Tumour genomic and microenvironmental heterogeneity as integrated predictors for prostate cancer recurrence: a retrospective study

Clinical prognostic groupings for localised prostate cancers are imprecise, with 30–50% of patients recurring after image-guided radiotherapy or radical prostatectomy. We aimed to test combined genomic and microenvironmental indices in prostate cancer to improve risk stratification and complement clinical prognostic factors

Identification of two genetic alterations in oral premalignancies and tumors using a novel fingerprinting assay

The identification of novel markers causal in oral tumorigenesis is critical for clinical diagnosis and intervention. The study of oral premalignancies is necessary in order to identify these early stage genetic events. As such tissues are typically available as archival formalin-fixed paraffin-embedded biopsies, we have modified the standard RAPD (Randomly Amplified Polymorphic DNA) fingerprinting technique to utilize this material. This modified assay SMAL (Scanning Minute Archival Lesions) enables reproducible high-density fingerprinting of specific cell populations dissected either manually or through LCM (Laser Capture Microdissection). 235 paraffin-embedded formalin-fixed oral tumor and premalignant biopsies were tested using the SMAL assay. Two novel genetic changes in oral cancer progression stages were identified. These changes were mapped to chromosomes 2p23 and 7q22. Genetic instability at these two loci was verified by LOH (Loss Of Heterozygosity) analysis. 72 oral specimens (38 tumors and 34 dysplasias) were used to define a minimal boundary of allelic imbalance at these loci by LOH. Candidate genes Anaplastic Lymphoma Kinase, Dynein, Phosphatidylinositol 3-Kinase and Fos-Like Antigen 2 have been identified.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

Posterior sternoclavicular joint dislocation: case report and discussion

The sternoclavicular joint is the most frequently mobilized non-axial, major joint, but is the least frequently dislocated. Most sternoclavicular dislocations are anterior. When posterior sternoclavicular joint dislocations do occur, they may present with a variety of signs and symptoms, including serious intrathoracic injuries. We discuss the case of a patient with a subacute posterior sternoclavicular dislocation who presented to the emergency department 2 months after being hit in the posterior neck. We also review the signs, symptoms and management of posterior sternoclavicular dislocation and the literature on this topic

An efficient method for the assessment of DNA quality of archival microdissected specimens

There will be an increasing need of methods for assessing the suitability of specimens for genetic-based assays as DNA markers become an integral part of molecular diagnosis. The targeting of specimens for specific analyses will require the ability to rapidly screen for DNA quality. Conventional methods such as Southern analysis and gene specific-polymerase chain reaction (PCR) often require quantities of material that represent a significant portion of the specimen, especially in microdissected samples. Here we describe a novel application of a commonly used PCR-based DNA-fingerprinting technology that requires minimal quantities of DNA to simultaneously assess multiple regions throughout the genome for DNA quality. Randomly amplified polymorphic DNA (RAPD) PCR generates DNA fragments of a broad size range with the product size reflecting the degree of sample fragmentation. Fourteen DNA samples extracted from cells microdissected from seven formalin-fixed, paraffin-embedded oral cancer biopsies were assessed for DNA quality using gene-specific PCR and RAPD-PCR. Although the more conventional assay required 2-ng DNA (or 300-cell equivalents) to examine DNA quality at a single locus, RAPD-PCR provided a more informative profile of DNA quality from the same microdissected archival specimens

4-1BB Aptamer-Based Immunomodulation Enhances the Therapeutic Index of Radiation Therapy in Murine Tumor Models

To report a novel strategy using oligonucleotide aptamers to 4-1BB as an alternate method for costimulation, and show that combinatorial therapy with radiation improves the therapeutic ratio over equivalent monoclonal antibodies. Subcutaneous 4T1 (mouse mammary carcinoma) tumors were established (approximately 100 mm(3)), and a radiation therapy (RT) dose/fractionation schedule that optimally synergizes with 4-1BB monoclonal antibody (mAb) was identified. Comparable tumor control and animal survival was observed when either 4-1BB antibody or aptamer were combined with RT using models of breast cancer and melanoma (4T1 and B16-F10). Off-target CD8(+) T-cell toxicity was evaluated by quantification of CD8(+) T cells in livers and spleens of treated animals. When combined with 4-1BB mAb, significant differences in tumor control were observed by varying RT dose and fractionation schedules. Optimal synergy between RT and 4-1BB mAb was observed at 5 Gy × 6. Testing 4-1BB mAb and aptamer independently using the optimal RT (5 Gy × 6 for 4T1/Balb/c and 12 Gy × 1 for B16/C57BL6J mouse models) revealed equivalent tumor control using 4-1BB aptamer and 4-1BB mAb. 4-1BB mAb, but not 4-1BB aptamer-treated animals, exhibited increased lymphocytic liver infiltrates and increased splenic and liver CD8(+) T cells. Radiation therapy synergizes with 4-1BB mAb, and this effect is dependent on RT dose and fractionation. Tumor control by 4-1BB aptamer is equivalent to 4-1BB mAb when combined with optimal RT dose, without eliciting off-target liver and spleen CD8(+) expansion. 4-1BB aptamer-based costimulation affords a comparable and less toxic strategy to augment RT-mediated tumor control

Abstract 4992: Development of a novel radiation-induced targeted immunotherapy strategy through oligonucleotide aptamer conjugation

Abstract Monoclonal antibodies (mAbs) produce dramatic anti-tumor responses in multiple solid tumors. Unfortunately, systemic administration can result in end organ retention and dose-limiting toxicities. Radiotherapy (RT) is a standard-of-care treatment modality that induces direct tumor cell kill through DNA damage. While studies assessing the synergy of RT-induced tumor cell kill with mAbs are ongoing, an additional unexploited effect of RT is the induction of stress response products in the tumor microenvironment, such as vascular endothelial growth factor (VEGF). Oligonucleotide aptamers are short pieces of nucleic acid that exhibit little to no immunogenicity compared to mAbs, making them ideal delivery vehicles for therapeutic mAbs. The purpose of this study was to assess the feasibility of a novel strategy for targeted tumor delivery of existing immunotherapeutic mAbs through conjugation to ligands that bind to radiation induced products. An antibody recognizing the costimulatory molecule 41BB was covalently modified with a 21-mer ssDNA oligonucleotide linker sequence that serves as an anchor site for the VEGF aptamer synthesized with a 3’ extension. Annealing of complementary strands formed a 41BB mAb-VEGF aptamer conjugate. First, VEGF aptamer conjugation to modified 41BB mAb was verified by polyacrylamide gel electrophoresis analysis which revealed the expected size shift of the 41BB mAb-VEGF aptamer conjugate versus the unannealed controls. Conjugation was then verified using flow cytometry. Recombinant mouse 41BB-Fc was immobilized on Protein A Dynabeads and 41BB mAb conjugated with 5’ biotinylated VEGF aptamer. 41BB mAb-VEGF aptamer conjugate retention on Dynabeads was confirmed by using streptavidin-PE 5’ biotin of aptamer attached to mAb in conjugate binding epitope target. Functionality of 41BB mAb-VEGF aptamer conjugate was confirmed in vitro using a CD8+ T cell costimulation assay. CD8+ T cells were isolated from 8 week old female C57BL6/J splenocytes, and exposed to sub-optimal levels of CD3 mAb. 41BB mAb-VEGF aptamer conjugate, unconjugated 41BB mAb and isotype control were added to activated cells. Elevated levels of interferon gamma were observed and equivalent in conjugated and unconjugated 41BB mAbs. These results indicate that conjugation of 41BB mAb-VEGF aptamer is feasible and that conjugation does not affect 41BB mAb functionality. Use of this novel targeting approach to improve the therapeutic index of immunotherapeutic mAbs requires validation in murine tumor models. Citation Format: Ana Paula Benaduce, Randall Brenneman, Diana Cardero, Brett Schrand, Eli Gilboa, Adrian Ishkanian. Development of a novel radiation-induced targeted immunotherapy strategy through oligonucleotide aptamer conjugation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4992.</jats:p

Methods for high throughput validation of amplified fragment pools of BAC DNA for constructing high resolution CGH arrays

The recent development of array based comparative genomic hybridization (CGH) technology provides improved resolution for detection of genomic DNA copy number alterations. In array CGH, generating spotting solution is a multi-step process where bacterial artificial chromosome (BAC) clones are converted to replenishable PCR amplified fragments pools (AFP) for use as spotting solution in a microarray format on glass substrate. With completion of the human and mouse genome sequencing, large BAC clone sets providing complete genome coverage are available for construction of whole genome BAC arrays. Currently, Southern hybridization, fluorescent in-situ hybridization (FISH), and BAC end sequencing methods are commonly used to identify the initial BAC clone but not the end product used for spotting arrays. The AFP sequencing technique described in this study is a novel method designed to verify the identity of array spotting solution in a high throughput manner. We show here that Southern hybridization, FISH, and AFP sequencing can be used to verify the identity of final spotting solutions using less than 10% of the AFP product. Single pass AFP sequencing identified over half of the 960 AFPs analyzed. Moreover, using two vector primers approximately 90% of the AFP spotting solutions can be identified. In this feasibility study we demonstrate that current methods for identifying initial BAC clones can be adapted to verify the identity of AFP spotting solutions used in printing arrays. Of these methods, AFP sequencing proves to be the most efficient for large scale identification of spotting solution in a high throughput manner