Rationale:
To establish a spatially exact co-registration procedure between
in vivo
multiparametric magnetic resonance imaging (mpMRI) and (immuno)histopathology of soft tissue sarcomas (STS) to identify imaging parameters that reflect radiation therapy response of STS.
Methods:
The mpMRI-Protocol included diffusion-weighted (DWI), intravoxel-incoherent motion (IVIM), and dynamic contrast-enhancing (DCE) imaging. The resection specimen was embedded in 6.5% agarose after initial fixation in formalin. To ensure identical alignment of histopathological sectioning and
in vivo
imaging, an
ex vivo
MRI scan of the specimen was rigidly co-registered with the
in vivo
mpMRI. The deviating angulation of the specimen to the
in vivo
location of the tumor was determined. The agarose block was trimmed accordingly. A second
ex vivo
MRI in a dedicated localizer with a 4 mm grid was performed, which was matched to a custom-built sectioning machine. Microtomy sections were stained with hematoxylin and eosin. Immunohistochemical staining was performed with anti-ALDH1A1 antibodies as a radioresistance and anti-MIB1 antibodies as a proliferation marker. Fusion of the digitized microtomy sections with the
in vivo
mpMRI was accomplished through nonrigid co-registration to the
in vivo
mpMRI. Co-registration accuracy was qualitatively assessed by visual assessment and quantitatively evaluated by computing target registration errors (TRE).
Results:
The study sample comprised nine tumor sections from three STS patients. Visual assessment after nonrigid co-registration showed a strong morphological correlation of the histopathological specimens with
ex vivo
MRI and
in vivo
mpMRI after neoadjuvant radiation therapy. Quantitative assessment of the co-registration procedure using TRE analysis of different pairs of pathology and MRI sections revealed highly accurate structural alignment, with a total median TRE of 2.25 mm (histology -
ex vivo
MRI), 2.22 mm (histology -
in vivo
mpMRI), and 2.02 mm (
ex vivo
MRI -
in vivo
mpMRI). There was no significant difference between TREs of the different pairs of sections or caudal, middle, and cranial tumor parts, respectively.
Conclusion:
Our initial results show a promising approach to obtaining accurate co-registration between histopathology and
in vivo
MRI for STS. In a larger cohort of patients, the method established here will enable the prospective identification and validation of
in vivo
imaging biomarkers for radiation therapy response prediction and monitoring in STS patients via precise molecular and cellular correlation.
We describe a sequential multistaining protocol for immunohistochemistry, immunofluorescence and CyTOF imaging for formalin-fixed, paraffin-embedded specimens (FFPE) in the formalin gas-phase (FOLGAS), enabling sequential multistaining, independent from the primary and secondary antibodies and retrieval. Histomorphologic details are preserved, and crossreactivity and loss of signal intensity are not detectable. Combined with a DAB-based hydrophobic masking of metal-labeled primary antibodies, FOLGAS allows the extended use of CyTOF imaging in FFPE sections.
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