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Loss of histone H3 lysine 36 trimethylation is associated with an increased risk of renal cell carcinoma-specific death.

Ho TH, Kapur P, Joseph RW, Serie DJ, Eckel-Passow JE, Tong P, Wang J, Castle EP, Stanton ML, Cheville JC, Jonasch E, Brugarolas J, Parker AS - Mod. Pathol. (2015)

Bottom Line: However, few data exist regarding the impact of loss of H3K36me3 on outcomes.In The Cancer Genome Atlas cohort, SETD2 DNA alterations or mRNA expression was not associated with overall survival (P>0.05).This association remains significant after stratifying for the SSIGN score, particularly among those patients with low-risk tumors.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology and Medical Oncology, Mayo Clinic, Scottsdale, AZ, USA.

ABSTRACT
Sequencing of clear cell renal cell carcinomas identified loss-of-function mutations of SETD2, a gene that encodes a nonredundant methytransferase responsible for histone H3 lysine 36 trimethylation (H3K36me3), and H3K36me3 is progressively deregulated in metastases. However, few data exist regarding the impact of loss of H3K36me3 on outcomes. We assessed the association of SETD2 DNA alterations and mRNA expression with overall survival using The Cancer Genome Atlas clear cell renal carcinoma data (N=411). Additionally, we assessed the association of H3K36 loss of methylation with renal cell carcinoma-specific survival and progression-free survival using an independent cohort at Mayo Clinic (N=1454). Overall survival, renal cell carcinoma-specific survival and progression-free survival were estimated using Kaplan-Meier method, and differences in survival across groups was compared using Cox regression models, adjusted for age and the Mayo SSIGN (stage, size, grade, and necrosis) score. In The Cancer Genome Atlas cohort, SETD2 DNA alterations or mRNA expression was not associated with overall survival (P>0.05). In the Mayo cohort, patients with H3K36me3-negative tumors were two times more likely to experience renal cell carcinoma-specific death than patients with H3K36me3-positive tumors (hazard ratio, 2.23; 95% confidence interval, 1.77-2.81); P<0.0001. After stratifying for the SSIGN score, H3K36me3-negative tumors in the low-risk SSIGN group had a worse renal cell carcinoma-specific survival (hazard ratio, 2.18; 95% confidence interval, 1.09-4.36); P=0.03. Although SETD2 DNA and mRNA alterations are not associated with overall survival, we provide evidence that deregulation of the H3K36me3 axis is associated with a higher risk of renal cell carcinoma-specific death. This association remains significant after stratifying for the SSIGN score, particularly among those patients with low-risk tumors.

No MeSH data available.


Related in: MedlinePlus

Analysis of H3K36me3 in Isogenic SETD2 Renal Cell Carcinoma CellLines and The Mayo Clinic Nephrectomy Registry. The SETD2wild-type 786-O cell line was transfected with zinc finger pairs that generate aSETD2 deletion. Single cell–derived clones wereanalyzed by fragment length analysis to identify clone SETD2zinc finger nuclease. SETD2 was sequenced and confirmed to havea 4 base deletion. A, Western blot confirming depletion of H3K36me3 with histoneH3 and actin as controls. B, Immunohistochemical staining of H3K36me3 comparingisogenic SETD2 cell lines. Representative images for H3K36me3immunohistochemical staining classifications: C, positive D, negative E, weakpositive, and F, focal negative in nephrectomy samples. Scale bar 50 μM.(Original magnification 400×; inset 1000×.) Kaplan-Meierestimate of G, renal cell carcinoma-specific death and H, progression-freesurvival in patients with H3K36me3 negative and positive tumors. H3K36me3indicates histone 3 lysine 36 trimethylation.
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Figure 2: Analysis of H3K36me3 in Isogenic SETD2 Renal Cell Carcinoma CellLines and The Mayo Clinic Nephrectomy Registry. The SETD2wild-type 786-O cell line was transfected with zinc finger pairs that generate aSETD2 deletion. Single cell–derived clones wereanalyzed by fragment length analysis to identify clone SETD2zinc finger nuclease. SETD2 was sequenced and confirmed to havea 4 base deletion. A, Western blot confirming depletion of H3K36me3 with histoneH3 and actin as controls. B, Immunohistochemical staining of H3K36me3 comparingisogenic SETD2 cell lines. Representative images for H3K36me3immunohistochemical staining classifications: C, positive D, negative E, weakpositive, and F, focal negative in nephrectomy samples. Scale bar 50 μM.(Original magnification 400×; inset 1000×.) Kaplan-Meierestimate of G, renal cell carcinoma-specific death and H, progression-freesurvival in patients with H3K36me3 negative and positive tumors. H3K36me3indicates histone 3 lysine 36 trimethylation.

Mentions: Setd2-knockout mice lack global H3K36me3(3), and in humans, loss-of-functionSETD2 mutations in various tumors are associated with lossof H3K36me3.(3, 5, 6) Toevaluate H3K36me3 expression as a dichotomized variable using animmunohistochemistry assay in which loss of H3K36me3 correlates withSETD2 mutations, we generated targetedSETD2 deletions in the 786-O renal cell carcinoma cell lineusing zinc-finger nucleases. In a clone, 786-O SETD2 zincfinger nuclease, a 4 base deletion in SETD2 was confirmed bySanger sequencing (Supplementary Figure S2). Consistent with our prior study in anindependent SETD2 zinc-finger nuclease clone,SETD2 deletion disrupts expression of H3K36me3 by Westernblot and immunohistochemistry assays (Figure 2A,B).(7) To validate H3K36me3expression as a dichotomized variable, H3K36me3 was scored by two pathologists(P.K. and M.L.S.), blinded to the SETD2 genotype. Of the 26SETD2-genotyped tumors, 21 (81%) were classified asH3K36me3 positive or negative, 4 as focal negative, and 1 as weak positive(Supplementary TableS1, Figure 2C–F). Of the15 tumors with a SETD2 wild-type genotype, 12 were classifiedas positive, 2 as focal negative, and 1 as weak positive. Of the 11 tumors witha SETD2 mutant genotype, 9 were classified as negative and 2 asfocal negative. Overall, 81% of the tumors using immunohistochemistryclassifications of negative or positive correlated with the knownSETD2 genotype; the SETD2genotype-H3K36me3 phenotype concordance improves to 100% after exclusionof the 5 heterogeneous staining tumors classified as weak positive or focalnegative.


Loss of histone H3 lysine 36 trimethylation is associated with an increased risk of renal cell carcinoma-specific death.

Ho TH, Kapur P, Joseph RW, Serie DJ, Eckel-Passow JE, Tong P, Wang J, Castle EP, Stanton ML, Cheville JC, Jonasch E, Brugarolas J, Parker AS - Mod. Pathol. (2015)

Analysis of H3K36me3 in Isogenic SETD2 Renal Cell Carcinoma CellLines and The Mayo Clinic Nephrectomy Registry. The SETD2wild-type 786-O cell line was transfected with zinc finger pairs that generate aSETD2 deletion. Single cell–derived clones wereanalyzed by fragment length analysis to identify clone SETD2zinc finger nuclease. SETD2 was sequenced and confirmed to havea 4 base deletion. A, Western blot confirming depletion of H3K36me3 with histoneH3 and actin as controls. B, Immunohistochemical staining of H3K36me3 comparingisogenic SETD2 cell lines. Representative images for H3K36me3immunohistochemical staining classifications: C, positive D, negative E, weakpositive, and F, focal negative in nephrectomy samples. Scale bar 50 μM.(Original magnification 400×; inset 1000×.) Kaplan-Meierestimate of G, renal cell carcinoma-specific death and H, progression-freesurvival in patients with H3K36me3 negative and positive tumors. H3K36me3indicates histone 3 lysine 36 trimethylation.
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Related In: Results  -  Collection

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Figure 2: Analysis of H3K36me3 in Isogenic SETD2 Renal Cell Carcinoma CellLines and The Mayo Clinic Nephrectomy Registry. The SETD2wild-type 786-O cell line was transfected with zinc finger pairs that generate aSETD2 deletion. Single cell–derived clones wereanalyzed by fragment length analysis to identify clone SETD2zinc finger nuclease. SETD2 was sequenced and confirmed to havea 4 base deletion. A, Western blot confirming depletion of H3K36me3 with histoneH3 and actin as controls. B, Immunohistochemical staining of H3K36me3 comparingisogenic SETD2 cell lines. Representative images for H3K36me3immunohistochemical staining classifications: C, positive D, negative E, weakpositive, and F, focal negative in nephrectomy samples. Scale bar 50 μM.(Original magnification 400×; inset 1000×.) Kaplan-Meierestimate of G, renal cell carcinoma-specific death and H, progression-freesurvival in patients with H3K36me3 negative and positive tumors. H3K36me3indicates histone 3 lysine 36 trimethylation.
Mentions: Setd2-knockout mice lack global H3K36me3(3), and in humans, loss-of-functionSETD2 mutations in various tumors are associated with lossof H3K36me3.(3, 5, 6) Toevaluate H3K36me3 expression as a dichotomized variable using animmunohistochemistry assay in which loss of H3K36me3 correlates withSETD2 mutations, we generated targetedSETD2 deletions in the 786-O renal cell carcinoma cell lineusing zinc-finger nucleases. In a clone, 786-O SETD2 zincfinger nuclease, a 4 base deletion in SETD2 was confirmed bySanger sequencing (Supplementary Figure S2). Consistent with our prior study in anindependent SETD2 zinc-finger nuclease clone,SETD2 deletion disrupts expression of H3K36me3 by Westernblot and immunohistochemistry assays (Figure 2A,B).(7) To validate H3K36me3expression as a dichotomized variable, H3K36me3 was scored by two pathologists(P.K. and M.L.S.), blinded to the SETD2 genotype. Of the 26SETD2-genotyped tumors, 21 (81%) were classified asH3K36me3 positive or negative, 4 as focal negative, and 1 as weak positive(Supplementary TableS1, Figure 2C–F). Of the15 tumors with a SETD2 wild-type genotype, 12 were classifiedas positive, 2 as focal negative, and 1 as weak positive. Of the 11 tumors witha SETD2 mutant genotype, 9 were classified as negative and 2 asfocal negative. Overall, 81% of the tumors using immunohistochemistryclassifications of negative or positive correlated with the knownSETD2 genotype; the SETD2genotype-H3K36me3 phenotype concordance improves to 100% after exclusionof the 5 heterogeneous staining tumors classified as weak positive or focalnegative.

Bottom Line: However, few data exist regarding the impact of loss of H3K36me3 on outcomes.In The Cancer Genome Atlas cohort, SETD2 DNA alterations or mRNA expression was not associated with overall survival (P>0.05).This association remains significant after stratifying for the SSIGN score, particularly among those patients with low-risk tumors.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology and Medical Oncology, Mayo Clinic, Scottsdale, AZ, USA.

ABSTRACT
Sequencing of clear cell renal cell carcinomas identified loss-of-function mutations of SETD2, a gene that encodes a nonredundant methytransferase responsible for histone H3 lysine 36 trimethylation (H3K36me3), and H3K36me3 is progressively deregulated in metastases. However, few data exist regarding the impact of loss of H3K36me3 on outcomes. We assessed the association of SETD2 DNA alterations and mRNA expression with overall survival using The Cancer Genome Atlas clear cell renal carcinoma data (N=411). Additionally, we assessed the association of H3K36 loss of methylation with renal cell carcinoma-specific survival and progression-free survival using an independent cohort at Mayo Clinic (N=1454). Overall survival, renal cell carcinoma-specific survival and progression-free survival were estimated using Kaplan-Meier method, and differences in survival across groups was compared using Cox regression models, adjusted for age and the Mayo SSIGN (stage, size, grade, and necrosis) score. In The Cancer Genome Atlas cohort, SETD2 DNA alterations or mRNA expression was not associated with overall survival (P>0.05). In the Mayo cohort, patients with H3K36me3-negative tumors were two times more likely to experience renal cell carcinoma-specific death than patients with H3K36me3-positive tumors (hazard ratio, 2.23; 95% confidence interval, 1.77-2.81); P<0.0001. After stratifying for the SSIGN score, H3K36me3-negative tumors in the low-risk SSIGN group had a worse renal cell carcinoma-specific survival (hazard ratio, 2.18; 95% confidence interval, 1.09-4.36); P=0.03. Although SETD2 DNA and mRNA alterations are not associated with overall survival, we provide evidence that deregulation of the H3K36me3 axis is associated with a higher risk of renal cell carcinoma-specific death. This association remains significant after stratifying for the SSIGN score, particularly among those patients with low-risk tumors.

No MeSH data available.


Related in: MedlinePlus