Development of synchronous VHL syndrome tumors reveals contingencies and constraints to tumor evolution

Abstract

Background: Genomic analysis of multi-focal renal cell carcinomas from an individual with a germline VHL mutation offers a unique opportunity to study tumor evolution.

Results: We perform whole exome sequencing on four clear cell renal cell carcinomas removed from both kidneys of a patient with a germline VHL mutation. We report that tumors arising in this context are clonally independent and harbour distinct secondary events exemplified by loss of chromosome 3p, despite an identical genetic background and tissue microenvironment. We propose that divergent mutational and copy number anomalies are contingent upon the nature of 3p loss of heterozygosity occurring early in tumorigenesis. However, despite distinct 3p events, genomic, proteomic and immunohistochemical analyses reveal evidence for convergence upon the PI3K-AKT-mTOR signaling pathway. Four germline tumors in this young patient, and in a second, older patient with VHL syndrome demonstrate minimal intra-tumor heterogeneity and mutational burden, and evaluable tumors appear to follow a linear evolutionary route, compared to tumors from patients with sporadic clear cell renal cell carcinoma.

Conclusions: In tumors developing from a germline VHL mutation, the evolutionary principles of contingency and convergence in tumor development are complementary. In this small set of patients with early stage VHL-associated tumors, there is reduced mutation burden and limited evidence of intra-tumor heterogeneity.

Figure 1

Multi-focal renal cell carcinoma tumors in a patient with VHL disease. Coronal sections from computed tomography scans show the spatial orientation of the tumors within the kidneys. (A) A macroscopically single tumor in the right kidney, but defined by exome sequencing as two tumors (one and two). (B) Two separate tumors in the left kidney, tumors three and four. Cysts typical of VHL disease are also present in both kidneys.

Figure 2

Intra-tumor heterogeneity in four germline tumors. (A,B) Heatmaps show the regional distribution of all non-silent mutations; presence (blue) or absence (grey) of each mutation is indicated for every tumor region in tumors from the right (A) and left kidneys (B). Whole exome sequencing defines four distinct tumors. In each, tumor evolution is linear rather than branched.

Figure 3

Copy number analysis in four germline tumors. (A) Copy number profiles of representative samples from each of the four tumors, with integer copy number on the x-axis. Loss of chromosome 3p is ubiquitous. (B) The chromosome 3p breakpoint locations for the four tumors. Each y-axis is logR, and the x-axis represents position along chromosome 3 in hg19. An ‘X’ near the x-axis marks the approximate position of the centromere.

Figure 4

Two distinct activating mutations in mTOR. Left: a schematic of the mTOR protein structure (cyan) in complex with mLST8 (dark blue) (PDB code 4JSP). Key residues and structural features are highlighted close to mTOR mutations relevant to the current study. The FRB and FAT domains are also labeled together with the FATC sub-domain. Right: a close-up of regions in proximity to the mTOR mutation site L2427 abutting the nucleotide-binding cleft and activation loop (upper right panel). Lower right panel shows the environment surrounding residue T1652 in the FAT domain.

Figure 5

Convergence upon the PI3K-AKT-mTOR pathway. Graphs showing the ratio of phosphorylated to total protein for intra-cellular kinases of the PI3-AKT-mTOR pathway. For tumors one, two and three, more than one region from each tumor was analyzed.