An alternative approach to histopathological validation of PET imaging for radiation therapy image-guidance: a proof of concept.

PURPOSE: In radiotherapy, PET images can be used to guide the delivery of selectively escalated doses to biologically relevant tumour subvolumes. Validation of PET for such applications requires demonstration of spatial coincidence between PET tracer uptake pattern and the histopathologically confirmed target. This study introduces a novel approach to histopathological validation of PET image segmentation for radiotherapy guidance. METHODS AND MATERIALS: Sequential tissue sections from surgically excised whole-tumour specimens were used to acquire full 3D-sets of both histopathological images (microscopy) and PET tracer distribution images (autoradiography). After these datasets were accurately registered, a full 3D autoradiographic distribution of PET tracer was reconstructed and used to obtain synthetic PET images (sPET) by simulating the image deterioration induced by processes involved in PET image formation. To illustrate the method, sPET images were used in this study to investigate spatial coincidence between high FDG uptake areas and the distribution of viable tissue in two small animal tumour models. RESULTS: The reconstructed 3D autoradiographic distribution of the PET tracer was spatially coherent, as indicated by the high average value of the normalised pixel-by-pixel correlation of intensities between successive slices (0.84 ± 0.05 and 0.94 ± 0.02). The loss of detail in the sPET images versus the 3D autoradiography was significant as indicated by Dice coefficient values corresponding to the two tumours (0 and 0.1 at 70% threshold). The maximum overlap between the FDG segmented volumes and the extent of the viable tissue as indicated by Dice coefficient values, was 0.8 for one tumour (for the image thresholded at 22% of max intensity) and 0.88 for the other (threshold of 14% of max intensity). CONCLUSION: It was demonstrated that the use of synthetic PET images for histopathological validation allows for bypassing a technically challenging and error-prone step of registering non-invasive PET images with histopathology.

Tumour microenvironment heterogeneity affects the perceived spatial concordance between the intratumoural patterns of cell proliferation and 18F-fluorothymidine uptake.

BACKGROUND AND PURPOSE: PET imaging with (18)F-fluorothymidine ((18)F-FLT) can potentially be used to identify tumour subvolumes for selective dose escalation in radiation therapy. The purpose of this study is to analyse the co-localization of intratumoural patterns of cell proliferation with (18)F-FLT tracer uptake. MATERIALS AND METHODS: Mice bearing FaDu or SQ20B xenograft tumours were injected with (18)F-FLT, and bromodeoxyuridine (proliferation marker). Ex vivo images of the spatial pattern of intratumoural (18)F-FLT uptake and that of bromodeoxyuridine DNA incorporation were obtained from thin tumour tissue sections. These images were segmented by thresholding and Relative Operating Characteristic (ROC) curves and Dice similarity indices were evaluated. RESULTS: The thresholds at which maximum overlap occurred between FLT-segmented areas and areas of active cell proliferation were significantly different for the two xenograft tumour models, whereas the median Dice values were not. However, ROC analysis indicated that segmented FLT images were more specific at detecting the proliferation pattern in FaDu tumours than in SQ20B tumours. CONCLUSION: Highly dispersed patterns of cell proliferation observed in certain tumours can affect the perceived spatial concordance between the spatial pattern of (18)F-FLT uptake and that of cell proliferation even when high-resolution ex vivo autoradiography imaging is used for (18)F-FLT imaging.

Comprehensive approach to coregistration of autoradiography and microscopy images acquired from a set of sequential tissue sections.

UNLABELLED: Histopathologic validation of a PET tracer requires assessment of colocalization of the tracer with its intended biologic target. Using thin tissue section autoradiography, it is possible to visualize the spatial distribution of the PET tracer uptake and compare it with the distribution of the intended biologic target (as visualized with immunohistochemistry). The purpose of this study was to develop and evaluate an objective methodology for deformable coregistration of autoradiography and microscopy images acquired from a set of sequential tissue sections. METHODS: Tumor-bearing animals were injected with 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT), (14)C-FDG, and other markers of tumor microenvironment including Hoechst 33342 (blood-flow surrogate). After sacrifice, tumors were excised, frozen, and sectioned. Multiple stacks of sequential 8 µm sections were collected from each tumor. From each stack, the middle (reference) sections were used to obtain images of (18)F-FLT and (14)C-FDG uptake distributions using dual-tracer autoradiography. Sections adjacent to the reference were used to acquire all histopathologic data (e.g., images of cell proliferation, hematoxylin and eosin). Hoechst images were acquired from all sections. To correct for deformations and misalignments induced by tissue processing and image acquisition, the Hoechst image of each nonreference section was deformably registered to the reference Hoechst image. This transformation was then applied to all images acquired from the same tissue section. In this way, all microscopy images were registered to the reference Hoechst image. The Hoechst-to-autoradiography image registration was done using rigid point-set registration based on external markers visible in both images. RESULTS: The mean error of Hoechst to (18)F-FLT autoradiography registration (both images acquired from the same section) was 30.8 ± 20.1 µm. The error of Hoechst-based deformable registration of histopathologic images (acquired from sequential tissue sections) was 23.1 ± 17.9 µm. Total error of registration of autoradiography images to the histopathologic images acquired from adjacent sections was evaluated at 44.9 µm. This coregistration precision supersedes current rigid registration methods with reported errors of 100-200 µm. CONCLUSION: Deformable registration of autoradiography and histopathology images acquired from sequential sections is feasible and accurate when performed using corresponding Hoechst images.