, no association was found between SLNB and either LRFS (HR, 1.26; 95% CI, 0.37-4.30; P = .71) or DFS (HR, 1.92; 95% CI, 0.86-4.32; P = .11). In addition, RT was not associated with LRFS (HR, 0.33; 95% CI, 0.09-1.24; P = .10) or DFS (HR, 0.99; 95% CI, 0.46-2.10; P = .97). Subgroup analysis showed that stratification by tumor grade or comorbidity was not associated with LRFS or DFS. Low absolute rates of recurrence were observed when comparing the groups that received SLNB (3.5%) and those that did not (4.5%) as well as the groups that received RT (2.7%) and those that did not (5.5%).

Conclusions And Relevance: This study found that receipt of SLNB or RT was not associated with improved LRFS or DFS in older patients with ER-positive, clinically node-negative breast cancer. Despite limited follow-up time and wide 95% CIs, this study supports the continued deimplementation of both SLNB and RT in accordance with the Choosing Wisely and National Comprehensive Cancer Network guidelines.
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http://dx.doi.org/10.1001/jamanetworkopen.2021.6322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050744PMC
April 2021

Exosomes in Breast Cancer - Mechanisms of Action and Clinical Potential.

Mol Cancer Res 2021 06 24;19(6):935-945. Epub 2021 Feb 24.

Women's Cancer Research Center, UPMC Hillman Cancer Center, Magee-Womens Research Institute, Pittsburgh, Pennsylvania.

Extracellular vesicles (EV) are a heterogeneous group of cell-derived membrane vesicles comprising apoptotic bodies, microvesicles, and small EVs also called as exosomes. Exosomes when initially identified were considered as a waste product but the advancement in research techniques have provided insight into the important roles of exosomes in cell-cell communication, various biological processes and diseases, including cancer. As an important component of EVs, exosomes contain various biomolecules such as miRNAs, lipids, and proteins that largely reflect the characteristics of their parent cells. Notably, cancer cells generate and secrete many more exosomes than normal cells. A growing body of evidence suggests that exosomes, as mediators of intercellular cross-talk, play a role in tumorigenesis, cancer cell invasion, angiogenesis, tumor microenvironment (TME) formation, and cancer metastasis. As we gain more insights into the association between exosomes and cancer, the potential of exosomes for clinical use is becoming more intriguing. This review is focused on the role of exosomes in breast cancer, in terms of breast cancer biology, mechanism of action, potential as biomarkers, and therapeutic opportunities.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-0952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178200PMC
June 2021

Comparative analysis of the AIB1 interactome in breast cancer reveals MTA2 as a repressive partner which silences E-Cadherin to promote EMT and associates with a pro-metastatic phenotype.

Oncogene 2021 02 8;40(7):1318-1331. Epub 2021 Jan 8.

Endocrine Oncology Research Group, Department of Surgery, Royal College of Surgeons in Ireland, Dublin, Ireland.

Steroid regulated cancer cells use nuclear receptors and associated regulatory proteins to orchestrate transcriptional networks to drive disease progression. In primary breast cancer, the coactivator AIB1 promotes estrogen receptor (ER) transcriptional activity to enhance cell proliferation. The function of the coactivator in ER metastasis however is not established. Here we describe AIB1 as a survival factor, regulator of pro-metastatic transcriptional pathways and a promising actionable target. Genomic alterations and functional expression of AIB1 associated with reduced disease-free survival in patients and enhanced metastatic capacity in novel CDX and PDX ex-vivo models of ER metastatic disease. Comparative analysis of the AIB1 interactome with complementary RNAseq characterized AIB1 as a transcriptional repressor. Specifically, we report that AIB1 interacts with MTA2 to form a repressive complex, inhibiting CDH1 (encoding E-cadherin) to promote EMT and drive progression. We further report that pharmacological and genetic inhibition of AIB1 demonstrates significant anti-proliferative activity in patient-derived models establishing AIB1 as a viable strategy to target endocrine resistant metastasis. This work defines a novel role for AIB1 in the regulation of EMT through transcriptional repression in advanced cancer cells with a considerable implication for prognosis and therapeutic interventions.
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http://dx.doi.org/10.1038/s41388-020-01606-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892341PMC
February 2021

Acquired mutations and transcriptional remodeling in long-term estrogen-deprived locoregional breast cancer recurrences.

Breast Cancer Res 2021 01 6;23(1). Epub 2021 Jan 6.

Women's Cancer Research Center, UPMC Hillman Cancer Center, Pittsburgh, PA, USA.

Background: Endocrine therapy resistance is a hallmark of advanced estrogen receptor (ER)-positive breast cancer. In this study, we aimed to determine acquired genomic changes in endocrine-resistant disease.

Methods: We performed DNA/RNA hybrid-capture sequencing on 12 locoregional recurrences after long-term estrogen deprivation and identified acquired genomic changes versus each tumor's matched primary.

Results: Despite being up to 7 years removed from the primary lesion, most recurrences harbored similar intrinsic transcriptional and copy number profiles. Only two genes, AKAP9 and KMT2C, were found to have single nucleotide variant (SNV) enrichments in more than one recurrence. Enriched mutations in single cases included SNVs within transcriptional regulators such as ARID1A, TP53, FOXO1, BRD1, NCOA1, and NCOR2 with one local recurrence gaining three PIK3CA mutations. In contrast to DNA-level changes, we discovered recurrent outlier mRNA expression alterations were common-including outlier gains in TP63 (n = 5 cases [42%]), NTRK3 (n = 5 [42%]), NTRK2 (n = 4 [33%]), PAX3 (n = 4 [33%]), FGFR4 (n = 3 [25%]), and TERT (n = 3 [25%]). Recurrent losses involved ESR1 (n = 5 [42%]), RELN (n = 5 [42%]), SFRP4 (n = 4 [33%]), and FOSB (n = 4 [33%]). ESR1-depleted recurrences harbored shared transcriptional remodeling events including upregulation of PROM1 and other basal cancer markers.

Conclusions: Taken together, this study defines acquired genomic changes in long-term, estrogen-deprived disease; highlights the importance of longitudinal RNA profiling; and identifies a common ESR1-depleted endocrine-resistant breast cancer subtype with basal-like transcriptional reprogramming.
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http://dx.doi.org/10.1186/s13058-020-01379-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788918PMC
January 2021

ADAM22/LGI1 complex as a new actionable target for breast cancer brain metastasis.

BMC Med 2020 11 19;18(1):349. Epub 2020 Nov 19.

Endocrine Oncology Research Group, Department of Surgery, Royal College of Surgeons in Ireland, Dublin 2, Ireland.

Background: Metastatic breast cancer is a major cause of cancer-related deaths in woman. Brain metastasis is a common and devastating site of relapse for several breast cancer molecular subtypes, including oestrogen receptor-positive disease, with life expectancy of less than a year. While efforts have been devoted to developing therapeutics for extra-cranial metastasis, drug penetration of blood-brain barrier (BBB) remains a major clinical challenge. Defining molecular alterations in breast cancer brain metastasis enables the identification of novel actionable targets.

Methods: Global transcriptomic analysis of matched primary and metastatic patient tumours (n = 35 patients, 70 tumour samples) identified a putative new actionable target for advanced breast cancer which was further validated in vivo and in breast cancer patient tumour tissue (n = 843 patients). A peptide mimetic of the target's natural ligand was designed in silico and its efficacy assessed in in vitro, ex vivo and in vivo models of breast cancer metastasis.

Results: Bioinformatic analysis of over-represented pathways in metastatic breast cancer identified ADAM22 as a top ranked member of the ECM-related druggable genome specific to brain metastases. ADAM22 was validated as an actionable target in in vitro, ex vivo and in patient tumour tissue (n = 843 patients). A peptide mimetic of the ADAM22 ligand LGI1, LGI1MIM, was designed in silico. The efficacy of LGI1MIM and its ability to penetrate the BBB were assessed in vitro, ex vivo and in brain metastasis BBB 3D biometric biohybrid models, respectively. Treatment with LGI1MIM in vivo inhibited disease progression, in particular the development of brain metastasis.

Conclusion: ADAM22 expression in advanced breast cancer supports development of breast cancer brain metastasis. Targeting ADAM22 with a peptide mimetic LGI1MIM represents a new therapeutic option to treat metastatic brain disease.
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http://dx.doi.org/10.1186/s12916-020-01806-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677775PMC
November 2020

Estrogen Receptor Alpha Mutations in Breast Cancer Cells Cause Gene Expression Changes through Constant Activity and Secondary Effects.

Cancer Res 2021 02 12;81(3):539-551. Epub 2020 Nov 12.

Department of Oncological Sciences, University of Utah, Salt Lake City, Utah.

While breast cancer patients with tumors that express estrogen receptor α (ER) generally respond well to hormone therapies that block ER activity, a significant number of patients relapse. Approximately 30% of these recurrences harbor activating mutations in the ligand binding domain (LBD) of ER, which have been shown to confer ligand-independent function. However, much is still unclear regarding the effect of mutant ER beyond its estrogen independence. To investigate the molecular effects of mutant ER, we developed multiple isogenic ER-mutant cell lines for the most common LBD mutations, Y537S and D538G. These mutations induced differential expression of thousands of genes, the majority of which were mutant allele specific and were not observed upon estrogen treatment of wild-type (WT) cells. These mutant-specific genes showed consistent differential expression across ER-mutant lines developed in other laboratories. WT cells with long-term estrogen exposure only exhibited some of these transcriptional changes, suggesting that mutant ER causes novel regulatory effects that are not simply due to constant activity. While ER mutations exhibited minor effects on ER genomic binding, with the exception of ligand independence, ER mutations conferred substantial differences in chromatin accessibility. Mutant ER was bound to approximately a quarter of mutant-enriched accessible regions that were enriched for other DNA binding factors, including FOXA1, CTCF, and OCT1. Overall, our findings indicate that mutant ER causes several consistent effects on gene expression, both indirectly and through constant activity. SIGNIFICANCE: This study demonstrates the multiple roles of mutant ER in breast cancer progression, including constant ER activity and secondary regulatory effects on gene expression and chromatin accessibility. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/539/F1.large.jpg. .
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854489PMC
February 2021

Steroid Hormone Receptor and Infiltrating Immune Cell Status Reveals Therapeutic Vulnerabilities of -Mutant Breast Cancer.

Cancer Res 2021 02 12;81(3):732-746. Epub 2020 Nov 12.

Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Mutations in that confer constitutive estrogen receptor alpha (ER) activity in the absence of ligand are acquired by ≥40% of metastatic breast cancers (MBC) resistant to adjuvant aromatase inhibitor (AI) therapy. To identify targetable vulnerabilities in MBC, we examined steroid hormone receptors and tumor-infiltrating immune cells in metastatic lesions with or without ER mutations. ER and progesterone receptor (PR) were significantly lower in metastases with wild-type (WT) ER compared with those with mutant ER, suggesting that metastases that evade AI therapy by mechanism(s) other than acquiring ER mutations lose dependency on ER and PR. Metastases with mutant ER had significantly higher T regulatory and Th cells, total macrophages, and programmed death ligand-1 (PD-L1)-positive immune-suppressive macrophages than those with WT ER. Breast cancer cells with CRISPR-Cas9-edited ER (D538G, Y537S, or WT) and patient-derived xenografts harboring mutant or WT ER revealed genes and proteins elevated in mutant ER cells, including androgen receptor (AR), chitinase-3-like protein 1 (CHI3L1), and IFN-stimulated genes (ISG). Targeting these proteins blunted the selective advantage of ER-mutant tumor cells to survive estrogen deprivation, anchorage independence, and invasion. Thus, patients with mutant ER MBC might respond to standard-of-care fulvestrant or other selective ER degraders when combined with AR or CHI3L1 inhibition, perhaps with the addition of immunotherapy. SIGNIFICANCE: Targetable alterations in MBC, including AR, CHI3L1, and ISG, arise following estrogen-deprivation, and ER-mutant metastases may respond to immunotherapies due to elevated PD-L1 macrophages..
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854521PMC
February 2021

Single-Cell Transcriptomic Heterogeneity in Invasive Ductal and Lobular Breast Cancer Cells.

Cancer Res 2021 01 4;81(2):268-281. Epub 2020 Nov 4.

Women's Cancer Research Center, UPMC Hillman Cancer Center, Magee-Womens Research Institute, Pittsburgh, Pennsylvania.

Invasive lobular breast carcinoma (ILC), one of the major breast cancer histologic subtypes, exhibits unique features compared with the well-studied ductal cancer subtype (IDC). The pathognomonic feature of ILC is loss of E-cadherin, mainly caused by inactivating mutations, but the contribution of this genetic alteration to ILC-specific molecular characteristics remains largely understudied. To profile these features transcriptionally, we conducted single-cell RNA sequencing on a panel of IDC and ILC cell lines, and an IDC cell line (T47D) with CRISPR-Cas9-mediated E-cadherin knockout (KO). Inspection of intracell line heterogeneity illustrated genetically and transcriptionally distinct subpopulations in multiple cell lines and highlighted rare populations of MCF7 cells highly expressing an apoptosis-related signature, positively correlated with a preadaptation signature to estrogen deprivation. Investigation of E-cadherin KO-induced alterations showed transcriptomic membranous systems remodeling, elevated resemblance to ILCs in regulon activation, and increased sensitivity to IFNγ-mediated growth inhibition via activation of IRF1. This study reveals single-cell transcriptional heterogeneity in breast cancer cell lines and provides a resource to identify drivers of cancer progression and drug resistance. SIGNIFICANCE: This study represents a key step towards understanding heterogeneity in cancer cell lines and the role of E-cadherin depletion in contributing to the molecular features of invasive lobular breast carcinoma.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856056PMC
January 2021

Estrogen Regulation of mTOR Signaling and Mitochondrial Function in Invasive Lobular Carcinoma Cell Lines Requires WNT4.

Cancers (Basel) 2020 Oct 12;12(10). Epub 2020 Oct 12.

Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.

Invasive lobular carcinoma of the breast (ILC) is strongly estrogen-driven and represents a unique context for estrogen receptor (ER) signaling. In ILC, ER controls the expression of the Wnt ligand WNT4, which is critical for endocrine response and anti-estrogen resistance. However, signaling mediated by WNT4 is cell type- and tissue-specific, and has not been explored in ILC. We utilized reverse phase protein array (RPPA) to characterize ER and WNT4-driven signaling in ILC cells and identified that WNT4 mediates downstream mTOR signaling via phosphorylation of S6 Kinase. Additionally, ER and WNT4 control levels of MCL-1, which is associated with regulation of mitochondrial function. In this context, WNT4 knockdown led to decreased ATP production and increased mitochondrial fragmentation. WNT4 regulation of both mTOR signaling and MCL-1 were also observed in anti-estrogen resistant models of ILC. We identified that high WNT4 expression is associated with similar mTOR pathway activation in ILC and serous ovarian cancer tumors, suggesting that WNT4 signaling is active in multiple tumor types. The identified downstream pathways offer insight into WNT4 signaling and represent potential targets to overcome anti-estrogen resistance for patients with ILC.
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http://dx.doi.org/10.3390/cancers12102931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650584PMC
October 2020

Pan-Cancer Analysis of and Genomic Alterations and Their Association With Genomic Instability as Measured by Genome-Wide Loss of Heterozygosity.

JCO Precis Oncol 2020 30;4:442-465. Epub 2020 Apr 30.

Foundation Medicine, Cambridge, MA.

Purpose: BRCA1 or BRCA2 loss of function results in homologous recombination deficiency (HRD), which is targetable by poly (ADP-ribose) polymerase (PARP) inhibitors and other DNA-damaging agents. In cancers associated with germline alterations (-associated cancers: breast, ovarian, pancreatic, prostate), alterations result in HRD and are biomarkers for PARP inhibitor use. In other (non--associated) cancer types, the association between alteration and HRD is less clear.

Methods: A total of 234,154 tumor samples were sequenced by hybrid capture-based comprehensive genomic profiling. Somatic, germline, and zygosity status was determined computationally. alterations were classified as predicted germline/somatic and biallelic/monoallelic. Genome-wide loss of heterozygosity (gLOH) was evaluated as a marker of HRD.

Results: alterations were observed at a 4.7% frequency. mutations were predicted germline in 57.4% of -associated and 37.2% of non--associated cancers. The fraction of -altered cases that were biallelic was 68.7%, with a higher biallelic fraction in -associated (89.9%) versus non--associated cancers (43.6%). Differences in tissue distribution of biallelic versus alterations were noted, including a higher rate of biallelic alteration in prostate cancer. Biallelic alteration was observed at a 3.2% frequency (-associated cancers, 8.9%; non--associated cancers, 1.3%) and > 1% frequency in at least 13 cancer types. Across cancer types, biallelic alteration was associated with increased gLOH versus monoallelic or wild-type ; predicted germline or somatic mutations were both associated with elevated gLOH.

Conclusion: Biallelic alterations were associated with elevated gLOH in diverse cancer types, including those not traditionally associated with cancer syndromes. Biomarker development for PARP inhibitors should integrate methods to distinguish biallelic from monoallelic status, and biallelic alteration should be broadly evaluated across cancer types as a biomarker for underlying HRD and PARP inhibitor sensitivity.
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http://dx.doi.org/10.1200/po.19.00345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446440PMC
April 2020

Targetable ERBB2 mutation status is an independent marker of adverse prognosis in estrogen receptor positive, ERBB2 non-amplified primary lobular breast carcinoma: a retrospective in silico analysis of public datasets.

Breast Cancer Res 2020 08 11;22(1):85. Epub 2020 Aug 11.

Nottingham Breast Cancer Research Centre, Nottingham Biodiscovery Institute, School of Medicine, University of Nottingham, Nottingham, UK.

Background: Invasive lobular carcinoma (ILC) accounts for 10-15% of primary breast cancers and is typically estrogen receptor alpha positive (ER+) and ERBB2 non-amplified. Somatic mutations in ERBB2/3 are emerging as a tractable mechanism underlying enhanced human epidermal growth factor 2 (HER2) activity. We tested the hypothesis that therapeutically targetable ERBB2/3 mutations in primary ILC of the breast associate with poor survival outcome in large public datasets.

Methods: We performed in silico comparison of ERBB2 non-amplified cases of ER+ stage I-III primary ILC (N = 279) and invasive ductal carcinoma (IDC, N = 1301) using METABRIC, TCGA, and MSK-IMPACT information. Activating mutations amenable to HER2-directed therapy with neratinib were identified using existing functional data from in vitro cell line and xenograft experiments. Multivariate analysis of 10-year overall survival (OS) with tumor size, grade, and lymph node status was performed using a Cox regression model. Differential gene expression analyses by ERBB2 mutation and amplification status was performed using weighted average differences and an in silico model of response to neratinib derived from breast cancer cell lines.

Results: ILC tumors comprised 17.7% of all cases in the dataset but accounted for 47.1% of ERBB2-mutated cases. Mutations in ERBB2 were enriched in ILC vs. IDC cases (5.7%, N = 16 vs. 1.4%, N = 18, p < 0.0001) and clustered in the tyrosine kinase domain of HER2. ERBB3 mutations were not enriched in ILC (1.1%, N = 3 vs. 1.8%, N = 23; p = 0.604). Median OS for patients with ERBB2-mutant ILC tumors was 66 months vs. 211 months for ERBB2 wild-type (p = 0.0001), and 159 vs. 166 months (p = 0.733) for IDC tumors. Targetable ERBB2 mutational status was an independent prognostic marker of 10-year OS-but only in ILC (hazard ratio, HR = 3.7, 95% CI 1.2-11.0; p = 0.021). Findings were validated using a novel ERBB2 mutation gene enrichment score (HR for 10-year OS in ILC = 2.3, 95% CI 1.04-5.05; p = 0.040).

Conclusions: Targetable ERBB2 mutations are enriched in primary ILC and their detection represents an actionable strategy with the potential to improve patient outcomes. Biomarker-led clinical trials of adjuvant HER-targeted therapy are warranted for patients with ERBB2-mutated primary ILC.
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http://dx.doi.org/10.1186/s13058-020-01324-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422515PMC
August 2020

Sulforaphane Diminishes the Formation of Mammary Tumors in Rats Exposed to 17β-Estradiol.

Nutrients 2020 Jul 30;12(8). Epub 2020 Jul 30.

Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15261, USA.

Elevated levels of estrogen are a risk factor for breast cancer. In addition to inducing DNA damage, estrogens can enhance cell proliferation as well as modulate fatty acid metabolism that collectively contributes to mammary tumorigenesis. Sulforaphane (SFN) is an isothiocyanate derived from broccoli that is currently under evaluation in multiple clinical trials for prevention of several diseases, including cancer. Previous studies showed that SFN suppressed DNA damage and lipogenesis pathways. Therefore, we hypothesized that administering SFN to animals that are co-exposed to 17β-estradiol (E2) would prevent mammary tumor formation. In our study, 4-6 week old female August Copenhagen Irish rats were implanted with slow-release E2 pellets (3 mg x 3 times) and gavaged 3x/week with either vehicle or 100 μmol/kg SFN for 56 weeks. SFN-treated rats were protected significantly against mammary tumor formation compared to vehicle controls. Mammary glands of SFN-treated rats showed decreased DNA damage while serum free fatty acids and triglyceride species were 1.5 to 2-fold lower in SFN-treated rats. Further characterization also showed that SFN diminished expression of enzymes involved in mammary gland lipogenesis. This study indicated that SFN protects against breast cancer development through multiple potential mechanisms in a clinically relevant hormonal carcinogenesis model.
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http://dx.doi.org/10.3390/nu12082282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468750PMC
July 2020

Proteomic and transcriptomic profiling identifies mediators of anchorage-independent growth and roles of inhibitor of differentiation proteins in invasive lobular carcinoma.

Sci Rep 2020 07 13;10(1):11487. Epub 2020 Jul 13.

Women's Cancer Research Center, University of Pittsburgh Medical Center (UPMC) Hillman Cancer Center (HCC), Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, PA, 15213, USA.

Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer with distinct molecular and clinical features from the more common subtype invasive ductal carcinoma (IDC). ILC cells exhibit anchorage-independent growth in ultra-low attachment (ULA) suspension cultures, which is largely attributed to the loss of E-cadherin. In addition to anoikis resistance, herein we show that human ILC cell lines exhibit enhanced cell proliferation in ULA cultures as compared to IDC cells. Proteomic comparison of ILC and IDC cell lines identified induction of PI3K/Akt and p90-RSK pathways specifically in ULA culture in ILC cells. Further transcriptional profiling uncovered unique upregulation of the inhibitors of differentiation family transcription factors ID1 and ID3 in ILC ULA culture, the knockdown of which diminished the anchorage-independent growth of ILC cell lines through cell cycle arrest. We find that ID1 and ID3 expression is higher in human ILC tumors as compared to IDC, correlated with worse prognosis uniquely in patients with ILC and associated with upregulation of angiogenesis and matrisome-related genes. Altogether, our comprehensive study of anchorage independence in human ILC cell lines provides mechanistic insights and clinical implications for metastatic dissemination of ILC and implicates ID1 and ID3 as novel drivers and therapeutic targets for lobular breast cancer.
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http://dx.doi.org/10.1038/s41598-020-68141-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359337PMC
July 2020

A sequential methodology for the rapid identification and characterization of breast cancer-associated functional SNPs.

Nat Commun 2020 07 3;11(1):3340. Epub 2020 Jul 3.

Aging Institute, University of Pittsburgh, Pittsburgh, PA, 15219, USA.

GWAS cannot identify functional SNPs (fSNP) from disease-associated SNPs in linkage disequilibrium (LD). Here, we report developing three sequential methodologies including Reel-seq (Regulatory element-sequencing) to identify fSNPs in a high-throughput fashion, SDCP-MS (SNP-specific DNA competition pulldown-mass spectrometry) to identify fSNP-bound proteins and AIDP-Wb (allele-imbalanced DNA pulldown-Western blot) to detect allele-specific protein:fSNP binding. We first apply Reel-seq to screen a library containing 4316 breast cancer-associated SNPs and identify 521 candidate fSNPs. As proof of principle, we verify candidate fSNPs on three well-characterized loci: FGFR2, MAP3K1 and BABAM1. Next, using SDCP-MS and AIDP-Wb, we rapidly identify multiple regulatory factors that specifically bind in an allele-imbalanced manner to the fSNPs on the FGFR2 locus. We finally demonstrate that the factors identified by SDCP-MS can regulate risk gene expression. These data suggest that the sequential application of Reel-seq, SDCP-MS, and AIDP-Wb can greatly help to translate large sets of GWAS data into biologically relevant information.
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http://dx.doi.org/10.1038/s41467-020-17159-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334201PMC
July 2020

Differential Regulation and Targeting of Estrogen Receptor α Turnover in Invasive Lobular Breast Carcinoma.

Endocrinology 2020 09;161(9)

Women's Cancer Research Center, UPMC Hillman Cancer Center, Magee Womens Research Institute, Pittsburgh, Pennsylvania.

Invasive lobular breast carcinoma (ILC) accounts for 10% to 15% of breast cancers diagnosed annually. Evidence suggests that some aspects of endocrine treatment response might differ between invasive ductal carcinoma (IDC) and ILC, and that patients with ILC have worse long-term survival. We analyzed The Cancer Genome Atlas dataset and observed lower levels of ESR1 mRNA (P = 0.002) and ERα protein (P = 0.038) in ER+ ILC (n = 137) compared to IDC (n = 554), and further confirmed the mRNA difference in a local UPMC cohort (ILC, n = 143; IDC, n = 877; P < 0.005). In both datasets, the correlation between ESR1 mRNA and ERα protein was weaker in ILC, suggesting differential post-transcriptional regulation of ERα. In vitro, 17β-estradiol (E2) decreased the rate of degradation and increased the half-life of ERα in ILC cell lines, whereas the opposite was observed in IDC cell lines. Further, E2 failed to induce robust ubiquitination of ERα in ILC cells. To determine the potential clinical relevance of these findings, we evaluated the effect of 2 selective estrogen receptor downregulators (SERDs), ICI 182,780 and AZD9496, on ERα turnover and cell growth. While ICI 182,780 and AZD9496 showed similar effects in IDC cells, in ILC cell lines, AZD9496 was not as effective as ICI 182,780 in decreasing ERα stability and E2-induced proliferation. Furthermore, AZD9496 exhibited partial agonist activity in growth assays in ILC cell lines. Our study provides evidence for a distinct ERα regulation by SERDs in ILC cell lines, and therefore it is important to include ILC models into preclinical and clinical testing of novel SERDs.
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http://dx.doi.org/10.1210/endocr/bqaa109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438704PMC
September 2020

Patient treatment and outcome after breast cancer orbital and periorbital metastases: a comprehensive case series including analysis of lobular versus ductal tumor histology.

Breast Cancer Res 2020 06 26;22(1):70. Epub 2020 Jun 26.

University of Pittsburgh School of Medicine, Department of Medicine, Division of Hematology/Oncology, UPMC Hillman Cancer Center, Pittsburgh, PA, USA.

Background: Breast cancer is the most common malignancy to spread to the orbit and periorbit, and the invasive lobular carcinoma (ILC) histologic subtype of breast cancer has been reported to form these ophthalmic metastases (OM) more frequently than invasive ductal carcinomas (IDC). We herein report our single academic institution experience with breast cancer OM with respect to anatomical presentation, histology (lobular vs. ductal), treatment, and survival.

Methods: We employed the natural language processing platform, TIES (Text Information Extraction System), to search 2.3 million de-identified patient pathology and radiology records at our institution in order to identify patients with OM secondary to breast cancer. We then compared the resultant cohort, the "OM cohort," to two other representative metastatic breast cancer patient (MBC) databases from our institution. Histological analysis of selected patients was performed.

Results: Our TIES search and manual refinement ultimately identified 28 patients who were diagnosed with breast cancer between 1995 and 2016 that subsequently developed OM. Median age at diagnosis was 54 (range 28-77) years of age. ER, PR, and HER2 status from the 28 patients with OM did not differ from other patients with MBC from our institution. The relative proportion of patients with ILC was significantly higher in the OM cohort (32.1%) than in other MBC patients in our institution (11.3%, p = 0.007). Median time to first OM in the OM cohort was 46.7 months, and OM were the second most frequent first metastases after bony metastases. After diagnosis of the first distant metastasis of any kind, median survival of patients with ILC (21.4 months) was significantly shorter than that of patients with IDC (55.3 months, p = 0.03). Nine patients developed bilateral OM. We observed a significant co-occurrence of OM and central nervous system metastases (p = 0.0053). The histological analysis revealed an interesting case in which the primary tumor was of a mixed ILC/IDC subtype, while only ILC was present in the OM.

Conclusions: OM from breast cancer are illustrative of the difference in metastatic behavior of ILC versus IDC and should be considered when treating patients with ILC, especially in those with complaints of visual acuity changes.
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http://dx.doi.org/10.1186/s13058-020-01309-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318761PMC
June 2020

FGFR4: A promising therapeutic target for breast cancer and other solid tumors.

Pharmacol Ther 2020 10 31;214:107590. Epub 2020 May 31.

Women's Cancer Research Center, UPMC Hillman Cancer Center, Pittsburgh, PA, USA; Magee-Women's Research Institute, Magee-Women's Research Hospital of University of Pittsburgh Medical Center, Pittsburgh, PA, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA. Electronic address:

The fibroblast growth factor receptor (FGFR) signaling pathway has long been known to cancer researchers because of its role in cell survival, proliferation, migration, and angiogenesis. Dysregulation of FGFR signaling is frequently reported in cancer studies, but most of these studies focus on FGFR1-3. However, there is growing evidence implicating an important and unique role of FGFR4 in oncogenesis, tumor progression, and resistance to anti-tumor therapy in multiple types of cancer. Importantly, there are several novel FGFR4-specific inhibitors in clinical trials, making FGFR4 an attractive target for further research. In this review, we focus on assessing the role of FGFR4 in cancer, with an emphasis on breast cancer. First, the structure, physiological functions and downstream signaling pathways of FGFR4 are introduced. Next, different mechanisms reported to cause aberrant FGFR4 activation and their functions in cancer are discussed, including FGFR4 overexpression, FGF ligand overexpression, FGFR4 somatic hotspot mutations, and the FGFR4 G388R single nucleotide polymorphism. Finally, ongoing and recently completed clinical trials targeting FGFRs in cancer are reviewed, highlighting the therapeutic potential of FGFR4 inhibition for the treatment of breast cancer.
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http://dx.doi.org/10.1016/j.pharmthera.2020.107590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494643PMC
October 2020

The Dysregulated Pharmacology of Clinically Relevant Mutants is Normalized by Ligand-activated WT Receptor.

Mol Cancer Ther 2020 07 7;19(7):1395-1405. Epub 2020 May 7.

Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina.

The estrogen receptor (ER/) is expressed in a majority of breast cancers and drugs that inhibit ER signaling are the cornerstone of breast cancer pharmacotherapy. Currently, aromatase inhibitors are the frontline endocrine interventions of choice although their durability in metastatic disease is limited by activating point mutations within the ligand-binding domain of that permit ligand-independent activation of the receptor. It has been suggested that the most commonly occurring mutations would likely compromise the clinical activity of selective estrogen receptor downregulators and selective estrogen receptor modulators (SERMs) when used as second-line therapies. It was unclear, however, how these mutations, which are likely coexpressed in cells with ER, may impact response to ER ligands in a clinically meaningful manner. To address this issue, we dissected the molecular mechanism(s) underlying -mutant pharmacology in models relevant to metastatic disease. These studies revealed that the response of mutations to ligands was dictated primarily by the relative coexpression of ER in cells. Specifically, dysregulated pharmacology was only evident in cells in which the mutants were overexpressed relative to ligand-activated ER; a finding that highlights the role of allelism in determining ER-mutant pharmacology. Importantly, we demonstrated that the antagonist activity of the SERM, lasofoxifene, was not impacted by mutant status; a finding that has led to its clinical evaluation as a treatment for patients with advanced ER-positive breast cancer whose tumors harbor mutations.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-1148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335351PMC
July 2020

Immune Landscape of Viral- and Carcinogen-Driven Head and Neck Cancer.

Immunity 2020 01 7;52(1):183-199.e9. Epub 2020 Jan 7.

Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA; Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA 15232, USA. Electronic address:

Head and neck squamous cell carcinoma (HNSCC) arises through exposure to environmental carcinogens or malignant transformation by human papillomavirus (HPV). Here, we assessed the transcriptional profiles of 131,224 single cells from peripheral and intra-tumoral immune populations from patients with HPV and HPV HNSCC and healthy donors. Immune cells within tumors of HPV and HPV HNSCC displayed a spectrum of transcriptional signatures, with helper CD4 T cells and B cells being relatively divergent and CD8+ T cells and CD4+ regulatory T cells being relatively similar. Transcriptional results were contextualized through multispectral immunofluorescence analyses and evaluating putative cell-cell communication based on spatial proximity. These analyses defined a gene expression signature associated with CD4 T follicular helper cells that is associated with longer progression-free survival in HNSCC patients. The datasets and analytical approaches herein provide a resource for the further study of the impact of immune cells on viral- and carcinogen-induced cancers.
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http://dx.doi.org/10.1016/j.immuni.2019.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201194PMC
January 2020

Metastatic breast cancers have reduced immune cell recruitment but harbor increased macrophages relative to their matched primary tumors.

J Immunother Cancer 2019 10 18;7(1):265. Epub 2019 Oct 18.

Womens Cancer Research Center, UPMC Hillman Cancer Center, Magee Womens Research Institute, Pittsburgh, USA.

The interplay between the immune system and tumor progression is well recognized. However, current human breast cancer immunophenotyping studies are mostly focused on primary tumors with metastatic breast cancer lesions remaining largely understudied. To address this gap, we examined exome-capture RNA sequencing data from 50 primary breast tumors (PBTs) and their patient-matched metastatic tumors (METs) in brain, ovary, bone and gastrointestinal tract. We used gene expression signatures as surrogates for tumor infiltrating lymphocytes (TILs) and compared TIL patterns in PBTs and METs. Enrichment analysis and deconvolution methods both revealed that METs had a significantly lower abundance of total immune cells, including CD8+ T cells, regulatory T cells and dendritic cells. An exception was M2-like macrophages, which were significantly higher in METs across the organ sites examined. Multiplex immunohistochemistry results were consistent with data from the in-silico analysis and showed increased macrophages in METs. We confirmed the finding of a significant reduction in immune cells in brain METs (BRMs) by pathologic assessment of TILs in a set of 49 patient-matched pairs of PBT/BRMs. These findings indicate that METs have an overall lower infiltration of immune cells relative to their matched PBTs, possibly due to immune escape. RNAseq analysis suggests that the relative levels of M2-like macrophages are increased in METs, and their potential role in promoting breast cancer metastasis warrants further study.
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http://dx.doi.org/10.1186/s40425-019-0755-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798422PMC
October 2019

FGFR4 overexpression and hotspot mutations in metastatic ER+ breast cancer are enriched in the lobular subtype.

NPJ Breast Cancer 2019 27;5:19. Epub 2019 Jun 27.

1Women's Cancer Research Center, UPMC Hillman Cancer Center, Pittsburgh, PA USA.

Invasive lobular carcinoma (ILC) is an understudied subtype of breast cancer that requires novel therapies in the advanced setting. To study acquired resistance to endocrine therapy in ILC, we have recently performed RNA-Sequencing on long-term estrogen deprived cell lines and identified FGFR4 overexpression as a top druggable target. Here, we show that FGFR4 expression also increases dramatically in endocrine-treated distant metastases, with an average fold change of 4.8 relative to the paired primary breast tumor for ILC, and 2.4-fold for invasive ductal carcinoma (IDC). In addition, we now report that FGFR4 hotspot mutations are enriched in metastatic breast cancer, with an additional enrichment for ILC, suggesting a multimodal selection of FGFR4 activation. These data collectively support the notion that FGFR4 is an important mediator of endocrine resistance in ILC, warranting future mechanistic studies on downstream signaling of overexpressed wild-type and mutant FGFR4.
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http://dx.doi.org/10.1038/s41523-019-0114-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597581PMC
June 2019

Frequent amplifications of ESR1, ERBB2 and MDM4 in primary invasive lobular breast carcinoma.

Cancer Lett 2019 Oct 20;461:21-30. Epub 2019 Jun 20.

Women's Cancer Research Center, Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, PA, USA; UPMC Hillman Cancer Center, Pittsburgh, PA, USA; Department of Pharmacology and Chemical Biology; University of Pittsburgh, Pittsburgh, PA, USA.

Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer following invasive ductal carcinoma (IDC). To identify potential genetic drivers of ILC progression, we used NanoString nCounter technology to investigate the DNA copy number (CN) in 70 well-curated primary ILC samples. We confirmed prior observations of frequent amplification of CCND1 (33%), and MYC (17%) in ILC, but additionally identified a substantial subset of ILCs with ESR1 and ERBB2 (19%) amplifications. Of interest, tumors with ESR1 CN gains (14%) and amplification (10%) were more likely to recur compared to those with normal CN. Finally, we observed that MDM4 (MDMX) was amplified in 17% of ILC samples. MDM4 knockdown in TP53 wild-type ILC cell lines caused increased apoptosis, decreased proliferation associated with cell cycle arrest, and concomitant activation of TP53 target genes. Similar effects were seen in TP53 mutant cells, indicting a TP53-independent role for MDM4 in ILC. To conclude, amplification of ESR1 and MDM4 are potential genetic drivers of ILC. These amplifications may represent actionable, targetable tumor dependencies, and thus have potential clinical implications and warrant further study.
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http://dx.doi.org/10.1016/j.canlet.2019.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682463PMC
October 2019

SNAIL is induced by tamoxifen and leads to growth inhibition in invasive lobular breast carcinoma.

Breast Cancer Res Treat 2019 Jun 23;175(2):327-337. Epub 2019 Feb 23.

Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261, USA.

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 G/G. 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.
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http://dx.doi.org/10.1007/s10549-019-05161-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625318PMC
June 2019

Network-guided prediction of aromatase inhibitor response in breast cancer.

PLoS Comput Biol 2019 02 11;15(2):e1006730. Epub 2019 Feb 11.

Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.

Prediction of response to specific cancer treatments is complicated by significant heterogeneity between tumors in terms of mutational profiles, gene expression, and clinical measures. Here we focus on the response of Estrogen Receptor (ER)+ post-menopausal breast cancer tumors to aromatase inhibitors (AI). We use a network smoothing algorithm to learn novel features that integrate several types of high throughput data and new cell line experiments. These features greatly improve the ability to predict response to AI when compared to prior methods. For a subset of the patients, for which we obtained more detailed clinical information, we can further predict response to a specific AI drug.
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http://dx.doi.org/10.1371/journal.pcbi.1006730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386390PMC
February 2019

Targeted mutation detection in breast cancer using MammaSeq™.

Breast Cancer Res 2019 02 8;21(1):22. Epub 2019 Feb 8.

Department of Pharmacology and Chemical Biology, and Human Genetics, UPMC Hillman Cancer Center, Magee-Womens Research Institute, University of Pittsburgh, 204 Craft Avenue, Pittsburgh, PA, 15213, USA.

Background: Breast cancer is the most common invasive cancer among women worldwide. Next-generation sequencing (NGS) has revolutionized the study of cancer across research labs around the globe; however, genomic testing in clinical settings remains limited. Advances in sequencing reliability, pipeline analysis, accumulation of relevant data, and the reduction of costs are rapidly increasing the feasibility of NGS-based clinical decision making.

Methods: We report the development of MammaSeq, a breast cancer-specific NGS panel, targeting 79 genes and 1369 mutations, optimized for use in primary and metastatic breast cancer. To validate the panel, 46 solid tumors and 14 plasma circulating tumor DNA (ctDNA) samples were sequenced to a mean depth of 2311× and 1820×, respectively. Variants were called using Ion Torrent Suite 4.0 and annotated with cravat CHASM. CNVKit was used to call copy number variants in the solid tumor cohort. The oncoKB Precision Oncology Database was used to identify clinically actionable variants. Droplet digital PCR was used to validate select ctDNA mutations.

Results: In cohorts of 46 solid tumors and 14 ctDNA samples from patients with advanced breast cancer, we identified 592 and 43 protein-coding mutations. Mutations per sample in the solid tumor cohort ranged from 1 to 128 (median 3), and the ctDNA cohort ranged from 0 to 26 (median 2.5). Copy number analysis in the solid tumor cohort identified 46 amplifications and 35 deletions. We identified 26 clinically actionable variants (levels 1-3) annotated by OncoKB, distributed across 20 out of 46 cases (40%), in the solid tumor cohort. Allele frequencies of ESR1 and FOXA1 mutations correlated with CA.27.29 levels in patient-matched blood draws.

Conclusions: In solid tumor biopsies and ctDNA, MammaSeq detects clinically actionable mutations (OncoKB levels 1-3) in 22/46 (48%) solid tumors and in 4/14 (29%) of ctDNA samples. MammaSeq is a targeted panel suitable for clinically actionable mutation detection in breast cancer.
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http://dx.doi.org/10.1186/s13058-019-1102-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368740PMC
February 2019
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