Citations

Abstract Background: Genomic assessment of cancer has been revolutionized by Next-Generation sequencing and is increasingly being applied to guide clinical prognostic and therapeutic decision-making. Initial clinical applications have been limited to gene panels and whole exome based strategies due to challenges with time to reporting of results, specimen quantity, analyte quality, and ethical-legal-social issues (ELSI). Methods: Excisional or core tumor biopsies or bone marrow biopsies were obtained from consenting participants. Nucleic acid was extracted from fresh or frozen samples followed by WGS/WES/RNASeq on the Illumina HiSeq2000 or HiSeq2500 and bioinformatics analysis. Therapeutic targets were then prioritized by a multi-disciplinary Genomic Tumor Board (GTB) and CLIA validated using Sanger sequencing, RT-qPCR, FISH and/or IHC. Treatment was delivered using on/off-label FDA approved drugs, available clinical trials and single patient INDs. Results: We consented 64 and enrolled 35 patients with advanced, treatment-refractory cancers. Median age was 59 (range 27-91) with 62% male. The majority had ECOG scores of 1 (94%). A median of 79 (range 28-8891) potentially functional somatic point mutations were identified in each case. One to two mutations/case were further identified as therapeutically targetable in 60% of the cases. The median time from tissue acquisition to CLIA validated results was 116 days (range 42-282) with CLIA validation of targets achieved in 21 of 22 patients. Genomic, target directed treatment was ultimately instituted in 13 patients utilizing: on/off label FDA approved drugs (n = 9), clinical trial (n = 3) and single patient IND (n = 1). Preliminary clinical efficacy was noted in 5 patients (2 PR, 3 SD). Integration of WES, long-insert WGS and RNA-Seq identified a case with an ERRFI1 mutant allele present in only 11% of the DNA reads while 82% of the RNA-Seq reads had the mutant transcript. The patient was treated with erlotinib and achieved a partial response by RECIST criteria. In another case, a fusion between FGFR2-MGEA5 was observed in WES and RNA-Seq data leading to prioritization as a drug target. Reasons for patients not receiving targeted therapy included: inability to access treatment (n = 1), death prior to intended treatment (n = 3), results returned after death (n = 3), no targets identified (n = 2) and decision to pursue alternate therapy (n = 5). Conclusions: Integrating whole genome analysis in a CLIA setting is not only feasible, but also valuable, in the prioritization and selection of potential targeted therapies for patients with advanced tumors. Continued barriers to broad application include the need for shorter time to reporting as well as broad availability of therapies through basket studies. Citation Format: Mitesh J. Borad, Jan Egan, Mia Champion, Katherine Hunt, Robert McWilliams, Ann McCullough, Jessica Aldrich, Sara Nasser, Winnie Liang, Michael Barrett, David Craig, Ramesh Ramanathan, John Carpten, A. Keith Stewart, Alan Bryce. Implementation of CLIA enabled integrated whole genome (WGS)/exome (WES)/transcriptome (RNAseq) next-gen sequencing to identify therapeutically relevant targets in advanced cancer patients. [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 CT112. doi:10.1158/1538-7445.AM2015-CT112

Abstract Introduction: Relapsed and refractory multiple myeloma (MM) remains a significant clinical challenge and drugs with new mechanisms of action to overcome resistance are needed. Constitutive activation of the PI3K/Akt/mTOR pathway in MM promotes tumorigenesis through propagation of the cell cycle, protein synthesis, and inhibition of apoptosis. Anti-MM effects of rapalogs are limited due to feedback activation of the PI3K/Akt signaling pathway. Agents capable of targeting PI3K and mTORC1 or both mTORC1/C2 are able to partially overcome such resistance mechanisms. However, the mTORC2 mediated increase in pAkt (Ser473) after PI3K/mTORC1 inhibition and the activation of PI3K after mTORC1/C2 inhibition could still contribute to resistance to such agents. We sought to investigate the effects of SPR965 (synthesized and provided by Sphaera Pharma, Singapore under an MTA), an orally bioavailable novel small molecule with inhibition of class 1 PI3 kinase and mTORC1/C2 kinases on MM cell lines and patient cells. Results: SPR965 induced cytotoxicity and inhibited proliferation in all MM cell lines examined with IC50 values between 25-500nM. To understand if SPR965 induced apoptotic cell death, annexin/PI staining followed by flow cytometric assays were performed, which showed a clear increase in cells undergoing apoptosis. Western blots showed increase in caspase-3, -9, and PARP cleavage confirming apoptotic induction by SPR965. Importantly, SPR965 caused potent increase in apoptosis in cytogenetically distinct MM patient cells. Next, we examined the mechanism of action of SPR965. SPR965 caused potent mTORC1 inhibition evidenced by decreased p4E-BP1, p-p70S6K, and pS6 at doses as low as 25nM. SPR965 was able to inhibit PI3K activity as shown by decreased pPDK1 and pBTK at slightly higher concentrations of 75nM. Phosphorylation of Akt S473, a TORC2 substrate, increased at initial low concentrations of SPR965, but was attenuated at concentrations of 100 nM and above, demonstrating dose-dependent inhibition of mTORC2. Such effects were also observed when we performed western blots on MM patient derived primary plasma cells. Increased levels of p27 (KIP1) were observed suggestive of G1 growth arrest, which was confirmed on cell cycle analysis. Since SPR965 caused an increase in pAkt (both T308 and S473) at doses up to 75nM, we examined if SPR965 was able to synergize with an Akt inhibitor MK2206 at doses lower than 75nM. Our results showed potent synergy suggesting that the pAkt up regulation contributes to partial resistance, which is inhibited by SPR965 at doses of 100nM and higher, doses that are still clinically achievable. Conclusion: Our findings demonstrate SPR965 induces apoptosis of MM cells through the inhibition of PI3K and mTORC1/C2 activity. Further studies are underway to better characterize the mechanism of action of SPR965, all of which will be informative for the drug to be used as a single agent or in combination with other agents in relapsed MM. Citation Format: Jeremy T. Larsen, Vijay Ramakrishnan, Jessica Haug, Teresa Kimlinger, Somdutta Sen, Dinesh Mahajan, Sundeep Dugar, S. Vincent Rajkumar, Shaji K. Kumar. Preclinical activity of dual PI3K/mTOR inhibitor SPR965 in multiple myeloma. [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 2653. doi:10.1158/1538-7445.AM2015-2653

AbstractIntroductionAfrican‐American (AA) individuals have a higher risk for late‐onset Alzheimer's disease (LOAD) than Americans of primarily European ancestry (EA). Recently, the largest genome‐wide association study in AAs to date confirmed that six of the Alzheimer's disease (AD)‐related genetic variants originally discovered in EA cohorts are also risk variants in AA; however, the risk attributable to many of the loci (e.g., APOE, ABCA7) differed substantially from previous studies in EA. There likely are risk variants of higher frequency in AAs that have not been discovered.MethodsWe performed a comprehensive analysis of genetically determined local and global ancestry in AAs with regard to LOAD status.ResultsCompared to controls, LOAD cases showed higher levels of African ancestry, both globally and at several LOAD relevant loci, which explained risk for AD beyond global differences.DiscussionExploratory post hoc analyses highlight regions with greatest differences in ancestry as potential candidate regions for future genetic analyses.

Background Disability, mortality and healthcare burden from fractures in older people is a growing problem worldwide. Observational studies suggest that aspirin may reduce fracture risk. While these studies provide room for optimism, randomised controlled trials are needed. This paper describes the rationale and design of the ASPirin in Reducing Events in the Elderly (ASPREE)-Fracture substudy, which aims to determine whether daily low-dose aspirin decreases fracture risk in healthy older people. Methods ASPREE is a double-blind, randomised, placebo-controlled primary prevention trial designed to assess whether daily active treatment using low-dose aspirin extends the duration of disability-free and dementia-free life in 19 000 healthy older people recruited from Australian and US community settings. This substudy extends the ASPREE trial data collection to determine the effect of daily low-dose aspirin on fracture and fall-related hospital presentation risk in the 16 500 ASPREE participants aged ≥70 years recruited in Australia. The intervention is a once daily dose of enteric-coated aspirin (100 mg) versus a matching placebo, randomised on a 1:1 basis. The primary outcome for this substudy is the occurrence of any fracture—vertebral, hip and non-vert-non-hip—occurring post randomisation. Fall-related hospital presentations are a secondary outcome. Discussion This substudy will determine whether a widely available, simple and inexpensive health intervention—aspirin—reduces the risk of fractures in older Australians. If it is demonstrated to safely reduce the risk of fractures and serious falls, it is possible that aspirin might provide a means of fracture prevention. Trial registration number The protocol for this substudy is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12615000347561).

This quality improvement project demonstrates that RN Care Managers, in a chronic pain programme, can assess for a family history of substance abuse in 5–10 min. Information informs treatment based on specific high risk criteria. Benefits include heightened awareness of the genetic and environmental risks associated with a family history of substance abuse, an opportunity to participate in motivational interventions to prevent or minimize consequences of substance use disorders, and likely substantial overall health‐care cost savings.

PurposeQuantification of tissue stiffness may facilitate identification of abnormalities in orofacial muscle tone and thus contribute to differential diagnosis of dysarthria. Tissue stiffness is affected by muscle tone as well as age-related changes in muscle and connective tissue.MethodThe Myoton-3 measured tissue stiffness in 40 healthy adults, including equal numbers of men and women in each of two age groups: 18–40 years and 60+ years. Data were collected from relaxed muscles at the masseter, cheek, and lateral tongue surfaces in two positions: reclined on the side and seated with head tilted.ResultsTissue stiffness differed across age, sex, and measurement site with multiple interaction effects. Overall, older subjects exhibited higher stiffness coefficients and oscillation frequency measures than younger subjects whereas sex differences varied by tissue site. Effects of body position were inconsistent across tissue site and measurement.ConclusionsAlthough older subjects were expected to have lower muscle tone, age-related nonmuscular tissue changes may have contributed to yield a net effect of higher stiffness. These data raise several considerations for the development of accurate normative data and for future diagnostic applications of tissue stiffness assessment.

Abstract The three-dimensional (3D) structure of the cell and its nucleus is intricately associated with cellular function. Aberrations and heterogeneity in cellular and nuclear morphology serve as diagnostically important cues to identify malignant transformation. However, current cytopathology practice is predominantly based on a qualitative, 2D assessment of cellular morphology that may bias the diagnosis. We utilized the Cell-CT® platform to perform single-cell, optical tomography imaging and generate 3D cell images with isotropic, sub-micron spatial resolution. We developed custom image analysis tools to accurately quantify 3D cell and nuclear morphology. We used these measures to characterize the morphological heterogeneity within and among epithelial cell populations, and to identify morphological descriptors that are reliably predictive of cell state. We assessed the efficacy of our approach relative to conventional 2D assessment (at 40x magnification) to detect malignant transformation in immortalized and primary normal-cancer cell line pairs from human esophagus and breast epithelia. We observed the 3D method of assessment to provide a consistently superior performance (sensitivity and specificity) in predicting malignant transformation. Further, some widely used diagnostic parameters, such as nuclear size or nucleus-to-cytoplasm ratio, did not always exhibit the highest predictive power in 3D space. The availability of 3D morphological information also provided novel insights on intra-population morphological heterogeneity in both normal and cancer cell populations. Our results emphasize the need to incorporate 3D morphological information into standard pathological practice. Citation Format: Vivek Nandakumar, Stephanie Helland, Jessica H. Han, Geriann Davis, Brian A. Ashcroft, Thai H. Tran, Ting Li, Karen S. Anderson, Rahul Pannala, Joseph R. Mikhael, David E. Fleischer, Roger H. Johnson, Kimberly J. Bussey, Deirdre R. Meldrum. Quantifying three-dimensional cellular morphology and its heterogeneity in epithelial cancers by single-cell optical tomography. [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 4297. doi:10.1158/1538-7445.AM2014-4297

To perform a range of ventilatory and nonventilatory behaviors, the diaphragm muscle (DIAm) must be able to generate sufficient forces throughout the lifespan. We hypothesized that sarcopenia impacts DIAm force generation and thus limits performance of expulsive, higher force, nonventilatory behaviors. Male and female mice ( n = 79) at 6 and 24 mo of age (100 vs. 70–75% survival, respectively) were used to examine transdiaphragmatic pressure (Pdi) generation across motor behaviors in vivo and in vitro DIAm specific force. We found a significant effect of age on maximum Pdi (20–41% decline during tracheal occlusion and bilateral phrenic nerve stimulation), maximum DIAm specific force (30% decline), and DIAm fatigue resistance (15% increase). There were no differences between sexes in these age effects on DIAm performance. These results support our hypothesis that sarcopenia primarily impacts higher force, nonventilatory motor behaviors of the DIAm. Such functional limitations may have negative implications in the ability of the DIAm to generate forces needed for airway clearance in old age and thereby contribute to age-related respiratory complications.