Loss of Histone H3 Lysine 36 Trimethylation Is Associated With an Increased Risk of Renal Cell Carcinoma-specific Death

Thai H Ho; Payal Kapur; Richard W Joseph; Daniel J Serie; Jeanette E Eckel-Passow; Pan Tong; Jing Wang; Erik P Castle; Melissa L Stanton; John C Cheville; Eric Jonasch; James Brugarolas; Alexander S Parker


Mod Pathol. 2016;29(1):34-42. 

In This Article

Materials and Methods

Patient Selection

After Mayo Clinic Institutional Review Board approval, we identified 1465 patients treated with radical nephrectomy or nephron-sparing surgery for clear cell renal cell carcinoma between 1990 and 2009 from the Mayo Clinic Nephrectomy Registry with representative paraffin-embedded tissue blocks available for immunohistochemistry staining and data on renal cell carcinoma-specific death. After review of the entire case, one representative slide was selected with the highest Fuhrman grade and tumor content for immunohistochemistry staining. A genitourinary pathologist (JC) reviewed all the tumors, which allowed for standardized clinicopathological variables.

Assessment of H3K36me3, PBRM1, and BAP1 by Immunohistochemistry Staining

Standard immunohistochemistry staining procedures for H3K36me3, PBRM1, and BAP1 were performed using the Dako (Carpinteria, USA) autostainer and Ventana (Tucson, USA) Benchmark XT automated stainer. After heat-induced epitope retrieval with Cell Conditioning Solution 1 (Ventana), sections were incubated with the appropriate primary antibody: H3K36me3 (Abcam 9050, Cambridge, USA) at 1:9000 (15 min); PBRM1 (Bethyl Laboratories A301–591 A, Montgomery, USA) at 1:250 (32 min), BAP1 (Santa Cruz Biotechnology sc-28383, Dallas, USA) at 1:50 (60 min). We previously validated immunohistochemistry assays to evaluate H3K36me3, PBRM1, and BAP1 protein expression in which negative staining correlated with loss-of-function mutations in SETD2, PBRM1, and BAP1 genes, respectively.[6,10] Samples were excluded from analysis if positive nuclear staining was not observed in background stromal cells or lymphocytes (internal control). Positivity (2+ staining intensity) was indicated by diffuse nuclear staining in tumor cells (≥10%); cytoplasmic staining was not analyzed. Samples with little-to-no tumor nuclei staining were classified as negative. Samples with positive nuclei in the internal control tissue (stroma and/or lymphocytes) and faint tumor nuclei staining were classified as weak positive (1+ staining intensity). Focal negatives had positive nuclei in the internal control tissue and had loss of tumor nuclear staining only in subclonal populations (<10% of total tumor nuclei). For the purposes of dichotomizing the H3K36me3 classifications (positive, negative, weak positive, focal negative), we classified weak positive as positive and focal negative as negative based on our immunohistochemistry results in the tumors with a defined SETD2 genotype. With respect to H3K36me3 classification, the genitourinary pathologists (PK and MLS) were blinded to all clinical outcomes and SETD2 genotypes.

Statistical Analyses

The Fisher exact or Chi-square tests, as appropriate, were used to compare categorical variables across molecular groups. Cox proportional hazards models and hazard ratio with 95% confidence interval were used to assess the association of H3K36me3, PBRM1, and BAP1 expression with outcome after adjusting for age and/or the Mayo Clinic SSIGN score. Overall survival was assessed in The Cancer Genome Atlas data, whereas renal cell carcinoma-specific survival and progression-free survival were assessed in the Mayo Clinic data. Renal cell carcinoma-specific survival analyses tracked time from nephrectomy to death owing to renal cell carcinoma, whereas progression-free survival considered either the first metastasis or renal cell carcinoma-death as an end point. In both analyses, patients who died from other causes or were lost to follow-up were censored at those respective times. We calculated concordance index values to assess predictive ability. All reported concordance indices were generated using the bootstrap methodology and represent optimism-corrected estimates of concordance.[11] The Kaplan–Meier method was used to estimate time to renal cell carcinoma-specific death and progression-free survival. Statistical analyses were performed using R, version 2.15. For all analyses, a P-value <0.05 was evidence of statistical significance.