Ho et al -11
Supplementary Information
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Supplementary Methods
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Mayo Clinic Nephrectomy Registry
Vital status for patients in the Nephrectomy Registry is updated annually; a visit
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to our institution within six months of the date of death for metastatic renal cell
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carcinoma is considered valid documentation that renal cell carcinoma was the cause of
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death. If a patient has not been seen at our institution in the previous year, the patient is
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sent a questionnaire. If there is evidence of disease progression in this questionnaire,
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the date, location, and treatment are verified with the patient's local physician. If a
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patient has died in the previous year, a death certificate is ordered to determine the
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cause of death. If the death certificate does not support this, the medical history is
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reviewed by a urologist to determine the cause of death. If a death certificate cannot be
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obtained, the cause of death must be verified with the patient’s family or local physician.
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Loss to follow-up is <5%.
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Analysis of SETD2 DNA Alterations and mRNA Expression in The Cancer
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Genome Atlas Dataset
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RNA-Sequencing by Expectation Maximization, a software package for
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estimating gene and isoform expression levels from RNA-Sequencing data, was used to
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estimate transcripts abundance (log2(RNA-Sequencing by Expectation Maximization
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+1)). The Genomic Identification of Significant Targets in Cancer output was used for
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copy number analyses: -2 denote homozygous deletion, -1 heterozygous deletion, 0
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copy number neutral, 1 low-level gain, and 2 high-level amplification.
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Generation of SETD2-Null Cells Using Zinc Finger Nucleases
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The 786-O renal cell carcinoma cell lines were purchased in 2014 from American
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Type Culture Collection and cultured in Roswell Park Memorial Institute
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1640 (Mediatech, Manassas, USA), supplemented with fetal bovine serum and
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penicillin/streptomycin. Zinc finger nuclease pairs (Primer F 5′-3′, ACT TTC CAA AAC
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AGG CCA GA; Primer R 5′-3′, TCC ATC CTG TTG ATC CCA AT) specific for the
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SETD2 gene were designed, assembled, and validated in a construct (Sigma-Aldrich,
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St. Louis, USA) designed to co-express green fluorescent protein with one half of the
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pair and red fluorescent protein with the other pair. Cells were nucleofected with 2 µg
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green/red fluorescent protein-zinc finger nuclease mRNA; green fluorescent protein and
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red fluorescent protein co-expressing cells were enriched by fluorescent activated cell
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sorting. Single cell–derived clones were analyzed by fragment length analysis to identify
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those with frameshifts due to deletions or insertions at the zinc finger nuclease cleavage
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site. Frameshifts in alleles were confirmed by sequencing and by immunohistochemistry
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staining using an H3K36me3 antibody. Cell line identity was reauthenticated using short
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tandem repeats at 17 loci by Promega’s (Madison, USA) Cell Line Authentication
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Service in May 2015.
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Data Availability
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The Cancer Genome Atlas kidney renal clear cell carcinoma datasets were
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downloaded from Broad GDAC Firehose
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(https://confluence.broadinstitute.org/display/GDAC/Home), including RNA sequencing
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for mRNA (Level 3), Genomic Identification of Significant Targets in Cancer
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(https://www.broadinstitute.org/cancer/cga/gistic) output for copy number (Level 3), and
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mutation data (Level 3). The clinical information was downloaded from The Cancer
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Genome Atlas data portal on August 29, 2014 (https://tcga-data.nci.nih.gov/tcga/).
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Supplementary Figure S1. Kaplan-Meier Survival Plots for SETD2 DNA alterations in
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The Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma Dataset. ‘SETD2 DNA
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altered’ includes both SETD2 copy number loss and mutations. No association was
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observed between any SETD2 alterations and overall survival (odds ratio [95%
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confidence interval], 1.45 [0.82-2.57]; (P=.23, log-rank test).
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Supplementary Figure S2. Sequencing confirmation of 4 base SETD2 deletion in 786-
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O cell line nucleofected with zinc finger nucleases. The bottom sequence highlighted in
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blue is the reference sequence.
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Supplementary Figure S3. Flow Diagram of Histone 3 Lysine 36 Trimethylation
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(H3K36me3) Analysis (Mayo Clinic Rochester Nephrectomy Registry). H3K36me3 was
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successfully stained in 1,454 of 1,465 slides (99.2%). For the purposes of dichotomizing
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the H3K36me3 classifications (positive, negative, weak positive, focal negative), we
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classified weak positive as positive and focal negative as negative. We previously
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assessed BAP1 and PBRM1 expression on this cohort. Our mean duration of follow-up
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is 8.1 years (median, 8.3 years; range, 0-23.5 years; 6 patients had no follow-up).
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Supplementary Figure S4. Kaplan-Meier Estimate of Progression-Free Survival in
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Patients With PBRM1 Positive and Negative Tumors by H3K36me3
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Immunohistochemistry Classification. A, PBRM1 positive. B, PBRM1 negative.
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H3K36me3 indicates histone 3 lysine 36 trimethylation.
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Supplementary Table S1. Correlation of Mutant SETD2 Genotype with Loss of Histone
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3 Lysine 36 Trimethylation Immunohistochemistry Classification
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Sample ID
SETD2 Genotype
Immunohistochemistry
Classification
MCA9
MCA10
MCA1
MCA2
MCA3
MCA8
MCA13
MCA14
MCA15
MCA16
UTSW1
UTSW2
UTSW3
UTSW4
MCA7
UTSW7
UTSW8
MCA4
MCA5
MCA6
MCA12
MCA17
MCA18
MCA11
UTSW5
UTSW6
wild-type
wild-type
wild-type
wild-type
wild-type
wild-type
wild-type
wild-type
wild-type
wild-type
wild-type
wild-type
wild-type
wild-type
wild-type
mutant
mutant
mutant
mutant
mutant
mutant
mutant
mutant
mutant
mutant
mutant
focal negative
focal negative
positive
positive
positive
positive
positive
positive
positive
positive
positive
positive
positive
positive
weak positive
focal negative
focal negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
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Supplementary Table S2. Association of H3K36me3 (Samples with Weak Positive and
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Focal Negative Staining Excluded) with Renal Cell Carcinoma-Specific Survival or
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Progression-Free Survival, Adjusted for Age
H3K36me3 (Positive vs
Negative, Excluding Weak
Positive and Focal Negative)
(N=1,205)
Adjusted for
age
H3K36me3
positive
H3K36me3
negative
Renal Cell Carcinoma-Specific
Survival (225 Events)
Progression-Free Survival
(303 Events)
Hazard ratio (95%
Confidence
interval)
P Value
Hazard ratio (95%
Confidence
interval)
P Value
1.0 (reference)
NA
1.0 (reference)
NA
3.10 (2.36-4.07)
<0.0001
2.87 (2.26-3.65)
<0.0001
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Abbreviations: H3K36me3, histone 3 lysine 36 trimethylation; SSIGN, stage, size,
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grade, and necrosis.
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90
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Supplementary Table S3. Association of H3K36me3 with Renal Cell Carcinoma-
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Specific Survival or Progression-Free Survival
Renal Cell Carcinoma-Specific Survival
(295 events)
Cohort
P-value
Conc
ordan
ceindex
Hazard ratio (95%
Confidence interval)
P-value
Conc
ordan
ceindex
1.024 (1.014 - 1.035)
<0.0001
0.58
1.014 (1.005 - 1.023)
0.002
0.54
2.23 (1.77 - 2.81)
<0.0001
0.849
1.51 (1.46 - 1.57)
<0.0001
0.838
H3K36me3
Positive
1.0 (reference)
NA
1.0 (reference)
NA
H3K36me3
Negative
2.23 (1.77 - 2.81)
<0.0001
2.12 (1.74 - 2.60)
<0.0001
H3K36me3
Positive
1.0 (reference)
NA
1.0 (reference)
NA
H3K36me3
Negative
1.26 (0.99 - 1.60)
0.06
1.30 (1.06 - 1.59)
0.01
H3K36me3
Positive
1.0 (reference)
NA
1.0 (reference)
NA
H3K36me3
Negative
1.22 (0.96 - 1.55)
0.10
1.29 (1.05 - 1.58)
0.02
(N=14541)
Age2
SSIGN2
Adjusted
for Age3
Adjusted
for
SSIGN3
Adjusted
for Age
and
SSIGN3
Progression-Free Survival (398 events)4
Hazard ratio (95%
Confidence interval)
0.632
0.852
0.856
0.611
0.840
0.842
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Abbreviations: H3K36me3, histone 3 lysine 36 trimethylation; NA, not applicable;
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SSIGN, stage, size, grade, and necrosis.
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1
Weak positive was classified as positive and focal negative was classified as negative
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2
Hazard ratio for continuous variables (age, SSIGN)
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3 Hazard
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4
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years) and non-progressors (N=1056) is 8.9 years (range, 0-23.5 years).
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ratio for dichotomous variables (positive, negative)
Our median duration of follow-up in progressors (N=398) is 4.7 years (range, 0-19.6
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Supplementary Table S4. Association of PBRM1, BAP1 and H3K36me3 Expression
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by Immunohistochemistry Staining Assay1
H3K36me3
Negative
PBRM1 expression
negative
positive
missing/weak positive/
focal negative
BAP1 expression
negative
positive
missing/weak positive/
focal negative
H3K36me3
Positive
<0.0001
150 (69.8%)
63 (30.2%)
387 (43.6%)
500 (56.4%)
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91
0.31
25 (11.6%)
190 (88.4%)
86 (9.2%)
845 (90.8%)
12
47
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Abbreviations: H3K36me3, histone 3 lysine 36 trimethylation.
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1 Weak
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2
positive and focal negative staining were excluded
Fisher test
P-value2