would have no effect on Bap1 or Pbrm1 genes. In contrast,
loss of 3p in humans results not only in the loss of a VHL
allele, but also one allele of both BAP1 and PBRM1. Therefore,
the arrangement of tumor suppressor genes in the genome
likely accounts for differences in tumor predis-
position across species.19,25,37
In summary, somatic mutations in either PBRM1 or BAP1
tend to be mutually exclusive and activate distinct gene expression
programs in tumors leading to differentiated pathological
features, ultimately causing divergent clinical
outcomes. As such, these discoveries resulted in the first molecular
classification of ccRCC. Several unanswered questions
remain, however. For instance, what other events
cooperate with VHL in tumors that are seemingly wild-type
for BAP1 and PBRM1? Or how do mutations in TCEB1 and
CUL2, which are located on chromosome 8 and 10 respectively,
lead to ccRCC formation, and what are the specific cooperating
events?
Next Generation Sequencing Supports
Comprehensive Integrated Analysis of RCC
The development of NGS and improved bioinformatics
tools allowed the collection and integration of data from
WES, copy-number analyses, DNA-methylation analyses,
and messenger RNA and microRNA sequencing, from individual
samples at a large scale.23,33 Through integrated analyses
of ~400 ccRCC samples, The Cancer Genome Atlas
(TCGA) consortium not only confirmed previous findings,
but significantly expanded on them through the identification
of altered subnetworks. Initial genome-wide copy number
analysis, using next-generation sequencing technol-
ogies, identified 3p and 14q loss, as well as 5q gain as the
three most common somatic copy number alterations
(SCNA) in ccRCC (43, 44). These were verified in a larger
TCGA cohort - 3p loss (91%), 14q loss (45%) and 5q gain
(67%).33 The most frequently mutated genes involved chromatin
remodeling pathways. These included genes encoding
the SWI/SNF family proteins PBRM1, SMARCA4 and
ARID1A (47.1%), the histone methyltransferases SETD2 and
MLL (23.8%), and the histone deubiquitinase BAP1 (12.1%).
Alterations in PTEN, TSC1, and other components of the
PI3K/AKT/mTOR pathway were observed in 28% of
ccRCCs.33,45,46 Other findings of significance included loss
of one CDKN2A allele in 16.2% of ccRCCs (mostly through
deletion of 9p21.3), and mutation of TP53 in 2.6% of
cases.24,46
In a similar analysis of non-ccRCC, we found distinct
mutated genes and SCNA alterations highlighting the diverse
molecular landscape of RCC.47 In pRCC, we identified
10 significantly mutated genes including MET, SETD2, and
NF2. Activating MET mutations were identified in 15%
(10/65) of pRCC analyzed, including 4 previously unreported
mutations. Furthermore, 70% of pRCC samples had amplification
of chromosome 7, containing MET, and these
samples had higher levels of MET expression, consistent
with the role of MET in type I pRCC.47 These findings were
confirmed in a subsequent analysis of the pRCC TCGA cohort,
where activating MET mutations were identified in
17% (14/75) of type I pRCC samples, which exhibited near
universal gain of chromosomes 7 and 17.48 In contrast, copy
number analysis of type II pRCC revealed two distinct subtypes,
one with relatively few SCNA and another with a
high degree of aneuploidy and frequent chromosome 9p
loss.48 DNA methylation studies revealed a subset of pRCC
(9/160) referred to as the CpG Island Methylator Phenotype
96 Kidney Cancer Journal
(CIMP), eight of which were categorized as type II pRCC histologically.
CIMP tumors were characterized by universal
hypermethylation of CDKN2A, frequent fumarate hydratase
(FH) mutations (including somatic mutations; 57%), and
worse survival relative to non-CIMP pRCC.48 Loss of FH and
the subsequent accumulation of fumarate has been demonstrated
to result in deregulation of the nuclear factor erythroid
2-related factor (NRF2)/antioxidant response element
(ARE) pathway through direct effects of fumarate on KEAP1,
an inhibitor of NRF2.49 This was first demonstrated in hereditary
leiomyomatosis and renal cell cancer (HLRCC), an
inherited form of type II pRCC arising from germline inactivation
of FH. Consistent with the notion that FH loss leads
to deregulation of the NRF2/ARE pathway, expression of
NQO1, a canonical transcriptional target of NRF2, was highest
in CIMP tumors.24,48 Targeted sequencing in sporadic
type II pRCC revealed activating mutations in the
NRF2/ARE pathway in four of five cases.50 Genomic analysis
of the TCGA cohort supported this finding with activating
mutations in the NRF2/ARE pathway in 16.7% (10/60) of
cases.48 An NRF2/ARE gene transcription program is a distinguishing
feature of type II pRCC.48
Analysis of chRCC revealed distinct molecular alterations
defining this subgroup. chRCC can be classified histologically
into classical and eosinophilic subtypes, the latter
characterized by abundant eosinophilic cytoplasm and densely
packed mitochondria.51 Analysis of the TCGA cohort
of 66 chRCC identified molecular distinctions between
these two subgroups.51,52 All 47 cases of classical chRCC in
the TCGA cohort demonstrated characteristic chromosomal
losses of chromosomes 1, 2, 6, 10, 13, and 17 whereas only
half (10/19) of the eosinophil variants did.47, 52 Studies from
TCGA and our own group found that TP53 mutations were
significantly enriched (P = 2.3E-5) in the classic chRCC subtype.
47,52 The two most frequently mutated genes across
chRCC variants are TP53 (31.1%) and PTEN (9%).47 Mutually
exclusive mutations in genes of the PI3K/AKT/mTOR
pathway were observed in 23% of cases.47,52 Interestingly,
analysis of mitochondrial DNA in chRCC revealed recurring
mutations in genes encoding components of complex 1 of
the electron transport chain (ETC), although these mutations
were not associated with changes in the expression of
genes implicated in oxidative phosphorylation.52 chRCC are
particularly enriched in mutations involving metabolic
genes, including deleterious mutations in PDHB (which encodes
a critical component of the pyruvate dehydrogenase
complex) and PRKAG2 (encoding one of three subunits of
AMP-Kinase (AMPK)). These findings reinforce the longstanding
implication that metabolic derangements in RCC
can contribute to oncogenesis.
Integrated transcriptome and protein expression analysis
(using reverse protein phase arrays) in TCGA revealed
distinct metabolic patterns both across and within histological
subtypes of RCC.24,5. ccRCC was characterized by overexpression
of glycolytic and fatty acid synthesis enzymes as
well as suppression of Krebs and ETC programs. This contrasts
with pRCC and chRCC, which generally expressed intermediate
and high levels of Krebs and ETC proteins,
respectively.24 Decreased expression of AMPK (which inhibits
fatty acid synthesis and mTOR), and increased ribose
sugar metabolism, correlated with higher stage and worse
prognosis in ccRCC.33 The CIMP subtype of pRCC demonstrated
the highest level of ribose sugar metabolism across
all RCC subtypes. A metabolically divergent subset of
chRCC was identified, which demonstrated decreased levels