Brain Metastasis In Human Breast Cancer Research Articles

Abstract 5973: The brain penetrant CDK4/6 Inhibitor, PRT3645, is highly effective in combination with other targeted therapies in preclinical models of NSCLC, CRC, and HER2-positive breast cancer

Abstract Cell cycle deregulation is a hallmark of cancer and the hyperactivation and overexpression of CDKs are often drivers of cancer pathogenesis. Cyclin-dependent kinase 4 and 6 (CDK4)/(CDK6) are critical mediators of cellular transition into S phase and important for the initiation, growth, and survival of many cancers. Activated CDK4/CDK6 complexes phosphorylate Rb1, reduce their binding affinities and release Rb1-containing transcription repressor complexes from E2F transcription factors, resulting in activation of E2F controlled cell cycle genes and progression of the cell cycle. At present three CDK4/CDK6 inhibitors are approved for the treatment of ER+/HER2- breast cancer, and are being explored in other cancer indications as well. Previously we described a novel brain penetrant CDK4/CDK6 inhibitor, PRT3645, that exhibits single digit nanomolar biochemical potency against CDK4/CDK6 and >2000-fold selectivity against CDK1, CDK2 and CDK9. PRT3645 inhibits cellular phosphorylation of Rb and exhibits a protein binding-adjusted cellular IC50 of <300 nM. PRT3645 exhibits favorable in-vitro safety pharmacology and ADME properties, including increased brain penetration, and demonstrates oral bioavailability across rodents, dog, and non-human primates. In addition to robust monotherapy activity observed in preclinical models of ER+/HER2- breast cancer, we explored the activity of PRT3645 in other tumor types as well as in combination with other targeted therapies. In NSCLC, PRT3645 treatment resulted in significant inhibition of cell lines that harbor activation of the RAS/MEK/ERK pathway in proliferation assays and demonstrated comparable high synergy scores when combined with clinically approved covalent KRAS G12C inhibitors. In-vivo, oral PRT3645 was well tolerated and induced anti-tumor efficacy in two KRAS G12C mutant xenograft models that harbor the CDKN2A (p16) deletion. Anti-tumor efficacy was further improved when PRT3645 was combined with KRAS/MEK inhibitors in xenograft models and the combination therapy was well tolerated. In addition, we explored combinations of PRT3645 with a brain penetrant receptor tyrosine kinase inhibitor (TKI), an approved treatment for patients with advanced HER2+ breast cancer, including patients with brain metastases. In a HER2+ orthotopic human breast cancer brain metastasis model, PRT3645 was highly efficacious in combination with HER2 kinase inhibition and enhanced median survival significantly. In summary, PRT3645 demonstrates an excellent balance of potency, selectivity, PK parameters across species and brain penetrance. In preclinical studies, PRT3645 was highly efficacious when combined with KRAS/MEK inhibitors, and with a brain penetrant HER2 receptor TKI, both in-vitro and in-vivo. PRT3645 has advanced into Phase 1 clinical trials (NCT05538572). Citation Format: Yue Zou, Srijita Dhar, Kirsten Gallagher, Andrew Buesking, Sarah Pawley, Ryan Holmes, Xiaowei Wu, Katarina Rohlfing, Min Wang, Joseph Rager, Tom Emm, Stefan Ruepp, Miles Cowart, Jing Ni, Jean Zhao, Bruce Ruggeri, Andrew Combs, Kris Vaddi, Sandy Geeganage, Ashish Juvekar, Sang Hyun Lee, Peggy Scherle. The brain penetrant CDK4/6 Inhibitor, PRT3645, is highly effective in combination with other targeted therapies in preclinical models of NSCLC, CRC, and HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5973.

Impact of prior systemic therapy on lymphocytic infiltration in surgically resected breast cancer brain metastases.

Tumor-infiltrating lymphocytes (TILs) have been positively correlated with response to systemic therapy for triple-negative and HER2 + subtypes and improved clinical outcomes in early breast cancer (BC). Less is known about TILs in metastatic sites, particularly brain metastases (BM), where unique immune regulation governs stromal composition. Reactive glial cells actively participate in cytokine-mediated T cell stimulation. The impact of prior medical therapy (chemotherapy, endocrine, and HER2-targeted therapy) on the presence of TILs and gliosis in human breast cancer brain metastases (BCBM) has not been previously reported. We examined prior treatment data for 133 patients who underwent craniotomy for resection of BMs from the electronic medical record. The primary endpoint was overall survival (OS) from the time of BM diagnosis. We examined the relationship between prior systemic therapy exposure and the histologic features of gliosis, necrosis, hemorrhage, and lymphocyte infiltration (LI) in BCBMs resected at subsequent craniotomy in univariate analyses. Complete treatment data were available for 123 patients. BCBM LI was identified in 35 of 116 (30%) patients who had received prior systemic treatment versus 5 of 7 (71.4%) who had not {significant by Fisher's exact test p = 0.045}. There were no statistically significant relationships between prior systemic therapy and the three other histologic variables examined. This observation suggests that systemic therapy may interfere with the immune response to BCBMs and cause exhaustion of anti-tumor immunity. This motivates clinical investigation of strategies to enhance LI for therapeutic benefit to improve outcomes for patients with BCBMs.

Abstract 2300: Brain penetrant CDK4/6 inhibitor PRT3645 demonstrates anti-tumor activity and enhances survival in glioblastoma and breast cancer brain metastasis models

Abstract Cell cycle deregulation is a hallmark of cancer and CDK inhibitors, specifically inhibiting CDK4/6 and blocking cells transition from the G1 to the S phase of the cell cycle are the first and only class of highly specific CDK inhibitors approved for cancer treatment to date. CDK4/6 inhibitors have transformed the treatment paradigm of estrogen receptor-positive (ER+), HER2- breast cancer with three CDK4/6 inhibitors currently FDA approved. Brain metastasis commonly arises in patients with breast, lung, melanoma and other cancer types, is associated with poor survival outcomes and poses distinct challenges in clinical management. Due to advances in imaging technologies, the detection of brain metastases is increasing and there is a dearth of novel therapies to combat brain metastatic cancers and impact patient survival. Here, we describe a novel brain penetrant CDK4/6 inhibitor, PRT3645 exhibiting single digit nanomolar biochemical potency against CDK 4/6 and >2000-fold selectivity against other CDK family members (CDK1, CDK2, and CDK9). In cellular assays, PRT3645 inhibits cellular phosphorylation of RB with low nanomolar activity. Consistent with this, PRT3645 treatment resulted in concentration-dependent inhibition of cell proliferation in glioblastoma (GBM) cell lines and in HER2- and HER2+ breast cancer lines (EC50 values < 125 nM). Furthermore, PRT3645 demonstrated additive in vitro activity with fulvestrant or tucatinib in ER+ and HER2+ breast cancer lines. PRT3645 exhibits favorable in vitro safety pharmacology and ADME profiles, including brain exposure in rodents at steady state, and demonstrates oral bioavailability across rodents, dog and nonhuman primates. In vivo, oral PRT3645 was well tolerated and highly efficacious in a dose-dependent manner in subcutaneous xenograft models of GBM and breast cancer and in orthotopic human breast cancer brain metastasis (BCBM) and GBM models in mice as a monotherapy. PRT3645 showed tumor regression as single agent in the MCF7 ER+ breast cancer model and a combinatorial benefit with the estrogen receptor blocker, fulvestrant. In a HER2+ BT474-luc orthotopic model, similarly efficacious single agent activity of PRT3645 was achieved, as well as a significant combinatorial benefit on tumor growth and median survival when administered with the brain penetrant HER2 kinase inhibitor, tucatinib. PRT3645 was highly efficacious in a U87-luc GBM orthotopic model and demonstrated enhanced median survival benefit when combined with an orally active brain penetrant PRMT5 inhibitor. In summary, PRT3645 demonstrates an excellent balance of potency, selectivity, PK parameters across species, brain penetrance and favorable tissue distribution relative to brain exposure, and currently has advanced into IND-enabling preclinical studies. Citation Format: Ashish Juvekar, Yang Zhang, Andrew Buesking, Min Wang, Dave Rominger, Joseph Rager, Stefan Ruepp, Kirsten Gallagher, Yue Zou, Miles Cowart, Xiaowei Wu, Sarah Pawley, Ryan Holmes, William Gowen-MacDonald, Kris Vaddi, Andrew Combs, Bruce Ruggeri, Peggy Scherle. Brain penetrant CDK4/6 inhibitor PRT3645 demonstrates anti-tumor activity and enhances survival in glioblastoma and breast cancer brain metastasis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2300.

Myocyte Enhancer Factor 2C as a New Player in Human Breast Cancer Brain Metastases.

Myocyte enhancer factor 2C (MEF2C) is increasingly expressed in mice along with breast cancer brain metastases (BCBM) development. We aim to ascertain MEF2C expression in human BCBM, establish the relationship with disease severity, disclose the involvement of vascular endothelial growth factor receptor-2 (VEGFR-2) and β-catenin, also known as KDR and CTNNB1, respectively, and investigate if matched primary tumors express the protein. We studied resected BCBM for the expression of MEF2C, VEGFR-2, and ß-catenin, as well as proliferation (Ki-67) and epithelial (pan Cytokeratin) markers, and related experimental and clinical data. MEF2C expression was further assessed in matched primary tumors and non-BCBM samples used as controls. MEF2C expression was observed in BCBM, but not in controls, and was categorized into three phenotypes (P): P1, with extranuclear location; P2, with extranuclear and nuclear staining, and P3, with nuclear location. Nuclear translocation increased with metastases extension and Ki-67-positive cells number. P1 was associated with higher VEFGR-2 plasma membrane immunoreactivity, whereas P2 and P3 were accompanied by protein dislocation. P1 was accompanied by β-catenin membrane expression, while P2 and P3 exhibited β-catenin nuclear translocation. Primary BC samples expressed MEF2C in mammary ducts and scattered cells in the parenchyma. MEF2C emerges as a player in BCBM associated with disease severity and VEGFR-2 and β-catenin signaling.

Abstract A89: Microglial galectin-3 enhances the metastatic phenotype of breast cancer cells in brain metastases

Abstract Background: Breast cancer brain metastases have poor prognosis and few treatment options. When breast cancer cells invade the brain, they interact with the vasculature and resident brain cells, including microglia and astrocytes. We hypothesize that brain cells produce factors that enhance the growth and invasion of breast cancer cells in the brain. Methods: Human breast cancer cell lines (MDA-MB231BR-HER2 and HCC1954) were inoculated intracranially in athymic rats as xenograft models of brain metastasis. Brains were analyzed for protumorigenic factor expression in the tumor microenvironment using fluorescent immunohistochemistry. In vitro assays assessed factors involved in breast cancer cell proliferation, morphology, and migration. Results: The brain tumor xenografts showed infiltration into the perivascular space. Galectin-3 (Gal3) colocalized with reactive microglia in and around the tumors. In vitro, Gal3 increased HCC1954 cell proliferation and migration. In transwell coculture assays, BV2 microglial cells increased the migration of HCC1954 breast cancer cells >25 fold, which was prevented by cilengitide, an inhibitor of alpha(v)beta(3)-integrin cell adhesion protein. ELISA analysis demonstrated that BV2 microglia secrete Gal3 in the presence of HCC1954 cells. Gal3 is known to bind and induce clustering of alpha(v)beta(3)-integrin, which is expressed on metastatic breast cancer cells. Immunohistochemistry of clinical specimens revealed that Gal3 is expressed in/around human breast cancer brain metastases. Conclusions: These data suggest that factors produced in the tumor microenvironment promote the growth and migration of breast cancer cells in the brain. Gal3, produced and secreted by activated microglia in vitro and expressed in and around brain metastases, increases the invasive capability of breast cancer cells. The interactions of neoplastic cells with the brain environment may provide a target to improve therapy of brain metastases. Citation Format: Cymon N. Kersch, DreeAnna F. Morris, Leslie L. Muldoon, Edward A. Neuwelt. Microglial galectin-3 enhances the metastatic phenotype of breast cancer cells in brain metastases [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A89.

Downregulation of circulating miR 802-5p and miR 194-5p and upregulation of brain MEF2C along breast cancer brain metastasization.

Breast cancer brain metastases (BCBMs) have been underinvestigated despite their high incidence and poor outcome. MicroRNAs (miRNAs), and particularly circulating miRNAs, regulate multiple cellular functions, and their deregulation has been reported in different types of cancer and metastasis. However, their signature in plasma along brain metastasis development and their relevant targets remain undetermined. Here, we used a mouse model of BCBM and next‐generation sequencing (NGS) to establish the alterations in circulating miRNAs during brain metastasis formation and development. We further performed bioinformatics analysis to identify their targets with relevance in the metastatic process. We additionally analyzed human resected brain metastasis samples of breast cancer patients for target expression validation. Breast cancer cells were injected in the carotid artery of mice to preferentially induce metastasis in the brain, and samples were collected at different timepoints (5 h, 3, 7, and 10 days) to follow metastasis development in the brain and in peripheral organs. Metastases were detected from 7 days onwards, mainly in the brain. NGS revealed a deregulation of circulating miRNA profile during BCBM progression, rising from 18% at 3 days to 30% at 10 days following malignant cells’ injection. Work was focused on those altered prior to metastasis detection, among which were miR‐802‐5p and miR‐194‐5p, whose downregulation was validated by qPCR. Using targetscan and diana tools, the transcription factor myocyte enhancer factor 2C (MEF2C) was identified as a target for both miRNAs, and its expression was increasingly observed in malignant cells along brain metastasis development. Its upregulation was also observed in peritumoral astrocytes pointing to a role of MEF2C in the crosstalk between tumor cells and astrocytes. MEF2C expression was also observed in human BCBM, validating the observation in mouse. Collectively, downregulation of circulating miR‐802‐5p and miR‐194‐5p appears as a precocious event in BCBM and MEF2C emerges as a new player in brain metastasis development.

TMIC-10. THE ROLE OF THE BRAIN MICROENVIRONMENT IN THE MIGRATION OF BREAST CANCER CELLS IN BRAIN METASTASES

Abstract BACKGROUND Breast cancer brain metastases have poor prognosis and few treatment options. When breast cancer cells invade the brain they interact with the vasculature and resident brain cells including microglia and astrocytes. We hypothesize that brain cells produce factors that enhance the growth and invasion of breast cancer cells in the brain. METHODS Human breast cancer cell lines (MDA-MB231BR-HER2 and HCC1954) were inoculated intracranially in athymic rats as xenograft models of brain metastasis. Brains were analyzed for pro-tumorigenic factor expression in the tumor microenvironment using fluorescent immunohistochemistry. In vitro assays assessed factors involved in breast cancer cell proliferation, morphology, and migration. RESULTS The tumor xenografts showed infiltration intothe perivascular space. Galectin-3 (Gal3), heparin-binding epidermal growth factor (HB-EGF), and Neuregulin (NRG1), factors known to interact with receptors expressed by breast cancer cells, co-localized with reactive microglia (Gal3) and astrocytes (HB-EGF and NRG1) in and around xenografts. In vitro, these factors individually increased HCC1954 cell proliferation and/or migration. In transwell co-culture assays, BV2 microglial cells increased the migration of HCC1954 cells >25 fold, which was prevented by cilengitide, an inhibitor of αvβ3-integrin cell adhesion protein. ELISA analysis demonstrated that BV2 microglia secrete Gal3 in the presence of HCC1954 cells. Gal3 is known to bind and induce clustering of αvβ3-integrin which is expressed on metastatic breast cancer cells. Immunohistochemistry of clinical specimens revealed that Gal3 is expressed in/around human breast cancer brain metastases. CONCLUSIONS These data suggest that factors produced in the tumor microenvironment promote the growth and migration of breast cancer cells in the brain. Gal3, produced and secreted by activated microglia in vitro and expressed in and around brain metastases, increases the invasive capability of breast cancer cells. The interactions of neoplastic cells with the brain environment may provide a target to improve therapy of brain metastases.

Expression of Angiopoietin-like 4 Fibrinogen-Like Domain increases risk of brain metastases in women with breast cancer

Abstract Background Brain metastases challenge daily clinical practice, and the mechanisms by which cancer cells cross the blood-brain barrier remain largely undeciphered. Angiopoietin-like 4 (ANGPTL4) proteolytic fragments have controversial biological effects on endothelium permeability. Here, we studied the link between ANGPTL4 and the risk of brain metastasis in cancer patients. Methods From June 2014 to June 2015, serum samples from 86 cancer patients were prospectively collected, and ANGPTL4 concentrations were assessed. Using a murine model of brain metastases, we investigated the roles of nANGPTL4 and cANGPTL4 fragments in the occurrence of brain metastases. Results A positive ANGPTL4 serum concentration over 0.1ng/mL was associated with decreased overall-survival. Multivariate analyses found that only breast cancer brain metastases were significantly associated with elevated ANGPTL4 serum concentrations. 4T1 murine breast cancer cells were transfected with either nANGPTL4- or cANGPTL4-encoding cDNAs. Compared to mice injected with wild-type 4T1 cells, mice injected with nANGPTL4 cells had shorter median survival (p Conclusions This study opens the way for further translational research on the role of cANGPTL4 fragment in human breast cancer brain metastases.

Human brain metastatic stroma attracts breast cancer cells via chemokines CXCL16 and CXCL12

The tumor microenvironment is composed of heterogeneous populations of cells, including cancer, immune, and stromal cells. Progression of tumor growth and initiation of metastasis is critically dependent on the reciprocal interactions between cancer cells and stroma. Through RNA-Seq and protein analyses, we found that cancer-associated fibroblasts derived from human breast cancer brain metastasis express significantly higher levels of chemokines CXCL12 and CXCL16 than fibroblasts from primary breast tumors or normal breast. To further understand the interplay between cancer cells and cancer-associated fibroblasts from each site, we developed three-dimensional organoids composed of patient-derived primary or brain metastasis cancer cells with matching cancer-associated fibroblasts. Three-dimensional CAF aggregates generated from brain metastasis promote migration of cancer cells more effectively than cancer-associated fibroblast aggregates derived from primary tumor or normal breast stromal cells. Treatment with a CXCR4 antagonist and/or CXCL16 neutralizing antibody, alone or in combination, significantly inhibited migration of cancer cells to brain metastatic cancer-associated fibroblast aggregates. These results demonstrate that human brain metastasis cancer-associated fibroblasts potently attract breast cancer cells via chemokines CXCL12 and CXCL16, and blocking CXCR6-CXCL16/CXCR4-CXCL12 receptor–ligand interactions may be an effective therapy for preventing breast cancer brain metastasis.

ATNT-22A PHASE 1 STUDY OF TPI 287 CONCURRENT WITH FRACTIONATED STEREOTACTIC RADIOTHERAPY (FSRT) IN TREATMENT OF BRAIN METASTASES FROM ADVANCED BREAST AND NON-SMALL CELL LUNG (NSCL) CANCER

BACKGROUND: TPI 287, a member of taxanes diterpenoid (taxoid) family, is a microtubule-stabilizer with significant cytotoxic activity. In vivo studies have demonstrated that TPI 287 penetrates the blood brain barrier and significantly reduces the formation of large brain metastases in models of human breast cancer brain metastasis. The radiosensitizing effect of taxanes has been well established. METHODS: This phase I study (3 + 3 design) explores the safety, recommended phase II dose (RP2D), pharmacokinetics (PK), and antitumor activity (local control rate, distant intra-cranial control rate, progression-free survival) of TPI 287 administered concurrently with FSRT (NCT02187822). Patients with up to 3 untreated brain metastases (maximum diameter of each brain lesion ≤ 5 cm, maximum tumor volume ≤ 120 cc) from breast cancer or NSCLC are eligible. Eligible patients are treated with FSRT to target brain metastases (25 Gy in 5 daily fractions). TPI 287 is administered intravenously once per week, for total of 3 doses. The first dose of TPI 287 is given concurrently with the first fraction of FSRT. Once the RP2D is determined, an additional 10 patients will be enrolled in an expansion safety cohort, for a planned total enrollment of 36 patients. Effect of treatment on quality of life measures will be evaluated. RESULTS: As of June 2015, a total of 4 patients have been enrolled in the first two dose escalation cohorts (14 and 28 mg/m2). No treatment-related grade 3/4 adverse events (AEs) have been observed. No dose limiting toxicity has been reported, and maximal tolerated dose has not been reached. TPI 287 dose escalation continues. CONCLUSIONS: Preliminary data to date indicate that administration of TPI 287 concurrent with FSRT is well tolerated. Updated safety and efficacy results will be presented.

Abstract 3779: Treatment of experimental brain metastasis of human breast cancer by macitentan, a dual antagonist of endothelin receptors combined with paclitaxel

Abstract Purpose: Breast cancer is a leading cause of cancer-related mortality among women in the United States. The major cause of death is due to metastasis that is resistant to conventional therapy. The median survival of women with brain metastases is measured in months due to resistance to all standard therapies. Thus, the development of effective treatment options for these patients is mandatory. We have reported that astrocytes can protect tumor cells from chemotherapeutic drugs by a mechanism involving the phosphorylation of endothelin receptors on tumor cells (Kim et al, AACR, 2012). Moreover, we observed a heterogeneous expression of endothelin receptors A and B in clinical samples of breast cancer brain metastasis. We therefore examined whether blockade of the endothelin axis by using macitentan, a dual endothelin antagonist, combined with paclitaxel can produce therapeutic effects in an orthotopic model of human breast cancer brain metastasis. Materials and Methods: Twenty thousand MDA-MB-231 human breast cancer cells were injected into the internal carotid artery of female nude mice to produce orthotopic brain metastasis. Two weeks later, the mice were randomized into 4 groups (n=10): (1) control group - mice receiving vehicle (p.o., daily; i.p. injection once per week); (2) paclitaxel group - mice receiving paclitaxel (8 mg/kg; i.p. injection once per week) and vehicle (p.o.; daily); (3) macitentan group - mice receiving macitentan (10 mg/kg; p.o.; daily) and vehicle (i.p. injection, once per week); and (4) paclitaxel plus macitentan group. Moribund mice were euthanized, days of survival were recorded, and the brains were harvested and processed for histology and immunohistochemical analyses. The study was repeated with luciferase-labeled MDA-MB-231 cells and produced similar therapeutic results supported by IVIS imaging. Results: After 130-140 days of treatment, the survival was significantly higher in mice treated with the combination of paclitaxel and macitentan (80%) as compared with control (20%), paclitaxel (20%), and macitentan alone (40%) (p<0.0001, log-rank test). Immunohistochemical analyses revealed that the phosphorylation of endothelin receptors A and B on tumor cells and tumor-associated endothelial cells were inhibited by macitentan. Treatment with macitentan also suppressed the expression of survival proteins such as pAKT, pMAPK, BCL2L1, TWIST1, and GSTA5 in tumor cells. More importantly, only the combination therapy induced significant apoptosis of tumor cells and tumor-associated endothelial cells and reduced the number of KI67 positive tumor cells. IVIS imaging confirmed regression of brain lesions. Conclusion: Treatment with macitentan, a dual endothelin receptor antagonist, combined with paclitaxel leads to regression of experimental breast cancer brain metastasis and therefore should be considered for clinical development. Citation Format: Ho Jeong Lee, Sun Jin Kim, Seung Wook Kim, Junqin He, Qiuyu Wu, Erica J. Lawson, Francois Lehembre, Urs Regenass, Isaiah J. Fidler. Treatment of experimental brain metastasis of human breast cancer by macitentan, a dual antagonist of endothelin receptors combined with paclitaxel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3779. doi:10.1158/1538-7445.AM2014-3779

Capecitabine and lapatinib uptake in surgically resected brain metastases from metastatic breast cancer patients: a prospective study.

Breast cancer brain metastases (BCBM) are challenging complications that respond poorly to systemic therapy. The role of the blood-tumor barrier in limiting BCBM drug delivery and efficacy has been debated. Herein, we determined tissue and serum levels of capecitabine, its prodrug metabolites, and lapatinib in women with BCBM resected via medically indicated craniotomy. Study patients with BCBM requiring surgical resection received either single-dose capecitabine (1250 mg/m(2)) 2-3 h before surgery or 2-5 doses of lapatinib (1250 mg) daily, the last dose 2-3 h before surgery. Serum samples were collected serially on the day of surgery. Drug concentrations were determined in serum and BCBM using liquid chromatography tandem mass spectrometry. Twelve patients were enrolled: 8 for capecitabine and 4 for lapatinib. Measurable drug levels of capecitabine and metabolites, 5'-deoxy-5-fluorocytidine, 5'-deoxy-5-fluorouridine, and 5-fluorouracil, were detected in all BCBM. The ratio of BCBM to serum was higher for 5-fluorouracil than for capecitabine. As for lapatinib, the median BCBM concentrations ranged from 1.0 to 6.5 µM. A high variability (0.19-9.8) was noted for lapatinib BCBM-to-serum ratio. This is the first study to demonstrate that capecitabine and lapatinib penetrate to a significant though variable degree in human BCBM. Drug delivery to BCBM is variable and in many cases appears partially limiting. Elucidating mechanisms that limit drug concentration and innovative approaches to overcome limited drug uptake will be important to improve clinical efficacy of these agents in the central nervous system. Trial registration ID: NCT00795678.

Abstract A64: Identification of p75NTR/TrkB-BDNF axis in breast cancer brain metastasis

Abstract Metastasis of breast cancer to the brain is increasing and is a significant cause of morbidity and mortality for breast cancer patients. In order to understand the molecular mechanisms controlling metastasis to the brain, we have generated two mouse models of breast cancer brain metastasis (4T1.luc2.Br5, Eo771.Br5) via serial selection of these cells through the brains of syngeneic mice following carotid artery injection. In vitro, the 4T1.luc2.Br5 cells exhibit increased proliferation and invasion through basement membrane proteins, while both models demonstrate increased ability to adhere to brain microvascular endothelial cells. Transcriptional profiling followed by Western analysis determined that the p75NTR neurotrophin receptor and its ligand BDNF are highly upregulated in brain-metastatic 4T1.luc2.Br5 cells. The TrkB neurotrophin receptor for BDNF is expressed by these cells as well. Knockdown of p75NTR abolishes the increased invasive capacity of brain-selected 4T1.luc2.Br5 in vitro, confirming a role for p75NTR signaling in invasion. While p75NTR expression in primary breast epithelial cells is reportedly low, we observed that 8 of 12 (67%) human breast cancer brain metastasis specimens express high levels of p75NTR, further suggesting an important role in metastasis to brain. Interestingly, while the brain metastatic Eo771.Br5 cells do not express p75NTR in vitro, both the TrkB receptor and its BDNF ligand are upregulated compared to parentals. Together these data suggest a role for activation of p75NTR/TrkB-BDNF signaling in breast cancer brain metastatic cells, and that targeting this pathway may be a means to prevent or treat breast cancer brain metastasis. Citation Format: Wenhong Chen, Christine N. McMahan, Keith D. Barlow, Dennis Juarez, Linda J. Metheny-Barlow. Identification of p75NTR/TrkB-BDNF axis in breast cancer brain metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A64.

β-catenin-independent WNT signaling in basal-like breast cancer and brain metastasis

A role of WNT signaling for primary breast cancers of the basal-like subtype and as a predictor of brain metastasis has been described. However, a responsible WNT ligand has not been identified. To further clarify this question, we comparatively investigated 22 human breast cancer brain metastases as well as the highly invasive human breast cancer cell line MDA-MB-231 and the weakly motile MCF-7 as models for the basal-like and the luminal A subtype. WNT5A and B were found overexpressed in MDA-MB-231 cells as compared with MCF-7. This corresponded to reduction of MDA-MB-231 invasiveness by WNT inhibitors, whereas MCF-7 invasion was enhanced by recombinant WNT5B and abolished by WNT and Jun-N-terminal kinase antagonists. Expression and subcellular distribution of β-catenin remained uninfluenced. Consistently, β-catenin was not localized in the nuclei of brain metastases while there was strong nuclear c-Jun staining. Similar to MDA-MB-231, metastases showed expression of WNT5A/B and the alternative WNT receptors ROR1 and 2. These findings were validated using external gene expression datasets (Gene Expression Omnibus) of different breast cancer subtypes and brain metastases. Hierarchical cluster analysis yielded a close relation between basal-like cancers and brain metastases. Gene set enrichment analyses confirmed WNT pathway enrichment not only in basal-like primaries but also in cerebral metastases of all subtypes. In conclusion, WNT signaling seems highly relevant for basal-like and other subtypes of breast cancers metastasizing into the brain. β-catenin-independent WNT signaling, presumably via ROR1-2, plays a major role in this context.

Correlation of hexokinase-2 expression with overall survival and potential as a therapeutic target in the treatment of breast cancer brain metastases

1005 Background: The prevalence of brain metastasis from breast cancer (BCA) is increasing. We have identified several potential therapeutic targets through gene expression profiling of human BCA brain metastases. One potential target, hexokinase II (HK-2), is up-regulated in brain metastases as compared to unlinked primary tumors. HK-2 plays a vital role in the generation of many components required for cell growth and inhibits apoptosis through mitochondrial binding. We hypothesized that HK-2 promotes metastatic cell proliferation in the brain and that its increased expression would negatively impact patient survival post-resection. Methods: The gene expression profile of surgically resected human BCA brain metastases was compared to that of non-related unlinked primary invasive ductal carcinomas using cDNA microarray analysis. In a separate retrospective study, we generated survival curves dividing a cohort of 113 patients based on HK-2 immunoreactivity in resected brain metastases. To evaluate the fun...