Feasibility of multiple immunoexpression assay for immune tumor micrornvironment (I-TME) on matched metastatic and primary renal cell carcinoma (RCC) for patient prognostication and predictiveness to immunotherapy (preliminary analyses of the Meet URO 18 study).
Authors: Brunelli Matteo , Rebuzzi Sara Elena , Vellone Valerio Gaetano , Sbaraglia Marta , Gaggero Gabriele , Fassan Matteo , Maruzzo Marco , Basso Umberto , Bersanelli Melissa , Damassi Alessandra , Martelli Valentino , Catalano Fabio , Pierantoni Francesco , Casartelli Chiara , Merler Sara , Businello Gianluca , Milella Michele , Buti Sebastiano , Fornarini Giuseppe 
Published: June 2021

e16545

Background: The Meet-URO 18 study is ongoing to assess the prognostic role of I-TME in advanced RCC patients treated with ≥second line nivolumab divided into two cohorts according to clinical benefit [progression-free survival ≥ 12 and ≤ 3 months]. We primarily assessed the feasibility of multiple antibody testing related to I-TME on matched metastases and primary tumor. Methods: Immunohistochemical analyses were used for the TME assessment of T-lineage (CD3, CD4, CD8), FOXP-3, granulocytes (CD15), macrophage-lineage (CD68), natural killer (NK)-cells (CD56), tumor cells (TCs) (CD56), B-lineage (CD20) and phosphorylated mTOR (phmTOR). TCs were quantitatively assessed for CD15, CD56 and phmTOR positivity. For T-, B- and CD68 cells within TC nests, the number of immunoreactive cells were counted with a microscopic field of x200 (0.933 mm2). Results: Overall, 42 tumor tissue samples (primary tumors, metastases) were available and for 17 patients both metastatic and primary tumor tissues were assessable for matched analyses. Among these patients, 12 had clear cell, 1 papillary and 4 mucinous tubular and spindle cell histotype according to WHO 2016 classification. Intratumoral T/CD8 cells ranged from 32 to >400 spots (mean 240; >400 in 7 samples) and intratumoral T/CD4 cells from 4 to >400 spots (mean 168; >400 in 5 samples). Nine samples showed absence of phmTOR expression, while 8 ranged from 10% to 90% of positive TCs. We did not observe countable NK-cells, whereas CD56 was visible in 5 samples (mean 55% of positive TCs). Intratumoral CD68 cells ranged from 34 to >400 spots (mean 175, >400 in 3 patients). Agreement of CD15 method of reporting granulocytic presence was high, thus only CD15 neoplastic expression was reported and ranged from 12% to 55% (mean 30%) in 15 patients. TME multiple analysis resulted equally clustered in 8 patients (<20% variability of single immuno-test) whereas the remaining 9 patients showed significant differences as percentage of immuno-tissue expression in at least one of the 5 immuno-indicators (T/CD8-CD4, C15, CD68, CD56, phmTOR). The remaining 8 samples of patients without matched analyses were used to test the feasibility of multiple analyses; among all antibodies exclusion of the CD20 and FOXP-3 final evaluation was needed, due to technical standardization. According to the 5 immuno-indicators, double-triple positive or penta-positive TME indicators may be identified and graded. Conclusions: Providing multiple immunoexpression platforms on a single specimen may be used as routine workflow. Profiling I-TME, especially CD56, CD15 on TCs and CD68 cells and phmTOR, deserves investigation with extensive control groups. A validation cohort will be tested at tissue level and in correlation with peripheral blood markers.