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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 antitumor immunity in TNBC BrM is warranted. [ABSTRACT FROM AUTHOR]

Background: Early in the pandemic, we designed a SARS-CoV-2 peptide vaccine containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation. The rationale for this design was to drive both humoral and cellular immunity with high specificity while avoiding undesired effects such as antibody-dependent enhancement (ADE). Methods: We explored the set of computationally predicted SARS-CoV-2 HLA-I and HLA-II ligands, examining protein source, concurrent human/murine coverage, and population coverage. Beyond MHC affinity, T cell vaccine candidates were further refined by predicted immunogenicity, sequence conservation, source protein abundance, and coverage of high frequency HLA alleles. B cell epitope regions were chosen from linear epitope mapping studies of convalescent patient serum, followed by filtering for surface accessibility, sequence conservation, spatial localization near functional domains of the spike glycoprotein, and avoidance of glycosylation sites. Results: From 58 initial candidates, three B cell epitope regions were identified. From 3730 (MHC-I) and 5045 (MHC-II) candidate ligands, 292 CD8+ and 284 CD4+ T cell epitopes were identified. By combining these B cell and T cell analyses, as well as a manufacturability heuristic, we proposed a set of 22 SARS-CoV-2 vaccine peptides for use in subsequent murine studies. We curated a dataset of ~ 1000 observed T cell epitopes from convalescent COVID-19 patients across eight studies, showing 8/15 recurrent epitope regions to overlap with at least one of our candidate peptides. Of the 22 candidate vaccine peptides, 16 (n = 10 T cell epitope optimized; n = 6 B cell epitope optimized) were manually selected to decrease their degree of sequence overlap and then synthesized. The immunogenicity of the synthesized vaccine peptides was validated using ELISpot and ELISA following murine vaccination. Strong T cell responses were observed in 7/10 T cell epitope optimized peptides following vaccination. Humoral responses were deficient, likely due to the unrestricted conformational space inhabited by linear vaccine peptides. Conclusions: Overall, we find our selection process and vaccine formulation to be appropriate for identifying T cell epitopes and eliciting T cell responses against those epitopes. Further studies are needed to optimize prediction and induction of B cell responses, as well as study the protective capacity of predicted T and B cell epitopes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

Aim: Particle Replication in Nonwetting Templates (PRINT®) PLGA nanoparticles of docetaxel and acid-labile C2-dimethyl-Si-Docetaxel were evaluated with small molecule docetaxel as treatments for non-small-cell lung cancer brain metastases. Materials & methods: Pharmacokinetics, survival, tumor growth and mice weight change were efficacy measures against intracranial A549 tumors in nude mice. Treatments were administered by intravenous injection. Results: Intracranial tumor concentrations of PRINT-docetaxel and PRINT-C2-docetaxel were 13- and sevenfold greater, respectively, than SM-docetaxel. C2-docetaxel conversion to docetaxel was threefold higher in intracranial tumor as compared with nontumor tissues. PRINT-C2-docetaxel increased median survival by 35% with less toxicity as compared with other treatments. Conclusion: The decreased toxicity of the PRINT-C2-docetaxel improved treatment efficacy against non-small-cell lung cancer brain metastasis. [ABSTRACT FROM AUTHOR]

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, highthroughput 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 standardof- care approaches. This work shows that targeted sequencing is an attractive approach for clinical use in melanoma. [ABSTRACT FROM AUTHOR]

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. Environ. Mol. Mutagen. 55:457-471, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Shows that splicing pathways can be modified in vivo in a sequence specific manner by antisense oligonucleotides using cationic liposomes as a carrier in the repair of thalassemic beta-globin mRNA. Construction of two cell lines; Treatment with 2'-O-methyl phosphotioates; Analysis of the total protein from oligonucleotide-treated cells.