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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. Mol Cancer Ther; 14(4); 920–30. ©2015 AACR.

This file contains additional details on the microarray analysis and normalization.

This file contains the compiled Supplemental Figures S1-S9. Figure S1 is a flowchart describing the process of normalizing the microarray data for the SUM149 model for the different scanners used. Figure S2 depicts the scanner differences in the microarray data for the SUM149 model before and after normalization. Figure S3 is a flowchart depicting the analytical process used to analyze and compare the SUM149 and MDA-MB-436 models microarray results, and Figure S4 shows the difference in scale between models before and after the scaling process for visual comparison of the SUM149 and MDA-MB-436 models. Figure S5 demonstrates the lack of survival benefit of Carboplatin +/- ABT88 in the two BRCA-wt models, MDA-MB-468 and MDA-MB-231BR. Figure S6 contains representative images of IHC cC3 staining for the BRCA-wt model, MDA-MB-468. Figure S7 shows the unsupervised clustering of the BRCA-mut SUM149 and MDA-MB-436 models with treatment. Figure S8 contains the supervised clustering based on carboplatin treatment in these two models. Figure S9 demonstrates the expression levels of the 38 commonly regulated genes in both models with carboplatin treatment.

This file contains the differentially expressed genes and enriched pathways lists from the gene expression microarray analyses in Supplemental Table S1, with sample information in S1a. Lists are for the following comparisons: SUM149 Control vs. ABT888 (S1b), MDA-MB-436 Control vs. ABT888 (S1c), SUM149 Control vs. Carboplatin (S1d), MDA-MB-436 Control vs. Carboplatin (S1e), SUM149 Control vs Carboplatin+ABT888 (S1f), MDA-MB-436 Control vs Carboplatin+ABT888 (S1g), SUM149 Carboplatin vs Carboplatin+ABT888 (S1h), MDA-MB-436 Carboplatin vs Carboplatin+ABT888 (S1i). DAVID pathway enrichment analysis results are shown for the significantly up-regulated (S1j) and down-regulated (S1k) genes with Carboplatin treatment for both models.

This file contains the figure legends for Supplemental Figures S1-S9.

Breast cancer metastasis remains a clinical challenge, even within a single patient across multiple sites of the disease. Genome-wide comparisons of both the DNA and gene expression of primary tumors and metastases in multiple patients could help elucidate the underlying mechanisms that cause breast cancer metastasis. To address this issue, we performed DNA exome and RNA sequencing of matched primary tumors and multiple metastases from 16 patients, totaling 83 distinct specimens. We identified tumor-specific drivers by integrating known protein-protein network information with RNA expression and somatic DNA alterations and found that genetic drivers were predominantly established in the primary tumor and maintained through metastatic spreading. In addition, our analyses revealed that most genetic drivers were DNA copy number changes, the TP53 mutation was a recurrent founding mutation regardless of subtype, and that multiclonal seeding of metastases was frequent and occurred in multiple subtypes. Genetic drivers unique to metastasis were identified as somatic mutations in the estrogen and androgen receptor genes. These results highlight the complexity of metastatic spreading, be it monoclonal or multiclonal, and suggest that most metastatic drivers are established in the primary tumor, despite the substantial heterogeneity seen in the metastases.

Glioma is a unique neoplastic disease that develops exclusively in the central nervous system (CNS) and rarely metastasizes to other tissues. This feature strongly implicates the tumor-host CNS microenvironment in gliomagenesis and tumor progression. We investigated the differences and similarities in glioma biology as conveyed by transcriptomic patterns across four mammalian hosts: rats, mice, dogs, and humans. Given the inherent intra-tumoral molecular heterogeneity of human glioma, we focused this study on tumors with upregulation of the platelet-derived growth factor signaling axis, a common and early alteration in human gliomagenesis. The results reveal core neoplastic alterations in mammalian glioma, as well as unique contributions of the tumor host to neoplastic processes. Notable differences were observed in gene expression patterns as well as related biological pathways and cell populations known to mediate key elements of glioma biology, including angiogenesis, immune evasion, and brain invasion. These data provide new insights regarding mammalian models of human glioma, and how these insights and models relate to our current understanding of the human disease. NIH [T32 CA154274, F30-CA200345-01, R01CA139099, K12 NS080223, K08 NS090430]; Wallace Coulter Foundation; Passano Foundation; American Cancer Society-Research Scholar Grant [128970-RSG-16-012-01-CDD] This study was supported by NIH T32 CA154274 (NC), NIH F30-CA200345-01 (MBS), NIH R01CA139099 (JHR); the Wallace Coulter Foundation (JHR); the Passano Foundation (GW); NIH K12 NS080223 Neurosurgeon Research Career Development Program (GW), NIH K08 NS090430 (GW), American Cancer Society-Research Scholar Grant 128970-RSG-16-012-01-CDD (GW).

Background Gliomas are diverse neoplasms with multiple molecular subtypes. How tumor-initiating mutations relate to molecular subtypes as these tumors evolve during malignant progression remains unclear. Methods We used genetically engineered mouse models, histopathology, genetic lineage tracing, expression profiling, and copy number analyses to examine how genomic tumor diversity evolves during the course of malignant progression from low- to high-grade disease. Results Knockout of all 3 retinoblastoma (Rb) family proteins was required to initiate low-grade tumors in adult mouse astrocytes. Mutations activating mitogen-activated protein kinase signaling, specifically KrasG12D, potentiated Rb-mediated tumorigenesis. Low-grade tumors showed mutant Kras-specific transcriptome profiles but lacked copy number mutations. These tumors stochastically progressed to high-grade, in part through acquisition of copy number mutations. High-grade tumor transcriptomes were heterogeneous and consisted of 3 subtypes that mimicked human mesenchymal, proneural, and neural glioblastomas. Subtypes were confirmed in validation sets of high-grade mouse tumors initiated by different driver mutations as well as human patient-derived xenograft models and glioblastoma tumors. Conclusion These results suggest that oncogenic driver mutations influence the genomic profiles of low-grade tumors and that these, as well as progression-acquired mutations, contribute strongly to the genomic heterogeneity across high-grade tumors.

Fixed drug eruption (FDE) is a type IV hypersensitivity reaction characterized by recurrence of lesions at identical sites with each exposure to the offending medication. After morbilliform exanthems, FDE is the most common cutaneous drug reaction.1 Generalized bullous fixed drug eruption (GBFDE) is a variant of FDE that can present rarely with significant, life-threatening body surface involvement akin to Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN).2 There are few reported cases of GBFDE occurring after the influenza vaccine, and all occurred after decades of receiving the vaccine.3, 4 Here we report a rare case of GBFDE occurring as a result of administration of the influenza vaccine.

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).

INTRODUCTION: Approximately 50% of patients with metastatic triple negative breast cancer (TNBC) will develop brain metastases (BM). Routinely treated with radiotherapy and/or surgery, survival is generally less than one year. There are no approved systemic therapies to treat TNBC BM. We characterized the genomic and immune landscape of TNBC BM to foster the development of effective brain permeable anti-cancer agents, including immunotherapy. EXPERIMENTAL PROCEDURES: A clinically-annotated BCBM biobank of archival tissues was created under IRB approval. DNA (tumor/normal) and RNA (tumor) were extracted from TNBC primaries and BM; whole exome (WES) and RNA sequencing (RNASeq) was performed. Mutations were determined from WES as those co-identified by two variant callers (Strelka|Cadabra). Immune gene signature expression, molecular subtype identification, and T cell receptor repertoires were inferred from RNAseq. RESULTS: 32 TNBC patient tissues (14 primaries, 18 BCBM, 6 primary-BCBM matched), characterized as basal-like by PAM50, were analyzed. Top exome mutation calls included ten genes in ≥19% of BCBMs including TP53, ATM, and PIK3R1, and four genes in ≥18% of primaries including TP53 and PIK3R1. Many immune gene signatures were lower in BM compared to primaries including B cell, dendritic cell, regulatory T cell, and IgG cluster (p< 0.05). A signature of PD-1 inhibition responsiveness was higher in BM compared with primaries (p< 0.05). BCBM T cell receptor repertoires showed higher evenness and lower read count (both p < 0.01) compared to primaries. CONCLUSIONS: TNBC BM compared to primaries that metastasize to the brain show lower immune gene signature expression, higher PD-1 inhibition response signature expression, and T cell receptor repertoire features less characteristic of an active antigen-specific response. Mutations common to TNBC BM and primaries include TP53 and PIK3R1. Given that non-BCBM (i.e. lung and melanoma) show response to checkpoint inhibitors, these findings collectively support further study of immunotherapy for TNBC BM.

Although targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models of breast cancer that faithfully recapitulate the disparate clinical responses in these micro-environments, we observed that brain metastases evade phosphatidylinositide 3-kinase (PI3K) inhibition despite drug accumulation in the brain lesions. In comparison to extracranial disease, we observed increased HER3 expression and phosphorylation in brain lesions. HER3 blockade overcame the resistance of HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, resulting in marked tumor growth delay and improvement in mouse survival. These data provide a mechanistic basis for therapeutic resistance in the brain microenvironment and identify translatable treatment strategies for HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases.

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.

Background Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Prognosis remains poor despite multimodal therapy. Developing alternative treatments is essential. Drugs targeting kinases within the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) effectors of receptor tyrosine kinase (RTK) signaling represent promising candidates. Methods We previously developed a non-germline genetically engineered mouse model of GBM in which PI3K and MAPK are activated via Pten deletion and KrasG12D in immortalized astrocytes. Using this model, we examined the influence of drug potency on target inhibition, alternate pathway activation, efficacy, and synergism of single agent and combination therapy with inhibitors of these 2 pathways. Efficacy was then examined in GBM patient-derived xenografts (PDX) in vitro and in vivo. Results PI3K and mitogen-activated protein kinase kinase (MEK) inhibitor potency was directly associated with target inhibition, alternate RTK effector activation, and efficacy in mutant murine astrocytes in vitro. The kinomes of GBM PDX and tumor samples were heterogeneous, with a subset of the latter harboring MAPK hyperactivation. Dual PI3K/MEK inhibitor treatment overcame alternate effector activation, was synergistic in vitro, and was more effective than single agent therapy in subcutaneous murine allografts. However, efficacy in orthotopic allografts was minimal. This was likely due to dose-limiting toxicity and incomplete target inhibition. Conclusion Drug potency influences PI3K/MEK inhibitor-induced target inhibition, adaptive kinome reprogramming, efficacy, and synergy. Our findings suggest that combination therapies with highly potent, brain-penetrant kinase inhibitors will be required to improve patient outcomes.

[This corrects the article DOI: 10.1371/journal.pmed.1002174.].

Orphan GPCRs provide an opportunity to identify potential pharmacological targets, yet their expression patterns and physiological functions remain challenging to elucidate. Here, we have used a genetically engineered knockin reporter mouse to map the expression pattern of the Gpr182 during development and adulthood. We observed that Gpr182 is expressed at the crypt base throughout the small intestine, where it is enriched in crypt base columnar stem cells, one of the most active stem cell populations in the body. Gpr182 knockdown had no effect on homeostatic intestinal proliferation in vivo, but led to marked increases in proliferation during intestinal regeneration following irradiation-induced injury. In the ApcMin mouse model, which forms spontaneous intestinal adenomas, reductions in Gpr182 led to more adenomas and decreased survival. Loss of Gpr182 enhanced organoid growth efficiency ex vivo in an EGF-dependent manner. Gpr182 reduction led to increased activation of ERK1/2 in basal and challenge models, demonstrating a potential role for this orphan GPCR in regulating the proliferative capacity of the intestine. Importantly, GPR182 expression was profoundly reduced in numerous human carcinomas, including colon adenocarcinoma. Together, these results implicate Gpr182 as a negative regulator of intestinal MAPK signaling-induced proliferation, particularly during regeneration and adenoma formation.

Background Metastasis is the main cause of cancer patient deaths and remains a poorly characterized process. It is still unclear when in tumor progression the ability to metastasize arises and whether this ability is inherent to the primary tumor or is acquired well after primary tumor formation. Next-generation sequencing and analytical methods to define clonal heterogeneity provide a means for identifying genetic events and the temporal relationships between these events in the primary and metastatic tumors within an individual. Methods and Findings We performed DNA whole genome and mRNA sequencing on two primary tumors, each with either four or five distinct tissue site-specific metastases, from two individuals with triple-negative/basal-like breast cancers. As evidenced by their case histories, each patient had an aggressive disease course with abbreviated survival. In each patient, the overall gene expression signatures, DNA copy number patterns, and somatic mutation patterns were highly similar across each primary tumor and its associated metastases. Almost every mutation found in the primary was found in a metastasis (for the two patients, 52/54 and 75/75). Many of these mutations were found in every tumor (11/54 and 65/75, respectively). In addition, each metastasis had fewer metastatic-specific events and shared at least 50% of its somatic mutation repertoire with the primary tumor, and all samples from each patient grouped together by gene expression clustering analysis. TP53 was the only mutated gene in common between both patients and was present in every tumor in this study. Strikingly, each metastasis resulted from multiclonal seeding instead of from a single cell of origin, and few of the new mutations, present only in the metastases, were expressed in mRNAs. Because of the clinical differences between these two patients and the small sample size of our study, the generalizability of these findings will need to be further examined in larger cohorts of patients. Conclusions Our findings suggest that multiclonal seeding may be common amongst basal-like breast cancers. In these two patients, mutations and DNA copy number changes in the primary tumors appear to have had a biologic impact on metastatic potential, whereas mutations arising in the metastases were much more likely to be passengers., Charles Perou and colleagues report on somatic mutations in primary tumors and metastases from two patients with basal-like breast cancer., Author Summary Background In the United States, 40,000 women die of breast cancer each year, thus making it the second leading cause of cancer-related deaths in women. Breast cancer mortality is caused by metastasis, the spread of the cancer beyond the breast to distant tissue sites, including the lungs, brain, and liver. Triple-negative breast cancer, defined by lack of expression of the estrogen and progesterone receptors and absent amplification of the HER2 gene, and the basal-like molecular subtype defined by RNA gene expression have earlier occurrences of metastasis, worsened survival, and fewer therapeutic options compared to other breast cancer subtypes. Why Was This Study Done? This study was done to gain an understanding of the underlying genetics leading to breast cancer metastasis and when these changes occur temporally. Previous reports of the evolution of breast cancer metastasis have reported one to two matched metastasis sites and did not focus on triple-negative breast cancer, the subtype with the greatest clinical need. What Did the Researchers Do and Find? We identified two patients with triple-negative and basal-like breast cancer with available tissue from the primary breast cancer and multiple matched metastases, and performed DNA whole genome sequencing and RNA sequencing on all tumors to identify the genetic landscape of each tumor and define the genomic evolution of metastases from the primary disease. We demonstrate that multiclonal seeding from the primary tumor to the metastases can occur, indicating that metastatic cancers can originate from a collection of different subclones that together seed the metastatic site, rather than each metastasis forming from a single cell. We also demonstrate that the majority of functional mutations, those expressed and likely to be driving metastasis, are established in the primary tumor rather than being acquired during the spread of disease. What Do These Findings Mean? This study demonstrates examples of multiclonal seeding of metastases from multiple cell populations in the original breast tumor of a patient with basal-like breast cancer. The high degree of similarity between the primary tumor and its metastases gives hope that targetable drivers of metastasis are present in the primary tumor and, if effectively treated, could prevent metastasis. A larger cohort of matched primaries with multiple sites of metastases per patient is needed to understand the generalizability of these results and possible evolutionary differences of metastasis across the different subtypes of breast cancer.

Introduction: Triple negative breast cancer (TNBC) is an aggressive subset of BC with high metastatic potential. Once metastatic, half of patients (pts) with TNBC will develop brain metastases (BM), commonly with progressive extracranial disease. While TNBC BM are routinely treated with radiotherapy, survival is generally less than one year. There are no approved systemic therapies to treat TNBC BM. Both the blood brain barrier and paucity of data on the biologic underpinnings and immune response of BCBM contribute to inadequate therapies for this disease. We sought to characterize the genomic and immune landscape of TNBC BM to foster the development of effective brain permeable anti-cancer agents. Experimental Procedures: A clinically-annotated BCBM biobank of archival brain metastases and primary BC that eventually metastasize to the brain was created under IRB approval (LCCC1419). DNA (tumor/normal) and RNA (tumor only) was extracted. Following library preparation, whole exome (WES) and RNA sequencing (RNASeq) was performed. Common mutations were determined from WES as those co-identified by two variant callers (Strelka, Cadabra), while immune gene signature expression, molecular subtype identification, and B and T cell receptor repertoires were inferred from RNAseq data. Results: Of the 26 enrolled pts, median age at BCBM diagnosis was 52 years (35-72); 23% were African American and 57% Caucasian. Additional non-brain metastatic sites included bone (23%), liver (20%), and lung (35%); 23% had a solitary BCBM. 35% of pts received chemotherapy after BCBM diagnosis, while 92% received radiation to the brain (21% WBRT, 5% SRS, 25% SRS plus WBRT and resection). The median survival from BCBM was 6 months. 40 (93%) of the tissues (21 primaries, 22 BCBM) were characterized as basal-like by PAM50; 1 HER2-enriched and 2 Luminal A. 34 genes were mutated in ≥20% of BCBMs, while only 8 were mutated in at least 25% of primaries. Commonly mutated BCBM genes included TP53, ATM and MYH9; in primaries only TP53. Many immune gene signatures were lower in BCBM compared to primary BC including B cell, dendritic cell, regulatory T cell, and IgG cluster (p Conclusions: TNBC BM compared to primary BC that metastasize to the brain had more significantly mutated genes, lower immune gene signature expression, higher PD1 inhibition response signature expression, and T cell receptor repertoire features less characteristic of an active antigen-specific response. Given that checkpoint inhibitors are showing response in non-BCBM (i.e. lung and melanoma), these findings indicate that immunotherapy to treat patients with TNBC BM is worthy of exploration. Citation Format: Benjamin G. Vincent, Maria Sambade, Shengjie Chai, Marni B. Siegel, Luz Cuaboy, Alan Hoyle, Joel Parker, Charles M. Perou, Carey K. Anders. Genomic and immune characterization of triple-negative breast cancer brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4055.

The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07–0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11–1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion.

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. Mol Cancer Ther; 14(4); 920–30. ©2015 AACR.

Brain metastases remain a significant challenge in the treatment of breast cancer patients due to the unique environment posed by the central nervous system. A better understanding of the biology of breast cancer cells that have metastasized to the brain is required to develop improved therapies. A recent Proceedings of the National Academy of Sciences article demonstrates that breast cancer cells in the brain microenvironment express γ-aminobutyric acid (GABA)-related genes, enabling them to utilize GABA as an oncometabolite, thus gaining a proliferative advantage. In this viewpoint, we highlight these findings and their potential impact on the treatment of breast cancer brain metastases.

Introduction: Nearly half of metastatic triple negative breast cancer (TNBC) patients develop brain metastases (BMs) and face a poor prognosis. There are no FDA-approved systemic therapies to treat TNBC BM, due in part to the blood-brain barrier. TNBC and breast cancer BMs exhibit both activation of the PI3K and MEK pathways, but attempts to target them in preclinical models have shown limited efficacy due to innate and acquired resistance to kinase inhibition in TNBC. In this study, we identify several clinically relevant rational combination therapies based on synthetic lethality and evaluate the efficacy of combined brain-penetrant, clinically-available kinase inhibitors in intracranial TNBC models. Methods: An siRNA screen against 720 kinase genes was used to identify synthetic enhancers of lethality with pan-PI3K inhibitor (BKM120) or MEK1/2 inhibitor (AZD6244) treatment in vitro using TNBC models capable of growing in mouse brain (SUM149, MDA-MB-231Br). The efficacy of these and other brain-penetrant drugs of interest based on the screen were assessed for efficacy in vitro, alone (IC50s) and combined (synergy). Some combinations were evaluated in vivo in mice bearing intracranial TNBC tumors for their effects on survival and tumor burden. Tumor burden was monitored via bioluminescence. IC tumors from treated mice were extracted, fresh frozen, and analyzed for the activation state of the kinome using multiplexed kinase inhibitor bead (MIB) enriched mass spectrometry (MS). Results: The screen identified the following combinations as synthetic lethal pairs: PI3K+MEK, MEK+PDGFR, PI3K+AURKA, MEK+BRAF. Pharmacological synergy of combined treatments was confirmed in vitro between PI3K(/mTOR)+MEK, MEK+PDGFR, and PI3K+AURKA, in TNBC cells (SUM149, 231Br) using brain-penetrant drugs in clinical development (mTOR inhibitor Everolimus, dual PI3K/mTOR inhibitor GNE317, PDGFR inhibitor Pazopanib, AURKA inhibitor MLN8237, BRAF inhibitor Dabrafenib). For some combinations, particularly PI3K+AURKA inhibition, sequencing of the drugs significantly altered the combined effects and synergy. Combinations which were synthetically lethal and synergistic at physiologically relevant doses in vitro demonstrated enhanced efficacy in vivo, including PI3K+MEK, MEK+PDGFR, and PI3K+AURKA. In contrast, other combinations (i.e. PI3K+mTOR) did not significantly improve survival or tumor burden in vivo. Despite improved survival with some combination treatments, mice eventually succumbed to tumor burden as tumors eventually grew. Kinome analysis of IC tumors treated with PI3K (BKM120) and/or MEK1/2 (AZD6244) inhibitors for 2 weeks identified several potential resistance markers, including INSR, IGF1R, and FGFR2, which may be targetable clinically. Conclusions: Synthetic lethality screens identified multiple rational combination therapies based on PI3K and/or MEK inhibition in TNBC cells, particularly PI3K+MEK, MEK+PDGFR, and PI3K+AURKA. Combined use of brain-penetrant, clinically available inhibitors against these targets showed promising efficacy in intracranial TNBC mouse models. Rational combinations of brain-penetrant kinase inhibitors are promising strategies for a patient population with few options. In vivo studies assessing the efficacy of other identified combinations, as well as more extensive characterization of potential resistance mechanisms, in intracranial TNBC mouse models are warranted to provide the translational foundation for future clinical studies. Citation Format: Amanda E.D. Van Swearingen, Maria J. Sambade, Marni B. Siegel, Shivani Sud, Samantha M. Bevill, Brian T. Golitz, Ryan E. Bash, Charlene M. Santos, David B. Darr, Joel S. Parker, C. Ryan Miller, Gary L. Johnson, Carey K. Anders. Several rational combination kinase inhibitor treatments identified by synthetic lethality screens are efficacious in intracranial triple negative breast cancer models [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A03.

Background: The non-basal triple negative breast cancer (Non Basal-TNBC) tumors, also called the luminal androgen receptor (LAR) subtype, make up ~20% of all new TNBC cases and is limited to multi-agent chemotherapy. TNBC is considered one of the leading causes of cancer-associated mortality amongst women in the USA with a high number of patients dying of metastatic disease. Some metastases that arise from luminal primary tumors lose their luminal features and become a HER2-enriched (HER2E) subtype, but remain clinically HER2 negative (HER2E/HER2-). The molecular features that drive these HER2E/HER2- tumors may also represent key checkpoints of metastatic progression. Within this HER2E/HER2- subtype, besides the androgen receptor (AR), FGFR4 has been shown to be expressed, but its functional role remains unknown. FGFR4 is a tyrosine kinase receptor involved in proliferation, survival and migration during embryonic development and cancer. We hypothesize that in addition to AR, that FGFR4 may be a driver of HER2E/HER2- tumors and that the therapeutic targeting of this gene/protein might improve patient outcomes. Objective: Our objective is to identify the mechanism of action of FGFR4 in HER2E/HER2- tumors as well as additional genetic drivers of HER2E TNBC subtype. Methods: We performed computational analysis of databases (TCGA, METABRIC) and the analysis of matched breast cancer primary tumors and metastases to detect genetic drivers. We ran microarrays on two FGFR4 stably transfected luminal HER2-/ER+ cell lines (MCF7 and T47D) and also treated a Luminal B cell line (MDA-MB-453; HER2high/AR+/ER-) and HER2E cell line (CAMA-1; HER2low/ARmed/ER+/low) with a FGFR4 inhibitor using a time course strategy. Results: Through the analysis of microarrays using all 4 cell lines, we identified a gene expression signature of FGFR4-activity enriched in genes involved in proliferation, cell junctions and cell differentiation. The FGFR4-associated signature of upregulated genes was enriched in HER2E and Basal-like subtypes and the downregulated genes enriched in the Luminal subtypes. To identify predictors of chemotherapy response, we evaluated FGFR4-associated gene signatures in the diagnostic (pre-treatment) samples of MDACC neoadjuvant dataset of 500 tumors. The high expression of the FGFR4- induced gene signature predicted pathological complete response (T.test P Conclusions: FGFR4 may play an important role in the progression of Luminal disease and has been identified as a new genetic driver of HER2E/HER2- subtype making it an attractive option as a therapeutic target. We are further testing these genetic predictions using Patient Derived Xenograft (PDX) mouse models. Citation Format: Susana Garcia Recio, Cheng Fan, Marni Siegel, Joel S. Parker, Gina N. Duronio, Aleix Prat, Charles M. Perou. Identification of genetic drivers in HER2enriched/HER2negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3119. doi:10.1158/1538-7445.AM2017-3119

Brain metastases represent a devastating progression of luminal breast cancer. While targeted therapies are often effective systemically, they fail to adequately control brain metastases. In preclinical models that faithfully recapitulate the disparate clinical responses in these microenvironments, we observed that brain metastases evade PI3K inhibition despite efficient drug delivery. In comparison to extracranial disease, there is increased HER3 expression and phosphorylation in the brain lesions. HER3 blockade overcomes the resistance of both HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases to PI3K inhibitors, leading to striking tumor growth delay and significant improvement of mouse survival. Collectively, these data provide a mechanistic basis underlying therapeutic resistance in the brain microenvironment and identify rapidly translatable treatment strategiesfor HER2-amplified and/or PIK3CA-mutant breast cancer brain metastases. Citation Format: Gino B. Ferraro, David P. Kodack, Vasileios Askoxylakis, Qing Sheng, Mark Badeaux, Shom Goel, Xiaolong Qi, Ram Shankaraiah, Alexander Z. Cao, Rakesh R. Ramjiawan, Divya Bezwada, Bhushankumar Patel, Youngchul Song, Carlotta Costa, Kamila Naxerova, Christina Wong, Jonas Kloepper, Rita Das, Angela Tam, Jantima Tanboon, Dan G. Duda, Ryan C. Miller, Marni B. Siegel, Carey K. Anders, Melinda Sanders, Valeria M. Estrada, Robert Schlegel, Carlos L. Arteaga, Elena Brachtel, Alan Huang, Dai Fukumura, Jeffrey A. Engelman, Rakesh K. Jain. The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5008. doi:10.1158/1538-7445.AM2017-5008

1011 Background: HER2+ BC is an aggressive subset of BC with high rates of BM and poor survival. Two-thirds of BCBM demonstrate activation of the PI3K/mTOR pathway driving resistance to anti-HER2 therapy (Rx). This phase II study evaluated everolimus (E), a brain permeable mTOR inhibitor, added to trastuzumab (T) and vinorelbine (V) in patients (pts) with HER2+ BCBM. Methods: Eligible pts had progressing HER2+ BCBM. Pts received E (5mg PO QD), T (2mg/kg IV weekly) and V (25mg/m2 IV d1, 8 of 21d cycle). The primary endpoint was intracranial response rate (RR [CR+PR], modified RECIST); secondary objectives (CNS clinical benefit rate [CBR, CR+PR+SD], extracranial RR, time to progression (TTP), overall survival (OS), and correlative studies). Targeted DNA sequencing of 20 tissues from 18 pts was performed. We used a two-stage design to distinguish ORR of 5% vs 20%. Results: 32 pts were evaluable for toxicity; 26 for efficacy. Median age was 53 (28–70 yrs). 31/32 pts had prior radiation: 13 (42%) WBRT, 8 (26%) radiosurgery, 9 (29%) both. Median prior lines of metastatic Rx was 2 (0–7). 30 (94%) received anti-HER2 Rx: 91% T, 69% lapatinib, 38% pertuzumab, 25% TDM1. Intracranial RR was 4% (1 PR, 6 SD > 6 mos, 10 SD > 3 mos, 9 PD). CNS CBR (6 mos) was 27%; CNS CBR (3 mos) was 65%. Extracranial RR was 46%. Median TTP was 4 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%). DNA sequencing showed heterogeneous HER2 copy number amplification (CN amp): only 11/20 show HER2 CN amp (median 40X v 0.7X; 5/11 BC, 4/4 BCBM, 1/2 liver mets, 1/3 LN). BCBM exhibited high-level HER2 CN amp (median 49X) vs other sites (16X). While 11/20 show both HER2 CN amp and PI3K pathway mutation, this was not associated TTP; RAD21 CN amp was associated with TTP < 3 mos. Lack of HER2 CN amp plus a HER2 Tyr-kinase domain mutation was seen in a pt with nonresponse and short OS. Conclusions: While intracranial RR to ETV was low in pts with HER2+ BCBM, a substantial proportion had CNS CBR; OS was 1 year for this pt population. No new toxicity signals were observed. Further evaluation of DNA heterogeneity, including degree of HER2 CN amp in HER2+ BCBM, and association with outcome is warranted. Clinical trial information: NCT01305941.

Malignant melanoma is characterized by a propensity for early lymphatic and hematogenous spread. The hypoxia-inducible factor (HIF) family of transcription factors is upregulated in melanoma by key oncogenic drivers. HIFs promote the activation of genes involved in cancer initiation, progression, and metastases. Hypoxia has been shown to enhance the invasiveness and metastatic potential of tumor cells by regulating the genes involved in the breakdown of the ECM as well as genes that control motility and adhesion of tumor cells. Using a Pten -deficient, Braf -mutant genetically engineered mouse model of melanoma, we demonstrated that inactivation of HIF1α or HIF2α abrogates metastasis without affecting primary tumor formation. HIF1α and HIF2α drive melanoma invasion and invadopodia formation through PDGFRα and focal adhesion kinase–mediated (FAK-mediated) activation of SRC and by coordinating ECM degradation via MT1-MMP and MMP2 expression. These results establish the importance of HIFs in melanoma progression and demonstrate that HIF1α and HIF2α activate independent transcriptional programs that promote metastasis by coordinately regulating cell invasion and ECM remodeling.

Background: The molecular characterization of primary breast cancers has led to signatures identifying risk of future metastasis and survival; however the underlying biology driving metastasis development is largely unknown. Methods: Utilizing a Rapid Autopsy Program, we have collected 61 metastatic breast cancer tumors from 7 individuals (4 triple negative, 2 HER2+, 1 ER+/HER2-) including primary tumors and 3-6 metastases/patient. We performed mRNA and DNA exome sequencing. We next used DawnRank, a novel network-based method that integrates DNA and RNA data to identify computationally determined "driver" genes (i.e. a DNA variant that significantly alters its gene expression-network) in each individual sample. Phylogenetic tree and clonal analysis were also performed, with the computationally determined drivers mapped onto these trees. Results: The breast cancer primaries were molecularly subtyped as 5 Basal-like, 1 HER2-Enriched, and 1 Luminal A; in all cases, the metastases clustered immediately adjacent to their primary tumor by hierarchical clustering analysis. Widespread DNA copy number alterations identified in the primary tumors were typically maintained throughout metastasis. On average, 1.9 ± 1.3% of DNA copy number altered genes, and 2.4 ± 0.95% of the somatic mutations per tumor were identified as "drivers" by DawnRank. There were an average of 199 ± 72 total drivers per tumor due to copy number alterations (amplifications or deletions) and 12 ± 23 drivers per tumor from somatic mutations. Phylogenetic tree analysis demonstrated that the majority of DNA copy number events occurred early in tumor development. Founding clones were defined as genetic events present in the primary and all matched metastases. Chr5q13 loss and TP53 mutation were the only consistent alterations in the founding clones of all 7 patients. Drivers on chr5q13 identified in this cohort include CCNB1, CDK7, and TAF9. Among the basal-like patients, all 5 patients' TP53 mutations were identified as a driver by DawnRank. 39% and 20% of drivers from copy number gains and losses, respectively, were identified in the primary tumor, while another 34% and 30% were not seen in the primary but were present in more than 1 metastasis within each patient. Metastasis-enriched copy number drivers not seen in any primary included FLT1, MAP2KR, and ARNT. 38% of the drivers resulting from somatic mutations were established in the primary and maintained in metastases. Of the remaining drivers from somatic mutation, only 18% were shared among metastases but not seen in the primary while 47% were not seen in any other tumor within a given patient (i.e. private to a single sample). TP53, PSEN1, CDC27, HDAC1, and BRCA1 were somatic mutation drivers established early in metastatic development, while CCNH was a consistent late driver. Conclusions: We present a novel computationally determined genetic "driver" analysis of matched breast cancer primaries and multi-organ metastases. In this cohort, our results suggest that most genetic drivers in a single tumor are based on copy number aberrations, are established early, and are maintained in metastases. In contrast to copy number, drivers from somatic mutations are acquired later, and most of the metastases continued to acquire new genetic driving features. Citation Format: Siegel MB, He X, Chen M, Hou JP, Garrett AL, Dye JB, Silva GO, Usary JE, Moylan VJ, Brady CM, Ma J, Thorne LB, Hoadley KA, Parker JS, Anders CK, Carey LA, Perou CM. Identification of early versus late drivers of breast tumors and metastasis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S4-01.

Introduction: Nearly half of metastatic triple negative breast cancer (TNBC) patients develop brain metastases (BM) and face a poor prognosis. The blood-brain barrier (BBB) prevents many treatments from reaching intracranial tumors, and there are no FDA-approved systemic therapies to treat TNBC BM. In this study, we evaluated the efficacy of BBB-permeable, clinically-available inhibitors of MEK and identified rational co-target pathways in preclinical models of intracranial (IC) TNBC. Methods: In vitro IC50s, synergy, and siRNA screens (700 kinase genes) were conducted in 4 human-derived TNBC lines (SUM149, MDA-MB-468, MDA-MB-436, MDA-MB-231Br). We evaluated the efficacy of the MEK1/2 inhibitor AZD6244 (AZD), the pan-PI3K inhibitor BKM120 (BKM), and the PDGFR inhibitor Pazopanib (Pazo) in IC TNBC mouse models. Tumor burden was monitored via bioluminescence, and IC tumors were frozen for gene expression analyses using custom human 4×44K Agilent microarrays or of kinome activity profiles using multiplex kinase inhibitor beads and mass spectrometry. To determine drivers of AZD sensitivity, DNA copy number data (Broad CCLE) was analyzed using SWITCHplus to identify copy number alterations that differ between sensitive (n = 8) vs. resistant (n = 12) TNBC lines based on their IC50s (Sanger Cancerxgene). Results: In vitro, SUM149 and 231Br TNBC cells exhibited lower (40 uM). Several genes synthetically enhanced lethality in SUM149 and 231Br cells: PI3K genes and PDGFRα/β with AZD, and MAPK/MAP2K/MAP3K genes with BKM, suggesting MEK+PI3K and MEK+PDGFR inhibition as rational combinations. AZD plus BKM or Pazo were synergistic in vitro in sensitive cell lines. In vivo, AZD reduced tumor burden and improved survival in the SUM149 (72 vs. 45 days in controls, p < 0.005) and 231Br (37 vs. 30 days, p < 0.02) models, with no benefit in the other two models. Single agent BKM or Pazo resulted in little to no improvement in any model. However, in AZD-sensitive models, combined AZD+BKM inhibition increased survival (SUM149: 87 vs 45 days, p < 0.0001; MDA-MB-231Br: 52 vs 30 days, p < 0.001) as did AZD+Pazo (SUM149: 88 vs. 35 days, p < 0.0001). Several DNA segments were significantly altered in sensitive vs. resistant TNBC cell lines. Notably, MEK-pathway genes were lost in the resistant lines. Ongoing work will complete characterization of therapies in all models in vitro and in vivo and will compare genetic, transcriptional, and kinome activity alterations. Conclusions: TNBC models exhibit different innate sensitivities to the BBB-permeable MEK inhibitor AZD6244. In sensitive models, AZD improves survival and reduces intracranial tumor burden, and rational combined inhibition of PI3K or PDGFR further increases survival. Identification of predictive biomarkers will enable translation of our results to biomarker-driven clinical trials for patients with TNBC BM. Citation Format: Amanda E.D. Van Swearingen, Marni B. Siegel, Maria J. Sambade, Shivani Sud, Samantha M. Miller, Grace Silva, Ryan E. Bash, Charlene M. Santos, David B. Darr, Brian Golitz, Joel S. Parker, C. Ryan Miller, Gary L. Johnson, Carey K. Anders. Combination therapy with MEK inhibition is efficacious in intracranial triple negative breast cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2579. doi:10.1158/1538-7445.AM2015-2579

Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype which lacks the classical clinical biomarkers for hormone receptors (HR) and HER2. Nearly half of metastatic TNBC patients develop brain metastases and these patients face a poor prognosis. Most new effective treatments for breast cancer target hyperactivated HER2 and HR pathways, which TNBC tumors lack. The blood-brain barrier (BBB) also prevents many treatments from reaching the brain and any developing tumors therein. There is currently no FDA-approved systemic chemotherapy for the treatment of TNBC brain metastases. Tissue-based studies show activation of the PI3K pathway in breast cancer brain metastases and in vivo models of extracranial TNBC are sensitive to PI3K and MEK inhibitors. BBB-permeable inhibitors targeting the PI3K and MEK pathways are in clinical development. In this study, we evaluated the efficacy of two such inhibitors, the pan-PI3K inhibitor BKM120 and the MEK1/2 inhibitor AZD6244, in preclinical models of intracranial (IC) TNBC. Methods: The efficacy of PI3K and MEK inhibition by BKM120 and AZD6244, respectively, alone and in combination was determined in orthotopic mouse models of basal-like TNBC through IC implantation of SUM149 or MDA-MB-468 cell lines. Drugs were administered at the maximum tolerated doses via chow: 30 mg/kg BKM, 37 mg/kg AZD, or 25 mg/kg BKM + 18 mg/kg AZD. Tumor burden was monitored via weekly bioluminescence imaging, and brain tumors were frozen at sacrifice for gene expression analyses. To explore potential resistance mechanisms and inform rational combination therapies, in vitro IC50s, combination synergy determinations, and single-agent synthetic lethal siRNA screens were conducted. Results: In the SUM149 model, median survival was 45 days (control), 53.5 days (BKM), 53.5 days (BKM+AZD), and 76 days (AZD). AZD alone and with BKM reduced tumor burden via bioluminescence imaging. In vitro, 3-day dose response curves showed that BKM and AZD were similarly potent but BKM was more effective (IC50; maximal % cell reduction: BKM: 1.3 uM, >90%; AZD: 816 nM, >50%). Several genes were identified as synthetically lethal “hits” in an initial kinome screen by two-class SAM. AURKA was a hit with BKM120, while BRAF and several PI3K, AMPK, and CDK genes were synthetically lethal with AZD6244. Ongoing work will include survival studies in the 468 model and gene expression changes in IC tumors in response to therapy. Conclusions: BKM120 and AZD6244 both improved survival in an IC TNBC SUM149 mouse model, with single agent AZD6244 being most efficacious. The siRNA screens indicate that combined treatment with BKM120 and AZD6244 should be synthetically lethal, suggesting that combination therapy may have underperformed due to toxicity. Ongoing in vitro and in vivo studies (including dosing schedules) will further characterize the effects of these drugs in intracranial TNBC. Citation Format: Amanda E.D. Van Swearingen, Marni B. Siegel, Ryan Bash, Brian Golitz, Charlene Santos, David Darr, Joel Parker, Gary L. Johnson, C. Ryan Miller, Carey K. Anders. PI3K and MEK inhibition in intracranial triple negative breast cancer: Efficacy of BKM120 and AZD6244 in preclinical mouse models. [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 5449A. doi:10.1158/1538-7445.AM2014-5449A

In the United States, several states have made policy decisions regarding whether and how to use familial searching of the Combined DNA Index System (CODIS) database in criminal investigations. Familial searching pushes DNA typing beyond merely identifying individuals to detecting genetic relatedness, an application previously reserved for missing persons identifications and custody battles. The intentional search of CODIS for partial matches to an item of evidence offers law enforcement agencies a powerful tool for developing investigative leads, apprehending criminals, revitalizing cold cases and exonerating wrongfully convicted individuals. As familial searching involves a range of logistical, social, ethical and legal considerations, states are now grappling with policy options for implementing familial searching to balance crime fighting with its potential impact on society. When developing policies for familial searching, legislators should take into account the impact of familial searching on select populations and the need to minimize personal intrusion on relatives of individuals in the DNA database. This review describes the approaches used to narrow a suspect pool from a partial match search of CODIS and summarizes the economic, ethical, logistical and political challenges of implementing familial searching. We examine particular US state policies and the policy options adopted to address these issues. The aim of this review is to provide objective background information on the controversial approach of familial searching to inform policy decisions in this area. Herein we highlight key policy options and recommendations regarding effective utilization of familial searching that minimize harm to and afford maximum protection of US citizens.