Spectrum of diverse genomic alterations define non–clear cell renal carcinoma subtypes

Authors

Steffen Durinck

Eric W Stawiski

Andrea Pavía-Jiménez

Zora Modrusan

Payal Kapur

Bijay S Jaiswal

James Brugarolas

Somasekar Seshagiri

Doi

10.1038/ng.3146

PMID: 25401301 · DOI: 10.1038/ng.3146 · Journal: Nature Genetics (2015)

TL;DR

Durinck et al. performed integrated exome, RNA-seq, and SNP copy-number profiling on 167 primary non–clear cell renal cell carcinomas (nccRCC) — 67 papillary RCC, 49 chromophobe RCC, 35 renal oncocytoma, 6 translocation RCC, 8 unclassified and 2 sarcomatoid — to define the molecular taxonomy of subtypes that are largely excluded from clinical trials. They identified ten significantly mutated genes in pRCC (including MET mutated in 15% of samples with previously unreported activating kinase-domain alterations) and six in chRCC (TP53, PTEN, PDHB, PRKAG2, among others). A five-gene RNA-seq classifier (ASB1, GLYAT, PDZK1IP1, PLCG2, SDCBP2) separates pRCC, chRCC and renal oncocytoma at 95.3% accuracy. RNA-seq also uncovered novel fusions including a transforming ACTG1-MITF fusion in pRCC and CLTC-TFEB in an unclassified case, defining a broader “MiTF-high” subtype in which the anti-apoptotic gene BIRC7 is upregulated as a potential drug target.

Cohort & data

  • 167 primary nccRCC tumors with patient-matched normals from the University of Texas Southwestern Kidney Cancer Program: 67 [[PRCC|pRCC]], 49 [[CHRCC|chRCC]] (36 classic, 12 eosinophilic, 1 mixed), 35 [[ROCY|renal oncocytomas]], 8 unclassified RCCs, 6 [[TRCC|tRCCs]], 2 sarcomatoid.
  • Dataset: nccrcc_genentech_2014 (cBioPortal study id; raw data deposited at EGA EGAS00001000926).
  • Assays: whole-exome sequencing (Agilent SureSelect All Exon 50Mb, Illumina HiSeq 2000, ~82× mean coverage) on 140 tumor-normal pairs + 5 tumor-only; targeted SPET capture using NuGEN Ovation Cancer Panel (344 genes) on 19 pRCCs; rna-seq on 159 tumors (TruSeq, HiSeq 2000, ~68M PE reads/sample); SNP-based copy-number profiling (Illumina HumanOmni2.5-8 arrays); orthogonal validation by sanger-sequencing (96% indel validation rate), mass-spectrometry genotyping (92% SNV validation rate) and FISH for amplifications and translocations.
  • Reads aligned with BWA to GRCh37/hg19; mutational signatures derived using the Genentech/Huber pipeline.

Key findings

  • Mutational burden: pRCC carried significantly more protein-coding alterations than ccRCC (mean 61 ± 31 vs 45 ± 19; P = 3.6 × 10⁻⁷), and substantially more than chRCC and renal oncocytoma.
  • Validation: 90% (4,280/4,751) of protein-altering mutations were novel relative to COSMIC; mass-spec genotyping validated 92% (164/178) of selected SNVs and Sanger validated 96% (127/132) of indels. RNA-seq confirmed expression of 1,412 somatic variants.
  • Mutational signatures: Five distinct signatures (S1–S5) identified across nccRCC. pRCC dominated by S1+S3; chRCC and renal oncocytoma dominated by S3+S4. Kidney cancer subtypes co-clustered by signature.
  • Significantly mutated genes in pRCC (q ≥ 1, FDR ≤ 10%): MET, SLC5A3, NF2, PNKD, CPQ, LRP2, CHD3, NHERF1 (SLC9A3R1), SETD2, CRTC1.
  • Significantly mutated genes in chRCC: TP53, PTEN, FAAH2, PDHB, PDXDC1, ZNF765.
  • Significantly mutated genes in renal oncocytoma: ERCC2, C2CD4C.
  • MET in pRCC: mutated in 15% (10/65) of pRCC, up from a previously reported 8% (9/121). Nearly all mutations were in the kinase domain. Four newly identified activating alterations (p.Val1088Ala, p.Ile1095Thr, recurrent p.Phe1218Ile, and the extracellular p.Asp153Tyr) were experimentally validated — they conferred elevated MET phosphorylation and anchorage-independent growth of NIH3T3 cells over wild-type MET. MET mutations were mutually exclusive with NF2, PTEN, TSC1 and MTOR mutations (low frequency, not statistically significant).
  • PDHB / PRKAG2 in chRCC: PDHB (pyruvate dehydrogenase E1β) mutations p.Phe222fs*35 and p.Arg105Leu; PRKAG2 p.Ile388Val (in pseudosubstrate sequence) and recurrent p.Arg531Gln (analogous to the GSC-causing mutation), plus an analogous PRKAG1 p.Arg299Gln in an independent chRCC dataset — implicating metabolic deregulation (AMPK + PDH) in a subtype of chRCC.
  • TP53 in chRCC: significantly enriched in the chRCC classic subtype (P = 2.3 × 10⁻⁵); essentially absent from eosinophilic chRCC.
  • Five-gene RNA-seq classifier: ASB1, GLYAT, PDZK1IP1, PLCG2 and SDCBP2 distinguish pRCC, chRCC and renal oncocytoma. Leave-one-out k-NN on a held-out 43-sample validation cohort (16 chRCC, 10 renal oncocytoma, 17 pRCC) reached 95.3% accuracy (41/43); replicated on an independent expression cohort (4 ROCY, 4 chRCC, 19 pRCC).
  • Copy-number changes: pRCC frequently amplified whole chromosomes 3, 7, 12, 16, 17, 20; ~70% had chr7 amplification (containing MET). Sample 16864T showed a 22-Mb chr7 focal amplification with the highest MET expression in the cohort. Sample 1216T had a 490-kb chr6 amplification encompassing TFEB with the highest TFEB expression (re-reviewed as low-grade RCC with papillary/oncocytic features). Classic chRCCs showed frequent loss of chr1, 2, 6, 8, 10, 13, 17, 21; eosinophilic chRCCs were near-diploid; renal oncocytomas showed mostly chr1 loss only.
  • MiTF gene fusions: known ASPSCR1-TFE3 and PRCC-TFE3 fusions confirmed in tRCCs (validated by FISH). Two tRCCs (14336T, PtS1T) had elevated TFE3 expression without TFE3 fusion or amplification; sample 14336T harbored a novel MIDN-SBNO2 fusion (chr19p13.3 inversion) placing full-length SBNO2 under the MIDN promoter, with the two highest SBNO2 expression levels seen in these two samples.
  • Novel CLTC-TFEB fusion identified in an unclassified case (8432T) — encodes an in-frame fusion protein containing the bHLH domain of TFEB; validated by FISH. Together with a PRCC-TFE3 fusion in another unclassified case (20825T1), this led to reclassification of both as tRCC after pathology review.
  • Novel transforming ACTG1-MITF fusion in pRCC sample 159T (with oncocytic/papillary features): replaces the first 118 aa of MITF with the N-terminal 121 aa of ACTG1. The fusion is more stable than wild-type MITF, induces HIF1A, MET and APEX1 target genes (P < 0.01) in HEK293T cells, and confers significantly greater anchorage-independent growth in NIH3T3 cells than wild-type MITF.
  • BIRC7 in MiTF-high tumors: 6/7 samples with MiTF family fusion or amplification overexpressed BIRC7, an anti-apoptotic MITF target gene — proposed as a diagnostic marker and therapeutic vulnerability for the “MiTF-high” nccRCC subtype.

Genes & alterations

  • MET — recurrent activating kinase-domain mutations (p.Val1088Ala, p.Ile1095Thr, p.Phe1218Ile) and an extracellular p.Asp153Tyr in 15% of pRCC; chr7 amplification of the MET locus in ~70% of pRCCs; induced as a downstream target of the ACTG1-MITF fusion. Major therapeutic rationale for MET inhibitors in pRCC.
  • NF2, SLC5A3, PNKD, CPQ, LRP2, CHD3, NHERF1, SETD2, CRTC1 — newly nominated significantly mutated genes in pRCC. SLC5A3 (SMIT) shows three frameshift LoF + one predicted-damaging point mutation, suggesting a tumor-suppressor role linked to mitochondrial alterations.
  • TP53 — significantly enriched mutations in the chRCC classic subtype only (P = 2.3 × 10⁻⁵).
  • PTEN, FAAH2, PDHB, PDXDC1, ZNF765 — significantly mutated in chRCC.
  • PRKAG2 — p.Ile388Val (pseudosubstrate, CBS2) and p.Arg531Gln (CBS4, AMP-binding) substitutions in chRCC, predicted activating by analogy to glycogen storage cardiomyopathy mutants. Also strongly upregulated in chRCC/oncocytoma vs pRCC (~3.4 log fold; P < 5 × 10⁻⁹⁰).
  • PRKAG1 — p.Arg299Gln in an independent chRCC cohort, analogous to PRKAG2 p.Arg531Gln.
  • TSC1, TSC2, MTOR — recurrent mutations in chRCC, suggesting mTORC1 addiction and possible responsiveness to mTORC1 inhibitors.
  • ARID1A — recurrently mutated in chRCC (not reaching q-score significance).
  • ERCC2, C2CD4C — significantly mutated in renal oncocytoma (low overall mutation rate).
  • IDH2, JAK2, MTOR — among 60 genes with COSMIC hotspot mutations identified as potential drug targets across nccRCC.
  • ACTG1-MITF fusion — novel transforming fusion in pRCC sample 159T; activates HIF1A, MET, APEX1; greater protein stability and transforming activity than wild-type MITF.
  • CLTC-TFEB fusion — novel; encodes in-frame fusion preserving the TFEB bHLH domain (sample 8432T, reclassified tRCC).
  • ASPSCR1-TFE3 / PRCC-TFE3 fusions — recovered as expected in tRCCs.
  • MIDN-SBNO2 fusion — novel chr19p13.3 inversion in a TFE3-fusion-negative tRCC (14336T), driving high SBNO2 expression.
  • TFEB focal amplification — 490-kb chr6 amplicon with highest TFEB expression in sample 1216T, expanding the spectrum of TFEB alterations beyond translocation.
  • BIRC7 — overexpressed in 6/7 MiTF-fusion/amplification samples; proposed therapeutic target via BIRC7 inhibitors.
  • VHL, FH, FLCN, SDHB, SDHC, SDHD, BAP1 — referenced as known germline kidney-cancer predisposition genes against which nccRCC somatic profiles are contrasted.

Clinical implications

  • MET-targeted therapy in pRCC: the authors recommend stratifying pRCC patients by MET alterations (mutation, amplification, and/or overexpression) in trials of MET inhibitors, noting prior responsiveness of germline-MET pRCC patients to broad-spectrum TKIs.
  • mTORC1 inhibition in chRCC: recurrent TSC1 / TSC2 / MTOR mutations in chRCC nominate mTORC1 inhibitors (rapalogs) as a candidate therapy.
  • AMPK / metabolic axis in chRCC: combined PRKAG2 and PDHB mutations define a chRCC subtype with metabolic deregulation, potentially actionable via AMPK or PDH modulators.
  • Five-gene RNA-seq classifier (ASB1, GLYAT, PDZK1IP1, PLCG2, SDCBP2): deployable to distinguish pRCC, chRCC and renal oncocytoma in challenging diagnostic cases (e.g. needle core biopsy where chRCC vs oncocytoma is histologically ambiguous). Requires prospective validation.
  • “MiTF-high” subtype: tumors with MITF, TFE3 or TFEB fusions, amplifications, or overexpression form a coherent biological group beyond classical tRCC. BIRC7 expression may serve as a diagnostic biomarker and a therapeutic target via BIRC7-directed apoptosis-sensitizing agents in clinical development.
  • TFEB beyond translocation: focal TFEB amplification (sample 1216T) is identified as an additional cancer-relevant TFEB lesion to evaluate when classical translocation FISH is negative.

Limitations & open questions

  • Many of the newly nominated significantly mutated genes (e.g., SLC5A3, PNKD, CPQ, FAAH2, PDHB, PDXDC1, ZNF765, C2CD4C) lack functional characterization in cancer — their oncogenic vs passenger status is unestablished.
  • Renal oncocytoma had very low mutation rates; the authors note that larger cohorts and functional follow-up are needed to confirm ERCC2 and C2CD4C as drivers.
  • The five-gene classifier was validated on a 43-sample held-out cohort and an external 27-sample expression dataset, but the authors call for prospective clinical validation before deployment.
  • Mechanism of TFE3 upregulation in fusion-negative, amplification-negative tRCCs (e.g. PtS1T) remains unresolved; whether SBNO2 modulates other MiTF family members is hypothesized but not demonstrated.
  • The functional consequence of PDHB p.Arg105Leu — inferred from a homologous germline lactic-acidosis mutation — is not directly validated.
  • The cohort is single-institution (UTSW) and excludes patients with HIV/HBV/HCV/TB; generalizability to broader nccRCC populations is untested.
  • No drug-response data accompany the genomic findings; therapeutic hypotheses (MET inhibitors, mTORC1 inhibitors, BIRC7 inhibitors) await clinical testing in nccRCC subsets.

Citations from this paper used in the wiki

  • “We identified ten significantly mutated genes in pRCC, including MET, NF2, SLC5A3, PNKD and CPQ. MET mutations occurred in 15% (10/65) of pRCC samples and included previously unreported recurrent activating mutations.” (Abstract)
  • “In chRCC, we found TP53, PTEN, FAAH2, PDHB, PDXDC1 and ZNF765 to be significantly mutated.” (Abstract)
  • “Among the nccRCC samples analyzed, TP53 mutations were found to be significantly enriched in the chRCC classic subtype (P = 2.3 × 10⁻⁵).” (Mutated genes section)
  • “A leave-one-out cross-validation using a k nearest-neighbors classifier correctly classified 41 of the 43 samples (95.3%)” — five-gene RNA-seq classifier validation.
  • “We identified an ACTG1-MITF gene fusion (Fig. 4b) in a pRCC sample (159T)… Cells expressing ACTG1-MITF had a significantly higher number of anchorage-independent colonies than cells expressing wild-type protein.” (MITF fusion section)
  • “We found that a majority (6/7) of the samples with MiTF fusion or amplification had elevated BIRC7 expression… Small molecule BIRC7 inhibitors that sensitize cancer cells to apoptosis are in clinical development and might prove effective.” (Discussion)
  • “Given these observations, it would be relevant to stratify patients on the basis of MET alterations in trials involving MET inhibitors.” (Discussion)

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