Craig Cummings

Processing of radical prostatectomy specimens for correlation of data from histopathological, molecular biological, and radiological studies : a new whole organ technique

Aims: To develop a method of processing non-formalin fixed prostate specimens removed at radical prostatectomy to obtain fresh tissue for research and for correlating diagnostic and molecular results with preoperative imaging. Methods/Results: The method involves a prostate slicing apparatus comprising a tissue slicer with a series of juxtaposed planar stainless steel blades linked to a support, and a cradle adapted to grip the tissue sample and receive the blades. The fresh prostate gland is held in the cradle and the blades are moved through the cradle slits to produce multiple 4 mm slices of the gland in a plane perpendicular to its posterior surface. One of the resulting slices is preserved in RNAlaterTM. The areas comprising tumour and normal glands within this preserved slice can be identified by matching it to the haematoxylin and eosin stained sections of the adjacent slices that are formalin fixed and paraffin wax embedded. Intact RNA can be extracted from the identified tumour and normal glands within the RNAlater preserved slice. Preoperative imaging studies are acquired with the angulation of axial images chosen to be similar to the slicing axis, such that stained sections from the formalin fixed, paraffin wax embedded slices match their counterparts on imaging. Conclusions: A novel method of sampling fresh prostate removed at radical prostatectomy that allows tissue samples to be used both for diagnosis and molecular analysis is described. This method also allows the integration of preoperative imaging data with histopathological and molecular data obtained from the prostate tissue slices.

Exploring the Biomechanical Properties of Brain Malignancies and Their Pathologic Determinants In Vivo with Magnetic Resonance Elastography

Malignant tumors are typically associated with altered rigidity relative to normal host tissue. Magnetic resonance elastography (MRE) enables the noninvasive quantitation of the mechanical properties of deep-seated tissue following application of an external vibrational mechanical stress to that tissue. In this preclinical study, we used MRE to quantify (kPa) the elasticity modulus Gd and viscosity modulus Gl of three intracranially implanted glioma and breast metastatic tumor models. In all these brain tumors, we found a notable softness characterized by lower elasticity and viscosity than normal brain parenchyma, enabling their detection on Gd and Gl parametric maps. The most circumscribed tumor (U-87 MG glioma) was the stiffest, whereas the most infiltrative tumor (MDA-MB-231 metastatic breast carcinoma) was the softest. Tumor cell density and microvessel density correlated significantly and positively with elasticity and viscosity, whereas there was no association with the extent of collagen deposition or myelin fiber entrapment. In conclusion, although malignant tumors tend to exhibit increased rigidity, intracranial tumors presented as remarkably softer than normal brain parenchyma. Our findings reinforce the case for MRE use in diagnosing and staging brain malignancies, based on the association of different tumor phenotypes with different mechanical properties.

Abstract 1488: In vivo magnetic resonance elastography in pediatric brain tumor models

Refined imaging strategies that could improve diagnosis and management of children with brain malignancies are urgently required. Pediatric brain malignancies possess distinct underlying biologies that discriminate them from adult tumors, even within a common neuropathological diagnosis such as glioblastoma (GBM). Magnetic resonance elastography (MRE) exploits the ability of MRI to visualize the propagation of shear waves resulting from vibrations applied to the cranium to map and quantify (kPa) brain tissue elasticity (Gd) and viscosity (Gl) in vivo. The altered viscoelastic properties of tumors, combined with the sensitivity of MRE for differences in tumor microstructure, establishes MRE as an attractive modality for the detection and differential diagnosis of brain malignancies. Here we aimed to determine whether the potential of MRE in the neuroradiological management of patients with brain malignancies could be extended to the pediatric population. The viscoelastic properties of two pediatric brain tumor models that faithfully emulate high risk childhood disease were investigated: i) Orthotopic D-212 MG pediatric supratentorial giant cell GBM (H3F3A wildtype, 11 year old patient) xenografts propagated in NCr nu/nu mice and ii) GTML/Trp53KI/KI transgenic mice that spontaneously develop aggressive, MYCN driven, p53 depleted, medulloblastomas. D-212 MG and GTML/Trp53KI/KI tumors were less elastic (lower Gd) and viscous (lower Gl), and therefore softer, than the surrounding brain tissue. Both tumor types were also significantly less elastic (D-212 MG Gd = 3.9±0.2; GTML/Trp53KI/KI Gd = 3.5±0.1) than the soft thalamic parenchyma in non-tumor-bearing mice (Gd = 5.9±0.2; p = 0.001 and p = 0.02, respectively, Mann-Whitney), in addition to being less viscous. Interestingly, GTML/Trp53KI/KI tumors demonstrated a bimodal distribution of Gd, which reflected the more marked transition between their relatively stiffer rim and the softer core compared with D-212 MG tumors. We demonstrate that two representative models of major high risk pediatric brain malignancies share the unique softness previously observed in adult brain tumor models, allowing their detection by MRE. This supports observations that pediatric GBMs are morphologically indistinguishable from adult GBMs. Although median Gd values were not sufficient to discriminate between the tumor types, the marked bimodal distribution of Gd in the GTML/Trp53KI/KI tumors was not apparent in D-212 MG GBM tumors. Whilst ongoing histopathological investigation into growth patterns, vascular, cellular and extracellular networks will aid elucidation of the pathological determinants of the bimodal signature, to date unique to GTML/Trp53KI/KI, these data reinforce the potential of MRE for the detection and differential diagnosis of pediatric brain malignancies based on their mechanical properties. Citation Format: Jin Li, Jessica K.R. Boult, Maria Vinci, Sergey Popov, Karen Barker, Zai Ahmad, Yann Jamin, Craig Cummings, Suzanne A. Eccles, Jeffrey C. Bamber, Ralph Sinkus, Louis Chesler, Chris Jones, Simon P. Robinson. In vivo magnetic resonance elastography in pediatric brain tumor models. [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 1488. doi:10.1158/1538-7445.AM2015-1488

Abstract 5017: T-cell repertoire turnover induced by anti-CTLA-4 antibody treatment in cancer patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA While treatment with anti-CTLA-4 antibody can induce clinical responses in advanced cancer patients, its effects on the breadth of the T cell response is unknown. We used a next-generation sequencing-based method to assess T cell repertoire diversity in 46 patients with metastatic castration resistant prostate cancer or metastatic melanoma. Peripheral blood mononuclear cells were obtained from patients prior to and during treatment with anti-CTLA-4 antibody. mRNA was amplified using locus-specific primer sets for T cell receptor (TCR) beta, and the amplified product was sequenced. Sequence reads were used to quantitate absolute TCR frequencies using standardized clonotype determination algorithms with normalization by spiked reference TCR sequences. Following clonotype quantitation, repertoire differences between serial samples were assessed by the Morisita index, a statistical measure of population dispersion. 97 paired samples were assessed, of which 46 (47%) had increases and 22 (23%) had decreases in TCR diversity by more than 2-fold. By comparison, none of 9 untreated sample pairs underwent more than a 2-fold change in diversity (P = 0.005, Fishers exact test, two tailed). TCR repertoire differences between monthly samples were markedly higher than for time-matched controls. After the first treatment, median Morisita index between samples was 0.197 for treated samples versus 0.039 for untreated (P = 0.0005, Mann-Whitney U test). The median number of clones that significantly changed in abundance was 421 for treated versus 45 for controls. In patients with multiple time points, this rapid clonotype evolution continued through treatment. Whereas the number of clonotypes that increased with treatment was not associated with clinical outcome, improved overall survival was associated with maintenance of high frequency clones present at baseline. In contrast the highest frequency clonotypes fell with treatment in patients with shorter overall survival. Stably maintained clonotypes included T cells possessing high avidity TCR such as CMV-reactive T cells. Together, these results suggest that CTLA-4 blockade induces T-cell repertoire evolution and diversification. Moreover, improved clinical outcomes are associated with less clonotype loss, consistent with the maintenance of high frequency TCR clonotypes during treatment. These clones may represent the presence of pre-existing high avidity T cells that may be relevant in the anti-tumor response. Citation Format: Lawrence H. Fong, Edward Cha, Mark Klinger, Yafei Hou, Craig Cummings, Antoni Ribas, Malek Faham. T-cell repertoire turnover induced by anti-CTLA-4 antibody treatment in cancer patients. [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 5017. doi:10.1158/1538-7445.AM2014-5017