Somatic Mutations of TSC2 or MTOR Characterize a Morphologically Distinct Subset of Sporadic Renal Cell Carcinoma With Eosinophilic and Vacuolated Cytoplasm

Abstract

The differential diagnosis of renal cell neoplasms with solid or nested architecture and eosinophilic cytoplasm has become increasingly complex. Despite recent advances in classifying a number of entities exhibiting this morphology, some tumors remain in the unclassified category. Here we describe a morphologically distinct group of sporadic renal cell carcinoma (RCC) with predominantly nested architecture, eosinophilic, and remarkably vacuolated cytoplasm retrospectively identified from a cohort of previously unclassified tumors. We examined the clinicopathologic and immunohistochemical features of these tumors and investigated their mutational and copy number alterations using a targeted next-generation sequencing platform. The study included 7 patients with a mean age of 54 years (range: 40 to 68 y) and a male to female ratio of 3:4. All patients presented with a solitary renal mass and had no prior medical or family history raising concern for syndromic conditions. Tumors were well-circumscribed, unencapsulated, and comprised of nests of eosinophilic cells in a hypocellular and often edematous stroma. Tumor cells had round nuclei with prominent nucleoli and granular cytoplasm with striking vacuolization. Thick-walled vessels and calcifications were also frequently present, whereas increased mitotic activity, necrosis, foamy histiocytes or lymphocytic infiltrates were not identified. All cases were positive for PAX8, had retained expression of SDHB and FH, and exhibited a CK7-/CK20- phenotype. While cathepsin-K was positive in 5 cases, none exhibited immunoreactivity to HMB45 or Melan A, or TFE3 immunostaining. Next-generation sequencing identified somatic inactivating mutations of TSC2 (3/5 tumors tested) or activating mutations of MTOR (2/5) as the primary molecular alterations, consistent with hyperactive mTOR complex 1 signaling which was further demonstrated by phospho-S6 and phospho-4E-BP1 immunostaining. Copy number analysis revealed a loss of chromosome 1 in both cases with MTOR mutation. These tumors represent a novel subset of sporadic RCC characterized by alterations in TSC1-TSC2 complex or the mTOR complex 1 pathway. Recognition of their characteristic morphologic and immunophenotypic features will allow them to be readily identified and separated from the unclassified RCC category.

Figure 1.

Macroscopic and microscopic features of a distinct group of RCC with eosinophilic and vacuolated cytoplasm. (A) Tumors are well-circumscribed with tan-yellow to tan-brown, mostly solid cut surfaces. Arrow marks intratumoral vessels with medium to large caliber; arrowheads mark the boundary between tumor and adjacent renal parenchyma. (B-C) Tumors consist of nests of eosinophilic cells in a hypocellular and often edematous stroma. Dispersed single or minute clusters of cells are also present. Note an absence of foamy histiocytes or lymphocytic infiltrates in the stroma. (D-F) Tumor cells show round nuclei with conspicuous to prominent nucleoli and eosinophilic, granular and vacuolated cytoplasm. Vacuolization varies from numerous small intracytoplasmic vesicles to large spaces almost occupying the entire cytoplasm. (D) Case 1, (E) Case 4, (F) Case 6.

Figure 2.

Additional histologic features include: (A) lack of tumor capsule and entrapped renal tubules (arrow); (B) extensive calcifications; (C) microscopic calcifications and perinuclear cytoplasmic clearing, mimicking chromophobe RCC; (D) marked cytoplasmic eosinophilia with filamentous material and focal cytoplasmic stippling; (E) cribriform or sieve-like appearance in areas with tightly packed nests and extensive vacuolization; (F) abrupt transition between tumor cells with prominent vacuolization and those with dense, granular cytoplasm.

Figure 3.

Immunohistochemical features. (A) Positive nuclear staining for PAX8. (B-C) Tumor cells negative for CK7 (B) and CK20 (C) except for scattered rare cells/vacuoles; arrow in (B) marks an entrapped renal tubule. (D) Weak membranous staining for CD117; arrow marks a mast cell. (E) Diffuse cathepsin-K immunoreactivity. (F) Retained SDHB expression.

Figure 4.

Representative images of p-S6 (A) and p-4EBP1 (B) immunostains.

Figure 5.

FACETS analysis in 4 cases. The integer copy number (copy number call corrected for tumor purity and ploidy) is plotted on the y-axis. Diploid corresponds to n = 2. Chromosomes 1–22 are plotted on the x-axis. Black line - total copy number, red line - minor allele. (A-B) Cases 4 (A) and 5 (B) (MTOR p.L2427R) show recurrent chromosome 1 loss (solid box). (C-D) Cases 2 (C) and 6 (D) (biallelic TSC2 mutations) have no shared gains/losses. Dashed boxes in (C) mark copy number changes detected in minor clone(s) with an estimated clone fraction ≤ 9%.