Translational outcomes
tumor
TUMOR
Flow cytometry analyses of immune cells were conducted according to local routine (Supplementary S7) utilizing BD FACSCanto II (3 lasers) and BD FACSDiva software v.8.0.1 (BD Biosciences).Dedicated uropathologists re-examined the formalin-fixed paraffin-embedded (FFPE) primary diagnostic biopsies, assigning all samples a histologic subtype and International Society of Urological Pathology (ISUP) grade group. Furthermore, FFPE hematoxylin- and eosin-stained slides from all eight study biopsies, collected prior to CryoIT from each participant, were examined and graded (Supplementary S8).For each participant, the study biopsy with the most tumor tissue and the highest Gleason pattern was selected for immunohistochemistry analyses for T cells. The corresponding fresh frozen biopsy was selected for the two genetic panels and the T-cell receptor (TCR) sequencing (Supplementary S8 and S10).The biopsied tissue was stained for the four MMR proteins: MSH2, MSH6, PMS2 and MLH1, using the platform Ventana BenchMark Ultra (Roche, Basel, Switzerland) and detection system OptiView (Supplementary S9). MSI was diagnosed when minimum one of four MMR proteins was unstained in tumor nuclei, while positive staining < 10% classified as equivocal.DNA was isolated from each sample using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany). Targeted parallel sequencing for library preparation (Agilent SureSelect XT-kit, Agilent) was performed on DNA from tumor tissue and matched peripheral blood. For the 360 gene panel targeted enrichment was performed using RNA baits (SureSelect, Agilent) against the coding regions of 360 cancer-related genes [TCR clonotypes were enumerated according to sequence counts. The 200 clonotypes with the highest sequence counts two and six weeks after CryoIT were compared separately with results from pre-treatment samples. Novel clonotypes were those that were undetectable prior to, but detectable after the CryoIT. For pre-treatment samples, the maximum clonal frequencies were used. Expanded clonotypes were pre-treatment clonotypes that expanded > fivefold after treatment. The sum of the frequencies of all identified TCR clonotypes in the entire data set defined the clonal space, while the clonal space percentage of a clonotype was calculated as the percentage of the total number of identified clonotypes.