Examination and prognostic implications of the unique microenvironment of breast cancer brain metastases.

PURPOSE: Brain metastases (BM) are a complication of advanced breast cancer (BC). Histology of melanoma BM offers prognostic value; however, understanding the microenvironment of breast cancer brain metastases (BCBM) is less characterized. This study reports on four histological biomarkers, gliosis, immune infiltrate, hemorrhage, necrosis, and their prognostic significance in BCBM. METHODS: A biobank of 203 human tissues from patients who underwent craniotomy for BCBM was created across four academic institutions. Degree of gliosis, immune infiltrate, hemorrhage, and necrosis were identified and scored via representative H&E stain (0-3+). Overall survival (OS) was estimated using the Kaplan-Meier method. Cox proportional hazards regression evaluated prognostic value of the biomarkers in the context of standard clinical characteristics. RESULTS: BCBM subtype (available for n = 158) was 36% Her2+, 26% hormone receptor (HR)+/Her2- 38% HR-/Her2- (triple negative, TN). Gliosis was observed in 82% (116/141) of BCBM, with immune infiltrate 44% (90/201), hemorrhage 82% (166/141), and necrosis 87% (176/201). Necrosis was significantly higher in TNBC (p < 0.01). Presence of gliosis, immune infiltrate, and hemorrhage correlated with improved OS (p = 0.03, p = 0.03, p = 0.1), while necrosis correlated with inferior OS (p = 0.01). Improved OS was associated with gliosis in TN (p = 0.02), and immune infiltrate (p = 0.001) and hemorrhage (p = 0.07) in HER2+. In a multivariable model for OS, incorporating these biomarkers with traditional clinical variables improved the model fit (p < 0.001). CONCLUSION: Gliosis confers superior prognosis in TNBC BM; immune infiltrate and hemorrhage correlate with superior prognosis in HER2+ BCBM. Understanding the metastatic microenvironment of BCBM refines prognostic considerations and may unveil novel therapeutic strategies.

LCCC 1025: a phase II study of everolimus, trastuzumab, and vinorelbine to treat progressive HER2-positive breast cancer brain metastases.

PURPOSE: HER2 + breast cancer (BC) is an aggressive subtype with high rates of brain metastases (BCBM). Two-thirds of HER2 + BCBM demonstrate activation of the PI3K/mTOR pathway driving resistance to anti-HER2 therapy. This phase II study evaluated everolimus (E), a brain-permeable mTOR inhibitor, trastuzumab (T), and vinorelbine (V) in patients with HER2 + BCBM. PATIENTS AND METHODS: Eligible patients had progressive HER2 + BCBM. The primary endpoint was intracranial response rate (RR); secondary objectives were CNS clinical benefit rate (CBR), extracranial RR, time to progression (TTP), overall survival (OS), and targeted sequencing of tumors from enrolled patients. A two-stage design distinguished intracranial RR of 5% versus 20%. RESULTS: 32 patients were evaluable for toxicity, 26 for efficacy. Intracranial RR was 4% (1 PR). CNS CBR at 6 mos was 27%; at 3 mos 65%. Median intracranial TTP was 3.9 mos (95% CI 2.2-5). OS was 12.2 mos (95% CI 0.6-20.2). Grade 3-4 toxicities included neutropenia (41%), anemia (16%), and stomatitis (16%). Mutations in TP53 and PIK3CA were common in BCBM. Mutations in the PI3K/mTOR pathway were not associated with response. ERBB2 amplification was higher in BCBM compared to primary BC; ERBB2 amplification in the primary BC trended toward worse OS. CONCLUSION: While intracranial RR to ETV was low in HER2 + BCBM patients, one-third achieved CNS CBR; TTP/OS was similar to historical control. No new toxicity signals were observed. Further analysis of the genomic underpinnings of BCBM to identify tractable prognostic and/or predictive biomarkers is warranted. CLINICAL TRIAL: (NCT01305941).

Comprehensive Analysis of the Immunogenomics of Triple-Negative Breast Cancer Brain Metastases From LCCC1419.

Background: Triple negative breast cancer (TNBC) is an aggressive variant of breast cancer that lacks the expression of estrogen and progesterone receptors (ER and PR) and HER2. Nearly 50% of patients with advanced TNBC will develop brain metastases (BrM), commonly with progressive extracranial disease. Immunotherapy has shown promise in the treatment of advanced TNBC; however, the immune contexture of BrM remains largely unknown. We conducted a comprehensive analysis of TNBC BrM and matched primary tumors to characterize the genomic and immune landscape of TNBC BrM to inform the development of immunotherapy strategies in this aggressive disease. Methods: Whole-exome sequencing (WES) and RNA sequencing were conducted on formalin-fixed, paraffin-embedded samples of BrM and primary tumors of patients with clinical TNBC (n = 25, n = 9 matched pairs) from the LCCC1419 biobank at UNC-Chapel Hill. Matched blood was analyzed by DNA sequencing as a comparison for tumor WES for the identification of somatic variants. A comprehensive genomics assessment, including mutational and copy number alteration analyses, neoantigen prediction, and transcriptomic analysis of the tumor immune microenvironment were performed. Results: Primary and BrM tissues were confirmed as TNBC (23/25 primaries, 16/17 BrM) by immunohistochemistry and of the basal intrinsic subtype (13/15 primaries and 16/19 BrM) by PAM50. Compared to primary tumors, BrM demonstrated a higher tumor mutational burden. TP53 was the most frequently mutated gene and was altered in 50% of the samples. Neoantigen prediction showed elevated cancer testis antigen- and endogenous retrovirus-derived MHC class I-binding peptides in both primary tumors and BrM and predicted that single-nucleotide variant (SNV)-derived peptides were significantly higher in BrM. BrM demonstrated a reduced immune gene signature expression, although a signature associated with fibroblast-associated wound healing was elevated in BrM. Metrics of T and B cell receptor diversity were also reduced in BrM. Conclusions: BrM harbored higher mutational burden and SNV-derived neoantigen expression along with reduced immune gene signature expression relative to primary TNBC. Immune signatures correlated with improved survival, including T cell signatures. Further research will expand these findings to other breast cancer subtypes in the same biobank. Exploration of immunomodulatory approaches including vaccine applications and immune checkpoint inhibition to enhance anti-tumor immunity in TNBC BrM is warranted.

Evaluating the efficacy of a priming dose of cyclophosphamide prior to pembrolizumab to treat metastatic triple negative breast cancer.

PURPOSE: Triple negative breast cancer (TNBC) is characterized by the presence of immune cells in the tumor microenvironment, however, the response to single-agent immune checkpoint inhibitor (ICI) therapy is modest. Preclinical models have demonstrated that intratumoral regulatory T cells (Tregs) dampen the antitumor response to ICI. We performed a single-arm phase II trial to evaluate the efficacy of a single low dose of cyclophosphamide (Cy) to deplete Tregs administered before initiating pembrolizumab. PATIENTS AND METHODS: 40 patients with pretreated metastatic TNBC were enrolled. The primary endpoints were progression-free survival (PFS) and change in peripheral blood Tregs after Cy. Secondary endpoints included overall response rate (ORR), duration of response, overall survival, treatment-related adverse events (AEs), and correlative evaluations. RESULTS: Median PFS was 1.8 months, and the ORR was 21%. Tregs were not significantly decreased after Cy prior to ICI (-3.3%, p=0.19), and increased significantly after the first cycle of therapy (+21% between cycles 1 and 2, p=0.005). Immune-related AEs were similar to historical pembrolizumab monotherapy, and were associated with response to therapy (p=0.02). Patients with pretreatment tumors harboring increased expression of B cell metagene signatures and increased circulating B cell receptor repertoire diversity were associated with clinical response and immune-related toxicity (IRT). CONCLUSIONS: Among patients with heavily pretreated TNBC, Cy prior to pembrolizumab did not significantly deplete Tregs, and in those with decreased numbers there was rapid recovery following therapy. Increased B cell gene expression in baseline samples was associated with clinical response and IRT.

Efficacy and pharmacodynamics of niraparib in BRCA-mutant and wild-type intracranial triple-negative breast cancer murine models.

BACKGROUND: Despite the poor prognosis of triple-negative breast cancer (TNBC) brain metastases, there are no approved systemic therapies. We explored the DNA-damaging poly(ADP-ribose) polymerase inhibitor (PARPi) niraparib in intracranial mouse models of breast cancer susceptibility protein (BRCA)-mutant TNBC. METHODS: Mice bearing intracranial human-derived TNBC cell lines (SUM149, MDA-MB-231Br, or MDA-MB-436) were treated with niraparib and monitored for survival; intracranial tissues were analyzed for PAR levels and niraparib concentration by mass spectrometry. RNASeq data of primary breast cancers using The Cancer Genome Atlas were analyzed for DNA damage signatures. Combined RAD51 and PARP inhibition in TNBC cell lines was assessed in vitro by colony-forming assays. RESULTS: Daily niraparib increased median survival and decreased tumor burden in the BRCA-mutant MDA-MB-436 model, but not in the BRCA-mutant SUM149 or BRCA-wild-type MDA-MB-231Br models despite high concentrations in intracranial tumors. RAD51 inhibitor B02 was shown to sensitize all cell lines to PARP inhibition (PARPi). In the analysis of BRCA-mutant primary human TNBCs, gene expression predictors of PARPi sensitivity and DNA repair signatures demonstrate widespread heterogeneity, which may explain the differential response to PARPi. Interestingly, these signatures are significantly correlated to RAD51 expression including PARPi sensitivity (R 2 = 0.602, R 2= 0.758). CONCLUSIONS: Niraparib penetrates intracranial tumor tissues in mouse models of TNBC with impressive single-agent efficacy in BRCA-mutant MDA-MB-436. Clinical evaluation of niraparib to treat TNBC brain metastases, an unmet clinical need desperate for improved therapies, is warranted. Further compromising DNA repair through RAD51 inhibition may further augment TNBC's response to PARPi.

Combined kinase inhibitors of MEK1/2 and either PI3K or PDGFR are efficacious in intracranial triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC), lacking expression of hormone and human epidermal growth factor receptor 2 receptors, is an aggressive subtype that frequently metastasizes to the brain and has no FDA-approved systemic therapies. Previous literature demonstrates mitogen-activated protein kinase kinase (MEK) pathway activation in TNBC brain metastases. Thus, we aimed to discover rational combinatorial therapies with MEK inhibition, hypothesizing that co-inhibition using clinically available brain-penetrant inhibitors would improve survival in preclinical models of TNBC brain metastases. METHODS: Using human-derived TNBC cell lines, synthetic lethal small interfering RNA kinase screens were evaluated with brain-penetrant inhibitors against MEK1/2 (selumetinib, AZD6244) or phosphatidylinositol-3 kinase (PI3K; buparlisib, BKM120). Mice bearing intracranial TNBC tumors (SUM149, MDA-MB-231Br, MDA-MB-468, or MDA-MB-436) were treated with MEK, PI3K, or platelet derived growth factor receptor (PDGFR; pazopanib) inhibitors alone or in combination. Tumors were analyzed by western blot and multiplexed kinase inhibitor beads/mass spectrometry to assess treatment effects. RESULTS: Screens identified MEK+PI3K and MEK+PDGFR inhibitors as tractable, rational combinations. Dual treatment of selumetinib with buparlisib or pazopanib was synergistic in TNBC cells in vitro. Both combinations improved survival in intracranial SUM149 and MDA-MB-231Br, but not MDA-MB-468 or MDA-MB-436. Treatments decreased mitogen-activated protein kinase (MAPK) and PI3K (Akt) signaling in sensitive (SUM149 and 231Br) but not resistant models (MDA-MB-468). Exploratory analysis of kinome reprogramming in SUM149 intracranial tumors after MEK ± PI3K inhibition demonstrates extensive kinome changes with treatment, especially in MAPK pathway members. CONCLUSIONS: Results demonstrate that rational combinations of the clinically available inhibitors selumetinib with buparlisib or pazopanib may prove to be promising therapeutic strategies for the treatment of some TNBC brain metastases. Additionally, effective combination treatments cause widespread alterations in kinase pathways, including targetable potential resistance drivers.

Efficacy of Carboplatin Alone and in Combination with ABT888 in Intracranial Murine Models of BRCA-Mutated and BRCA-Wild-Type Triple-Negative Breast Cancer.

Patients with breast cancer brain metastases have extremely limited survival and no approved systemic therapeutics. Triple-negative breast cancer (TNBC) commonly metastasizes to the brain and predicts poor prognosis. TNBC frequently harbors BRCA mutations translating to platinum sensitivity potentially augmented by additional suppression of DNA repair mechanisms through PARP inhibition. We evaluated brain penetrance and efficacy of carboplatin ± the PARP inhibitor ABT888, and investigated gene-expression changes in murine intracranial TNBC models stratified by BRCA and molecular subtype status. Athymic mice were inoculated intracerebrally with BRCA-mutant: SUM149 (basal), MDA-MB-436 (claudin-low); or BRCA-wild-type (wt): MDA-MB-468 (basal), MDA-MB-231BR (claudin-low). TNBC cells were treated with PBS control [intraperitoneal (IP), weekly], carboplatin (50 mg/kg/wk, IP), ABT888 (25 mg/kg/d, oral gavage), or their combination. DNA damage (γ-H2AX), apoptosis (cleaved caspase-3, cC3), and gene expression were measured in intracranial tumors. Carboplatin ± ABT888 significantly improved survival in BRCA-mutant intracranial models compared with control, but did not improve survival in BRCA-wt intracranial models. Carboplatin + ABT888 revealed a modest survival advantage versus carboplatin in BRCA-mutant models. ABT888 yielded a marginal survival benefit in the MDA-MB-436, but not in the SUM149 model. BRCA-mutant SUM149 expression of γ-H2AX and cC3 proteins was elevated in all treatment groups compared with control, whereas BRCA-wt MDA-MB-468 cC3 expression did not increase with treatment. Carboplatin treatment induced common gene-expression changes in BRCA-mutant models. Carboplatin ± ABT888 penetrates the brain and improves survival in BRCA-mutant intracranial TNBC models with corresponding DNA damage and gene-expression changes. Combination therapy represents a potential promising treatment strategy for patients with TNBC brain metastases warranting further clinical investigation.

Mechanisms of chromosomal instability in melanoma.

A systems biology approach was applied to investigate the mechanisms of chromosomal instability in melanoma cell lines. Chromosomal instability was quantified using array comparative genomic hybridization to identify somatic copy number alterations (deletions and duplications). Primary human melanocytes displayed an average of 8.5 alterations per cell primarily representing known polymorphisms. Melanoma cell lines displayed 25 to 131 alterations per cell, with an average of 68, indicative of chromosomal instability. Copy number alterations included approximately equal numbers of deletions and duplications with greater numbers of hemizygous (-1,+1) alterations than homozygous (-2,+2). Melanoma oncogenes, such as BRAF and MITF, and tumor suppressor genes, such as CDKN2A/B and PTEN, were included in these alterations. Duplications and deletions were functional as there were significant correlations between DNA copy number and mRNA expression for these genes. Spectral karyotype analysis of three lines confirmed extensive chromosomal instability with polyploidy, aneuploidy, deletions, duplications, and chromosome rearrangements. Bioinformatic analysis identified a signature of gene expression that was correlated with chromosomal instability but this signature provided no clues to the mechanisms of instability. The signature failed to generate a significant (P = 0.105) prediction of melanoma progression in a separate dataset. Chromosomal instability was not correlated with elements of DNA damage response (DDR) such as radiosensitivity, nucleotide excision repair, expression of the DDR biomarkers γH2AX and P-CHEK2, nor G1 or G2 checkpoint function. Chromosomal instability in melanoma cell lines appears to influence gene function but it is not simply explained by alterations in the system of DDR.

Targeted next generation sequencing identifies clinically actionable mutations in patients with melanoma.

Somatic sequencing of cancers has produced new insight into tumorigenesis, tumor heterogeneity, and disease progression, but the vast majority of genetic events identified are of indeterminate clinical significance. Here, we describe a NextGen sequencing approach to fully analyzing 248 genes, including all those of known clinical significance in melanoma. This strategy features solution capture of DNA followed by multiplexed, high-throughput sequencing and was evaluated in 31 melanoma cell lines and 18 tumor tissues from patients with metastatic melanoma. Mutations in melanoma cell lines correlated with their sensitivity to corresponding small molecule inhibitors, confirming, for example, lapatinib sensitivity in ERBB4 mutant lines and identifying a novel activating mutation of BRAF. The latter event would not have been identified by clinical sequencing and was associated with responsiveness to a BRAF kinase inhibitor. This approach identified focal copy number changes of PTEN not found by standard methods, such as comparative genomic hybridization (CGH). Actionable mutations were found in 89% of the tumor tissues analyzed, 56% of which would not be identified by standard-of-care approaches. This work shows that targeted sequencing is an attractive approach for clinical use in melanoma.

MERTK receptor tyrosine kinase is a therapeutic target in melanoma.

Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. Although recent therapeutic advances have prolonged patient survival, the prognosis remains dismal. C-MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that MERTK expression correlates with disease progression. MERTK expression was highest in metastatic melanomas, followed by primary melanomas, while the lowest expression was observed in nevi. Additionally, over half of melanoma cell lines overexpressed MERTK compared with normal human melanocytes; however, overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the MERTK ligand GAS6 resulted in the activation of several downstream signaling pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and diminished tumor volume by 60% in a human melanoma murine xenograft model. Treatment of melanoma cells with UNC1062, a novel MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of MERTK-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar, and inhibited invasion of melanoma cells. This work establishes MERTK as a therapeutic target in melanoma and provides a rationale for the continued development of MERTK-targeted therapies.

A prognostic signature of defective p53-dependent G1 checkpoint function in melanoma cell lines.

Melanoma cell lines and normal human melanocytes (NHM) were assayed for p53-dependent G1 checkpoint response to ionizing radiation (IR)-induced DNA damage. Sixty-six percent of melanoma cell lines displayed a defective G1 checkpoint. Checkpoint function was correlated with sensitivity to IR with checkpoint-defective lines being radio-resistant. Microarray analysis identified 316 probes whose expression was correlated with G1 checkpoint function in melanoma lines (P≤0.007) including p53 transactivation targets CDKN1A, DDB2, and RRM2B. The 316 probe list predicted G1 checkpoint function of the melanoma lines with 86% accuracy using a binary analysis and 91% accuracy using a continuous analysis. When applied to microarray data from primary melanomas, the 316 probe list was prognostic of 4-yr distant metastasis-free survival. Thus, p53 function, radio-sensitivity, and metastatic spread may be estimated in melanomas from a signature of gene expression.

Melanoma cells show a heterogeneous range of sensitivity to ionizing radiation and are radiosensitized by inhibition of B-RAF with PLX-4032.

PURPOSE: To assess the relative radiosensitivities of a large collection of melanoma cell lines and to determine whether pharmacologic inhibition of mutant B-RAF with PLX-4032 can radiosensitize B-Raf+ melanoma cells. MATERIALS AND METHODS: A large collection of melanoma cell lines (n=37) were treated with 0-8Gy IR and clonogenic survival assays used to generate survival curves to rank relative radiosensitivities among the cell lines. The ability of a B-RAF inhibitor, PLX-4032, to radiosensitize highly radioresistant B-Raf+ cells was also assessed by clonogenic cell survival and spheroid invasion assays and the effects of treatment on the cell cycle assessed by FACS. RESULTS: Melanoma cell lines displayed a very large, heterogeneous range of SF2 values (1.002-0.053) with a mean of 0.51. Cell lines with surviving fractions of 0.29 or less at SF2 and SF4 were observed at a high frequency of 18.9% and 70.2%, respectively. Treatment of B-Raf+ cells with the B-RAF inhibitor PLX-4032 in combination with radiation provided enhanced inhibition of both colony formation and invasion, and radiosensitized cells through an increase in G(1) arrest. CONCLUSIONS: Our data suggest that melanomas are not uniformly radioresistant with a significant subset displaying inherent radiosensitivity. Pharmacologic inhibition of B-RAF with PLX-4032 effectively radiosensitized B-Raf+ melanoma cells suggesting that this combination approach could provide improved radiotherapeutic response in B-Raf+ melanoma patients.

Lapatinib in combination with radiation diminishes tumor regrowth in HER2+ and basal-like/EGFR+ breast tumor xenografts.

PURPOSE: To determine whether lapatinib, a dual epidermal growth factor receptor (EGFR)/HER2 kinase inhibitor, can radiosensitize EGFR+ or HER2+ breast cancer xenografts. METHODS AND MATERIALS: Mice bearing xenografts of basal-like/EGFR+ SUM149 and HER2+ SUM225 breast cancer cells were treated with lapatinib and fractionated radiotherapy and tumor growth inhibition correlated with alterations in ERK1 and AKT activation by immunohistochemistry. RESULTS: Basal-like/EGFR+ SUM149 breast cancer tumors were completely resistant to treatment with lapatinib alone but highly growth impaired with lapatinib plus radiotherapy, exhibiting an enhancement ratio average of 2.75 and a fractional tumor product ratio average of 2.20 during the study period. In contrast, HER2+ SUM225 breast cancer tumors were highly responsive to treatment with lapatinib alone and yielded a relatively lower enhancement ratio average of 1.25 during the study period with lapatinib plus radiotherapy. Durable tumor control in the HER2+ SUM225 model was more effective with the combination treatment than either lapatinib or radiotherapy alone. Immunohistochemical analyses demonstrated that radiosensitization by lapatinib correlated with ERK1/2 inhibition in the EGFR+ SUM149 model and with AKT inhibition in the HER2+ SUM225 model. CONCLUSION: Our data suggest that lapatinib combined with fractionated radiotherapy may be useful against EGFR+ and HER2+ breast cancers and that inhibition of downstream signaling to ERK1/2 and AKT correlates with sensitization in EGFR+ and HER2+ cells, respectively.

Mechanism of lapatinib-mediated radiosensitization of breast cancer cells is primarily by inhibition of the Raf>MEK>ERK mitogen-activated protein kinase cascade and radiosensitization of lapatinib-resistant cells restored by direct inhibition of MEK.

BACKGROUND AND PURPOSE: We recently showed that lapatinib, an EGFR/HER2 inhibitor, radiosensitized breast cancer cells of the basal and HER2+ subtypes. The purpose of this study was to identify the downstream signaling pathways responsible for lapatinib-mediated radiosensitization in breast cancer. MATERIALS AND METHODS: Response of EGFR downstream signaling pathways was assessed by Western blot and clonogenic cell survival assays in breast tumor cells after irradiation (5Gy), lapatinib, CI-1040, or combined treatment. RESULTS: In SUM102 cells, an EGFR+ basal breast cancer cell line, exposure to ionizing radiation elicited strong activation of ERK1/2 and JNK, which was blocked by lapatinib, and weak/no activation of p38, AKT or STAT3. Direct inhibition of MEK1 with CI-1040 resulted in 95% inhibition of surviving colonies when combined with radiation while inhibition of JNK with SP600125 had no effect. Lapatinib-mediated radiosensitization of SUM102 cells was completely abrogated with expression of constitutively active Raf. Treatment of lapatinib-resistant SUM185 cells with CI-1040 restored radiosensitization with 45% fewer surviving colonies when combined with radiation. CONCLUSIONS: These data suggest that radiosensitization by lapatinib is mediated largely through inhibition of MEK/ERK and that direct inhibition of this pathway may provide an additional avenue of radiosensitization in EGFR+ or HER2+ breast cancers.