Weekly Intra-Amniotic IGF-1 Treatment Increases Growth of Growth-Restricted Ovine Fetuses and Up-Regulates Placental Amino Acid Transporters

Frequent treatment of the growth-restricted (IUGR) ovine fetus with intra-amniotic IGF-1 increases fetal growth. We aimed to determine whether increased growth was maintained with an extended dosing interval and to examine possible mechanisms. Pregnant ewes were allocated to three groups: Control, and two IUGR groups (induced by placental embolization) treated with weekly intra-amniotic injections of either saline (IUGR) or 360 µg IGF-1 (IGF1). IUGR fetuses were hypoxic, hyperuremic, hypoglycemic, and grew more slowly than controls. Placental glucose uptake and SLC2A1 (GLUT2) mRNA levels decreased in IUGR fetuses, but SLC2A3 (GLUT3) and SLC2A4 (GLUT4) levels were unaffected. IGF-1 treatment increased fetal growth rate, did not alter uterine blood flow or placental glucose uptake, and increased placental SLC2A1 and SLC2A4 (but not SLC2A3) mRNA levels compared with saline-treated IUGR animals. Following IGF-1 treatment, placental mRNA levels of isoforms of the system A, y+, and L amino acid transporters increased 1.3 to 5.0 fold, while the ratio of phosphorylated-mTOR to total mTOR also tended to increase. Weekly intra-amniotic IGF-1 treatment provides a promising avenue for intra-uterine treatment of IUGR babies, and may act via increased fetal substrate supply, up-regulating placental transporters for neutral, cationic, and branched-chain amino acids, possibly via increased activation of the mTOR pathway

Effect of alirocumab on mortality after acute coronary syndromes. An analysis of the ODYSSEY OUTCOMES randomized clinical trial

Background: Previous trials of PCSK9 (proprotein convertase subtilisin-kexin type 9) inhibitors demonstrated reductions in major adverse cardiovascular events, but not death. We assessed the effects of alirocumab on death after index acute coronary syndrome. Methods: ODYSSEY OUTCOMES (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab) was a double-blind, randomized comparison of alirocumab or placebo in 18 924 patients who had an ACS 1 to 12 months previously and elevated atherogenic lipoproteins despite intensive statin therapy. Alirocumab dose was blindly titrated to target achieved low-density lipoprotein cholesterol (LDL-C) between 25 and 50 mg/dL. We examined the effects of treatment on all-cause death and its components, cardiovascular and noncardiovascular death, with log-rank testing. Joint semiparametric models tested associations between nonfatal cardiovascular events and cardiovascular or noncardiovascular death. Results: Median follow-up was 2.8 years. Death occurred in 334 (3.5%) and 392 (4.1%) patients, respectively, in the alirocumab and placebo groups (hazard ratio [HR], 0.85; 95% CI, 0.73 to 0.98; P=0.03, nominal P value). This resulted from nonsignificantly fewer cardiovascular (240 [2.5%] vs 271 [2.9%]; HR, 0.88; 95% CI, 0.74 to 1.05; P=0.15) and noncardiovascular (94 [1.0%] vs 121 [1.3%]; HR, 0.77; 95% CI, 0.59 to 1.01; P=0.06) deaths with alirocumab. In a prespecified analysis of 8242 patients eligible for ≥3 years follow-up, alirocumab reduced death (HR, 0.78; 95% CI, 0.65 to 0.94; P=0.01). Patients with nonfatal cardiovascular events were at increased risk for cardiovascular and noncardiovascular deaths (P<0.0001 for the associations). Alirocumab reduced total nonfatal cardiovascular events (P<0.001) and thereby may have attenuated the number of cardiovascular and noncardiovascular deaths. A post hoc analysis found that, compared to patients with lower LDL-C, patients with baseline LDL-C ≥100 mg/dL (2.59 mmol/L) had a greater absolute risk of death and a larger mortality benefit from alirocumab (HR, 0.71; 95% CI, 0.56 to 0.90; Pinteraction=0.007). In the alirocumab group, all-cause death declined wit h achieved LDL-C at 4 months of treatment, to a level of approximately 30 mg/dL (adjusted P=0.017 for linear trend). Conclusions: Alirocumab added to intensive statin therapy has the potential to reduce death after acute coronary syndrome, particularly if treatment is maintained for ≥3 years, if baseline LDL-C is ≥100 mg/dL, or if achieved LDL-C is low. Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01663402

Abstract P4-07-01: DNA repair deficiency enhances immune response and correlates with excellent clinical outcome in triple negative breast cancer

Abstract Background: Mutations or epigenetic silencing of BRCA1/2 genes result in DNA repair deficiency in a large proportion of TNBC cases. Yet it is unclear whether this deficiency is associated with increased chemosensitivity and improved benefit from standard-of-care chemotherapy. We systematically evaluated BRCA deficiency in TNBC using integrated DNA and RNA sequencing data and its association clinical outcome, exploring the potential role of tumor immune response. Patients and Methods: Whole-exome, DNA methylation, copy number variation, and RNA sequencing data from 102 stage I-III TNBCs were retrieved from The Cancer Genome Atlas (TCGA). Almost all patients received adjuvant taxane-anthracycline-cyclophosphamide (T-AC) chemotherapy and had &amp;gt;30 days of follow-up. The number of predicted neoantigens and an estimate of the level of immune cell activity for 77 of these tumors were previously published. Deleterious germline or somatic BRCA1/2 mutations were identified by majority voting on predictions of 5 variant scoring algorithms. Definition of BRCA1/2 deficiency (BRCA-D) included carrier of deleterious BRCA1/2 mutations or BRACA1/2 normal (BRCA-N) with wild type BRCA1/2 expression less than the maximum observed in mutation carriers. Normalized genomic mutation rate and mutant allele tumor heterogeneity (MATH) were computed as broad measures of genomic instability. Characteristics of BRCA-D vs N tumors were compared using the Wilcoxon rank test. Results: Twenty tumors (19.6%) had mutations in BRCA1, 6 (5.8%) in BRCA2, and 2 (1.9%) in both. Based on the expanded definition, 39 cases (38%) were characterized as BRCA1 deficient, 5 (4.9%) as BRCA2 deficient and 4 (3.9%) as deficient in both. BRCA-D tumors (47%) were associated with a significantly higher mutation rate (P=8x10-4) but had similar clonal heterogeneity (P=0.55) as BRCA-N tumors. BRCA-D tumors had excellent 4-year overall survival (100%) compared to 79.5% (95%CI: 66.6- 94.9) for BRCA-N tumors (log-rank P=0.02). BRCA-D tumors also presented a significantly higher number of predicted neoantigents (P=0.003), which resulted in increased level of immune cell activity. In contrast, low immunogenic TNBC tumors were underrepresented in BRCA-D (p=0.05) and showed potential signs of immunoediting (observed/expected number of predicted neoantigens &amp;lt; 1; p=0.07). Conclusions: Deleterious mutations in BRCA1/2 genes occur in 25% of TNBC tumors, but parallel quantification of wild-type BRCA1/2 expression identifies 47% of TNBC samples with double strand break DNA repair deficiency. These BRCA-D TNBC tumors are characterized by a significantly higher mutation rate and present a significantly greater number of neoantigens that result in increased immune cell activity. Our analysis suggested that enhanced immune activation could explain to a large extent the excellent clinical outcome in patients with BRCA-D tumors treated with standard-of-care T-AC chemotherapy. Citation Format: Jiang T, Safonov A, Bianchini G, Shi W, Wali VB, Pusztai L, Hatzis C. DNA repair deficiency enhances immune response and correlates with excellent clinical outcome in triple negative breast cancer. [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 P4-07-01.</jats:p

Abstract P6-13-06: Novel combination therapies for triple negative breast cancer identified by high-throughput screening

Abstract Background: Finding effective novel therapies for triple negative breast cancer (TNBC) remains an unmet challenge. Our objective was to assess the sensitivity of TNBC to single drugs and to combinations using a systematic screen of existing FDA approved drugs combined with other approved or experimental agents and to characterize the mechanism of action of the most promising combinations. Methods: We conducted a comprehensive high-throughput drug combination screen in six TNBC cell lines with different genetic backgrounds. We tested a panel of 128 agents with known targets for their growth inhibitory potential individually and in pairwise combinations with six FDA approved anticancer drugs in MDA-MB-231, MDA-MB-436, MDA-MB-468, BT-20, BT-549, and HCC-38 TNBC cell lines. High-throughput assays were performed in 384-well plates and dose response curves were generated using 5 concentrations of the secondary drug, while keeping the first drug at fixed dose. Cell viability was measured 72h after treatment exposure using CellTiter-Glo®. Drug combinations were assessed for overall inhibitory effect and also for superadditivity, assessed as deviations from non-interaction using Bliss model of synergy. Selected synergistic and effective drug combinations identified from the high-throughput screen were subsequently validated in a 96-well low-throughput format in all TNBC cell lines. Results: Cell cycle and apoptosis regulators were more inhibitory as single agents across TNBC cell lines relative to other drug classes. Bortezomib, carfilzomib, YM155, Flavopiridol, KP372-1, and dactinomycin were highly toxic to all TNBC lines as single agents. Combinations with either everolimus or erlotinib were particularly effective in BT-20 and MDA-MB-468, and combinations with crizotinib in MDA-MB-231 cells. Bay-11-7082/erlotinib and MK-1775/everolimus were few of the promising combinations that elicited potentiated response in all the cell lines. ABT-263/crizotinib was one of the top synergistic combination most effective in MDA-MB-231 cells that express highest relative mRNA and protein levels of respective drug targets, Bcl-xL, and AXL. The combination treatment with ABT-263 and crizotinib also resulted in apoptosis in MDA-MB-231 cells, indicated by marked PARP cleavage in these cells, while MDA-MB-436 cells with lowest expression of Bcl-xL were resistant to apoptosis or growth inhibition, indicating a potential efficacy of this combination in a subset of TNBCs. Conclusions: This study reveals novel combinations of cell cycle or apoptosis regulators and crizotinib, everolimus, or erlotinib with enhanced anticancer activity. Combinations of Bay-11-7082 and erlotinib; and MK-1775 and everolimus had mild synergistic activity in all TNBC lines, while ABT-263 and Crizotinib showed large synergistic antiproliferative activity in a subset of TNBCs. These promising drug combinations have great potential to improve cure rates in TNBC patients. Since crizotinib, everolimus, and erlotinib are US FDA-approved while ABT-263 and MK-1775 are in advanced clinical trials, there is a rapid path for clinical translation for these drug-combinations after validation in animal studies. Citation Format: Wali VB, Langdon CG, Held MA, Platt JT, Safonov A, Aktas B, Stern DF, Pusztai L, Hatzis C. Novel combination therapies for triple negative breast cancer identified by high-throughput screening. [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 P6-13-06.</jats:p

Abstract P2-04-02: Comparison of DNA methylation patterns in normal breast tissue from women with and without breast cancer

Abstract BACKGROUND: Increasing evidence suggests that epigenetic mechanisms play critical roles in the development of breast cancer. However, precise DNA methylation signatures associated with breast cancer susceptibility remain unknown. We sought to compare DNA methylation changes in the normal breast tissue of women with and without breast cancer to identify patterns of aberrant DNA methylation in women with breast cancer. METHODS:Samples of normal breast tissue were collected from four cohorts of women: age &amp;lt; 50 years with and without breast cancer, and age ≥50 years with and without breast cancer. Normal breast tissue from healthy women was obtained from the Komen Tissue Bank at IU Simon Cancer Center and from women presenting for reduction mammoplasty at Yale New Haven Hospital. Normal breast tissue from women with breast cancer was obtained from patients undergoing adjuvant total mastectomy at Yale Breast Center. DNA was extracted using Qiagen AllPrep Universal kit. Raw data files in idat format were imported to Partek Genomics Suite 6.6 for normalization and differential methylation analysis. Raw intensities were normalized using With Array Normalization (SWAN) method. Principal component analysis (PCA) were performed as quality control. Differentially methylated loci (DML) between control and breast cancer groups were detected when False discovery rate (FDR) &amp;lt; 0.05 and fold change &amp;gt; 1.5. Functional enrichment analysis of genes with DML in the gene body were conducted using METACORE™. Pathways with FDR &amp;lt; 0.05 were selected. RESULTS: Ninety-three normal breast tissue samples from 89 subjects were analyzed (breast cancer=40, unaffected=53). Comparison of DNA methylation patterns between women with and without breast cancer revealed 200 DMLs. The majority of DMLs (186) were hyper-methylated in breast cancer patients, and 48 DMLs locate in enhancers of genes. 170 DMLs locate in 134 genes, enriched in two pathways: (1) Cell adhesion_Endothelial cell contacts by junctional mechanisms, and (2) Neurophysiological process_Constitutive and regulated NMDA receptor trafficking. Genes associated with cell adhesion and cell contacts included: ACTN2, GJA4, GJA7 and MAGI1. Two hyper-methylated loci were found in enhancers of ACTN2. In addition, one hyper-methylated locus in GJA4, one hyper-methylated and one hypo-methylated loci in GJA7, and two hyper-methylated loci in MAGI1 were detected in breast cancer patients. Genes associated with NMDA receptor trafficking include: TPK1, ADCY4 and LIN7C. One and two loci were found in TPK1 and ADCY4, respectively, that were hyper-methylated in normal breast tissue from cancer patients in the gene body, while a hypo-methylated locus in breast cancer patients was identified in LIN7C. CONCLUSIONS: Comparison of DNA methylation patterns of normal breast tissue from women with and without breast cancer reveal specific mechanistic pathways and genes that are differentially methylated in women with breast cancer. DNA methylation of normal breast tissue deserves further study as a potential biomarker for breast cancer risk stratification and may lend new insight into mechanisms of breast cancer development. Citation Format: Hofstatter EW, Zhu Y, Horvath S, Chagpar AB, Wali VB, Bossuyt V, Storniolo AM, Hatzis C, Patwardhan G, Von Wahlde M-K, Butler M, Epstein L, Stavris K, Sturrock T, Au A, Kwei S, Pusztai L. Comparison of DNA methylation patterns in normal breast tissue from women with and without breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-04-02.</jats:p