Late-onset common cancers in a kindred with an Arg213Gln TP53
Marie ¨ lle W.G. Ruijs1,2, Senno Verhoef1, Gea Wigbout1, Roelof Pruntel1, Arno N. Floore1, Daphne de
Jong1, Laura J. van ¢t Veer1and Fred H. Menko2
1Family Cancer Clinic, The Netherlands Cancer Institute, Amsterdam, The Netherlands;2Department of Clinical
Genetics and Human Genetics, VU University Medical Center, Amsterdam, The Netherlands
Received 20 February 2004; accepted in revised form 1 November 2005
Key words: FASAY, Li-Fraumeni syndrome, Li-Fraumeni-like syndrome, missense mutation, TP53
Li-Fraumeni syndrome (LFS) is an autosomal-dominant condition characterized by early-onset sarcoma, breast
cancer and other specific tumour types. In most LFS kindreds germline TP53 mutations have been identified. In
general, TP53 germline mutations are not associated with late-onset common cancers. We encountered a large
kindred in which a wide spectrum of tumour types occurred, including melanoma, breast, ovarian, colorectal,
stomach and renal cell cancer, without clear-cut early ages at onset of disease. An Arg213Gln TP53 germline
mutation was detected in 12 out of 15 affected family members whereas testing for other cancer susceptibility genes
in selected patients was negative. In vitro testing indicated that the specific TP53 mutation inactivates the protein
transcriptionally. Our findings suggest that this TP53 germline mutation is a causative factor in this family and that
specific TP53 germline mutations can be associated with relatively late-onset common cancers.
Tumour susceptibility syndromes were defined, until
recently, by their clinical phenotypes. Classical diag-
nostic criteria for Li-Fraumeni syndrome (LFS) are: a
proband diagnosed with sarcoma before 45 years of
age, a first-degree relative with cancer before 45 years of
age, and another first- or second-degree relative in the
same lineage with any cancer diagnosed under 45 or sarcoma
occurring at any age . Criteria for Li-Fraumeni-like
syndrome (LFL) as defined by Birch et al.  are: a proband
with any childhood cancer or sarcoma, brain tumour, or
adrenocortical carcinoma diagnosed before 45 years of age,
with one first- or second-degree relative with a typical LFS
cancer (sarcoma, breast cancer, brain tumour, leukaemia, or
adrenocortical carcinoma) diagnosed at any age, plus one
first- or second-degree relative in the same lineage with any
cancer diagnosed under age 60.
In 1990 germline mutations in the TP53 tumour
suppressor gene were shown to be causative of both LFS
and LFL syndrome [3, 4]. Since then pathogenic TP53
mutations–most of them missense mutations–have
been found in up to 75% of LFS families and 40% of
LFL kindreds [5–7].
Recently, the spectrum of tumours in 28 LFS/LFL
families with TP53 mutations was evaluated by Birch
et al. . These authors found the following malig-
nancies to be associated with germline TP53 muta-
tions: female breast cancer, tumours of the brain and
spinal cord, soft tissue sarcoma, osteosarcoma (except
Ewing’s sarcoma), adrenocortical carcinoma and Wil-
ms’ tumour. To a lesser degree, carcinoma of the
pancreas seemed related. Noticeably, there was no
association with the majority of other common carci-
nomas (apart from breast cancer). In general, the ages
at diagnosis of cancer in TP53 mutation carriers are
lower than for sporadic cancer. However, it has been
suggested that the types of malignancies and ages at
onset might be more variable than assumed thus far
Correspondence to: F.H. Menko, MD, PhD, Department of Clinical Genetics and Human Genetics, VU University Medical Center, Amsterdam,
The Netherlands. Tel.: +31-20-4440150; Fax: +31-20-4440769; E-mail: firstname.lastname@example.org
Familial Cancer (2006) 5:169–174
? Springer 2006
In any family with a novel TP53 germline mutation
the question arises whether the mutation is pathogenic or
represents a non-pathogenic polymorphism. Arguments
in favour of pathogenicity include segregation of the
mutation with the cancer phenotype in the kindred, the
type of mutation  and in vitro assays to determine its
functional effect [13, 14]. Several authors have studied
tumours from TP53 mutation carriers for loss of the
wild-type allele (loss of heterozygosity, LOH) and
accumulation of TP53 protein, with variable patterns
of LOH and immunohistochemical staining of TP53
protein in target organs [15, 16]. In general, the presence
of LOH and accumulation of TP53 protein strengthens
the belief of a mutation being pathogenic, albeit the
absence of LOH and TP53 staining cannot exclude
We describe a cancer family in which a germline
Arg213Gln TP53 mutation was identified. Twelve out of
15 affected family members were mutation carriers. The
carriers exhibited a variety of common cancers without
a clear-cut early onset of disease.
We evaluated the tumour spectrum, the functional
effect of the germline TP53 mutation, somatic genetic
alterations and the possible contribution of other genetic
defects in this family.
Patients and methods
The index patient (Figure 1, III-20) developed unilateral
breast cancer at the age of 42 years. Her maternal family
history was positive for various types of malignancy.
Medical data on affected family members, including
surgical and pathology reports, was collected. Tumours
for which tissue samples were available were reviewed.
Informed consent for DNA analysis of the TP53 gene,
and when applicable other genes, was obtained after
BRCA1, BRCA2 testing showed no mutation in the
index patient. After the identification of a germline
TP53 mutation in the proband, almost all other cancer
patients were tested for this mutation. Selected family
members were also tested for germline mutations in
CHEK2, MLH1, MSH2 and MSH6. DNA was isolated
from peripheral lymphocytes according to standard
procedures. From four deceased affected family mem-
bers DNA was isolated from archival tissue samples.
Mutation analysis of the TP53 gene was performed by
sequence analysis of all coding exons (2–11) and
flanking intron–exon boundaries of these exons using
probe amplification (MLPA) was performed on 5
samples, to exclude additional TP53 deletions or dupli-
cations (TP53 MLPA KIT of MRC-Holland) .
Details on primers and PCR conditions are available on
request. The frequency of the identified TP53 germline
mutation was assessed in a control group of 135
anonymous blood donors, using denaturing gradient
gel electrophoresis (DGGE).
The functional effect of the TP53 germline mutation
was evaluated in four family members–three TP53
mutation carriers and one affected family member
without the TP53 germline mutation–by functional
analysis of separated alleles in yeast (FASAY) using
RNA isolated from blood lymphocytes [13, 14].
LOH (loss of heterozygosity) studies were performed
on tumour tissue from five affected mutation-carriers.
The presence or absence of the wild-type TP53 allele was
determined by DNA sequencing of TP53 exon 6, the
allele ratio of the normal versus mutated allele in the
tumour compared to normal DNA. To determine
whether the other TP53 allele in the tumour was
mutated by a different independent mutation, other
codons were sequenced as well in one tumour showing
LOH. Immunohistochemical staining of the TP53 pro-
tein was performed in tumour and normal tissues from
nine affected mutation carriers, using the mouse mono-
clonal antibody DO7, according to standard procedures.
(Dako, Glostrup, Denmark). Staining of the TP53
protein stands for the presence of aberrant protein, in
a normal situation immunohistochemical staining of the
TP53 protein is absent.
Microsatellite instability was evaluated in the tumour
tissue of 4 individuals affected by colorectal cancer using
5 microsatellite markers as described  and immuno-
histochemical staining of the MLH1, MSH2 and MSH6
proteins was performed in these 4 individuals.
The pedigree data fulfil the criteria of Li-Fraumeni-like
syndrome as defined by Birch et al. . The pedigree of
the LFL family is depicted in Figure 1, with a summary
of the clinical data of affected family members given in
Table 1. Ten out of 13 tested family members affected
by cancer had a germline mutation in codon 213 of
TP53, exon 6: nt 638 G>A, Arg213Gln, two additional
affected family members were obligate carriers. The
three patients who did not exhibit the gene defect on
repeated examination were patient III-13, who had
colorectal cancer at the age of 43 years, patient II-8,
who had breast cancer at 64 and colorectal cancer at
83 years of age and patient III-6, who developed rectal
cancer at the age of 58 years. The father of the index
patient (partner of II-7) died without a previous history
of cancer, there was no history of cancer in his family.
No mutations were found in specific cancer suscepti-
bility genes (BRCA1, BRCA2, CHEK2, MLH1, MSH2
and MSH6) tested in selected family members (Table 1).
The mean age at diagnosis of TP53 mutation carriers
with a documented malignancy was 58.4 years (range
42–71 years). When we include the ages of patients at
diagnosis for whom only family history data are
available (I-1, I-2, II-2, II-4, II-9, II-10, II-13, II-15,
III-10, III-14 and IV-3) or mutation analysis could not
be performed (III-9), the mean age was 50.6 years (range
3–81 years). The mean age at diagnosis of the three
170M.W.G. Ruijs et al.
Figure 1. Pedigree of the family with the germline Arg213Gln TP53 mutation fi index patient; (n) male with documented malignancy; (h)
male unaffected; (d) female with documented malignancy; (s) female unaffected; (h) deceased; (F): diagnosis based on family history. Abd:
abdominal cancer, B: breast cancer, Br: brain tumour, C: colorectal cancer, K: kidney (renal cell) cancer, L: Lung cancer, M: melanoma,
Mes: mesothelioma, O: ovarian cancer, P: prostate cancer, St: stomach cancer, T: thyroid cancer; C42: age at diagnosis, d...: age at death. +:
mutation carrier; (+): obligate mutation carrier; ()): non-carrier.
Late-onset common cancers171
affected non-carriers was 62 years (range 43–83 years).
Of the 36 unaffected family members tested, 10 mutation
carriers were identified at a mean age of 41.4 years
(range 21–53 years).
No TP53 deletions or duplications were demon-
strated by MLPA in 5 family members tested (Table 1).
The Arg213Gln TP53 mutation was absent in 135
control individuals. Six controls carried a known silent
polymorphism, Arg213Arg, in the TP53 gene.
The yeast-based assay (FASAY), which can distin-
guish inactivating mutations from functionally silent
mutations in exons 4 to 10, showed that the mutated
allele lacks biological transcriptional activity (Table 1).
Loss of the TP53 wild-type allele was found for 2/5
tumours from mutation-carriers (Table 2). In the two
tumours showing LOH, all TP53 exons were sequenced.
Only in one sample, DNA was of sufficient quality to
evaluate, no (additional) somatic mutation was found.
Immunohistochemical TP53 staining was found for
all 9 tumours from mutation-carriers, 3 of the 9 adjacent
normal tissues stained positive, the other six showed
sporadic positive cells.
There was no microsatellite instability in the 4
colorectal tumours tested, in 2 colon tumours all 3
MMR proteins stained positive, in 1 colon tumour
MSH2 and MSH6 staining was negative and in the
rectal tumour, MLH1 staining was negative.
In the family reported here a wide variety of tumours
occurred. The pedigree data fulfil the criteria of
Li-Fraumeni-like syndrome . Several results suggest
that the germline Arg213Gln TP53 mutation is a
causative factor for the cancer phenotype in this family:
(1) this specific mutation was found previously in an
LFS family  and has also been reported as somatic
mutation in malignancies , (2) in the kindred under
investigation the mutation was found in 12/15 cancer
patients, (3) the specific mutation is located within
codon 213, which is one of the 6 codons harbouring
46% of all TP53 missense mutations , (4) an in vitro
test demonstrated that the mutation has a functional
effect, (5) the mutation was absent in healthy controls
and (6) tests for other cancer susceptibility genes in
selected patients were negative.
Many of the tumour types in the family we investi-
gated are common cancers including melanoma, breast,
ovarian, colorectal, stomach and renal cell carcinoma.
Table 1. Clinical data, TP53 germline mutation analysis, TP53 functional testing (fasay), MLPA and investigation of additional genes in
family members with documented malignancies.
(age at diagnosis, years)
FASAY MLPA Additional DNA
BRCA1/2 CHEK 2 MLH1 MSH2 MSH6
Ovarian adenocarcinoma (59) Carriera
Breast carcinoma (68)
Colon adenocarcinoma (71)
Breast carcinoma (64)
Colon adenocarcinoma (83)
Renal cell carcinoma (mixed
papillary/clear cell) (71)
Rectal adenocarcinoma (58)
Colon adenocarcinoma (43)
Renal cell (papillary)
carcinoma (47) Thyroid
Breast carcinoma (42)
Renal cell (clear cell)
Stable/ + / + / +
Stable/ + / + / +
II-14CarrierPos Neg Neg
Neg Neg Neg Stable/ ) / + / +
Neg NegNeg Stable/ + / ) / )
aIn normal tissue sample; FASAY: Pos = positive, meaning only 50% transcriptional activity, Neg = negative, meaning normal transcriptional
activity, MLPA: Neg = negative, meaning no TP53 deletions or duplications found, Additional DNA mutation testing: Neg = negative,
meaning no mutation found.
*MSI/IHC: Microsatellite instability testing and immunohistochemical staining of MLH1, MSH2, MSH6 protein in colorectal cancer;
Stable = no microsatellite instability, + = staining positive, ) = staining negative.
172 M.W.G. Ruijs et al.
There was no clear-cut early age at onset of disease. In
fact, 79% of all documented malignancies of the TP53
mutation carriers occurred after 50 years of age and
36% after 60 years of age. Some of the cancers may
simply be sporadic due to environmental influences and
chance, especially those malignancies which developed
above the population median ages. The picture is
somewhat biased towards adult tumours due to the fact
that we were not able to confirm the two brain tumours
occurring in childhood and the two colorectal tumours
occurring in early adulthood. Additionally, no mutation
analysis could be performed in the brain tumour
occurring at 28 years of age (III-9). The complete
pedigree data is suggestive for a predisposition not only
for adult-onset but also for childhood-onset malignan-
cies. The association of a TP53 germline mutation with
relatively late-onset common cancers has not been
documented before [8, 11]. It has been suggested,
however, that in some families with TP53 mutations
there may be a lower cancer risk and later onset of
tumours suggesting that deleterious TP53 mutations
may be more frequent in the population than generally
The fact that we detected the TP53 germline mutation
in cancer patients is not sufficient proof that the cancer is
in fact caused by the mutation. Therefore, we studied
tumours of mutation carriers to investigate this issue. We
found loss of heterozygosity for some tumours, immu-
nohistochemical TP53 staining for all cancers and TP53
staining of normal tissue in three mutation carriers.
Although these results should be interpreted with
caution, they are in tune with the TP53 germline
mutation being an underlying cause of disease in this
kindred (e.g., it is consistent with LOH found in about
50% of tumours of pathogenic TP53 mutation carriers
Family members who carried the TP53 germline
mutation, underwent screening procedures for occult
malignancies, including renal ultrasound. This led to the
diagnosis of renal cell cancer in II-16 and III-39. So far,
no other malignancies have been found by screening the
The specific missense mutation in this family leads
to relatively late onset malignancies, confirmed by the
fact that 10 mutation carriers are without any malig-
nancy. It must be noted however, that the average age
of these healthy mutation carriers is significantly lower
(independent samples t-test, P=0.013) than the average
age at diagnosis of cancer is this family. If the
penetrance of this mutation is reduced, the mutant
protein product might still have some residual functional
activity, although the functional test showed a lack of
transcriptional activity. However, the only other family
reported in the TP53 mutation database  with the
same mutation had, according to the limited information
provided, early-onset malignancies and fulfilled the classic
criteria for LFS. The fact that this specific mutation is
only rarely found in published high penetrance LFS/LFL
mutation families, could be explained by a low pene-
trance phenotype in other families not examined thus far.
Our data are not in agreement with the studied genotype-
phenotype correlations in LFS/LFL families by Birch
et al. , who found a higher penetrance and earlier age
at onset for missense point mutations in the core DNA
binding domain of TP53 in comparison with families
harbouring mutations leading to truncated or non-
The effect of the Arg213Gln TP53 mutation in the
family presented here may be modified by other genetic
and/or environmental factors. Alternatively, the picture
might be complicated by the existence of mutations in
other cancer susceptibility genes. Due to the occurrence
of breast, ovarian and colorectal cancer in the family we
indeed tested selected patients for mutations in other
susceptibility genes known to be associated with
these tumour types: BRCA1, BRCA2, CHEK2 and the
DNA-mismatch repair (MMR) genes MLH1, MSH2
and MSH6. BRCA1 and BRCA2 are associated primar-
ily with breast and ovarian cancer, MLH1, MSH2 and
MSH6 with HNPCC and CHEK2 with breast and
colorectal cancer . No germline alterations in these
genes were found. The colorectal cancers showed no
microsatellite instability; in 2 tumours the immunohis-
tochemical staining was abnormal but in three patients
tested (including the two patients with abnormal stain-
ing in tumour tissue), no MLH1, MSH2 or MSH6
mutations were found. Therefore, there is no positive
evidence for involvement of the MMR genes in these
cancers . BRCA1 and BRCA2 was only tested in the
youngest breast cancer patient. The other breast cancer
Table 2. Loss of heterozygosity and immunohistochemical staining of TP53 protein in tissues.
number (Figure 1)
Tumour typeLoss of wild-type TP53 allele Other somatic mutationsTP53 staining pattern
Tumour tissueNormal tissue
± (Sporadic cells)
Late-onset common cancers 173
patients developed breast cancer at an older age in Download full-text
combination with a different malignancy, not typical for
BRCA1 or BRCA2. Renal malignancies reported in
multiple family members have been associated with
many syndromes including Von Hippel-Lindau syn-
drome and Birt-Hogg-Dube ´ syndrome. In this family
there are no clinical signs which would suggest one of
On the basis of our observations, we propose that the
detected mutation contributes considerably to the can-
cer risk in this large family, although not as severely as
previously reported for Li-Fraumeni families or other
Li-Fraumeni-like families. If more such families with a
reduced penetrance and later average age of onset exist,
they might remain unnoticed due to the generally
applied restrictive policies for TP53 mutation testing.
More research is needed to confirm the existence of
(other) low penetrance TP53 germline mutations.
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