Validation of a Novel Three-Dimensional (3D Fusion) Gross Sampling Protocol for Clear Cell Renal Cell Carcinoma to Overcome Intratumoral Heterogeneity: The Meet-Uro 18 Study
Authors: Brunelli Matteo , Martignoni Guido , Malpeli Giorgio , Volpe Alessandro , Cima Luca , Raspollini Maria Rosaria , Barbareschi Mattia , Tafuri Alessandro , Masi Giulia , Barzon Luisa , Ammendola Serena , Villanova Manuela , Cerruto Maria Angela , Milella Michele , Buti Sebastiano , Bersanelli Melissa , Fornarini Giuseppe , Rebuzzi Sara Elena , Vellone Valerio Gaetano , Gaggero Gabriele , Procopio Giuseppe , Verzoni Elena , Bracarda Sergio , Fanelli Martina , Sabbatini Roberto , Passalacqua Rodolfo , Perrucci Bruno , Giganti Maria Olga , Donini Maddalena , Panni Stefano , Tucci Marcello , Prati Veronica , Ortega Cinzia , Caliò Anna , Eccher Albino , Alongi Filippo , Pappagallo Giovanni , Iacovelli Roberto , Mosca Alessandra , Umari Paolo , Montagnani Ilaria , Gobbo Stefano , Atzori Francesco , Munari Enrico , Maruzzo Marco , Basso Umberto , Pierconti Francesco , Patriarca Carlo , Colombo Piergiuseppe , Lapini Alberto , Conti Giario , Salvioni Roberto , Bollito Enrico , Cossarizza Andrea , Massari Francesco , Rizzo Mimma , Franco Renato , Zito-Marino Federica , Aberasturi Plata Yoseba , Galuppini Francesca , Sbaraglia Marta , Fassan Matteo , Dei Tos Angelo Paolo , Colecchia Maurizio , Moch Holger , Scaltriti Maurizio , Porta Camillo , Delahunt Brett , Giannarini Gianluca , Bortolus Roberto , Rescigno Pasquale , Banna Giuseppe Luigi , Signori Alessio , Obispo Miguel Angel Llaja , Perris Roberto , Antonelli Alessandro 
Published: May 2022

We aimed to overcome intratumoral heterogeneity in clear cell renal cell carcinoma (clearRCC). One hundred cases of clearRCC were sampled. First, usual standard sampling was applied (1 block/cm of tumor); second, the whole tumor was sampled, and 0.6 mm cores were taken from each block to construct a tissue microarray; third, the residual tissue, mapped by taking pieces 0.5 × 0.5 cm, reconstructed the entire tumor mass. Precisely, six randomly derived pieces of tissues were placed in each cassette, with the number of cassettes being based on the diameter of the tumor (called multisite 3D fusion). Angiogenic and immune markers were tested. Routine 5231 tissue blocks were obtained. Multisite 3D fusion sections showed pattern A, homogeneous high vascular density (10%), pattern B, homogeneous low vascular density (8%) and pattern C, heterogeneous angiogenic signatures (82%). PD-L1 expression was seen as diffuse (7%), low (33%) and absent (60%). Tumor-infiltrating CD8 scored high in 25% (pattern hot), low in 65% (pattern weak) and zero in 10% of cases (pattern desert). Grading was upgraded in 26% of cases (G3–G4), necrosis and sarcomatoid/rhabdoid characters were observed in, respectively, 11 and 7% of cases after 3D fusion (p = 0.03). CD8 and PD-L1 immune expressions were higher in the undifferentiated G4/rhabdoid/sarcomatoid clearRCC subtypes (p = 0.03). Again, 22% of cases were set to intermediate to high risk of clinical recurrence due to new morphological findings of all aggressive G4, sarcomatoid/rhabdoid features by using 3D fusion compared to standard methods (p = 0.04). In conclusion, we propose an easy-to-apply multisite 3D fusion sampling that negates bias due to tumor heterogeneity.