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

Abstract

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.

Atlas of PD-L1 for Pathologists: Indications, Scores, Diagnostic Platforms and Reporting Systems

“…Both TPS and CPS were studied: usually, a minority (7–15%) of clear cell RCCs harbor diffuse TPS scores (≥50%), while conversely, most of them (85%) show positive PD-L1 expression by using the CPS score (85%). The discrepancy is mainly due to the assessment of TIL and myeloid cells in the CPS, which increases the number of positive cells in the final score [ 68 ]. By focusing on the available clones, Ventana SP142 tends to label much more inflammatory than neoplastic cells, while Ventana SP263 mainly stains tumor cells, such as Dako 22C3.…”

Towards Personalized Sampling in Clear Cell Renal Cell Carcinomas

“…A comparison of the performance in detecting histological features of bad prognosis such as high grade and granular eosinophilic cells [15] with both methods in 38 CCRCC showed that MSTS was significantly more informative than routine sampling [16]. Aside from CCRCC, the usefulness of MSTS in CCRCC has been confirmed by subsequent histological, immunohistochemical, and molecular studies in ovarian carcinoma, mesothelioma, and head and neck squamous cell carcinoma [17][18][19][20]. Lakis et al [17] have analyzed 294 tumor sections from 70 treatment naïve patients who had undergone cytoreductive surgery of ovarian cancer and have observed not only the high histological variability of tumors across different regions, but also the irregular qualitative and quantitative distribution of tumor-associated lymphocyte, information with obvious prognostic and therapeutic implications.…”

“…The authors included in the comparison histological, immunohistochemical, and molecular parameters, and concluded that MSTS was more informative than routine sampling in detecting perineurial permeation, peritumoral vascular/lymphatic growth, necrosis, muscle invasion, PIK3CA mutations (exons 9 and 20), and CDKN2A promoter methylation. Brunelli et al [ 20 ] have recently compared a multi-regional sampling strategy called 3D fusion with routine sampling in 100 CCRCC analyzing the respective performance of both methods in the detection of angiogenic and immune markers. These authors confirm the superiority of 3D fusion sampling and agree that sampling one block/cm of tumor tissue diameter is inadequate to fully characterize ITH in CCRCC.…”

“…In a context of high diagnostic pressure, some pathologists may be reluctant to increase the time and cost needed to implement both 3D fusion [ 20 ] and the homogenization of the leftover residual tumor [ 22 ], a point that may limit their widespread implementation. By contrast, MSTS saves time because it is an all-in-one procedure, enabling at the same time the histological analysis with a genomic correlation to take place in the same paraffin block and preserving the formalin-fixed paraffin-embedded material for the future.…”

“…Jie et al [19] have compared the performance of routine sampling and MSTS in 182 oral and oropharyngeal squamous-cell carcinomas. The authors included in the comparison histological, immunohistochemical, and molecular parameters, and concluded that MSTS was more informative than routine sampling in detecting Aside from CCRCC, the usefulness of MSTS in CCRCC has been confirmed by subsequent histological, immunohistochemical, and molecular studies in ovarian carcinoma, mesothelioma, and head and neck squamous cell carcinoma [17][18][19][20]. Lakis et al [17] have analyzed 294 tumor sections from 70 treatment naïve patients who had undergone cytoreductive surgery of ovarian cancer and have observed not only the high histological variability of tumors across different regions, but also the irregular qualitative and quantitative distribution of tumor-associated lymphocyte, information with obvious prognostic and therapeutic implications.…”