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.

Keywords: angiogenesis; clear cell renal cell carcinoma; immunity; immunohistochemistry; intratumoral heterogeneity; tumor sampling.

Figure 1

Intratumoral heterogeneity in clear cell RCCs. Gross images of different slices of clear cell RCCs with heterogenous macroscopic appearance (A); usual block (white cassette) and multisite sampling with 6 pieces of tumor tissue per single block (green cassette) and 3D fusion multisite reconstruction (B,C); H&E staining (C).

Figure 2

Schematic representation of multisite 3D fusion sampling procedure. Fusion sampling was based on the size of the tumor. For each cm diameter of tumor, six blocks of tissue, each measuring 0.5 × 0.5 cm, were randomly selected and placed in an individual cassette.

Figure 3

Transcriptome investigation in 512 clear cell RCC cases from TCGA (PanCancer Atlas study). The mRNA levels are z-scores relative to diploid samples (RNA Seq V2 RSEM). Clusterization procedure was applied to the first 18 genes, then the PECAM1 (CD31), CD274 (PD-L1) and CD8A mRNA levels were added to the established case list order. A dashed line was added to separate mesenchymal and epithelial clearRCC cases. The heat map shows angiogenesis and immune signatures in the clearRCC epithelial and mesenchymal subtypes. Higher levels of angiogenic markers CD31 and CD34 and of the immune markers PD-L1 and CD8A come up predominantly in the mesenchymal subtype.

Figure 4

Classification of angiogenesis in clear cell RCCs after 3D fusion multisite tumor sampling. Diagram showing categorization of pattern of angiogenesis: pattern A, characterized by homogeneous high level of angiogenesis; pattern B, characterized by homogeneous low level of angiogenesis; and pattern C, characterized by a mixture of patterns.

Figure 5

Classification of PD-L1 immune activation in clear cell RCCs after 3D fusion multisite tumor sampling. Diagram showing categorization of immune patterns: score 2 strong expression; score 1, low/weak expression; score 0 with absence of any expression.

Figure 6

Distribution of PD-L1 immunohistochemical expression in clear cell RCCs after 3D fusion multisite tumor sampling.