Citations

Background Intracranial aneurysms ( IAs ), abdominal aortic aneurysms ( AAAs ), and thoracic aortic aneurysms ( TAAs ) all have a familial predisposition. Given that aneurysm types are known to co‐occur, we hypothesized that there may be shared genetic risk factors for IAs , AAAs, and TAAs . Methods and Results We performed a mega‐analysis of 1000 Genomes Project‐imputed genome‐wide association study ( GWAS ) data of 4 previously published aneurysm cohorts: 2 IA cohorts (in total 1516 cases, 4305 controls), 1 AAA cohort (818 cases, 3004 controls), and 1 TAA cohort (760 cases, 2212 controls), and observed associations of 4 known IA , AAA , and/or TAA risk loci (9p21, 18q11, 15q21, and 2q33) with consistent effect directions in all 4 cohorts. We calculated polygenic scores based on IA ‐, AAA ‐, and TAA ‐associated SNP s and tested these scores for association to case‐control status in the other aneurysm cohorts; this revealed no shared polygenic effects. Similarly, linkage disequilibrium–score regression analyses did not show significant correlations between any pair of aneurysm subtypes. Last, we evaluated the evidence for 14 previously published aneurysm risk single‐nucleotide polymorphisms through collaboration in extended aneurysm cohorts, with a total of 6548 cases and 16 843 controls ( IA ) and 4391 cases and 37 904 controls ( AAA ), and found nominally significant associations for IA risk locus 18q11 near RBBP 8 to AAA (odds ratio [ OR ]=1.11; P =4.1×10 −5 ) and for TAA risk locus 15q21 near FBN 1 to AAA ( OR =1.07; P =1.1×10 −3 ). Conclusions Although there was no evidence for polygenic overlap between IAs , AAAs , and TAAs , we found nominally significant effects of two established risk loci for IAs and TAAs in AAAs . These two loci will require further replication.

A cancer-promoting histone protein Mutations in the chromatin protein histone H3 are found in a number of pediatric cancers. The lysine-36–to–methionine (K36M) “oncohistone” mutation is seen in almost all chondroblastomas. Fang et al. show that the K36M mutant histones inhibit the normal methylation of this same residue in wild-type H3 histones. They do so by interfering with the enzymes that normally methylate this residue. The altered chromatin methylation patterns alter the expression of known cancer-related genes and impart cancer-related characteristics to the chondrocyte cells. Science , this issue p. 1344

Abstract Background ER+ BC is associated with activated CDK4/6. The CDK4/6 inhibitor palbociclib (P) markedly improves time to progression in advanced ER+HER2- BC. We conducted a neoadjuvant phase II trial to determine the activity of P in primary breast cancer as a prelude to adjuvant studies. Methods To assess molecular changes induced by anastrozole (A) or P+A, patients (pts) were treated initially with A alone (1mg PO daily) for 28 days in cycle 0 (C0) before the addition of P (125mg PO daily on D1-21 each cycle) on C1D1. P+A was administered for 4 28-day cycles followed by C5 with A alone for 2-4 weeks (wks) before surgery. P was added in C5 for 10-12 days immediately prior to surgery in the last 20 pts enrolled to assess molecular changes induced by A, either alone or in combination with P immediately prior to surgery, in resected tumor. Goserelin was added in premenopausal pts. Research tumor biopsies were obtained at baseline, C1D1, and C1D15. Central Ki67 analysis was performed at all timepoints, those with Ki67 >10% at C1D15 went off study treatment. The primary endpoint was complete cell cycle arrest (CCA), defined as Ki67 <2.7%, at C1D15. Patient stratification was based on PIK3CA mutation status with an initial focus on PIK3CA wild type (WT) disease. Pts with PIK3CA mutant (Mut) tumors enrolled to a separate cohort. A sample size of 33 pts in the PIK3CA WT cohort was chosen based on the Fleming's single-stage phase II design to test the hypothesis that P+A leads to > 50% improvement over A in CCA rate on C1D15 biopsy (44% with A alone based on historical data, vs 66% with P+A, power = 0.8, alpha=0.05). The primary endpoint is met if >20 pts achieved CCA in this cohort. Correlative endpoints included assessment of markers of proliferation, apoptosis, senescence, Rb, gene expression microarray, intrinsic subtype, and next generation sequencing of 83-gene panels, which will be reported at the meeting. Results Between 4/23/2013 and 4/24/2015, 50 pts (33 PIK3CA WT, 11 PIK3CA Mut, 2 pending, 4 tissue quantity or quality not sufficient for sequencing (QNS)) were enrolled to the study. Median age was 57.5 (range: 34.1–79.6) years. Four pts, all with WT PIK3CA, went off study due to Ki67 >10% on C1D15 biopsy, 26 pts completed treatment and surgery, 1 refused surgery, 3 withdrew study treatment in C1, and 16 continued to receive study drug (2 in C0, 3 in C1, 4 in C2, 5 in C3, 1 in C4, and 1 in C5). Among the 40 pts currently evaluable for the primary endpoint (C1D15 Ki67), CCA occurred in 34 (85%) pts, including 9 of 9 (100%) PIK3CA Mut, 22 of 28 (78.5%) WT, and 3 of 3 QNS pts. Preliminary analysis of available data indicated a significantly lower Ki67 value after 2 wks of P+A (C1D15) compared to that on A alone (C1D1) (p=0.034, n=18). Conclusion This study met the primary endpoint demonstrating that P+A is a highly effective anti-proliferative combination. The sequential biopsy design clearly demonstrated that P+A increased cell cycle control over A alone. P+A was effective regardless of PIK3CA mutation status and these results support the evaluation of this combination in the adjuvant setting for ER+HER2- BC. Citation Format: Ma CX, Gao F, Northfelt D, Goetz M, Forero A, Naughton M, Ademuyiwa F, Suresh R, Anderson KS, Margenthaler J, Aft R, Hobday T, Moynihan T, Gillanders W, Cyr A, Eberlein TJ, Hieken T, Krontiras H, Hoog J, Han J, Guo Z, Vij K, Mardis E, Al-Kateb H, Sanati S, Ellis MJ. A phase II trial of neoadjuvant palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, in combination with anastrozole for clinical stage 2 or 3 estrogen receptor positive HER2 negative (ER+HER2-) breast cancer (BC). [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 S6-05.

Abstract Background Activating mutations in PIK3CA occur in approximately 40% ER+BC. MK-2206 (M), a pan-AKT inhibitor, induced apoptosis of ER+ BC under estrogen deprivation in preclinical studies. We conducted this neoadjuvant trial to determine the pathologic complete response (pCR) rate of M plus anastrozole (A) for PIK3CA mutant (Mut) ER+ BC. Methods This single arm open label study of M+A used a 2-stage Simon phase II design (stage 1, n=16; stage 2, n=13, alpha=0.10, power=0.90) to test whether pCR rate <1% (based on historical data with A alone), against the alternative that pCR rate ≥15% in PIK3CA Mut ER+ BC. At least 1 pCR in stage 1 was required to proceed to stage 2. Eligible patients (pts) with clinical stage II or III ER+HER2- BC were pre-registered and proceeded to a research tumor biopsy for PIK3CA sequencing, followed by treatment with daily A monotherapy for 28 days (cycle 0). Pts with PIK3CA Mut BC were subsequently registered, underwent a second biopsy, and started M (150mg PO weekly) with daily A on cycle 1 day 1 (C1D1) for a maximum of four 28-day cycles followed by surgery. Goserelin was added for premenopausal pts. A tumor biopsy on C1D17, 17 days post the start of M, was performed. Those with C1D17 Ki67 >10% discontinued study treatment. pCR was defined as no invasive cancer in the breast and the lymph nodes. Tumor specimens collected at all timepoints are being analyzed for markers of proliferation, apoptosis, and PI3K pathway activity, gene expression microarray, intrinsic subtypes, and next generation sequencing of 83 genes. Results Of the 51 pts pre-registered, 35 pts did not register due to no PIK3CA mutation (n=22), inadequate specimen for testing (n=6), physician/pt decision (n=7). The remaining 16 pts (median age: 58, range: 40-77 years) received combination therapy. Three pts did not complete 4 cycles due to C1D17 Ki67 >10% (n=2) and intolerability (grade (Gr) 4 transaminase elevation in C1, n=1). Other severe toxicities possibly related to M included Gr 3 rash (25%) and pruritus (12.5%). Of the 13 pts completed study therapy and underwent surgery, all had residual disease in the breast and 7 also had positive nodes. Table 1 summarized changes in Ki67 during treatment. ComparisonsnAbsolute changes in Ki67 median (range)Wilcoxon signed rank p-valueC1D1 relative to pre-registration11-17.0% (-49.8 to 4.1%)0.0020C1D17 relative to pre-registration14-16.4% (-51.4 to 4.1%)0.0004C1D17 relative to C1D112-1.5% (-18.6 to 15.8%)0.9697C1D1, biopsy post 28 days of A alone; C1D17 biopsy post 17 days on combination therapy Although Ki67 levels post A monotherapy (C1D1) or M+A (C1D17) were significantly lower than that of pre-registration samples, Ki67 did not differ between C1D17 and C1D1 samples. Other correlative studies are ongoing and results will be presented. Conclusion Despite the small sample size, biomarker analysis on serial biopsy specimens demonstrated that M+A is unlikely to be more effective than A alone in PIK3CA Mut ER+ BC. This trial demonstrated the feasibility of genomic sequencing for pt selection and the value of a small, well-designed proof-of-principle neoadjuvant trial for the evaluation of targeted agents. Citation Format: Ma CX, Suman VJ, Goetz M, Northfelt D, Burkard M, Ademuyiwa F, Naughton M, Margenthaler J, Aft R, Gray R, Tavaarwerk A, Wilke L, Haddad T, Moynihan T, Loprinzi C, Hieken T, Hoog J, Guo Z, Han J, Vij K, Mardis E, Sanati S, Al-Kateb H, Doyle L, Erlichman C, Ellis MJ. A phase II neoadjuvant trial of MK-2206, an AKT inhibitor, in combination with anastrozole for clinical stage 2 or 3 PIK3CA mutant estrogen receptor positive HER2 negative (ER+HER2-) breast cancer (BC). [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 P5-13-04.

Hypothermia/rewarming (H/R) is poorly tolerated by the myocardium; however, the underlying intracellular basis of H/R-induced cardiac dysfunction remains elusive. We hypothesized that in cardiomyocytes, H/R disrupts excitation-contraction coupling by reducing myofilament Ca2+sensitivity due to an increase in cardiac troponin I (cTnI) phosphorylation. To test this hypothesis, isolated rat cardiomyocytes (13–15 cells from 6 rats per group) were electrically stimulated to evoke both cytosolic Ca2+([Ca2+]cyto) and contractile (sarcomere shortening) responses that were simultaneously measured using an IonOptix system. Cardiomyocytes were divided into two groups: 1) those exposed to hypothermia (15°C for 2 h) followed by rewarming (35°C; H/R); or 2) time-matched normothermic (35°C) controls (CTL). Contractile dysfunction after H/R was indicated by reduced velocity and extent of sarcomere length (SL) shortening compared with time-matched controls. Throughout hypothermia, basal [Ca2+]cytoincreased and the duration of evoked [Ca2+]cytotransients was prolonged. Phase-loop plots of [Ca2+]cytovs. contraction were shifted rightward in cardiomyocytes during hypothermia compared with CTL, indicating a decrease in Ca2+sensitivity. Using Western blot, we found that H/R increases cTnI phosphorylation. These results support our overall hypothesis and suggest that H/R disrupts excitation-contraction coupling of cardiomyocytes due to increased cTnI phosphorylation and reduced Ca2+sensitivity.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/hypothermiarewarming-disrupts-e-c-coupling/ .

Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (C t ) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98–100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score genotypes. ASQ is cost-effective because universal fluorescent probes negate the necessity of designing expensive probes for each locus.