The genomic landscape of pediatric renal cell carcinomas

Abstract

Pediatric renal cell carcinomas (RCC) differ from their adult counterparts not only in histologic subtypes but also in clinical characteristics and outcome. However, the underlying biology is still largely unclear. For this reason, we performed whole-exome and transcriptome sequencing analyses on a cohort of 25 pediatric RCC patients with various histologic subtypes, including 10 MiT family translocation (MiT) and 10 papillary RCCs. In this cohort of pediatric RCC, we find only limited genomic overlap with adult RCC, even within the same histologic subtype. Recurrent somatic mutations in genes not previously reported in RCC were detected, such as in CCDC168, PLEKHA1, VWF, and MAP3K9. Our papillary pediatric RCCs, which represent the largest cohort to date with comprehensive molecular profiling in this age group, appeared as a distinct genomic subtype differing in terms of gene mutations and gene expression patterns not only from MiT-RCC but also from their adult counterparts.

Keywords: Cancer; Genomics.

Graphical abstract

Figure 1

Genomic aberrations in pediatric renal cell carcinomas (pRCC, n = 25) Tumor mutational burden, number of SNV/Indels in WES, genes affected by recurrent nonsynonymous somatic aberrations^# and canonical adult RCC-associated genes bearing somatic aberrations, as well as gene fusions and germline predisposition genes in different histologic and clinical subgroups of the pRCC cohort. # Only the most important recurrent somatic aberrations are selected; in total 39 recurrent SNV/Indels were detected after exclusion of RCC987; complete list in Table S3. ∗ Cases without control blood DNA. § RCC380: initially histologically classified as “unclassified” subtype, but after sequencing data analysis recognized as harboring a germline FH-mutation with bi-allelic FH-inactivation, i.e., as a FH-deficient RCC subtype. ▪ Gray shaded bar: RCC987 is probably highly biased by FFPE artefacts. For this sample, all SNV/Indels shown in the somatic aberrations have been manually verified in IGV. A slightly higher SNV/Indel count is expectable, as this was the only case of a primarily localized papillary RCC with a fatal disease course following a disseminated metastatic relapse within 2 years (plus initial high LDH [3.047 U/l] and high ferritin/CrP level). For patients with more than one tumor sample taken from different locations of the tumor piece of the same disease episode, the mean of the SNV and indel number of these tumor samples was calculated for each patient and used for calculation of tumor mutational burden (TMB) and in this figure. RCC544_I/RCC_544_II: tumor Samples from the same patient with 10 years in between (RCC544_I from initial phase abdominal tumor material; RCC544_II from lung metastasis after 10 years). ∗ Acyl-CoA-related genes: ACSBG2 (3 SNVs) in RCC544_I, ACSM3 in RCC751, ACSM2B in RCC419, ACSL5 in RCC1230, ACBD7 in RCC987, ACOT13 and ACOX1 in RCC1230, ACADSB in RCC 419. ∗CHD: CHD1 in RCC1230, CHD4 in RCC751, CHD5 in RCC987, CHD6 in RCC419. ∗ MAPK: MAP3K9 in RCC996 und RCC1230, MAP2K5 in RCC1230, MAP2K7 in RCC419, MAP4K1 in RCC544_I, MAP4K3 in RCC751∗ SWI/SNF: SMARCA4 in RCC987, SMARCA2 in RCC419, SMARCC1 and SMARCC2 in RCC1230.

Figure 2

Fusion genes found for the first time in pediatric RCC and discovery of a new germline FH mutation (A) A complex gene fusion involving 3 different genes: TFE3, ASPSCR1, and SNX8 in RCC751. (B) Discovery of gene fusion CRK-PITPNA in papillary pediatric RCC (RCC1176), associated with the second highest CRK expression across all pRCC. (C) Germline structural variant within the FH gene (RCC380): duplication and intrachromosomal fusion of exon 1-7 with exon 2-10 and “second hit” copy-neutral LOH of the wild-type FH allele resulting in the lowest FH gene expression compared with all other pRCCs.

Figure 3

Copy-number profiles of the pediatric RCC cohort (A) Copy-number profiles of the papillary pediatric RCC (ppRCC, n = 10) and the MiT-family translocation pediatric RCC (MiT-pRCC, n = 9^#) shown as average CNA per group. ^#RCC4039 was excluded due to poor DNA quality. ppRCC displayed high CNA frequency affecting predominantly gains of chr. 2, 7, 12, 16, 17, and 20. MiT-pRCC showed mostly a low CNA frequency; a high CNA count was predominantly restricted to metastatic/progredient (=poor prognosis-) RCC in older patients including losses of 1p, 3p, chr.4 or 4q, 9p, chr.11 or 11q, chr.18 and, partially, gains of 17q. (B) Two metastatic MiT-pRCC cases selected from 3a displayed higher CNA frequency including losses of 3p, 9p, and chr.11 or 11q. (C) Copy-number profiles of the chromophobe-RCC (RCC1348), the ALK-rearrangement-associated RCC (RCC1429), the post-neuroblastoma RCC (RCC419), and a secondary-malignancy, histologically unclassified RCC (RCC1230), each presented as a single case (copy-number profile of RCC380 see Figure 2C).

Figure 4

Gene expression analysis (RNA-seq data) of pediatric RCC (pRCC; n = 18). Among the 18/25 pRCC cases with available RNA, 6 MiT-pRCC cases (4 with ASPSCR1-TFE3 fusion, 1 with TFE3-SFPQ fusion, and 1 with TFEB-CLTC fusion), 8 papillary pRCC (6/8 histologically papillary type 1), one chromophobe, one ALK-rearranged, one post-NB, and one FH-deficient RCC were included (A) Unsupervised hierarchical clustering of pRCC samples based on the top 1,000 most differentially expressed genes (DEGs). (B) Volcano-plot illustration of the significantly DEGs in MiT-pRCC versus papillary pRCC. (C) Volcano-plot illustration of the significantly DEGs in metastatic fatal MiT-pRCC versus nonmetastatic surviving MiT-pRCC. (D) MET and RET gene expression in MiT-pRCC and papillary pRCC (ppRCC) compared with Wilms tumor relapses (INFORM), primary Wilms tumors, and normal pediatric kidney (TARGET) as well as adult clear cell RCC, adult papillary RCC, and adult normal kidney (TCGA). MET gene expression significantly higher in ppRCC versus MiT-pRCC, log2FC = 1.6, p < 0.0001, q < 0.01 (Wald test). RET gene expression significantly higher in MiT-pRCC versus ppRCC, log2FC = 1.15, p = 0.013, q = 0.056 (Wald test).

Figure 5

Tumor immune microenvironment (TME) deconvolution and gene expression of immune-regulatory, checkpoint or immunosuppressive activity-related genes in pediatric RCC (pRCC; n = 18) (A) Fraction of different immune cell populations in each pRCC case. (B) Overview of immune cell composition (median value of each cell type) in all pRCC, MiT-pRCC, and papillary pRCC (ppRCC). (C) Fractions of selected immune cell types in all pRCC, MiT-pRCC, and ppRCC; dendritic cell fraction was significantly different between MiT-pRCC and ppRCC, p = 0.026 (Welch two sample t test). (D) Unsupervised hierarchical clustering based on gene expression profiles of 59 immune-regulatory, checkpoint or immunosuppression-associated genes (Samstein et al., 2020) in pRCC. (E) Anti-tumoral cytolytic activity (CYTscore) in all pRCC, MiT-pRCC, and ppRCC. (F) Gene expression of selected immune checkpoint genes in all pRCC, MiT-pRCC, and ppRCC; ∗PD1 (p < 0.001, Wald test), ∗PD-L1 (p < 0.001, Wald test), ∗CTLA4 (p = 0.01, Wald test), and ∗TIGIT (p = 0.002, Wald test) significantly different between MiT-pRCC and ppRCC.