We report novel whole-exome sequencing data derived from metachronous, bilateral ccRCC tumors in a patient with VHL disease. Multi-focal tumors developing from the same germline, and in the same organ, are of independent clonal origin in this patient, despite identical histopathological characterization. Furthermore, genomic analysis reveals the presence of two distinct tumors in the right kidney, from what appeared macro- and microscopically to be a single ccRCC tumor. Our findings are consistent with an earlier report of copy number variation in 36 germline VHL mutant primary ccRCC tumors; within each of 12 patients, metachronous tumors displayed different copy number profiles, to the extent that tumors from the same patient were as dissimilar to each other as they were to tumors from other patients with VHL disease .
This clinical situation allows a rare insight into tumor evolution from the VHL mutant germline state in ccRCC, where the initiating event is the same across multiple tumors within the same organ and similar microenvironments; the combination of somatic mutation and copy number data defines a unique series of abnormalities for each of the four tumors. It is evident that, from a common starting point, there are multiple, currently unpredictable, routes to tumor development. Mutation of VHL was assumed to be the first hit, because of its presence in the germline in blood and normal kidney tissue. Based on previous data establishing that chromosome 3p loss is one of the founder events in ccRCC [8,9], and the ubiquitous nature of this event within the four tumors subjected to M-seq in this study, 3p LOH is likely to be the second hit. However, copy number data indicate that even amongst tumors arising from the same germline and in an identical environment, each of the four tumors harbored a distinct secondary 3p LOH event. Conceivably, the consequence of a different second event in the four tumors is to drive each through divergent trajectories; thus, subsequent events might be contingent upon the nature of the 3p event in these tumors. The notion that the final result of evolution is determined by antecedent steps is consistent with Gould’s theory of historical contingency. Of course, it is possible that an alternative driver event, such as 5q gain or somatic mutation, was the contingent step; however, on the basis that the 3p LOH is the only other ubiquitous event observed in sporadic cases and is ubiquitous in all tumors studied here, this is also likely to be one of the earliest events in VHL syndrome clear cell carcinoma.
Gould used the re-interpretation of the fossils of the Burgess Shale to illustrate an alternative view of life’s history, and offered the term contingency to encompass the idea of evolution as a series of unpredictable and unrepeatable events, but a system in which the final outcome is causally dependent on the occurrence of each antecedent step . Gould referred to the concept of historical constraints within a lineage that limit the ability of natural selection to modify the future lineage; this notion of internal selective factors is cited by others in the field of developmental biology [39,40]. Our results support these statements by showing that histologically similar tumors derived from germline VHL mutation share 3p LOH as the second event but are then distinguished by their subsequent somatic genomic events. However, there may be constraints to their evolution, exemplified by the activation of the mTOR pathway in all tumor regions.
Naturally occurring or experimental evidence to support Gould’s claim of historical contingency is limited [41,42]. The molecular analysis of acute lymphoblastic leukemia in monozygotic twins provides support for his ideas [43–45]. Two separate twin studies now show that while the initiating event of a chromosomal fusion (even if not inherited) is the same, subsequent genomic aberrations differ substantially between cases of concordant and discordant acute lymphoblastic leukemia within monozygotic twin pairs, and this may have a profound bearing on clinical outcome. A second example from cancer medicine is in myelodysplastic syndromes, in which early driver mutations in different genes have been shown to direct disease evolution down ‘preferred’ trajectories, resulting in distinct clinical phenotypes . In a simply designed long-term evolution experiment using 12 identical populations of Escherichia coli established in 1988, Lenski and colleagues managed to re-play the ‘evolutionary tape’ and thus provide unique evidence for historical contingency [47,48]. Further evidence for the evolution of specific protein functions being contingent upon permissive, ancestral mutations has been reported recently . Results from our observational data support similar themes illustrated by these experiments: the development of a particular phenotype from an identical cell population and environment occurs via diverse, multi-step evolutionary pathways.
Conway Morris argued that evolution is constrained to such a degree that it converges upon a restricted range of options . In this case, despite parallel evolution of the four individual tumors, there is indeed evidence for convergence upon the PI3K-AKT-mTOR pathway with two independent MTOR mutations in tumors from the left kidney and proteomic evidence for pathway activation in all tumor regions. Our data add to a compelling body of evidence for convergence upon this pathway, both within and between individual patients. A recent analysis of 106 ccRCC cases reported that the MTOR gene was mutated in nearly 6% of cases, but that a quarter of cases harbored mutations in genes encoding signaling molecules in the PI3K-AKT-mTOR cascade, including PTEN, PIK3CA, TSC1 and TSC2 . Furthermore, the proportion of pathway mutations rises to 60% when calculated on a per patient, rather than per biopsy, basis .
Functional convergence on the mTOR pathway was evidenced by phosphorylation of downstream substrates S6K or 4E-BP1 in all four tumors relative to normal kidney, despite divergent somatic copy number and mutational events. In vitro experiments showed that the mutation in the FAT domain of mTOR was not activating; the functional significance of this mutation therefore remains unclear but theoretically it could influence mTOR-substrate interactions. It is likely that similar microenvironmental selection pressures across all four tumors contribute significantly to convergent signaling activity witnessed in this study. The identification of an ARID1A mutation in tumor one in the contralateral kidney to those tumors harboring MTOR mutations may further illustrate convergence upon the PI3K-AKT-mTOR pathway. Co-occurrence of mutations of this gene and those of the PI3K pathway are reported to occur in endometrial and head and neck cancers, and ARID1A mutations may impact upon PI3K pathway activity [32,50]. Although the mechanism is unknown, phosphorylation of PI3K downstream targets, including PDK1, AKT, TSC2 and S6K, was significantly upregulated in the presence of ARID1A mutations, and knockdown of wild-type ARID1A in three endometrial cancer cell lines resulted in significantly elevated phosphorylation of AKT . Pathway activation in tumor two could have been the result of gain of chromosome 5q, which has been identified as a driver in ccRCC, occurring in 67% of samples . Importantly, gain of amplicon 5q.35.3 results in alteration of two genes, GNB2L1 and SQSTM1, whose overexpression is associated with activation of the PI3K pathway [6,33–35]. It is noteworthy that mutations in MTOR and in GNB2L1 and SQSTM1, were shown to be mutually exclusive in ccRCC , consistent with pathway convergence as demonstrated by tumors one/two and three/four in this patient. Furthermore, we observed a strong tendency towards mutual exclusivity between mutations in ARID1A and PI3K pathway genes (MTOR and TSC1/2) . In summary, we suggest that dysregulation of the PI3K pathway in this patient was achieved in all tumors but mediated by diverse mutational or copy number events.
In two patients with ccRCC tumors arising from germline VHL mutation, ITH was minimal despite extensive sampling in both a morphologically heterogeneous stage 3 tumor and a morphologically homogeneous stage 1 tumor. Within one of these patients, two evaluable tumors appeared to follow a linear, rather than branched evolutionary path. Furthermore, there was a noticeable absence of mutations in driver ccRCC genes, or second hits, in genes such as SETD2, PBRM1 and BAP1 in both patients. These patterns, distinct from those observed in tumors from patients with sporadic ccRCC, might be a direct consequence of a critical mutational event in the germline. Alternatively, early stage, non-metastatic disease may harbor less genetic ITH than advanced later stage tumors, although our recent cohort of sporadic ccRCCs included one stage 2 and one stage 3 tumor and both demonstrated ITH and branched evolution; in particular, the high ITB index for the stage 2 tumor testifies to this . Further assessment of spatial ITH in early stage, node-negative, sporadic and germline VHL mutant tumors will be required to formally test these hypotheses.