The upper part of the resected right kidney contained a tumor with a maximum diameter of 10.5 cm. The cut surface of the specimen was tan to whitish or yellowish in color, solid, and showed hemorrhage and necrosis. The tumor invaded the capsule of the kidney (Figure 1C). The left retroperitoneal tumor had a maximum diameter of 21.5 cm. The cut section appeared dark red, and cystic changes, hemorrhage, and necrosis were observed. Distinct, confluent whitish to yellowish nodules were observed within the tumor mass (Figure 1D).
Histopathological examination of the right kidney tumor showed a typical World Health Organization (WHO) grade 3 CCRCC morphology, characterized by clear tumor cells with distinct cell membranes and focal necrosis (Figures 2A and B). Immunohistochemistry (IHC) showed characteristic positivity for PAX8 (Figure 2C), CAIX (Figure 2D), and EMA, as well as intact expression of fumarate hydratase and succinate dehydrogenase (SDHB). The results were negative for CK7 and TFE3 expression. The final diagnosis was CCRCC with WHO/ISUP grade 3.
Figure 2 Histopathological and immunohistochemiscal figures of the right kidney.
A: Typical clear cell renal cell carcinoma morphology of the right kidney tumor; B: Necrosis in the right kidney tumor; C: The tumor showed positive for PAX8; D: The tumor showed positive for CAIX.
Two components were observed in the left retroperitoneal tumor mass. The main tumor component (grossly dark-red areas) showed typical pheochromocytoma morphology with amphophilic tumor cells arranged in organoid nests, and cords and rich stromal capillary vasculature (Figures 3A and D). IHC showed positive staining for CgA (Figure 3G), Syn (Figure 3H), and intact expression of SDHB, but was negative for PAX8 (Figure 3E), CAIX (Figure 3F), CR, inhibin-α, MART(A103), TFE3, and EMA, with a Ki67 index of approximately 2%. The other tumor component from the whitish to yellowish areas showed CCRCC morphology similar to that of the right kidney tumor (Figure 3A and D), and was immunohistochemically positive for PAX8 (Figure 3E), CAIX (Figure 3F), EMA, and SDHB, but negative for CgA (Figure 3G), Syn (Figure 3H), S-100, CR, inhibin-α, MART-190 (A103), and TFE3, with a Ki67 index of approximately 5%, consistent with the diagnosis of metastatic CCRCC.
Figure 3 Histopathological and immunohistochemiscal figures showed the left retroperitoneal tumor mass contained two components.
A: One component was typical pheochromocytoma (PCC) with tumor cells arranged in organoid nests and cords; B: The other component exhibited clear cell renal cell carcinoma (CCRCC) morphology; C: The PCC nests at high magnification; D: The PCC nests mixed with CCRCC at their intersection; E: Immunohistochemistry (IHC) showed PAX8 was positive in the CCRCC tumor cells; F: IHC showed CAIX was positive in the CCRCC tumor cells; G: PCC tumor cells showed positive for CgA; H: PCC tumor cells showed positive for Syn.
WES was first performed on the CCRCC component tissue within the left retroperitoneal PCC, together with normal right kidney tissue, using a HiSeq next-generation sequencer (Illumina, San Diego, CA, United States) at GloriousMed Technology (Beijing, China). DNA extraction and quantification, library preparation, and sequencing were performed in accordance with the manufacturer’s protocol. Next generation sequencing results identified a c.529A>T (p.Arg177Ter) somatic mutation in the VHL gene.
Sanger sequencing was performed using the following primers: 5'- TCT GAA AGA GCG ATG CCT CC-3' (Forward primer) and 5'- TCT CCC ATC CGT TGA TGT GC-3' (Reverse primer). The 169bp PCR products were sequenced in both sense and antisense directions using an automated sequencer (ABI PRISM 3100 Genetic Analyzer; Applied Biosystems, United States). Sanger sequencing was performed on the right kidney CCRCC (Figure 4A), CCRCC tumor component within the left PCC tumor mass (Figure 4B), PCC (Figure 4C), and right normal kidney tissue (Figure 4D). These results confirmed that the right kidney CCRCC (Figure 4A) and left metastatic CCRCC tumor (Figure 4B) components had an identical VHL c.529A>T mutation. This mutation was not detected in the left retroperitoneal PCC (Figure 4C) or in normal tissue samples obtained from the right kidney resection specimen (Figure 4D).
Figure 4 Results of Sanger sequencing and fluorescence in situ hybridization.
A: Sanger sequencing showed identical VHL c.529A>T mutation and fluorescence in situ hybridization analysis showed the loss of 3p in the right kidney clear cell renal cell carcinoma (CCRCC); B: The VHL c.529A>T mutation and loss of 3p in metastatic CCRCC component within the left pheochromocytoma (PCC); C: The left retroperitoneal PCC did not carry VHL c.529A>T mutation and loss of 3p; D: The right normal kidney tissue did not carry VHL c.529A>T mutation and loss of 3p. CCRCC: Clear cell renal cell carcinoma; PCC: Pheochromocytoma.
We also analyzed the tissue samples using FISH, which showed a loss of chromosome 3p in the right kidney CCRCC (Figure 4A), as well as in the metastatic CCRCC component within the left PCC (Figure 4B). Loss of 3p was not observed in the left retroperitoneal PCC (Figure 4C) or in normal tissue samples obtained from the right kidney (Figure 4D).
Sequencing and FISH analyses demonstrated that identical molecular features were shared by the primary right kidney CCRCC and the CCRCC component within the left retroperitoneal PCC, further supporting the final diagnosis of the right kidney CCRCC metastasizing to PCC.