Hereditary leiomyomatosis and renal cell carcinoma syndrome-associated renal cell carcinomas (RCC) are difficult to diagnose prospectively. We used immunohistochemistry (IHC) to identify fumarate hydratase (FH)-deficient tumors (defined as FH negative, 2-succinocysteine [2SC] positive) in cases diagnosed as "unclassified RCC, high grade or with papillary pattern," or "papillary RCC type 2," from multiple institutions. A total of 124 tumors (from 118 patients) were evaluated by IHC for FH and 2SC. An FH deficiency was found in 24/124 (19%) cases. An indeterminate result (only 1 marker abnormal) was found in 27/124 (22%) cases. In a tissue microarray of 776 RCCs of different types, only 2 (0.5%) tumors, initially considered papillary type 2, were FH deficient. FH mutations were found in 19/21 FH-deficient tumors (with confirmed germline mutations in 9 of 9 tumors in which germline status could be assessed) and in 1/26 FH-indeterminate tumors identified by IHC. No FH mutations were found in 2/21 FH-deficient RCCs, 25/26 FH-indeterminate RCCs, and 10/10 RCCs demonstrating FH expression by IHC. Patients with FH-deficient RCC had a median age of 44 years (range, 21 to 65 y). Average tumor size was 8.2 cm (range, 0.9 to 18 cm). FH-deficient RCCs were characterized by at least focal macronucleoli and demonstrated 2 or more growth patterns in 93% cases. Papillary was the most common (74%) and dominant (59%) pattern, whereas other common patterns included: solid (44%), tubulocystic (41%), cribriform (41%), and cystic (33%). At presentation, 57% were stage ≥pT3, 52% had positive nodes, and 19% had distant metastases. After a mean follow-up of 27 months (range, 1 to 114 mo), 39% of patients were dead of disease, and 26% had disease progression. We conclude that FH and 2SC are useful IHC ancillary tools, which allow recognition of FH-deficient RCC.

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BACKGROUND Novel targeted treatments and immunotherapies have substantially changed therapeutic options for advanced and metastatic renal cell carcinomas (RCCs). However, accurate diagnostic tests for the identification of high-risk patients are urgently needed. Here, we analyzed SHOX2 mRNA expression in RCC tissues and SHOX2 gene body methylation quantitatively in circulating cell-free DNA (ccfDNA) and RCC tissues with regard to risk stratification. METHODS The clinical performance of SHOX2 methylation was tested retrospectively and prospectively in a training and testing cohort of RCC tissue samples (n = 760 in total). SHOX2 mRNA expression analysis was included in the training cohort. In matched blood plasma samples from the testing cohort (n = 100), we prospectively examined the capability of pretherapeutic quantitative SHOX2 ccfDNA methylation to assess disease stage and identify patients at high risk of death. RESULTS SHOX2 gene body methylation was positively correlated with mRNA expression in RCC tissues (training cohort: Spearman ρ = 0.23, P < 0.001). SHOX2 methylation in tissue and plasma strongly correlated with an advanced disease stage (training cohort: ρ = 0.28, P < 0.001; testing cohort/tissue: ρ = 0.40, P < 0.001; testing cohort/plasma: ρ = 0.34, P = 0.001) and risk of death after initial partial or radical nephrectomy [training cohort: hazard ratio (HR) = 1.40 (95% CI, 1.24–1.57), P < 0.001; testing cohort/tissue: HR = 1.16 (95% CI, 1.07–1.27), P = 0.001; testing cohort/plasma: HR = 1.50 (95% CI, 1.29–1.74), P < 0.001]. CONCLUSIONS Pretherapeutic SHOX2 ccfDNA methylation testing allows for the identification of RCC patients at high risk of death after nephrectomy. These patients might benefit from an adjuvant treatment or early initiation of a palliative treatment.

ObjectiveTo study the expression of adipophilin (PLIN2), a lipid storage-associated cell protein, in different subtypes of renal cell cancer and to elucidate its prognostic value.Materials and MethodsTwo-hundred-seventy-five patients with renal cell carcinoma (RCC) were included in this study. Immunohistochemistry with a polyclonal antibody to adipophilin was used on the tissue microarray (formalin-fixed, paraffin-embedded tissue) for detection of adipophilin. Median follow-up time was 91 (range 1-159) months in the whole cohort and 100 (1-159) months for patients with clear-cell RCC. Additional validation for adipophilin was performed using publicly available gene expression data for clear cell RCC from The Cancer Genome Atlas (TCGA).ResultsAdipophilin expression was detected in 14.3% of papillary RCC, in 0% of chromophobe RCC and in 58.7% of clear-cell RCC in the cytoplasm or at the membrane. Only membrane expression was correlated with other clinical parameters (pT-stage, pN-stage, R-status, sex) and showed a prognostic significance in univariate analysis with regard to overall survival of patients with clear cell subtype (HR 2.90, 95% CI 1.55-5.42, p=0.001), which failed significance on multivariate analysis. mRNA expression of PLIN2 on TCGA data using best selected cut-off was prognostically significant in both univariate (HR 1.76, 95% CI 1.28-2.42, p = 0.0005) and multivariate analyses (HR 1.46, 95% CI 1.05-2.04, p = 0.0257).ConclusionsAdipophilin is a novel and still understudied prognostic biomarker in clear cell renal cell carcinoma which deserves further study.

Optimal in vitro conditions are necessary for the development of a strong, well structured, and functional tissue engineered cardiovascular structure eventually designed for implantation. To further optimize in vitro conditions for cell proliferation and extracellular matrix formation in tissue engineering of cardiovascular structures, in this study, ascorbic acid and growth factors as additives to standard cell culture medium were evaluated for their effect on tissue development in vitro. Biodegradable polymer patches [polyglycolic acid (PGA) coated with poly-4-hydroxybutyrate (P4HB)] were seeded with human pediatric aortic cells and cultured for 7 and 28 days. Group A was cultured with standard medium (DMEM with 10% fetal calf serum and 1% antibiotics) supplemented with ascorbic acid; group B was cultured with standard medium plus ascorbic acid and basic fibroblast growth factor (bFGF); group C was cultured with standard medium adding ascorbic acid and transforming growth factor (TGF). Analysis of the cell seeded polymer constructs included DNA assay, collagen assay, and histologic and immunohistochemical examination for cell proliferation and collagen formation. After 7 and 28 days of culture, group B and group C showed a significantly higher DNA content compared with group A. The addition of bFGF (group B) led to a markedly higher collagen synthesis after 28 days of culture compared with the additives in groups C and A. The histologic and immunohistochemical examination also revealed a more dense, organized tissue development with pronounced matrix protein formation in the tissue engineered structures in group B after 28 days of culture. When seeded on to the polymeric scaffold, human vascular cells proliferate and form organized cell tissue after 28 days of culture. The addition of bFGF and ascorbic acid to the standard medium enhances cell proliferation and collagen synthesis on the biodegradable polymer, which leads to the formation of more mature, well organized tissue engineered structures.