Development of a registration framework to validate MRI with histology for prostate focal therapy.

PURPOSE: Focal therapy has been proposed as an alternative method to whole-gland treatment for prostate cancer when aiming to reduce treatment side effects. The authors recently validated a radiobiological model which takes into account tumor location and tumor characteristics including tumor cell density, Gleason score, and hypoxia in order to plan optimal dose distributions for focal therapy. The authors propose that this model can be informed using multiparametric MRI (mpMRI) and in this study present a registration framework developed to map prostate mpMRI and histology data, where histology will provide the "ground truth" data regarding tumor location and biology. The authors aim to apply this framework to a growing database to develop a prostate biological atlas which will enable MRI based planning for prostate focal therapy treatment. METHODS: Six patients scheduled for routine radical prostatectomy were used in this proof-of-concept study. Each patient underwent mpMRI scanning prior to surgery, after which the excised prostate specimen was formalin fixed and mounted in agarose gel in a custom designed sectioning box. T2-weighted MRI of the specimen in the sectioning box was acquired, after which 5 mm sections of the prostate were cut and histology sections were microtomed. A number of image processing and registration steps were used to register histology images with ex vivo MRI and deformable image registration (DIR) was applied to 3D T2w images to align the in vivo and ex vivo MRI data. Dice coefficient metrics and corresponding feature points from two independent annotators were selected in order to assess the DIR accuracy. RESULTS: Images from all six patients were registered, providing histology and in vivo MRI in the ex vivo MRI frame of reference for each patient. Results demonstrated that their DIR methodology to register in vivo and ex vivo 3D T2w MRI improved accuracy in comparison with an initial manual alignment for prostates containing features which were readily visible on MRI. The average estimated uncertainty between in vivo MRI and histology was 3.3 mm, which included an average error of 3.1 mm between in vivo and ex vivo MRI after applying DIR. The mean dice coefficient for the prostate contour between in vivo and ex vivo MRI increased from 0.83 before DIR to 0.93 after DIR. CONCLUSIONS: The authors have developed a registration framework for mapping in vivo MRI data of the prostate with histology by implementing a number of processing steps and ex vivo MRI of the prostate specimen. Validation of DIR was challenging, particularly in prostates with few or mostly linear rather than spherical shaped features. Refinement of their MR imaging protocols to improve the data quality is currently underway which may improve registration accuracy. Additional mpMRI sequences will be registered within this framework to quantify prostate tumor location and biology.

Conditions providing enhanced transfection efficiency in rat pheochromocytoma PC12 cells permit analysis of the activity of the far-upstream and proximal promoter of the brain creatine kinase gene.

While brain creatine kinase (CKB) is expressed at highest levels in the brain, where it functions in regenerating ATP, the gene elements and protein factors regulating CKB transcription in neuronal and glial cells have not been identified. To investigate the regulation of CKB in neuronal cells, we examined the expression of the promoter proximal and 5' far-upstream regions of the rat CKB gene transiently transfected into rat PC12 pheochromocytoma cells. Initially, these experiments were hampered by the extremely low transfection efficiency of PC12 cells. We increased efficiency by greater than 200-fold by employing CaPO4-precipitated DNA transfection into PC12 cells which were optimized for transient transfection by: (i) culturing cells in polylysine-coated dishes to insure attachment throughout transfection; (ii) exposing cells to transfected DNA for an optimal time and employing a glycerol shock; and, most importantly, (iii) dissociating the characteristic self-adhesive clumps of PC12 into mostly single cells. Use of the plasmid expressing green fluorescent protein allowed identification of the transfected cells that averaged 10-20% of the total. Analyses of CKB promoter-CAT gene constructs showed that in PC12 cells expression of the proximal (0.2 kb) CKB promoter was low while expression of the 1.4 kb promoter was three fold higher and the 2.9 kb promoter was ten fold higher, suggesting the presence of at least two upstream cis-acting, positive regulatory elements. In agreement, the steady-state CKB mRNA level was higher in PC12 than in other neuronal cell lines examined, possibly reflecting the effects of positive upstream factors. The results are discussed in relation to how this economical and straightforward transfection procedure may be useful in identify factors regulating the transcription of CKB and other genes expressed in neuronal cells.

Expression of the rat brain creatine kinase gene in C6 glioma cells.

We have recently shown that while brain creatine kinase (CKB) mRNA was detectable in RNA from cultured primary rat brain neurons, CKB mRNA was about 15-fold higher in primary astrocytes and 17-fold higher in oligodendrocytes (Molloy et al., J Neurochem 59:1925-1932, 1992). To begin to understand the molecular mechanisms responsible for brain glial cells containing the highest levels of CKB mRNA in the body, we have examined the expression of rat CKB mRNA in established C6 glioma cells. RNase-protection analysis showed the endogenous CKB mRNA levels in exponentially growing C6 were high and measured 50% of that in total RNA from rat brain lysate and 60% of that in cultured primary astrocytes and oligodendrocytes. The 5' and 3' ends of CKB mRNA in C6 were mapped to the same nucleotides as CKB mRNA from rat brain, indicating that the sites of in vivo transcription initiation and termination/polyadenylation of CKB mRNA in C6 are the same as in total rat brain RNA. The level of CKB enzyme activity in C6 whole cell lysates was among the highest of the glial cell lines which we measured. All creatine kinase enzyme activity present in C6 was found in the dimeric CKB isoform (BB), which is characteristic of CKB expression in the brain. A 2.9 kb gene fragment containing the basal CKB promoter and far-upstream 5' sequences was cloned upstream of the chloramphenicol acetyltransferase (CAT) gene and transfected into C6 cells. CAT activity was readily detectable in C6 and mapping of the 5' end of the CAT mRNA showed that transcription was directed from the correct initiation site. Since we found C6 cells were difficult to transfect, conditions were established which both maximized transfection efficiency and maintained normal C6 cell morphology. These results should permit the future identification of the nuclear trans-acting factors and the cognate cis-acting regulatory elements responsible for high CKB mRNA expression in brain glial cells.