Comprehensive molecular characterization of clear cell renal cell carcinoma

Author

The Cancer Genome Atlas Research Network

Doi

10.1038/nature12222

PMID: 23792563 · DOI: 10.1038/nature12222 · Journal: Nature (2013)

TL;DR

The Cancer Genome Atlas Research Network performed multi-platform molecular characterization of clear cell renal cell carcinoma (CCRCC) across more than 400 primary nephrectomy tumors, integrating whole-exome sequencing, DNA copy number arrays, mRNA and miRNA sequencing, DNA methylation arrays, and reverse phase protein arrays. They identified 19 significantly mutated genes — dominated by VHL, PBRM1, SETD2, KDM5C, PTEN, BAP1, MTOR, and TP53 — and showed that mutations in the SWI/SNF chromatin remodeling complex (PBRM1, ARID1A, SMARCA4) and the H3K36 methyltransferase SETD2 drive widespread DNA hypomethylation and pleiotropic transcriptional changes. The PI3K/AKT/mTOR pathway was altered in ~28% of tumors. Aggressive cancers showed a metabolic shift toward the Warburg phenotype — decreased AMPK and Krebs cycle activity, increased pentose phosphate pathway and fatty acid synthesis — that correlated with worse survival.

Cohort & data

  • 446 primary nephrectomy specimens with histologically confirmed clear cell renal cell carcinoma (CCRCC), ≥60% tumor nuclei by pathology review (median 85%); 372 samples had data across all platforms (“Core” set), 446 across at least one (“Extended” set).
  • Dataset: kirc_tcga_pub — TCGA Kidney Renal Clear Cell Carcinoma publication cohort.
  • Platforms: whole-exome sequencing (n=417 tumors yielding 36,353 putative somatic mutations), Affymetrix SNP arrays for copy number, RNA sequencing, miRNA sequencing, DNA methylation arrays (450K Human Methylation, n=224 for SETD2 analysis), and reverse phase protein arrays. Whole-genome sequencing on 22 patients calibrated and confirmed 83% of WES mutation calls.

Key findings

  • 19 significantly mutated genes (q<0.1): top 8 (q<0.00001) are VHL, PBRM1, SETD2, KDM5C, PTEN, BAP1, MTOR, and TP53. Only BAP1 mutation correlated with worse survival (Figure S27). Approximately 20% of cases had none of the 19 SMGs.
  • Chromosome 3p loss in 91% of samples, encompassing the four most commonly mutated genes (VHL, PBRM1, BAP1, SETD2). Arm-level 14q loss (with HIF1A) occurred in 45% of samples; 5q gain in 67%.
  • Focal amplifications implicated PRKCI and MECOM (3q26), MDM4 (1q32), MYC (8q24), and JAK2 (9p24); focal deletions affected CDKN2A (9p21), PTEN (10q23), NEGR1, QKI, CADM2, PTPRD, and NRXN3.
  • Recurrent SFPQ–TFE3 fusion in 5 samples (84 putative RNA fusions total, 11/13 validated → 85% true positive rate); all five fusion-positive tumors were VHL wildtype with X(p11) rearrangements visible by FISH in 3/3 evaluable cases.
  • Epigenetic silencing of VHL in ~7% of tumors, mutually exclusive with VHL mutation. UQCRH hypermethylated in 36% of tumors. Promoter hypermethylation frequency increased with stage and grade.
  • SETD2 mutation associated with regional loss of DNA methylation at non-promoter regions (FDR=0.001, |Δβ|>0.1, n=2,557 CpG loci differentially methylated between mutant and wild-type), consistent with H3K36me3 guiding DNMT3A-mediated methylation.
  • Four mRNA subtypes (m1–m4) and four miRNA subtypes (mi1–mi4) by unsupervised clustering. m1 corresponds to prior ccA classification with survival advantage; m4 captures previously unclassified tumors. m1 was enriched for PBRM1 mutations (39% vs 27%, p=0.027); m3 enriched for CDKN2A deletion (53% vs 26%, p<0.0001) and PTEN mutation (11% vs 1%, p<0.0001); m4 enriched for BAP1 (17% vs 7%, p=0.002) and MTOR (12% vs 4%, p=0.01) mutations.
  • PI3K/AKT/mTOR pathway altered in ~28% of tumors by MEMo mutual-exclusivity analysis; the module includes RACK1 (GNB2L1) and SQSTM1/p62 from the 5q35.3 amplicon, and frequent over-expression of EGFR correlated with receptor phosphorylation.
  • Metabolic shift correlates with worse survival: down-regulation of AMPK complex and Krebs cycle genes; up-regulation of pentose phosphate pathway (G6PD, PGLS, TALDO, TKT) and fatty acid synthesis (FASN, ACC); increased glutamine transporter expression. miR-21 promoter hypomethylation (higher miR-21 expression) and GRB10 promoter methylation patterns were prognostic, with both regulating PI3K signaling.
  • HotNet identified 25 subnetworks; the largest centered on VHL and partners, the second on the PBAF SWI/SNF complex (PBRM1, ARID1A, SMARCA4). TieDIE linked chromatin-modifier mutations to transcriptional hubs including HIF/ARNT, MYC/Max, SP1, FOXM1, JUN, and FOS, and to downstream effectors including ESR1, NFκB1, IL6, HIF1A, BRCA1 (via BAP1), CTNNB1, and TGFBR2.
  • Prognostic signatures built on discovery (n=193) and validated on n=253: mRNA, miRNA, and protein signatures all added prognostic power beyond clinical variables in multivariate Cox analysis. Top protein correlates of worse survival were reduced AMPK and increased acetyl-CoA carboxylase (ACC).

Genes & alterations

  • VHL — top significantly mutated gene; mutually exclusive with promoter methylation (~7% of tumors silenced epigenetically); central to HIF stabilization in CCRCC.
  • PBRM1 — significantly mutated (q<0.00001); SWI/SNF (PBAF) subunit; enriched in m1 subtype (39% vs 27%).
  • SETD2 — H3K36 methyltransferase; mutation associated with regional DNA hypomethylation at non-promoter CpGs (n=2,557 differentially methylated loci).
  • BAP1 — only SMG correlated with worse survival; enriched in m4 mRNA subtype (17% vs 7%, p=0.002).
  • KDM5C, PTEN, MTOR, TP53 — among the top eight most significant SMGs (q<0.00001). PTEN mutation enriched in m3 (11% vs 1%, p<0.0001); MTOR mutation enriched in m4 (12% vs 4%, p=0.01).
  • ARID1A, SMARCA4 — SWI/SNF complex members co-occurring in the HotNet PBAF subnetwork with PBRM1.
  • HIF1A — frequently lost via 14q deletion (45% of samples), predicted to drive aggressive disease.
  • CDKN2A — focal 9p21 deletion; enriched in m3 (53% vs 26%, p<0.0001).
  • MYC, MDM4, JAK2, PRKCI, MECOM — focal amplifications.
  • SFPQ–TFE3 — recurrent in-frame fusion in 5 samples, all VHL wildtype, often with X(p11) translocation by FISH.
  • EGFR — over-expressed in the PI3K/AKT/mTOR alteration module with corresponding phospho-receptor increase; prior link to lapatinib response cited.
  • RACK1 (GNB2L1), SQSTM1 — both in the 5q35.3 amplicon, both linked to PI3K activation; copy number gain correlated with mRNA expression and pathway alteration status.
  • GRB10 — promoter methylation pattern prognostic; tumor suppressor that negatively regulates PI3K/insulin signaling.
  • TSC2 — PI3K pathway inhibitor whose higher expression correlated with better survival.
  • UQCRH — hypermethylated in 36% of tumors, top inverse expression-vs-methylation correlate; not previously linked to CCRCC.
  • CTNNB1, TGFBR2, FASN, G6PD, NEGR1, QKI, CADM2, PTPRD, NRXN3 — downstream pathway and SCNA features highlighted in TieDIE / focal-deletion analyses.

Clinical implications

  • The authors explicitly nominate the PI3K/AKT/mTOR pathway as a therapeutic target in CCRCC, supporting use of MTOR and related inhibitors (citing prior trials of everolimus and temsirolimus in advanced RCC).
  • EGFR over-expression and phosphorylation in the PI3K-alteration module is linked by the authors to prior lapatinib response data in CCRCC (citing Ravaud et al. 2008 phase III trial).
  • BAP1 mutation is the only SMG independently prognostic for worse survival.
  • mRNA, miRNA, and protein signatures provide prognostic information beyond standard clinical variables (validated on n=253).
  • Metabolic biomarkers — AMPK, ACC, pentose phosphate pathway, miR-21 / GRB10 methylation — are proposed as both prognostic markers and candidate therapeutic-vulnerability indicators given the Warburg-like shift in aggressive tumors.

Limitations & open questions

  • Single-site sampling: tumors were sampled from a single region of the primary, but CCRCC is known to be regionally heterogeneous (citing Gerlinger 2012). Extent to which convergent evolution captures critical events at single sites remains open.
  • Tumor cellularity was lower and more variable than pathology review suggested (median 54% ± 14% vs 85% by review), reflecting stromal/endothelial contribution or heterogeneity; the authors argue mutation frequencies were nonetheless consistent with prior reports.
  • Primary-only cohort: samples reflect patients fit for nephrectomy; the metastatic landscape was not characterized and is flagged for future work.
  • Mechanistic uncertainty for chromatin modifiers: how PBRM1, SETD2, and BAP1 mutations mechanistically drive transformation and the metabolic shift remains to be defined.
  • Therapeutic translation: PI3K/AKT/mTOR targeting is supported by the genomics but specific predictive biomarkers tying alteration class to response were not tested in this study.

Citations from this paper used in the wiki

  • “We surveyed more than 400 tumors using different genomic platforms and identified 19 significantly mutated genes.” (Abstract)
  • “PBRM1, a subunit of the PBAF SWI/SNF chromatin remodeling complex, as well as histone deubiquitinase BAP1 and histone methyltransferase SETD2, were recently found to be altered in 41%, 15% and 12% of ccRCCs, respectively.” (Introduction)
  • “the most frequent arm-level events involved loss of chromosome 3p (91% of samples), encompassing all of the four most commonly mutated genes (VHL, PBRM1, BAP1 and SETD2).” (Somatic Alterations)
  • “the validated mutation data identified nineteen significantly mutated genes (SMGs) (q< 0.1), with VHL, PBRM1, SETD2, KDM5C, PTEN, BAP1, MTOR and TP53 representing the eight most extreme members (q<0.00001)” (Somatic Alterations)
  • “Among all SMGs, only mutation of BAP1 correlated with poor survival outcome” (Somatic Alterations)
  • “A recurrent SFPQ-TFE3 fusion (previously linked to non-clear cell translocation-associated RCC) was found in five samples, all of which were VHL wildtype” (Somatic Alterations)
  • “epigenetic silencing of VHL in about 7% of ccRCC tumors, which was mutually exclusive with mutation of VHL” (DNA Methylation Profiles)
  • “Mutations in SETD2, a non-redundant H3K36 methyltransferase, were associated with increased loss of DNA methylation at non-promoter regions” (DNA Methylation Profiles)
  • “the MEMo algorithm identified mutually exclusive patterns of alterations targeting multiple components of the PI3K/Akt/mTOR pathway in 28% of the tumors” (Integrative data analyses)
  • “frequent over-expression of EGFR, which correlates with increased phosphorylation of the receptor, and which has been previously associated with Lapatinib response in ccRCC” (Integrative data analyses)
  • “Poor prognosis correlated with down-regulation of AMPK complex and the Krebs cycle genes, and with up-regulation of genes involved in the pentose phosphate pathway (G6PD, PGLS, TALDO, TKT) and fatty acid synthesis (FASN, ACC).” (Correlations with survival)
  • “The PI3K/AKT pathway presents a strong therapeutic target in ccRCC, supporting the potential value of MTOR and/or related pathway inhibitor drugs for this cancer.” (Discussion)

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