Comprehensive Molecular Characterization of Papillary Renal-Cell Carcinoma

Abstract

Background: Papillary renal-cell carcinoma, which accounts for 15 to 20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist.

Methods: We performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis.

Results: Type 1 and type 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into three individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-antioxidant response element (ARE) pathway. A CpG island methylator phenotype (CIMP) was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase (FH).

Conclusions: Type 1 and type 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CIMP in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renal-cell carcinoma consisted of at least three subtypes based on molecular and phenotypic features. (Funded by the National Institutes of Health.).

Figure 1. Somatic alterations in papillary renal cell carcinoma underlie molecular differences between Type 1 and Type 2 cancers

(a) Unsupervised clustering of DNA copy profiles of papillary renal cell carcinoma, revealing three molecular subtypes, one of which is highly enriched for Type 1 tumors (light blue) and the other two, for Type 2 tumors (yellow). (b) Significantly mutated genes (SMGs) in papillary renal cell carcinoma, determined by considering all genes (q<0.1, range 0.0-1.0) or focusing on the set of 260 genes previously implicated in cancer by large-scale, pan-cancer exome analyses (q<0.1). p-values by MutSig 2CV algorithm. (c) Pathway-centric view of gene mutations in papillary renal cell carcinoma, involving key pathways and genes implicated in cancer, either in this present study or elsewhere as indicated. The tumors were classified by histological type from left to right: Type 1 (light blue), Type 2 (yellow) or unclassified papillary renal cell carcinoma (gray), and from top to bottom by gene or pathway altered. Pathways and genes represented include: MET, HIPPO pathway (NF2, SAV1, WWC1), NRF2 pathway (NFE2L2, KEAP1, CUL3, SIRT1, FH), chromatin modification (CREBBP, DOTL1, EHMT1/2, EP300, EZH1/2, KAT2A/B, KDM1A/B, KDM4A/B, KDM5A/B/C, KDM6A/B, MLL1/2/3/4/5, NSD1, SETD2, SMYD4, SRCAP), SWI/SNF complex (ACTB, ACTL6A/B, ARID1A/B, ARID2, BCL6A/B/C, BCL11A/B, BRD7/9, DPF1/2/3, PHF10, PBRM1, SMARCA2/4, SMARCB1, SMARCC1/2, SMARCD1/2/3, SMARCE1), mTOR pathway (MTOR, PIK3CA, PTEN, STK11, TSC1, TSC2), and p53 pathway (ATM, CDKN1A, CDKN2A, FBXW7, RB1, TP53). (d) Fusion gene analysis identified TFE3 or TFEB fusions in 8 PRCC tumors, including two novel gene fusion partners for TFE3, DVL2 and RBM10, and two novel gene fusion partners for TFEB, COL21A1 and CADM2. Schematic versions of these fusions demonstrate the exons and functional domains that are present within the different gene fusions and the position of potential miR binding sites in TFEB. The retained exons of TFE3 or TFEB are colored in shades of blue. Thin regions represent non-coding sequence, while thick regions represent the translated reading frame and white strips indicate the region is no longer to scale. AD = Strong Transcription Activation Domain, bHLH = Basic Helix-Loop-Helix Domain, LZ = Leucine Zipper Domain, MAD2L2 = Mitotic Arrest Deficient-Like 2 Interaction Domain, DIX = Dishevelled and Axin Domain, RMM = RNA-Recognition Motif.

Figure 2. Alterations in papillary renal cell carcinoma involving MET oncogene

(a) Schematic of somatic mutations in MET, along with germline variant H1112R previously implicated in hereditary papillary renal cell carcinoma , and the novel RNA transcript variant of MET lacking the canonical exons 1 and 2, but containing a novel exon 1 that splices to the canonical exon 3. (b) Crystal structure for the MET tyrosine kinase catalytic domain (RCSB-PDB 3I5N), on which are mapped the residues altered in papillary renal cell carcinoma. All amino acid numbering is based on the MET protein sequences.

Figure 3. DNA methylation profiling uncovers a subset of papillary renal cell carcinoma manifesting a CpG Methylator Phenotype (CIMP)

(a) Molecular subtyping by DNA methylation platform revealed three subtypes of papillary renal cell carcinoma, one of which showed widespread DNA hypermethylation patterns characteristic of CIMP tumors. Corresponding data tracks highlight molecular features associated with CIMP tumors (n=9 cases), including CDKN2A silencing, germline or somatic mutations of FH, Type 2 histological status, and expression of both cell cycle- and hypoxia-related genes. (b) Differences in patient age among the three DNA methylation-based subtypes. (c) Differences in patient overall survival among the three DNA methylation-based subtypes (p=1E-16, log-rank). (d) Differential mRNA expression patterns comparing papillary renal cell carcinoma CIMP, Type 1, non-CIMP Type 2, and normal kidney for key genes involved in metabolism. (e) Differential expression patterns of CIMP tumors versus Type 1 in metabolism-related pathways, focused on gene and protein expression patterns previously associated with Warburg-like effects in kidney cancer. p-values by t-test.

Figure 4. Multi-platform-based subtype discovery in papillary renal cell carcinoma

(a) Integration of subtype classifications from five “omic” data platforms using cluster of cluster analysis identified four major papillary renal cell carcinoma groups: C1 (Type 1-enriched), C2a and C2b (Type 2-enriched), and C2c (representing the CIMP papillary renal cell carcinomas). The blue and white heat map displays sample consensus, below which a second heat map displays the subtypes defined independently by DNA methylation (Pink), Chromosomal copy number (CN)(Black), miRNA expression (Red), mRNA expression (Blue), and protein (RPPA) expression (Green, Gray represents samples missing RPPA expression data). Clinical features associated with the multi-platform-based subtypes are shown near the bottom. Histological types are shown with Type 1 in light blue, Type 2 in yellow and unclassified papillary renal cell carcinoma in gray. Stage I and II tumors are shown in dark blue or light blue respectively, and Stage III and IV tumors are shown in light red or dark red respectively. (b) Differences in patient overall survival between COCA defined groups (overall, p<1E-16, log-rank). In comparison to the Type 1-enriched C1group, the overall survival of the Type 2-enriched C2a group was similar, while the C2b (* p=0.003) and CIMP (** p=1E-22) groups had worse overall survival.