A village in a dish model system for population-scale hiPSC studies.

The mechanisms by which DNA alleles contribute to disease risk, drug response, and other human phenotypes are highly context-specific, varying across cell types and different conditions. Human induced pluripotent stem cells are uniquely suited to study these context-dependent effects but cell lines from hundreds or thousands of individuals are required. Village cultures, where multiple induced pluripotent stem lines are cultured and differentiated in a single dish, provide an elegant solution for scaling induced pluripotent stem experiments to the necessary sample sizes required for population-scale studies. Here, we show the utility of village models, demonstrating how cells can be assigned to an induced pluripotent stem line using single-cell sequencing and illustrating that the genetic, epigenetic or induced pluripotent stem line-specific effects explain a large percentage of gene expression variation for many genes. We demonstrate that village methods can effectively detect induced pluripotent stem line-specific effects, including sensitive dynamics of cell states.

Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration.

There are currently no treatments for geographic atrophy, the advanced form of age-related macular degeneration. Hence, innovative studies are needed to model this condition and prevent or delay its progression. Induced pluripotent stem cells generated from patients with geographic atrophy and healthy individuals were differentiated to retinal pigment epithelium. Integrating transcriptional profiles of 127,659 retinal pigment epithelium cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identify 445 expression quantitative trait loci in cis that are asssociated with disease status and specific to retinal pigment epithelium subpopulations. Transcriptomics and proteomics approaches identify molecular pathways significantly upregulated in geographic atrophy, including in mitochondrial functions, metabolic pathways and extracellular cellular matrix reorganization. Five significant protein quantitative trait loci that regulate protein expression in the retinal pigment epithelium and in geographic atrophy are identified - two of which share variants with cis- expression quantitative trait loci, including proteins involved in mitochondrial biology and neurodegeneration. Investigation of mitochondrial metabolism confirms mitochondrial dysfunction as a core constitutive difference of the retinal pigment epithelium from patients with geographic atrophy. This study uncovers important differences in retinal pigment epithelium homeostasis associated with geographic atrophy.

Activin-A, Growth Differentiation Factor-11 and Transforming Growth Factor-ß as predictive biomarkers for platinum chemotherapy in advanced non-small cell lung cancer.

BACKGROUND: Despite advances in immunotherapy and targeted therapy, platinum-based chemotherapy remains crucial for many patients with advanced non-small cell lung cancer (NSCLC). Resistance to platinum chemotherapy is common, and predictive biomarkers are needed to tailor treatment to patients likely to respond. In vitro evidence implicates the transforming growth factor-ß (TGF-ß) superfamily ligands activin-A and growth differentiation factor 11 (GDF-11) in innate platinum resistance. We performed a validation study to assess their utility as predictive biomarkers of platinum chemotherapy response in advanced NSCLC. PATIENTS AND METHODS: Our study included 123 adult patients with advanced NSCLC without a driver mutation treated with platinum chemotherapy. 98 patients were from a retrospective cohort and 25 from a prospective cohort. We performed immunohistochemistry staining for Activin-A, GDF-11 and TGF-ß on tumour samples for each patient and analysed IHC expression with objective radiological response and overall survival. RESULTS: The overall median survival was 14.8 months. We performed statistical analysis around a cytoplasmic score of 8/18 for Activin-A and GDF-11 based on previously published work, and 110/30 for TGF-ß based on a calculated cutpoint for significance. No survival difference was detected between these groups for Activin-A (p=0.35), GDF-11 (p=0.57) or TGF-ß (p=0.34). There was no association between rates of progressive disease and high Activin-A expression (p=0.43), high GDF-11 expression (p=1.0) or high TGF-ß expression p=0.89). CONCLUSION: Within the confines of our study, Activin-A, GDF-11 and TGF-ß expression was not a predictor of objective radiological response to chemotherapy or overall survival.

Radiomics feature robustness as measured using an MRI phantom.

Radiomics involves high-throughput extraction of large numbers of quantitative features from medical images and analysis of these features to predict patients' outcome and support clinical decision-making. However, radiomics features are sensitive to several factors, including scanning protocols. The purpose of this study was to investigate the robustness of magnetic resonance imaging (MRI) radiomics features with various MRI scanning protocol parameters and scanners using an MRI radiomics phantom. The variability of the radiomics features with different scanning parameters and repeatability measured using a test-retest scheme were evaluated using the coefficient of variation and intraclass correlation coefficient (ICC) for both T1- and T2-weighted images. For variability measures, the features were categorized into three groups: large, intermediate, and small variation. For repeatability measures, the average T1- and T2-weighted image ICCs for the phantom (0.963 and 0.959, respectively) were higher than those for a healthy volunteer (0.856 and 0.849, respectively). Our results demonstrated that various radiomics features are dependent on different scanning parameters and scanners. The radiomics features with a low coefficient of variation and high ICC for both the phantom and volunteer can be considered good candidates for MRI radiomics studies. The results of this study will assist current and future MRI radiomics studies.

RET gene rearrangements occur in a subset of pancreatic acinar cell carcinomas.

Pancreatic acinar cell carcinoma is relatively rare (1 to 2% of pancreatic malignancies) but may be under-recognized. In contrast to pancreatic ductal adenocarcinoma, most acinar cell carcinomas lack mutations in KRAS, DPC, CDKN2A or TP53, but appear to have a high incidence of gene rearrangements, with up to 20% reported to be driven by BRAF fusions. With the development of a new class of RET-specific tyrosine kinase inhibitors, which appear to have particularly strong activity against RET gene rearranged tumours, there is now considerable interest in identifying RET gene rearrangements across a wide range of cancers. RET rearrangements have been reported to occur at a very low incidence (<1%) in all pancreatic carcinomas. We postulated that given its unique molecular profile, RET gene rearrangements may be common in acinar cell carcinomas. We performed fluorescent in-situ hybridization (FISH) studies on a cohort of 40 acinar cell spectrum tumours comprising 36 pure acinar cell carcinomas, three pancreatoblastomas and one mixed acinar-pancreatic neuroendocrine tumour. RET gene rearrangements were identified in 3 (7.5%) cases and BRAF gene rearrangements in 5 (12.5%). All gene rearranged tumours were pure acinar cell carcinomas. Our findings indicate that amongst all pancreatic carcinomas, acinar carcinomas are highly enriched for potentially actionable gene rearrangements in RET or BRAF. FISH testing is inexpensive and readily available in the routine clinical setting and may have a role in the assessment of all acinar cell carcinomas-at this stage to recruit patients for clinical trials of new targeted therapies, but perhaps in the near future as part of routine care.

MCL-1 antagonism enhances the anti-invasive effects of dasatinib in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies. It is phenotypically heterogeneous with a highly unstable genome and provides few common therapeutic targets. We found that MCL1, Cofilin1 (CFL1) and SRC mRNA were highly expressed by a wide range of these cancers, suggesting that a strategy of dual MCL-1 and SRC inhibition might be efficacious for many patients. Immunohistochemistry revealed that MCL-1 protein was present at high levels in 94.7% of patients in a cohort of PDACs from Australian Pancreatic Genome Initiative (APGI). High MCL1 and Cofilin1 mRNA expression was also strongly predictive of poor outcome in the TCGA dataset and in the APGI cohort. In culture, MCL-1 antagonism reduced the level of the cytoskeletal remodeling protein Cofilin1 and phosphorylated SRC on the active Y416 residue, suggestive of reduced invasive capacity. The MCL-1 antagonist S63845 synergized with the SRC kinase inhibitor dasatinib to reduce cell viability and invasiveness through 3D-organotypic matrices. In preclinical murine models, this combination reduced primary tumor growth and liver metastasis of pancreatic cancer xenografts. These data suggest that MCL-1 antagonism, while reducing cell viability, may have an additional benefit in increasing the antimetastatic efficacy of dasatinib for the treatment of PDAC.

Independent recalculation outperforms traditional measurement-based IMRT QA methods in detecting unacceptable plans.

PURPOSE: To evaluate the performance of an independent recalculation and compare it against current measurement-based patient specific intensity-modulated radiation therapy (IMRT) quality assurance (QA) in predicting unacceptable phantom results as measured by the Imaging and Radiation Oncology Core (IROC). METHODS: When institutions irradiate the IROC head and neck IMRT phantom, they are also asked to submit their internal IMRT QA results. Separately from this, IROC has previously created reference beam models on the Mobius3D platform to independently recalculate phantom results based on the institution's DICOM plan data. The ability of the institutions' IMRT QA to predict the IROC phantom result was compared against the independent recalculation for 339 phantom results collected since 2012. This was done to determine the ability of these systems to detect failing phantom results (i.e., large errors) as well as poor phantom results (i.e., modest errors). Sensitivity and specificity were evaluated using common clinical thresholds, and receiver operator characteristic (ROC) curves were used to compare across different thresholds. RESULTS: Overall, based on common clinical criteria, the independent recalculation was 12 times more sensitive at detecting unacceptable (failing) IROC phantom results than clinical measurement-based IMRT QA. The recalculation was superior, in head-to-head comparison, to the EPID, ArcCheck, and MapCheck devices. The superiority of the recalculation vs these array-based measurements persisted under ROC analysis as the recalculation curve had a greater area under it and was always above that for these measurement devices. For detecting modest errors (poor phantom results rather than failing phantom results), neither the recalculation nor measurement-based IMRT QA performed well. CONCLUSIONS: A simple recalculation outperformed current measurement-based IMRT QA methods at detecting unacceptable plans. These findings highlight the value of an independent recalculation, and raise further questions about the current standard of measurement-based IMRT QA.

Radiation Planning Assistant - A Streamlined, Fully Automated Radiotherapy Treatment Planning System.

The Radiation Planning Assistant (RPA) is a system developed for the fully automated creation of radiotherapy treatment plans, including volume-modulated arc therapy (VMAT) plans for patients with head/neck cancer and 4-field box plans for patients with cervical cancer. It is a combination of specially developed in-house software that uses an application programming interface to communicate with a commercial radiotherapy treatment planning system. It also interfaces with a commercial secondary dose verification software. The necessary inputs to the system are a Treatment Plan Order, approved by the radiation oncologist, and a simulation computed tomography (CT) image, approved by the radiographer. The RPA then generates a complete radiotherapy treatment plan. For the cervical cancer treatment plans, no additional user intervention is necessary until the plan is complete. For head/neck treatment plans, after the normal tissue and some of the target structures are automatically delineated on the CT image, the radiation oncologist must review the contours, making edits if necessary. They also delineate the gross tumor volume. The RPA then completes the treatment planning process, creating a VMAT plan. Finally, the completed plan must be reviewed by qualified clinical staff.

Developing and characterizing MR/CT-visible materials used in QA phantoms for MRgRT systems.

PURPOSE: Synthetic tissue equivalent (STE) materials currently used to simulate tumor and surrounding tissues for IROC-Houston's anthropomorphic head and thorax QA phantoms cannot be visualized using magnetic resonance (MR) imaging. The purpose of this study was to characterize dual MR/CT-visible STE materials that can be used in an end-to-end QA phantom for MR-guided radiotherapy (MRgRT) modalities. METHODS: Over 80 materials' MR, CT, and dosimetric STE properties were investigated for use in MRgRT QA phantoms. The materials tested included homogeneous and heterogeneous materials to simulate soft tissue/tumor and lung tissues. Materials were scanned on a Siemens' Magnetom Espree 1.5 T using four sequences, which showed the materials visual contrast between T1- and T2-weighted images. Each material's Hounsfield number and electron density data was collected using a GE's CT Lightspeed Simulator. Dosimetric properties were examined by constructing a 10 × 10 × 20 cm3 phantom of the selected STE materials that was divided into three sections: anterior, middle, and posterior. Anterior and posterior pieces were composed of polystyrene, whereas the middle section was substituted with the selected STE materials. EBT3 film was inserted into the phantom's midline and was irradiated using an Elekta's Versa 6 MV beam with a prescription of 6 Gy at 1.5 cm and varying field size of: 10 × 10 cm2 , 6 × 6 cm2 , and 3 × 3 cm2 . Measured film PDD curves were compared to planning system calculations and conventional STE materials' percent depth dose (PDD) curves. RESULTS: The majority of the tested materials showed comparable CT attenuation properties to their respective organ site; however, most of the tested materials were not visible on either T1- or T2-weighted MR images. Silicone, hydrocarbon, synthetic gelatin, and liquid PVC plastic-based materials showed good MR image contrast. In-house lung equivalent materials made with either silicone- or hydrocarbon-based materials had HUs ranging from: -978 to -117 and -667 to -593, respectively. Synthetic gelatin and PVC plastic-based materials resembled soft tissue/tumor equivalent materials and had HUs of: -175 to -170 and -29 to 32, respectively. PDD curves of the selected MR/CT-visible materials were comparable to IROC-Houston's conventional phantom STE materials. The smallest field size showed the largest disagreements, where the average discrepancies between calculated and measured PDD curves were 1.8% and 5.9% for homogeneous and heterogeneous testing materials, respectively. CONCLUSIONS: Gelatin, liquid plastic, and hydrocarbon-based materials were determined as alternative STE substitutes for MRgRT QA phantoms.

miR-139-5p Modulates Radiotherapy Resistance in Breast Cancer by Repressing Multiple Gene Networks of DNA Repair and ROS Defense.

Radiotherapy is essential to the treatment of most solid tumors and acquired or innate resistance to this therapeutic modality is a major clinical problem. Here we show that miR-139-5p is a potent modulator of radiotherapy response in breast cancer via its regulation of genes involved in multiple DNA repair and reactive oxygen species defense pathways. Treatment of breast cancer cells with a miR-139-5p mimic strongly synergized with radiation both in vitro and in vivo, resulting in significantly increased oxidative stress, accumulation of unrepaired DNA damage, and induction of apoptosis. Several miR-139-5p target genes were also strongly predictive of outcome in radiotherapy-treated patients across multiple independent breast cancer cohorts. These prognostically relevant miR-139-5p target genes were used as companion biomarkers to identify radioresistant breast cancer xenografts highly amenable to sensitization by cotreatment with a miR-139-5p mimetic.Significance: The microRNA described in this study offers a potentially useful predictive biomarker of radiosensitivity in solid tumors and a generally applicable druggable target for tumor radiosensitization. Cancer Res; 78(2); 501-15. ©2017 AACR.

Tailored first-line and second-line CDK4-targeting treatment combinations in mouse models of pancreatic cancer.

OBJECTIVE: Extensive molecular heterogeneity of pancreatic ductal adenocarcinoma (PDA), few effective therapies and high mortality make this disease a prime model for advancing development of tailored therapies. The p16-cyclin D-cyclin-dependent kinase 4/6-retinoblastoma (RB) protein (CDK4) pathway, regulator of cell proliferation, is deregulated in PDA. Our aim was to develop a novel personalised treatment strategy for PDA based on targeting CDK4. DESIGN: Sensitivity to potent CDK4/6 inhibitor PD-0332991 (palbociclib) was correlated to protein and genomic data in 19 primary patient-derived PDA lines to identify biomarkers of response. In vivo efficacy of PD-0332991 and combination therapies was determined in subcutaneous, intrasplenic and orthotopic tumour models derived from genome-sequenced patient specimens and genetically engineered model. Mechanistically, monotherapy and combination therapy were investigated in the context of tumour cell and extracellular matrix (ECM) signalling. Prognostic relevance of companion biomarker, RB protein, was evaluated and validated in independent PDA patient cohorts (>500 specimens). RESULTS: Subtype-specific in vivo efficacy of PD-0332991-based therapy was for the first time observed at multiple stages of PDA progression: primary tumour growth, recurrence (second-line therapy) and metastatic setting and may potentially be guided by a simple biomarker (RB protein). PD-0332991 significantly disrupted surrounding ECM organisation, leading to increased quiescence, apoptosis, improved chemosensitivity, decreased invasion, metastatic spread and PDA progression in vivo. RB protein is prevalent in primary operable and metastatic PDA and may present a promising predictive biomarker to guide this therapeutic approach. CONCLUSION: This study demonstrates the promise of CDK4 inhibition in PDA over standard therapy when applied in a molecular subtype-specific context.

Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis.

The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer.

Whole genomes redefine the mutational landscape of pancreatic cancer.

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.