The genomic landscape of testicular germ cell tumours: a timeline perspective from susceptibility
to treatment
Kevin Litchfield1, Max A Levy1, Robert A Huddart2, Janet Shipley3, Clare Turnbull1,4
1. Division of Genetics and Epidemiology, The Institute of Cancer Research, London, UK
2. Academic Radiotherapy Unit, Institute of Cancer Research, Sutton, Surrey, UK
3. Divisions of Molecular Pathology and Cancer Therapeutics, The Institute of Cancer Research,
London, UK
4. William Harvey Research Institute, Queen Mary University, London, UK
Correspondence to: Clare Turnbull, Division of Genetics and Epidemiology, The Institute of Cancer
Research, London, UK; Tel: ++44 (0) 208 722 4485; E-mail: [email protected]
Key words: Testicular germ cell tumours, TGCT, Predisposition, Cancer genomics
Running title: The genomic landscape of TGCT
SUMMARY
The genomic landscape of testicular germ cell tumour (TGCT) can be summarised through four
overarching hypotheses. Firstly that TGCT risk is dominated by inherited genetic factors, which
determine nearly half of all disease risk, and are highly polygenic in nature. Secondly KIT/KITLG
signalling is the major pathway implicated to date in TGCT formation, featuring both as a
predisposition risk factor and a somatic driver event. Results from GWAS have also consistently
implicated other closely related pathways, involving male germ cell development and sex
determination. Thirdly TGCT has a unique model of disease formation, with tumours universally
stemming from a non-invasive precursor lesion of likely foetal origins, which lies dormant through
childhood into adolescence and then eventually begins malignant growth in early-adulthood.
Formation of a 12p isochromosome, a hallmark of TGCT observed in nearly all tumours, is likely to be
a key triggering event for malignant transformation. Fourthly and finally TGCTs have been shown to
have a distinctive somatic mutational profile, with a low rate of point mutations contrasted by
frequent large-scale chromosomal gains. While these four hypotheses by no means constitute a
complete model that explains TGCT tumorigenesis, it is clear that recent advances in genomic
technologies have enabled significant progress in describing and understanding the disease. Further
advancing our understanding of the genomic basis of TGCT offers clear opportunity for clinical
benefit, in terms of preventing invasive cancer arising in young men, decreasing the burden of
chemotherapy-related survivorship issues and reducing mortality in the minority with treatmentrefractory disease state.
Introduction
Testicular germ cell tumour (TGCT) is the most common malignancy affecting young men, with a
mean age at diagnosis of 36 years1,2. In western industrialised nations the incidence of TGCT has
approximately doubled over the last four decades3, strongly motivating the need to understand its
biological and genetic basis. Whilst cure rates for TGCTs are typically high, due to the effectiveness
of platinum-based chemotherapy, survivorship comes with a burden of longer-term sequelae,
including increased risk of metabolic syndrome, infertility and secondary cancer4-6. Furthermore
there are limited options for patients who demonstrate platinum resistance, a group for whom fiveyear survival is only 10-15%7. The advent of genome-wide array and next-generation sequencing
technologies has heralded rich insights into TGCT oncogenesis, revealing genetic determinants of the
strong inherited risk, as well as a distinctive somatic mutational profile. In this review we discuss
these recent findings and outline the emerging genomic architecture of TGCT using a time-line
approach (figure 1), as well as highlighting the potential implications for disease treatment and drug
resistance.
Pre-Cancer: TGCT predisposition
Family, twin and migration studies support a strong inherited genetic basis to TGCT susceptibility,
with brothers of cases having a substantially increased risk of TGCT, estimates of which range from
4-5 to 8-10 fold increased8-11. Recent population-wide analysis of the Swedish family cancer
database has estimated the heritability of TGCT to be 48.9%12, suggesting nearly half of all TGCT risk
is determined by inherited genetic factors. Both of these values suggest a substantially higher
component of genetic susceptibility than most common tumour types, for which sibling relative risk
is typically ~2-fold increased and heritability in the region of 30%13,14. Consistent with a strong
inherited genetic component, disease incidence also varies considerably between ethnic groups,
with white American men being at a 5 times higher risk than their black American counterparts9.
Elucidation of these genetic risk factors was first approached through genome-wide linkage analysis,
which essentially yielded null results, concluding that a single high penetrance risk locus is unlikely to
exist 15,16. Through subsequent candidate association studies a 1.6Mb-deletion on chromosome Y
(designated gr/gr) was identified which confers a two-fold elevation of TGCT risk17. The frequency of
the gr/gr deletion is low however (observed in ~2% of cases), meaning it is likely to account for only
~0.5% of the total genetic (excess familial) risk of TGCT. Furthermore whole-exome sequencing,
which has been recently undertaken in >900 TGCT cases including >150 multiplex TGCT families by
our group and others, has also so far failed to identify high-risk TGCT predisposition genes of
significant frequency (unpublished data). Coding variants conferring intermediate-high risk of TGCT
may still exist however, but they are likely to each only account for a small proportion of TGCT
families. Indeed, a number of additional strands of evidence also instead support a highly polygenic
model of TGCT susceptibility, with disease risk enshrined in the co-inheritance of multiple risk
variants, many of which are common18. Indeed heritability estimates using genomic datasets suggest
that over three quarters of the inherited genetic risk is transmitted through common variation12.
Exposition of this common component has come from genome-wide association studies (GWAS),
which have so far identified 25 risk loci for TGCT (Table 1)19-28. Although each individual common
variant only makes a modest contribution to the genetic risk of TGCT, it is of note that the TGCT risk
SNPs identified to date include individual SNPs carrying per allele odds ratios (OR) in excess of 2.6,
among the highest reported in GWAS of any disease phenotype29.
In addition to high effect sizes, the associations identified by GWAS have provided novel biological
insights into the development of TGCT, highlighting in particular genes related to KIT/KITLG
signalling, other pathways of male germ cell development, sex determination and genomic integrity
(table1, figure 2). The first risk locus was identified at 12q21, containing KITLG, and this remains the
strongest genetic risk factor for TGCT (per allele odd ratio>2.6). KITLG encodes the ligand for the
membrane bound receptor tyrosine kinase KIT, which regulates the survival, proliferation and
migration of germ cells30. Prior to elucidation of this locus by GWAS, KIT signaling had already been
strongly implicated in TGCT, with mice null for either KIT or KITLG being shown to be infertile31 and
mice with a heterozygous deletion encompassing just the KITLG region having demonstrated a twofold elevated risk of TGCT32. Functional studies in human cell lines have elucidated the likely
mechanism of TGCT risk at this locus at 12q21, showing an allele-specific p53 binding effect and
subsequent upregulation of KITLG expression33. As well as 12q21, GWAS has identified three other
risk loci containing genes related to KIT, 5q31 (SPRY4), 6p21 (BAK1) and 11q14.1 (GAB2). SPRY4
inhibits the mitogen-activated protein kinase (MAPK3/MAPK1) pathway, which is in turn activated
by KIT signalling34, whilst apoptosis promoting protein BAK1 is also regulated by KIT35. GAB2, a
member of the GRB2-associated binding protein (GAB) family, also associates with KIT and forms a
critical part of this KIT/KITLG signalling cascade36. GWAS, together with wide-ranging functional
evidence, has established the KIT pathway as a central tenet in TGCT formation, activated not only
during overt tumourigenesis but also predefining disease risk.
As well as KIT, other pathways of male germ cell development have also been repeatedly highlighted
in TGCT GWAS, including genes at 3p24.2 (DAZL) and 8q13.3 (PRDM14). DAZL encodes an RNAbinding protein that has been shown in expression studies in human primordial germ cells to have a
central role in the early differentiation of primordial germ cells: DAZL (-/-) mice are rendered
infertile, with differentiation of the germ cells halted at the type A spermatogonia stage 37. PRDM14
encodes a transcriptional regulator and modulates primordial germ cell (PGC) specification through
control of expression of key pluripotency genes, such as POU5F1 (OCT4), NANOG and SOX238. A
distinct but closely related pathway also recurrently highlighted through GWAS is sex determination,
in particular through locus 9p24 which contains a single gene DMRT1. In many species, high
expression of DMRT1 is required for differentiation along the male lineage39; furthermore, DMRT1
deficiency is also associated with testicular cancer in mouse models: 90% of DMRT1 −/− 129Sv mice
develop teratomas compared to <1% of DMRT1 +/+ mice 40. Other biological pathways putatively
associated with TGCT through GWAS relate to genomic integrity, such as telomerase function,
centrosome cycle and DNA damage repair. It is important to note however, that while many of the
genes and pathways implicated through GWAS have extensive pre-existing evidence to support their
role in TGCT formation, a GWAS signal points only to a genomic block of linkage disequilibrium.
Hence further functional fine mapping is urgently required to validate and expand this putative
attribution of signal to particular genes.
The current known TGCT risk loci collectively explain ~25% of the excess sibling risk of TGCT and
accordingly there are likely to be multiple additional TGCT susceptibility loci still to be identified. This
notion is supported by genome-wide complex trait analysis41 recently conducted for TGCT42, which
suggested that at least 50 additional risk SNPs of OR ~ 1.2 are likely to exist. Or, more plausibly, with
a trailing set of effect sizes (OR = 1.01~1.20), the undiscovered set could be significantly larger. Thus
the prevailing evidence supports a genomic architecture of TGCT predisposition dominated by
multiple common risk loci, perturbing a consistent set of biological pathways.
In addition to genetic predisposition, research into environmental risk factors has been of increasing
interest, on account of the rapidly rising incidence of TGCT. Although there have been many
hypotheses explored around maternal and in utero exposures, to date there has been no compelling
evidence implicating specific environmental risk factors in the aetiology of TGCT43. The rise in TGCT
incidence has been accompanied by a concordant fall in global sperm counts, prompting the
hypothesis that TGCT may be aetiologically linked to male reproductive abnormalities as a part of
the so-called 'testicular dysgenesis syndrome’ (TDS)44. Indeed genetic studies have found shared risk
factors, such as the chromosome Y gr/gr deletion, which predisposes to infertility as well as TGCT17.
Large meta-analysis shows that the sub-fertile men have a 1.6-fold increased risk of TGCT and in a
Danish study reviewing >200 contralateral biopsy specimens in men after a first TGCT, showed
evidence of testicular dysgenesis in 25% of samples. Other recognised risk factors for TGCT include a
history of undescended testis (UDT) and other testicular abnormalities such as hypogonadism and
microlithiasis45,46. Elevated TGCT risk is also associated with Down’s (trisomy 21)47 and Klinefelter’s
(47XXY karyotype)48 syndromes and intriguingly somatic gains of both chromosomes 21 and X are
frequently observed in TGCTs49. Furthermore examples of ovarian germ cell tumour have also been
recurrently reported in pedigrees along with TGCT, suggesting shared oncogenic pathways50. Finally
epigenetic influences on TGCT predisposition have attracted widespread recent interest, due to both
the known vulnerability of epigenetic reprogramming to environmental exposures as well as the
established role of DNA methylation in the formation of many cancer types51. Proof of concept for
this has been demonstrated at GWAS loci 5q31 (SPRY4) in analysis of a Scandinavian dataset, with a
strong parent-of-origin effect with TGCT risk associated with maternal (OR = 1.72) but not paternal
(OR = 0.99) transmission of the risk allele52. This result is consistent with an imprinting effect,
whereby epigenetic mechanisms control gene expression and risk is silenced in paternally inherited
alleles.
Precursor state: tumour origin
It is widely accepted that TGCT arises from a non-invasive precursor lesion termed intratubular germ
cell neoplasia unclassified (ITGCN, formerly known as carcinoma in situ, CIS), located in the
spermatogonial niche of the seminiferous tubule of the adult human testis53. Molecular observations
of ITGCN suggest close phenotypic similarities to primordial germ cells (PGCs), or gonocytes, with the
possibility that during fetal development normal germ cells transform into ITGCN54-56. This is
consistent with the clinical finding of ITGCN in a limited number of mid-trimester fetuses57. The
incidence of ITGCN is equivalent to the lifetime risk of developing TGCT, which combined together
with longitudinal studies, have led to wide acceptance that ITGCN progresses to invasive TGCT in
most patients.
Expression of pluripotency factors OCT3/4 and NANOG, together with imprinting patterns,
characterise ITGCN cells as the malignant equivalent of PGCs/gonocytes. The genetic features of
ITGCN have not been rigorously profiled; however targeted sequencing studies have documented
examples of KIT mutation, which are well described in TGCT (see section below on somatic
genetics)58. Following a primary TGCT, men have a 12-fold elevated risk of disease in the
contralateral testicle59: a pertinent question is whether these bilateral tumours originate from a
common pre-invasive ancestor or whether other predisposing mechanisms cause multiple
independent TGCTs. In this context, the identification of concordant KIT mutations in bilateral TGCTs
was initially proposed as evidence of a common precursor lesion with mutation occurring during
early fetal development58. However in larger subsequent studies this pattern was not replicated60.
The question of mutational concordance in bilateral disease and tumour origin remain compelling
lines of inquiry, with potential to significantly inform TGCT aetiology.
Progression to cancer: tumour subtypes
It is widely accepted that the pre-invasive ITGCN cells lie dormant in the gonad during fetal
development, infant and childhood life. Progression towards invasive TGCT then occurs following
puberty, when ITGCN cells begin to proliferate, likely due to hormonal influences61,62. The presence
of ITGCN can be ascertained via testicular biopsy. Following detection of ITGCN, invasive TGCT is
observed in 50% of cases within 5 years, 70% of cases within 7 years and, eventually, in almost all
cases 63. ITGCN transforms into two main TGCT histological subtypes: seminomas, which resemble
undifferentiated PGCs, and non-seminomas, which show differing degrees of differentiation.
Seminomas generally arise later in life, with a mean age at diagnosis of 35 years compared with 25
years for non-seminomas. Transcriptional profiling has identified differing gene signatures for each
sub-type, with spermatogenesis-associated genes (such as PRAME, MAGEA4 and SPAG1) overexpressed in seminomas and regulatory genes (DNMT3B and SOX2) over-expressed in nonseminoma subtypes64,65. Despite these morphological differences however, several observations
suggest commonality of underlying oncogenic pathways and that histological differences are likely to
develop late in the tumour formation. For example 15% of TGCTs have a mixed pathology 66 and
discordant histologies are frequently reported in bilateral/familial cases 67,68. In addition GWAS
results, when stratified by histology, have been noteworthy for their consistent lack of difference in
genetic risk between seminoma vs non-seminoma cases, despite sufficient power for
detection19,22,24.
Clinical presentation: somatic mutational profile of TGCTs
Study of TGCT has revealed these tumours and their subtypes to have a similar mutational profile,
with a markedly low rate of somatic mutation. In fact recent whole exome sequencing studies49,69
have reported a mean rate of 0.5 somatic mutations per megabase (Mb) for TGCT, a rate
significantly lower than other adult solid tumours such as lung cancer (8.0/Mb) and melanoma
(11.0/Mb). Overall the TGCT mutation rate is observed to be some eight times lower than the pancancer average (reported at 4.0/Mb across 27 tumour types70), and only marginally greater than
paediatric cancers such as Ewing sarcoma (0.3/Mb) and Rhabdoid tumour (0.15/Mb). This low
mutational frequency is consistent with the hypothesised embryonic origins of TGCT54. The most
frequently mutated TGCT driver gene is KIT, where activating mutations are observed in 25-30% of
seminomas71,72. Mutations are found to cluster in the juxta membrane and kinase encoding domains
of KIT71,72, with by far the most frequent mutations being D816V/H. KIT mutation is also associated
with other cancer types including myeloid leukaemias73. In addition to KIT, mutations in KRAS have
also consistently been reported, albeit at a lower frequency of ~5-10%. Subsequent to the initial
targeted and hypothesis-driven analyses of individual genes/gene sets in TGCTs through which KIT
and KRAS mutations were reported, whole-exome profiling of TGCTs has been undertaken but no
other driver genes with higher mutational frequency have been observed. Several lower frequency
driver candidates have been proposed, however further large scale sequencing studies (>300
tumours) will be required to robustly assess this. TGCTs are also noted for their general absence of
p53 mutation, with wild-type (wt) p53 retained in >97% of testicular tumours, compared to only
~50% of all cancers overall74-76. However, p53 signalling may still be disrupted in TGCTs but through
indirect mechanisms such as overexpression of MDM2/MDMX or the activity of micro RNAs such as
miR-372 and miR-37377
In contrast to the low rate of point mutations, TGCTs are highly aneuploid, with large scale copy
number variations (CNV) frequently observed. This is proposed to be derived from an initial
tetraploidisation event followed by subsequent chromosome loss78. Gain of chromosomal material
from 12p is noted in virtually all tumours, and most commonly manifests as a 12p isochromosome
i(12p)79-81. i(12p) is widely regarded as the hallmark of TGCT and minimal mapping of the 12p region
involved in cases without an i(12p), undertaken to identify causal driver gene(s), has been described
in several studies82,83. Several candidates have been proposed, including KRAS at 12p11.2-p12.1 and
a cluster of stem cell associated genes such as NANOG at 12p13.31, all of which are overexpressed in
TGCTs82,83. However definitive evidence is still lacking as to both the exact driver gene(s) and
functional mechanism(s) underlying i(12p). In addition to minimal mapping, the study of i(12p) in
precursor ITGCN cells has also been of significant interest, with the majority of studies
demonstrating that pre-malignant ITGCN (i.e. with no adjacent invasive TGCT) does not contain
i(12p) but malignant ITGCN does84,85. Hence it is hypothesised that i(12p) is not required for ITGCN
formation but is the triggering event responsible for driving invasive growth. Overall i(12p) is widely
accepted as a critical step in TGCT pathogenesis.
As well as i(12p), recurring gain of chromosomes 7, 8, 21, 22, X and loss of chromosome Y are also
consistently reported at a frequency of 25%-40% TGCTs49,79-81,86-89. In addition to these large scale
CNVs (>1Mb), specific amplification of the KIT gene at 4q12 leads to overexpression and is an
alternative mechanism to activating mutations that enhances KIT signalling in seminomas71.
Recently two independent studies have sought to further profile focal CNVs (<1Mb) in TGCTs49,90,91,
with both studies reporting a recurrent small scale focal gain at 2q32.1, encompassing gene FSIP2,
present in 15-20% of each of the independent TGCT cohorts. FSIP2 encodes a protein which forms
part of the fibrous sheath (FS), a cytoskeletal structure located in the principle piece region of the
sperm flagellum. FSIP2 expression is testis-specific 92 and it is hypothesised to act as a linker protein,
binding AKAP4 to the FS92. Dysplasia of the FS and mutations in AKAP4 have also both been linked to
male infertility93,94. Aside from 2q32.1, these studies of focal CNVs also identified a number of other
candidate regions with recurring focal CNV which, conversely, failed to replicate across studies.
Finally the somatic mutational profile of TGCTs show strong similarities across histological subtypes,
with almost identical mutation rates in both seminomas and non-seminomas (0.50 and 0.49
mutations per Mb respectively) and i(12p) being an archetypical feature of both49. There are two
consistent differences known between the subtypes; firstly that KIT mutations are found to be
largely unique to seminomas and secondly CNV profiles show that seminomas are hypertriploid
whereas non-seminomas are hypotriploid. Additional more subtle differences between the subtypes
have been reported but larger in-depth studies are required to further clarify these95.The other
clinically associated feature of note is a correlation between the somatic mutational rate and patient
age, with a significantly higher rate in older patients, which are more frequently seminomas. This
suggests the majority of somatic TGCT mutations are passengers that simply accumulate with
patient age, following their early initiation of the disease.
Post-treatment: genetic characteristics and response to chemotherapy
Over recent decades there have been significant advances in the treatment of TGCT, owing to the
exceptional sensitivity of malignant testicular germ cells to platinum-based chemotherapies. Today a
cure is expected in over 95% of all patients and in around 80% of patients presenting with metastatic
disease96,97. Despite these high cure rates there are a minority of men who exhibit resistance to
platinum, and for whom treatment options are limited and long term survival prospects are poor.
Indeed the average age of death for these men is 32 years; hence an urgent clinical priority is the
identification of novel treatment options, efficacious in the platinum-refractory setting. Several
hypotheses have been proposed to explain both the exceptional sensitivity of TGCTs to platinum as
well as the corresponding mechanisms of resistance. The overall low somatic mutation rate of TGCTs
is likely to be a contributing factor to the hypersensitive chemo-response, with a reduced genetic
diversity and lower probability of pre-existing mutation in a resistance driving gene. Another model
proposes that treatment sensitivity is due to the inability of TGCTs to repair treatment-induced DNA
damage, due to the low expression of DNA repair genes such as ERCC198. Conversely it is proposed
that resistant TGCTs acquire an enhanced capacity to repair DNA and avoid apoptosis. It is
noteworthy therefore, that sensitivity to poly(ADPribose) polymerase (PARP) inhibition has been
observed in platinum resistant TGCT cell lines, suggesting these cells have deficiency in homologous
recombination pathways99. An additional feature which may in part explain TGCT platinum sensitivity
is that, unlike most other solid tumours, TP53 is rarely mutated in TGCTs. In fact p53 expression is
not only retained at normal levels in TGCTs but is frequently upregulated, which is proposed as a
major factor in explaining platinum hypersensitivity100. A variety of other models have also been
proposed, including a hypersensitive apoptotic response to platinum or alternatively mechanisms
related to epigenetics, which hypothesise that sensitivity is due to the hypomethylation status of
some TGCTs 101.
The study of platinum resistance has also been approached clinically, through comparison of
treatment sensitive/resistant patient tumour samples. Initial research102 highlighted BRAF mutation,
with the V600E variant present in 26% of resistant versus 1% of sensitive tumours, however this
finding failed to replicate in larger subsequent studies103. Hotspot mutational analysis identified
PIK3/AKT variants in 7% of resistant tumours (n=46), versus 0% in sensitive samples (n=24), as well
as mutations RAS, and FGFR3 genes103. Whole exome sequencing of two treatment resistant
tumours identified missense mutation in DNA repair gene XRCC249, suggesting an activation of DNA
repair pathways in response to treatment induced damage. Interestingly mutation of related gene
ERCC2 has been found to correlate with platinum response in a recent exome-wide study of 50
urothelial carcinomas104. Aside from XRCC2 mutation, the exome profile of resistant TGCT appears to
be comparable to sensitive tumours, with a consistent mutational load of 0.5 mutations per Mb,
albeit based on only two samples. The role of KIT has also been studied in treatment response;
however clinical trial of imatinib (which inhibits activated KIT) showed no anti-tumour activity
against KIT-positive platinum-refractory TGCTs105. CCND1 overexpression has also been associated
with cisplatin resistance in TGCTs as well as other tumour types106,107. As well as somatic research,
several germline pharmacogenomics studies have also been completed, with polymorphism of the
gene PAI-1 being shown to act as a predictor of negative platinum response, with homozygote risk
allele carriers having a hazard ratio of 2.69 for TGCT-related death108. However due to the difficulty
in obtaining large case sample sizes, with both available DNA and clinical data on treatment
response, the majority of germline biomarkers have not yet been validated in large cohorts. Thus the
mechanisms driving platinum resistance remain unclear and are likely to involve a sequence of
genetic/epigenetic changes, delineation of which will be critical to identifying improved treatment
options and prognostic biomarkers.
Post-cancer: long term survivorship issues
The success of treating TGCT has been accompanied by an increase in survivorship issues, caused by
the long-term consequences associated with platinum-based chemotherapy. A number of associated
morbidities have been identified, including neurotoxicity, hypogonadism, infertility and increased
risks of both cardiovascular disease and secondary cancer4-6,109. For example neuropathic symptoms
are reported in 20% of TGCT survivors at 2-years post combination chemotherapy, and cisplatin
treatment is associated with ototoxicity (high-frequency hearing loss and tinnitus). Genetic studies
have identified polymorphisms associated with predisposition to these side effects, such variants in
the gene GSTP1 which are associated with an increased risk of peripheral neuropathy and
ototoxicity110,111. In terms of hypogonadism, while a proportion of patients will already have low
testosterone levels pre-TGCT (through TDS), chemo- and radio-therapy treatments have been shown
to increase hypogonadism rates from 11% to 38%112. In addition the deleterious effects of
chemotherapy on spermatogenesis have been well documented5, with 20-30% of men being left
infertile post-TGCT treatment106. Moreover there is additional risk in men whose sperm counts do
recover, with sperm DNA damage after chemotherapy remaining elevated 2-years post treatment,
compromising gamete quality and causing potential risk for offspring113. Finally both cardiovascular
and secondary cancer risks are elevated following chemotherapy, with an increased incidence in
particular of metabolic syndrome (central obesity, dyslipidemia and hypertension) and secondary
leukemia. A large scale study of >40,000 TGCT survivors demonstrated a lifetime risk of secondary
cancer of 31% for seminoma and 36% for non-seminoma patients respectively, compared with 23%
for the general population114.
Other potential applications of genetics in clinical management of TGCT
Whilst there is currently no clinical application of genetics in predicting disease or stratifying clinical
care, a number of clinically-orientated studies are underway to explore potential utility. Disease
prevention has been a central focus, with the high effect-size of TGCT GWAS SNPs suggesting genetic
screening could be clinically useful29. Preliminary assessment of genetic profiling has been
approached by two groups20,115, using polygenic risk scoring (PRS) models to consider the combined
effect of all known SNPs on TGCT risk. The latest such model demonstrates that the top 1% of men
with the highest genetic risk of TGCT carry a 10.4-fold elevated disease risk compared to the
population average. Comparing this to equivalent PRS models for ovarian, breast and prostate,
where the top 1% of highest risk genotypes have only a 2-fold, 3-fold and 5-fold increased risk
respectively 116, shows the TGCT SNPs have useful power in terms of risk discrimination. However,
whilst the SNP set for TGCT is comparatively predictive for disease, the case for screening is weaker
as TGCT is of much lower frequency than these cancers and has an excellent prognosis. The rare
nature of TGCT (lifetime male Caucasian absolute risk of 0.5%) mean high relative risks (RR) translate
into only modest absolute risk: the 1% of men with the highest risk of TGCT have an impressive RR
of 10.4 but still only have a ~5% lifetime risk of TGCT. It may be possible to combine genetic/nongenetic risk factors to improve the discriminatory performance of the risk profiling. For example one
model testing this has predicted that men in the top 1% of genetic risk, and with a history of UDT,
would have a RR of 50 115, equating to a lifetime TGCT risk of ~25%. However this model assumes full
independence of effects between genetic and non-genetic factors, an assumption not yet validated
through the relevant modelling of large datasets combining clinical and genetic data. Currently men
at high risk of TGCT would proceed to bilateral testicular biopsy to assess for presence of the
premalignant ITGCN lesion, proceeding to orchidectomy if detected. The invasive nature of
testicular biopsy necessarily currently restricts development of TGCT screening programs.
Improvement in sensitivity and specificity of semen assay, an emerging non-invasive diagnostic tool
by which presence/absence of ITGCN is detected, may in the future impact on the viability of TGCT
screening117,118. Such an approach would be taking advantage of the unique characteristic of ITGCN
as a faithful precursor lesion of TGCT, clinically detectable from adolescence.
Future directions
Large-scale GWAS is expected to identify multiple additional TGCT risk variants, as sample sizes
rapidly increase. For example the largest TGCT meta-GWAS to date contains 3,556 cases and 13,969
controls, with a 7.2% power to detect common variants119 (defined as OR > 1.25 and MAF > 5%).
Sample sizes are expected to soon reach 10,000 cases, through large collaborative efforts such as
the Testicular Cancer Association Consortium (TECAC, http://www.tecac.org/). At the level of 10,000
cases, this will increase the power to detect common variants to 88% (based on the same OR/MAF
as above, with assumed 50,000 controls). Following these studies, it is expected that a significantly
higher proportion of heritable risk factors for TGCT will be identified, offering a greater potential for
personalised risk prediction. In addition the functional fine mapping of TGCT risk loci will become a
major focus moving forward, as genetic/functional evidence is becoming increasingly important for
drug target identification and validation. Indeed recent evidence directly from big pharma shows
that genetically validated targets are twice as likely to be successful in clinical development120. A
wide range of functional mapping techniques are likely to be informative, including: targeted
resequencing, expression quantitative trait loci analysis, transcription factor binding analysis,
chromosome conformation capture techniques, methylation profiling as well direct genotype
manipulation in cell line models. Utility of these techniques to build an integrated functional view of
oncogenic pathways will be critical, and likely to be particularly informative for TGCT, given the
strong commonality between historic biological evidence, recent genetic findings and overlapping
conditions such as TDS.
Aside from common SNPs, the influence of rare predisposition variants also remains yet to be
revealed. While a single high penetrance “TGCT gene” is unlikely to exist, higher-risk mutations may
yet be identified through large-scale NGS analyses of TGCT families. In terms of additional exposition
of somatic variation in TGCT, comprehensive profiling of 150 TGCTs has been undertaken by The
Cancer Genome Atlas (TCGA) project (http://cancergenome.nih.gov/). Exome sequencing data in the
TCGA project, when meta-analysed together with previous data49, have confirmed in this series the
low mutation rate and absence of other consistent driver genes except KIT and KRAS (AACR 2015
abstract 2986). Additional studies are underway to comprehensively study the genomics of platinum
resistance.
Historically there are two main animal models predisposed to developing TGCT: a zebrafish model
carrying BMPR1B mutations that disrupt BMP signalling, and 129Sv strain mice, with complete loss
of DMRT1, which display high incidence of teratoma40. While both these models provide relevant
biological insight into TGCT development, their relevance to human tumourigenesis is limited, as
genes such as BMPR1B have not been found to be mutated in human TGCT. More recently a new
zebrafish model with inactivating mutations in the ciliary protein LRCC50 has also been reported,
with 80% incidence of seminoma, suggesting an intriguing functional link between TGCT and cilia
function121. Despite the utility of these models and others, no model currently exists to reflect the
biology of human non-seminomas, such as embryonal carcinoma or yolk sac tumour. Given nonseminomas are generally more aggressive, with higher rates of platinum resistance, there is an
urgent need for new models representing these subtypes. Future development and utilisation of
these animal systems will provide novel insights into the underlying molecular mechanisms of TGCT,
as well as useful tools to test therapeutic strategies.
Given the exceptional sensitivity of TGCTs to platinum, near-term therapeutic advancements are
likely to focus on chemotherapy in combination with other synergistic compounds. A recent example
of this is a trial of bevacizumab together with high-dose chemotherapy, exploiting the over
expression of VEGF in metastatic TGCT122. Although small in size (n=37) this study demonstrated
promising results in otherwise refractory cases, with an overall survival rate of 58% at 46 months,
albeit with 11% of patients dying from treatment-related toxicities123. Further integrated
genomic/transcriptomic profiling of refractory TGCTs may inform additional rational treatment
combinations, of platinum together with other targeted molecules. In the longer term
immunotherapeutic strategies have been postulated as a less toxic treatment alternative, with a
recent study showing frequent expression of the programmed death receptor ligand 1 (PD-L1) in
TGCTs124. This finding is somewhat counterintuitive, given a high mutational load is hypothesised to
be a pre-requisite for immune-mediated response, as evidenced by the effectiveness of PD-L1
inhibition in highly mutated tumours such as melanoma and lung cancer. Clearly TGCT, with its low
mutation rate, does not fit this same profile; however increasing evidence now suggests that tumour
immune response is mediated by a more complex set of factors125.
Finally, attention will continue to focus on longstanding unresolved questions of TGCT oncogenesis,
such as tumour origin and the functional mechanisms of i(12p). With regard to tumour origin,
detailed profiling of bilateral tumours from the same case, together with the ITGCN precursor cells,
would comprehensively address the question of common ancestry, although a more immediately
tractable approach may come from use of model systems in the first instance. Lastly, insights into
the functional significance of i(12p) maybe forthcoming through a number of approaches, such as
integrated functional analysis utilising TCGA data, or long read / whole genome sequencing to finely
map the structure of i(12p), detecting any complex rearrangements or gene fusion events.
TABLES AND FIGURES LEGENDS
FIGURE 1 - The genomic features of TGCT, summarised using a timeline approach.
FIGURE 2 - Risk pathways implicated for TGCT through GWAS, in clockwise order staring from top
left panel: i) KIT/KITLG Signalling – all genes related to this pathway using STRING database126 with
genes implicated in TGCT risk loci are highlighted with a red circle, ii) Other pathways of male germ
cell development – genes implicated in TGCT risk loci are highlighted, iii) Sex determination – genes
implicated in TGCT risk loci are highlighted, iv) Chromosomal assembly/DNA Repair – genes
implicated in TGCT risk loci are highlighted.
TABLE 1 - TGCT predisposition loci identified to date, presented in chronological order with
functional grouping based126. For loci with multiple reported SNPs the marker listed is taken from
first study in the reference column.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Bray, F., Ferlay, J., Devesa, S.S., McGlynn, K.A. & Moller, H. Interpreting the international
trends in testicular seminoma and nonseminoma incidence. Nat Clin Pract Urol 3, 532-43
(2006).
Ruf, C.G. et al. Changes in epidemiologic features of testicular germ cell cancer: age at
diagnosis and relative frequency of seminoma are constantly and significantly increasing.
Urol Oncol 32, 33 e1-6 (2014).
Le Cornet, C. et al. Testicular cancer incidence to rise by 25% by 2025 in Europe? Modelbased predictions in 40 countries using population-based registry data. Eur J Cancer 50, 8319 (2014).
de Haas, E.C. et al. Early development of the metabolic syndrome after chemotherapy for
testicular cancer. Ann Oncol 24, 749-55 (2013).
Bujan, L. et al. Impact of chemotherapy and radiotherapy for testicular germ cell tumors on
spermatogenesis and sperm DNA: a multicenter prospective study from the CECOS network.
Fertil Steril 100, 673-80 (2013).
Rusner, C. et al. Risk of second primary cancers after testicular cancer in East and West
Germany: a focus on contralateral testicular cancers. Asian J Androl 16, 285-9 (2014).
Nitzsche, B. et al. Anti-tumour activity of two novel compounds in cisplatin-resistant
testicular germ cell cancer. Br J Cancer 107, 1853-63 (2012).
Swerdlow, A.J., De Stavola, B.L., Swanwick, M.A. & Maconochie, N.E. Risks of breast and
testicular cancers in young adult twins in England and Wales: evidence on prenatal and
genetic aetiology. Lancet 350, 1723-8 (1997).
McGlynn, K.A., Devesa, S.S., Graubard, B.I. & Castle, P.E. Increasing incidence of testicular
germ cell tumors among black men in the United States. J Clin Oncol 23, 5757-61 (2005).
Hemminki, K. & Li, X. Familial risk in testicular cancer as a clue to a heritable and
environmental aetiology. British Journal of Cancer 90, 1765-1770 (2004).
Kharazmi, E. et al. Cancer Risk in Relatives of Testicular Cancer Patients by Histology Type
and Age at Diagnosis: A Joint Study from Five Nordic Countries. Eur Urol 68, 283-9 (2015).
Litchfield, K. et al. Quantifying the heritability of testicular germ cell tumour using both
population-based and genomic approaches. Sci Rep 5, 13889 (2015).
Locatelli, I., Lichtenstein, P. & Yashin, A.I. The heritability of breast cancer: a Bayesian
correlated frailty model applied to Swedish twins data. Twin Res 7, 182-91 (2004).
Kampman, E. A first-degree relative with colorectal cancer: what are we missing? Cancer
Epidemiol Biomarkers Prev 16, 1-3 (2007).
Crockford, G.P. et al. Genome-wide linkage screen for testicular germ cell tumour
susceptibility loci. Hum Mol Genet 15, 443-51 (2006).
Rapley, E.A. et al. Localization to Xq27 of a susceptibility gene for testicular germ-cell
tumours. Nat.Genet. 24, 197-200 (2000).
Nathanson, K.L. et al. The Y deletion gr/gr and susceptibility to testicular germ cell tumor.
Am J Hum Genet 77, 1034-43 (2005).
Pathak, A. et al. Prospectively Identified Incident Testicular Cancer Risk in a Familial
Testicular Cancer Cohort. Cancer Epidemiol Biomarkers Prev 24, 1614-21 (2015).
Rapley, E.A. et al. A genome-wide association study of testicular germ cell tumor. Nat Genet
41, 807-10 (2009).
Turnbull, C. & Rahman, N. Genome-wide association studies provide new insights into the
genetic basis of testicular germ-cell tumour. Int J Androl 34, e86-96; discussion e96-7 (2011).
Kanetsky, P.A. et al. Common variation in KITLG and at 5q31.3 predisposes to testicular germ
cell cancer. Nat Genet 41, 811-5 (2009).
Turnbull, C. et al. Variants near DMRT1, TERT and ATF7IP are associated with testicular germ
cell cancer. Nat Genet 42, 604-7 (2010).
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
Kanetsky, P.A. et al. A second independent locus within DMRT1 is associated with testicular
germ cell tumor susceptibility. Hum Mol Genet 20, 3109-17 (2011).
Ruark, E. et al. Identification of nine new susceptibility loci for testicular cancer, including
variants near DAZL and PRDM14. Nat Genet 45, 686-9 (2013).
Bojesen, S.E. et al. Multiple independent variants at the TERT locus are associated with
telomere length and risks of breast and ovarian cancer. Nat Genet 45, 371-84, 384e1-2
(2013).
Chung, C.C. et al. Meta-analysis identifies four new loci associated with testicular germ cell
tumor. Nat Genet 45, 680-5 (2013).
Litchfield, K. et al. Multi-stage genome wide association study identifies new susceptibility
locus for testicular germ cell tumour on chromosome 3q25. Hum Mol Genet (2014).
Kristiansen, W. et al. Two new loci and gene sets related to sex determination and cancer
progression are associated with susceptibility to testicular germ cell tumor. Hum Mol Genet
(2015).
Chanock, S. High marks for GWAS. Nat Genet 41, 765-6 (2009).
Boldajipour, B. & Raz, E. What is left behind--quality control in germ cell migration. Sci STKE
2007, pe16 (2007).
Roskoski, R., Jr. Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.
Biochem Biophys Res Commun 337, 1-13 (2005).
Heaney, J.D., Lam, M.Y., Michelson, M.V. & Nadeau, J.H. Loss of the transmembrane but not
the soluble kit ligand isoform increases testicular germ cell tumor susceptibility in mice.
Cancer Res 68, 5193-7 (2008).
Zeron-Medina, J. et al. A polymorphic p53 response element in KIT ligand influences cancer
risk and has undergone natural selection. Cell 155, 410-22 (2013).
Sasaki, A. et al. Mammalian Sprouty4 suppresses Ras-independent ERK activation by binding
to Raf1. Cell Cycle 2, 281-2 (2003).
Yan, W., Samson, M., Jegou, B. & Toppari, J. Bcl-w forms complexes with Bax and Bak, and
elevated ratios of Bax/Bcl-w and Bak/Bcl-w correspond to spermatogonial and spermatocyte
apoptosis in the testis. Mol Endocrinol 14, 682-99 (2000).
Yu, M. et al. The scaffolding adapter Gab2, via Shp-2, regulates kit-evoked mast cell
proliferation by activating the Rac/JNK pathway. J Biol Chem 281, 28615-26 (2006).
Schrans-Stassen, B.H., Saunders, P.T., Cooke, H.J. & de Rooij, D.G. Nature of the
spermatogenic arrest in Dazl -/- mice. Biol Reprod 65, 771-6 (2001).
Tsuneyoshi, N. et al. PRDM14 suppresses expression of differentiation marker genes in
human embryonic stem cells. Biochem Biophys Res Commun 367, 899-905 (2008).
Smith, C.A., McClive, P.J., Western, P.S., Reed, K.J. & Sinclair, A.H. Conservation of a sexdetermining gene. Nature 402, 601-2 (1999).
Krentz, A.D. et al. The DM domain protein DMRT1 is a dose-sensitive regulator of fetal germ
cell proliferation and pluripotency. Proc Natl Acad Sci U S A 106, 22323-8 (2009).
Yang, J., Lee, S.H., Goddard, M.E. & Visscher, P.M. GCTA: a tool for genome-wide complex
trait analysis. Am J Hum Genet 88, 76-82 (2011).
Litchfield, K. Quantifying the heritability of testicular germ cell tumour using both genomic
and population-based approaches. Scientific Reports (In Press) (2015).
McGlynn, K.A. & Cook, M.B. Etiologic factors in testicular germ-cell tumors. Future Oncol 5,
1389-402 (2009).
Skakkebaek, N.E., Rajpert-De Meyts, E. & Main, K.M. Testicular dysgenesis syndrome: an
increasingly common developmental disorder with environmental aspects. Hum Reprod 16,
972-8 (2001).
Trabert, B., Zugna, D., Richiardi, L., McGlynn, K.A. & Akre, O. Congenital malformations and
testicular germ cell tumors. Int J Cancer 133, 1900-4 (2013).
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
Coffey, J. et al. Testicular microlithiasis as a familial risk factor for testicular germ cell
tumour. British Journal of Cancer 97, 1701-1706 (2007).
Nizetic, D. & Groet, J. Tumorigenesis in Down's syndrome: big lessons from a small
chromosome. Nat Rev Cancer 12, 721-32 (2012).
Hasle, H., Mellemgaard, A., Nielsen, J. & Hansen, J. Cancer incidence in men with Klinefelter
syndrome. Br J Cancer 71, 416-20 (1995).
Litchfield, K. et al. Whole-exome sequencing reveals the mutational spectrum of testicular
germ cell tumours. Nat Commun 6, 5973 (2015).
Stettner, A.R. et al. Familial ovarian germ cell cancer: Report and review. American Journal of
Medical Genetics 84, 43-46 (1999).
Kristensen, D.G., Skakkebaek, N.E., Rajpert-De Meyts, E. & Almstrup, K. Epigenetic features
of testicular germ cell tumours in relation to epigenetic characteristics of foetal germ cells.
Int J Dev Biol 57, 309-17 (2013).
Karlsson, R. et al. Investigation of six testicular germ cell tumor susceptibility genes suggests
a parent-of-origin effect in SPRY4. Hum Mol Genet 22, 3373-80 (2013).
Oosterhuis, J.W. & Looijenga, L.H. Testicular germ-cell tumours in a broader perspective. Nat
Rev Cancer 5, 210-22 (2005).
Kristensen, D.M. et al. Origin of pluripotent germ cell tumours: the role of microenvironment
during embryonic development. Mol Cell Endocrinol 288, 111-8 (2008).
Skakkebaek, N.E., Berthelsen, J.G., Giwercman, A. & Muller, J. Carcinoma-in-situ of the testis:
possible origin from gonocytes and precursor of all types of germ cell tumours except
spermatocytoma. Int J Androl 10, 19-28 (1987).
Rajpert-De Meyts, E. Developmental model for the pathogenesis of testicular carcinoma in
situ: genetic and environmental aspects. Hum Reprod Update 12, 303-23 (2006).
Jacobsen, G.K. & Henriques, U.V. A fetal testis with intratubular germ cell neoplasia (ITGCN).
Mod Pathol 5, 547-9 (1992).
Biermann, K. et al. c-KIT is frequently mutated in bilateral germ cell tumours and downregulated during progression from intratubular germ cell neoplasia to seminoma. J Pathol
213, 311-8 (2007).
Fossa, S.D. et al. Risk of contralateral testicular cancer: a population-based study of 29,515
U.S. men. J Natl Cancer Inst 97, 1056-66 (2005).
Coffey, J. et al. Somatic KIT mutations occur predominantly in seminoma germ cell tumors
and are not predictive of bilateral disease: report of 220 tumors and review of literature.
Genes Chromosomes Cancer 47, 34-42 (2008).
Horwich, A., Shipley, J. & Huddart, R. Testicular germ-cell cancer. Lancet 367, 754-65 (2006).
Rajpert-De Meyts, E. et al. The emerging phenotype of the testicular carcinoma in situ germ
cell. APMIS 111, 267-78; discussion 278-9 (2003).
Dieckmann, K.P., Kulejewski, M., Heinemann, V. & Loy, V. Testicular biopsy for early cancer
detection--objectives, technique and controversies. Int J Androl 34, e7-13 (2011).
Almstrup, K. et al. Genome-wide gene expression profiling of testicular carcinoma in situ
progression into overt tumours. Br J Cancer 92, 1934-41 (2005).
Biermann, K. et al. Genome-wide expression profiling reveals new insights into pathogenesis
and progression of testicular germ cell tumors. Cancer Genomics Proteomics 4, 359-67
(2007).
Gori, S. et al. Germ cell tumours of the testis. Crit Rev Oncol Hematol 53, 141-64 (2005).
Mai, P.L. et al. The International Testicular Cancer Linkage Consortium: a clinicopathologic
descriptive analysis of 461 familial malignant testicular germ cell tumor kindred. Urol Oncol
28, 492-9 (2010).
Forman, D. et al. Familial testicular cancer: a report of the UK family register, estimation of
risk and an HLA class 1 sib-pair analysis. Br J Cancer 65, 255-62 (1992).
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
Cutcutache, I. et al. Exome-wide Sequencing Shows Low Mutation Rates and Identifies Novel
Mutated Genes in Seminomas. Eur Urol 68, 77-83 (2015).
Lawrence, M.S. et al. Mutational heterogeneity in cancer and the search for new cancerassociated genes. Nature 499, 214-8 (2013).
McIntyre, A. et al. Amplification and overexpression of the KIT gene is associated with
progression in the seminoma subtype of testicular germ cell tumors of adolescents and
adults. Cancer Res 65, 8085-9 (2005).
Kemmer, K. et al. KIT mutations are common in testicular seminomas. Am J Pathol 164, 30513 (2004).
Kim, H.J. et al. KIT D816 mutation associates with adverse outcomes in core binding factor
acute myeloid leukemia, especially in the subgroup with RUNX1/RUNX1T1 rearrangement.
Ann Hematol 92, 163-71 (2013).
Bignell, G. et al. Sequence analysis of the protein kinase gene family in human testicular
germ-cell tumors of adolescents and adults. Genes Chromosomes Cancer 45, 42-6 (2006).
Heidenreich, A. et al. Immunohistochemical and mutational analysis of the p53 tumour
suppressor gene aml the bcl-2 oncogene in primary testicular germ cell, tumours. Apmis 106,
90-99 (1998).
Laumann, R., Jucker, M. & Tesch, H. Point Mutations in the Conserved Regions of the P53
Tumor Suppressor Gene Do Not Account for the Transforming Process in the Jurkat Acute
Lymphoblastic-Leukemia T-Cells. Leukemia 6, 227-228 (1992).
Voorhoeve, P.M. et al. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes
in testicular germ cell tumors. Cell 124, 1169-81 (2006).
Suijkerbuijk, R.F. et al. Overrepresentation of chromosome 12p sequences and karyotypic
evolution in i(12p)-negative testicular germ-cell tumors revealed by fluorescence in situ
hybridization. Cancer Genet Cytogenet 70, 85-93 (1993).
Atkin, N.B. & Baker, M.C. Specific Chromosome Change, I(12p), in Testicular-Tumors. Lancet
2, 1349-1349 (1982).
Atkin, N.B. & Baker, M.C. I(12p) - Specific Chromosomal Marker in Seminoma and Malignant
Teratoma of the Testis. Cancer Genetics and Cytogenetics 10, 199-204 (1983).
Sandberg, A.A., Meloni, A.M. & Suijkerbuijk, R.F. Reviews of chromosome studies in
urological tumors .3. Cytogenetics and genes in testicular tumors. Journal of Urology 155,
1531-1556 (1996).
Rodriguez, S. et al. Expression profile of genes from 12p in testicular germ cell tumors of
adolescents and adults associated with i(12p) and amplification at 12p11.2-p12.1. Oncogene
22, 1880-91 (2003).
Korkola, J.E. et al. Down-regulation of stem cell genes, including those in a 200-kb gene
cluster at 12p13.31, is associated with in vivo differentiation of human male germ cell
tumors. Cancer Research 66, 820-827 (2006).
Ottesen, A.M. et al. High-resolution comparative genomic hybridization detects extra
chromosome arm 12p material in most cases of carcinoma in situ adjacent to overt germ cell
tumors, but not before the invasive tumor development. Genes Chromosomes Cancer 38,
117-25 (2003).
Summersgill, B., Osin, P.S., Lu, Y.J., Huddart, R. & Shipley, J. Chromosomal imbalances
associated with carcinoma in situ and associated testicular germ cell tumours of adolescents
and adults. British Journal of Cancer 85, 213-219 (2001).
Henegariu, O., Vance, G.H., Heiber, D., Pera, M. & Heerema, N.A. Triple-color FISH analysis of
12p amplification in testicular germ-cell tumors using 12p band-specific painting probes. J
Mol Med (Berl) 76, 648-55 (1998).
Roelofs, H. et al. Restricted 12p amplification and RAS mutation in human germ cell tumors
of the adult testis. Am J Pathol 157, 1155-66 (2000).
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
Summersgill, B. et al. Molecular cytogenetic analysis of adult testicular germ cell tumours
and identification of regions of consensus copy number change. Br J Cancer 77, 305-13
(1998).
Zafarana, G. et al. 12p-amplicon structure analysis in testicular germ cell tumors of
adolescents and adults by array CGH. Oncogene 22, 7695-701 (2003).
LeBron, C. et al. Genome-wide analysis of genetic alterations in testicular primary seminoma
using high resolution single nucleotide polymorphism arrays. Genomics 97, 341-349 (2011).
Silveira, S.M. et al. Genomic screening of testicular germ cell tumors from monozygotic
twins. Orphanet J Rare Dis 9, 181 (2014).
Brown, P.R., Miki, K., Harper, D.B. & Eddy, E.M. A-kinase anchoring protein 4 binding
proteins in the fibrous sheath of the sperm flagellum. Biol Reprod 68, 2241-8 (2003).
Chemes, H.E., Brugo, S., Zanchetti, F., Carrere, C. & Lavieri, J.C. Dysplasia of the fibrous
sheath: an ultrastructural defect of human spermatozoa associated with sperm immotility
and primary sterility. Fertil Steril 48, 664-9 (1987).
Miki, K. et al. Targeted disruption of the Akap4 gene causes defects in sperm flagellum and
motility. Dev Biol 248, 331-42 (2002).
Vladusic, T. et al. Histological groups of human postpubertal testicular germ cell tumours
harbour different genetic alterations. Anticancer Res 34, 4005-12 (2014).
Oldenburg, J. et al. Testicular seminoma and non-seminoma: ESMO Clinical Practice
Guidelines for diagnosis, treatment and follow-up. Annals of Oncology 24, 125-132 (2013).
Siegel, R. et al. Cancer treatment and survivorship statistics, 2012. Ca-a Cancer Journal for
Clinicians 62, 220-241 (2012).
Usanova, S. et al. Cisplatin sensitivity of testis tumour cells is due to deficiency in
interstrand-crosslink repair and low ERCC1-XPF expression. Molecular Cancer 9(2010).
Cavallo, F. et al. Reduced Proficiency in Homologous Recombination Underlies the High
Sensitivity of Embryonal Carcinoma Testicular Germ Cell Tumors to Cisplatin and Poly (ADPRibose) Polymerase Inhibition. Plos One 7(2012).
Gutekunst, M. et al. p53 hypersensitivity is the predominant mechanism of the unique
responsiveness of testicular germ cell tumor (TGCT) cells to cisplatin. PLoS One 6, e19198
(2011).
Sheikine, Y. et al. Molecular genetics of testicular germ cell tumors. Am J Cancer Res 2, 15367 (2012).
Honecker, F. et al. Microsatellite instability, mismatch repair deficiency, and BRAF mutation
in treatment-resistant germ cell tumors. J Clin Oncol 27, 2129-36 (2009).
Feldman, D.R. et al. Presence of somatic mutations within PIK3CA, AKT, RAS, and FGFR3 but
not BRAF in cisplatin-resistant germ cell tumors. Clin Cancer Res 20, 3712-20 (2014).
Van Allen, E.M. et al. Somatic ERCC2 mutations correlate with cisplatin sensitivity in muscleinvasive urothelial carcinoma. Cancer Discov 4, 1140-53 (2014).
Einhorn, L.H., Brames, M.J., Heinrich, M.C., Corless, C.L. & Madani, A. Phase II study of
imatinib mesylate in chemotherapy refractory germ cell tumors expressing KIT. American
Journal of Clinical Oncology-Cancer Clinical Trials 29, 12-13 (2006).
Noel, E.E. et al. The association of CCND1 overexpression and cisplatin resistance in
testicular germ cell tumors and other cancers. Am J Pathol 176, 2607-15 (2010).
Garcia-Velasco, A. et al. Biological markers of cisplatin resistance in advanced testicular germ
cell tumours. Clin Transl Oncol 14, 452-7 (2012).
de Haas, E.C. et al. Association of PAI-1 gene polymorphism with survival and chemotherapyrelated vascular toxicity in testicular cancer. Cancer 116, 5628-36 (2010).
Singhera, M., Lees, K., Huddart, R. & Horwich, A. Minimizing toxicity in early-stage testicular
cancer treatment. Expert Rev Anticancer Ther 12, 185-93 (2012).
110.
111.
112.
113.
114.
115.
116.
Oldenburg, J. et al. Association between long-term neuro-toxicities in testicular cancer
survivors and polymorphisms in glutathione-s-transferase-P1 and-M1, a retrospective cross
sectional study. Journal of Translational Medicine 5(2007).
Peters, U. et al. Glutathione S-transferase genetic polymorphisms and individual sensitivity
to the ototoxic effect of cisplatin. Anticancer Drugs 11, 639-43 (2000).
Huddart, R.A. et al. Fertility, gonadal and sexual function in survivors of testicular cancer. Br J
Cancer 93, 200-7 (2005).
O'Flaherty, C., Hales, B.F., Chan, P. & Robaire, B. Impact of chemotherapeutics and advanced
testicular cancer or Hodgkin lymphoma on sperm deoxyribonucleic acid integrity. Fertil Steril
94, 1374-9 (2010).
Travis, L.B. et al. Second cancers among 40,576 testicular cancer patients: focus on longterm survivors. J Natl Cancer Inst 97, 1354-65 (2005).
Greene, M.H. et al. Familial testicular germ cell tumors (FTGCT) - overview of a
multidisciplinary etiologic study. Andrology 3, 47-58 (2015).
O, B. Risk prediction and population screening for breast, ovarian and
prostate cancers. Nat Genet http://www.nature.com/icogs/primer/risk-prediction-andpopulation-screening-for-breast-ovarian-and-prostate-cancers/( 2013).
117. Almstrup, K. et al. Screening of subfertile men for testicular carcinoma in situ by an
automated image analysis-based cytological test of the ejaculate. Int J Androl 34, e21-30;
discussion e30-1 (2011).
118. Hoei-Hansen, C.E. et al. Towards a non-invasive method for early detection of testicular
neoplasia in semen samples by identification of fetal germ cell-specific markers. Human
Reproduction 22, 167-173 (2007).
119. Skol, A.D., Scott, L.J., Abecasis, G.R. & Boehnke, M. Joint analysis is more efficient than
replication-based analysis for two-stage genome-wide association studies (vol 38, pg 209,
2006). Nature Genetics 38, 390-390 (2006).
120. Nelson, M.R. et al. The support of human genetic evidence for approved drug indications.
Nat Genet 47, 856-60 (2015).
121. Chen, K.S. & Amatruda, J.F. A big catch for germ cell tumour research. PLoS Genet 9,
e1003481 (2013).
122. Fukuda, S. et al. Expression of vascular endothelial growth factor in patients with testicular
germ cell tumors as an indicator of metastatic disease. Cancer 85, 1323-30 (1999).
123. Nieto, Y. et al. Bevacizumab/high-dose chemotherapy with autologous stem-cell transplant
for poor-risk relapsed or refractory germ-cell tumors. Ann Oncol (2015).
124. Fankhauser, C.D. et al. Frequent PD-L1 expression in testicular germ cell tumors. Br J Cancer
113, 411-3 (2015).
125. Gajewski, T.F., Schreiber, H. & Fu, Y.X. Innate and adaptive immune cells in the tumor
microenvironment. Nature Immunology 14, 1014-1022 (2013).
126. Litchfield, K., Shipley, J. & Turnbull, C. Common variants identified in genome-wide
association studies of testicular germ cell tumour: an update, biological insights and clinical
application. Andrology 3, 34-46 (2015).