International Journal of Clinical Medicine, 2013, 4, 44-50
Published Online December 2013 (
Open Access IJCM
TP53 Mutations and Chemotherapy Response to
Neoadjuvant Metotrexate, Cisplatin and Adryamicin
Chemotherapy in Resected Osteosarcoma
Ligia Richter1,2, Marcelo Buzzi2*, Carmela Dantas-Barbosa2,3
1Faculdade LS—Coordenação do Curso de Enfermagem, Setor D Sul, Lote 5, Taguatinga Sul, DF, Brasil; 2Laboratório de
Bioquimica Analítica, Patologia Molecular, Rede Sarah de Hospitais de Reabilitação, Brasília, DF, Brasil; 3Centre de Recherche en
Cancérologie de Lyon, UMR INSERM 1052—CNRS 5286, Centre Léon Bérard, Cheney D, 28 Rue Laënnec, Lyon, France.
Email:; *;
Received October 30th, 2013; revised November 28th, 2013; accepted December 10th, 2013
Copyright © 2013 Ligia Richter et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-
dance of the Creative Commons Attribution License all Copyrights © 2013 are reserved for SCIRP and the owner of the intellectual
property Ligia Richter et al. All Copyright © 2013 are guarded by law and by SCIRP as a guardian.
Osteosarcoma is a rare and highly malignant tumor that usually affects adolescents and young adults. Despite current
management protocols, up to half of patients succumb to the disease. Moreover, there is no well-characterized molecu-
lar marker for diagnosis and prognosis. TP53 alterations have been associated with a poor prognosis in many cancers.
The aim of this retrospective work was to find out whether TP53 functional status predicts response to neoadjuvant
chemotherapy and thus may help treatment decision for osteosarcoma patients. Seventeen biopsies of osteosarcoma
patients receiving primary metotrexate, cisplatin and adryamicin chemotherapy followed by surgery were analyzed.
TP53 exons 5 - 9 mutations were screened. Among 17 biopsies, 4 (23.5%) displayed TP53 mutations: 3 deletions and
one single-nucleotide substitution. The presence of TP53 gene mutation does not correlate with resistance to chemo-
therapy according to histological Rosen grade and nevertheless is associated with patient’s age in a significant manner
(p < 0.05). The presence of non-mutated TP53 is not entirely specific for a good prognosis. We found no evidence that
TP53 mutations predict chemoresistance in osteosarcoma patients more over the overall survival curve, followed for
more than 12 years, showing no difference between patients with tumors harboring wild type or mutated TP53 gene (p
< 0.5). Further analysis to identify other genes that can influence chemotherapy response and clinical outcome in os-
teosarcoma is needed to improve patient treatment.
Keywords: Osteosarcoma; Chemoresistance; TP53 Gene
1. Introduction
Osteosarcoma is the commonest primary bone cancer in
children and young adults. Osteosarcoma presents a peak
of incidence at the age of 15 - 19 years that coincides
with the growth spurt. The estimated incidence rate
worldwide is 4 million/year. It is highly malignant, char-
acterized by a high potential to metastasize [1]. These
tumors typically arise in the metaphyseal regions of long
bones. A preference for the distal femur, proximal tibia
and proximal humerus is observed [2]. Almost all osteo-
sarcomas are high grade and have a poor prognosis. Pa-
tients diagnosed with osteosarcoma usually present a
large tumor and numerous lung micrometastases which
markedly decrease the potential for cure [3]. The use of
multiple chemotherapy agents pre- and postoperatively
has significantly improved the outcome in the last 30
years, leading to a 5-year disease-free survival for pa-
tients with localized tumors in the range of 20% to 60%
range [4]. Despite these improvements, approximately
30% of patients with localized disease and 60% of pa-
tients with pulmonary metastases succumb to the illness.
Osteosarcoma is a complex neoplasia. Cytogenetic
analysis has revealed multiple chromosomal rearrange-
ments with a high degree of aneuploidy, gene amplifica-
*Corresponding author.
TP53 Mutations and Chemotherapy Response to Neoadjuvant Metotrexate, Cisplatin and Adryamicin
Chemotherapy in Resected Osteosarcoma
tion, and multiple unbalanced chromosomal rearrange-
ments without a typical translocation, as it can be seen in
other sarcomas [5]. Osteosarcoma markers are required to
further characterize this complex, multifactorial neoplasia
and improve diagnosis and prognosis.
The TP53 gene was defined as the tumor suppressor
gene due to its ability to suppress the malignant growth
of transformed cells as well as tumors [6]. The TP53
gene locates in chromosome 17 (17p13), a region fre-
quently lost in tumors (LOH) and extensive TP53 muta-
tion searches revealed that over 50% of human tumors
carry mutations in this gene [7]. The majority of the
TP53 mutations are missense mutations occurring in the
highly conserved DNA binding domain of TP53 (exons 5
- 8), plus exon 9 [8]. According to IARC TP53 mutation
database, exons 5, 7 and 8 encompass 30, 26 and 24% of
all TP53 mutations respectively. Exon 10 harbors only
1.1% of reported TP53 mutations [9]. Of Li-Fraumeni
family members, who present an increased risk for de-
velopment of cancers, including osteosarcoma, it was
observed a correlation between tumor type, family struc-
ture and TP53 mutation type [10].
TP53 is a transcription factor which responds to di-
verse cellular stresses to regulate target genes that induce
cell cycle arrest, apoptosis, senescence, DNA repair, or
changes in metabolism [11]. TP53 also fulfills functions
during development in normal tissues [12] and in respon-
se to inflammation [13]. The p53 protein is expressed at
lower levels in normal cells compared to transformed
cells where higher levels of p53 was observed, suggest-
ing that p53 can contribute to transformation and mali-
gnancy. Many attempts to establish TP53 mutations as a
tumor marker have been done and the results are often
contradictory. It is difficult to compare all the studies, be-
cause different methodologies have been employed. Im-
munohistochemistry does not allow the precise identifi-
cation of the mutation. DNA sequencing revealed that in
human cancers, >1800 distinct TP53 missense mutations
have been identified. In breast cancer, it was demonstra-
ted that TP53 mutation is a marker of biologically ag-
gressive disease [14] and a response to therapy and survi-
val [15]. The correlation between p53 protein functional
status and a response to neoadjuvant chemotherapy was
demonstrated in head and neck squamous cell carcinoma
(HNSCC) in the oral cavity. The results indicate that the
p53 loss of function may predict a significant low rate of
complete remission and suboptimal response to cisplatin-
based neoadjuvant chemotherapy in patients with oral
cavity SCC [15]. The purpose of this paper is to investi-
gate whether TP53 mutational status predicts a response
to neoadjuvant chemotherapy and thus may help treat-
ment decision in osteosarcoma patients.
2. Material and Methods
2.1. Patient’s Selection
Patients with a confirmed diagnosis of osteosarcoma, at
any age of both genders that had the biopsies effectuated
in our center (Sarah Network of Hospitals, Brasília, Bra-
zil), without previous treatment of chemotherapy or ra-
diotherapy, were eligible for the study. Both fresh tissue
and paraffin embedded biopsies were considered. To be
included patients should be submitted to the treatment
comporting neoadjuvant chemotherapy, followed by sur-
gery and adjuvant chemotherapy according to the proto-
col established in May 1996. Of the 33 eligible patients,
15 were excluded due to insufficient DNA quantity and
one patient left our institution before the chemotherapy
treatment. The remaining 17 patients were analyzed in
the present work. This study was previously approved by
the Sarah Hospital Ethics Committee.
After neoadjuvant chemotherapy, all patients were ra-
diologically re-evaluated and surgery of primary tumor
was performed. The type of surgery (amputation or limb
salvage) was chosen according to the location and exten-
sion of the tumor.
To evaluate chemotherapy response, pathologists re-
viewed histological material to determine the primary
tumor response according to a method previously descri-
bed [16]. Responses were defined as “good” (>95% tu-
mor necrosis) or “poor” (<95% tumor necrosis).
2.2. TP53 Gene Amplification
Genomic DNA was extracted from 50 mg of frozen tis-
sue or from 3 to 6 10 µM sections of formalin-fixed, par-
affin-embedded blocks using the QIAamp DNA Mini Kit
(Qiagen). PCR analysis of exons 5 to 9 was performed
using the primers shown in the Table 1. TP53 exons
were amplified during 30 cycles of 94˚C for 45 s, 65˚C
for 45 s and 72˚C for 1 min in a 25 l volume reaction
containing from 50 to 200 ng of genomic DNA, 1.5 mM
Table 1. TP53 primers sequence.
Primer Sequence 5’ 3’ Amplicon Size in bp
E5-53-Bgccccagctgctcaccatcgcta 211
E6-53-Bggccactgacaaccacccttaacc 185
E7-53-Bcaagtggctcctgacctggagtc 139
E8-53-Bgtcctgcttgcttacctcgcttagt 200
E9-53-Bcgcaagacttagtacctgaagggtg 102
Open Access IJCM
TP53 Mutations and Chemotherapy Response to Neoadjuvant Metotrexate, Cisplatin and Adryamicin
Chemotherapy in Resected Osteosarcoma
of MgCl2, 0.2 mM dNTP’s, 50 M of each primer and
0.3 U of Taq DNA Polymerase Platinum (Life Techno-
2.3. Single Strand Conformation Polymorphism
(SSCP) and Sequencing Analysis
The single strand conformation polymorphism (SSCP)
analysis was performed using the PhastSystem automat-
ed electrophoresis apparatus (GE Healthcare/Amersham
Pharmacia) and 12.5% and 20% polyacrilamid gels. The
run settings were adapted from Kurvinen and coworkers
(1995) [17]. Pre-run: 400 V at 10 mA, 2.5 W, 100 Avh;
gel loading: 400 V, 1 mA, 2.5 W, 2 Avh, separation: 400
V, 10 mA, 2.5 W, 400 Avh*. All runs were performed at
15˚C. *Avh (accumulated volts per hour) the run time
varies according to the DNA size. Exon 5: 300 Avh,
Exon 6: 350 Avh, Exon 7: 350 Avh, Exon 8: 300, Avh,
Exon 9: 200 Avh.
After electrophoresis the gels were silver stained with
the Plus-One DNA Silver Staining kit (GE Healthcare/
Amersham Pharmacia) following manufactures’ indica-
tions. Using a vacuum gel drying system the gels were
dry with Whatman paper. Samples with mutations were
identified by the presence of an abnormal electrophoretic
migration pattern in comparison with a control carrying
wild-type (wt) TP53 and were further excised from the
gel, for PCR reamplification. The excised band was
placed in a 1.5 mL tube, following the addition of 100 µl
of water the material were frozen, macerated and heated
at 100˚C for 10 minutes. Following a 10,000 g centrifu-
gation for 10 minutes the supernatant were transferred to
a new tube and 5 µL of it used for PCR reamplification
in only 20 PCR cycles. After checking in an agarose gel
for the presence of a PCR product, the material was
cloned into a pGEM-T Easy vector (Promega). Plasmid
purification was performed using the Kit Flexi Prep (GE
Healthcare) according to instructions.
The sequencing reactions were prepared using the
universal primer (Promega) and the Big Dye Terminator
kit (Applied Biosystems), following manufacturers’ in-
structions. Sequencing reactions were resolved in the
ABI310 automatic sequencer (Applied Biosystems) Se-
quence alignments and translations were made with
BioEdit software.
2.4. Statistical Analysis
The association between TP53 gene mutations and the
response to chemotherapy was calculated using the Fi-
sher’s two-tailed exact test. Students’ test was applied to
compare ages. The criterion for statistical significance
was = 0.05. Kaplan and Meier survival curves and sta-
tistical analyses were conducted with GraphPad Prism
software (GraphPad Software).
3. Results
Seventeen patients aged 10 - 75 years (median 17 years)
entered the study. Ten patients were male (59%) and 7
female (41%). Sixteen patients had an osteoblastic tumor
and only one patient had a chondroblastic subtype.
We selected only biopsies of primary tumors before
treatment aiming to correlate the TP53 status to the
neoadjuvant treatment response. The identified somatic
mutations associated with hotspot region from the tumor
suppressor gene TP53 are presented in Table 2. Among
the 17 biopsies, 4 (23.5%) displayed TP53 mutations. In
total, 6 mutations were identified in 4 osteosarcoma tu-
mor samples: The patient 2 presented deletion of exon 9,
Ex9 c.924_936del102, patient 14 presented a silent mu-
tation at position E x 7 c.697 C > T and a deletion at po-
sition E x 7 c.732del1 corresponding to codon 244 which
engender a frameshit. The patient 25 also harbors a silent
mutation at position E x 7 c.703 C > A single-nucleotide
substitution and a serine cysteine substitution at codon
241 (E x 7 c.722 C > G S241C). For these patients, the
mutations can lead to a loss of function of p53 protein.
The patient 30 presented a deletion in intron 7 at position
In7 c14115del1.
The patients included in this study were treated with
the protocol established in the Sarah Hospital in May
1996 that consisted of a neoadjuvant chemotherapy, sur-
gery and adjuvant chemotherapy. The drugs concentra-
tions were estimated after a detailed clinical evaluation
of patients. Parameters such as body surface, age, even-
tual diseases, clinical and laboratorial conditions were
taken in account before the beginning of treatment and
between each cycle. The goal of neoadjuvant chemo-
therapy is to shrink the cancer. The osteosarcoma neoad-
juvant chemotherapy is composed of a high-dose of Me-
totrexate (12 g/m2 of body surface area), cisplatin at 120
mg/ m2 of body surface area and Adriamycin at 75 mg/m2
of body surface area and the schedule was as follow: first
and second weeks: Metotrexate, third week: Cisplatin
and Adriamycin, 4th and 5th weeks: rest, during 3 cycles.
The four mutated samples presented the tumor necro-
sis rate under 95% (Table 3) that lead us to conclude that
none of those samples presented a good response to the
chemotherapy. Nevertheless, 10 other samples also pre-
sented a tumor necrosis rate under 95%, indicating a bad
response to the chemotherapy. Only 3 samples had a
good response (>95%). However there is no correlation
between TP53 mutation and response to chemotherapy, p
= 1.2 (p > 0.05), that means the difference is not signifi-
cant for this population. The response to the neoadjuvant
chemotherapy is summarized in Table 2. There is a sig-
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TP53 Mutations and Chemotherapy Response to Neoadjuvant Metotrexate, Cisplatin and Adryamicin
Chemotherapy in Resected Osteosarcoma
Open Access IJCM
Table 2. Clinical and molecular features of osteosarcoma patients.
N˚. Histological subtype Age
(years) Sex TP53 gene status Mutation
type Effect
type Tumoral
Necrosis % Status* Duration of follow
up (months)**
1 Osteoblastic 12 M WT 78 AWD 164
2 Osteoblastic 75 F Ex9 c.924_936del102deletion frameshift5 DOD 6
3 Osteoblastic 12 F WT 100 NED 183
4 Chondroblastic 23 M WT 73 NED 175
6 Osteoblastic central high grade 18 F WT 75 DOD 14
9 Osteoblastic central high grade 17 M WT 88 DOD 11
11 Osteoblastic 16 M WT 49 DOD 11
12 Osteoblastic 13 F WT 10 DOD 21
14 Osteoblastic 16 M Ex7 c.732del1 deletion frameshift70 AWD 140
22 Osteoblastic 20 M WT 30 LFU ---
23 Osteoblastic 10 F WT 95 NED 150
24 Osteoblastic 17 M WT 70 NED 130
25 Osteoblastic 24 M Ex7 c.722 C > G S241Csubstitutionmissense70 NED 130
28 Osteoblastic 17 M WT 95 NED 127
30 Osteoblastic 19 F
In7 c14115del1 deletion intronic87 AWD 123
31 Osteoblastic 13 F WT 89 AWD 153
33 Osteoblastic 18 M WT 85 AWD 140
*AWD = alive with disease, DOD = dead of disease, NED = no evolutive disease, LFU = lost of follow-up. **Duration of follow up until death for DOD or last
consultation for AWD and NED, in December 2011.
Table 3. Fischer test for variables: mutated and wild type
Tumoral necrosis rate
TP53 < of 95% > of 95% Total
Mutated 4 0 4
Wild type 10 3 13
Total 14 3 17
nificant correlation between patient age and TP53 muta-
tions. Patients harboring TP53 mutations are older (me-
dian 21.5 years) than those that do not present TP53 mu-
tations (median 17 years), p = 0.03 (p < 0.05) with Stu-
dent’s test.
Figure 1. Kaplan-Meier overall survival curves stratified by
TP53 mutation status (N = 17).
lish an association with a favorable prognosis related to a
molecular alteration. Even considered as a rare disorder
0.4/100,0001 in the Sarah Hospital, osteosarcoma is the
most frequent neoplasia. One retrospective study effectu-
ated from 1982 to 1996 revealed that the event free sur-
vival of 5 years is 54% for patients without metastasis at
the diagnostic, and 29% for those presenting metastases.
These data differ significantly from those related in the
It is worth to note that our follow up of patients
reached 12 years or more. It is very rare to find such a
long last follow up in the literature. Considering this, we
have observed that Kaplan-Meier overall survival curve
for patients with TP53 mutations are similar to those for
individuals with wild type TP53, in conclusion there is
no difference in survival rat between patients harboring
TP53 wild type or mutated forms (Log-rank test, p =
0.8872). Nevertheless, considering our small sample size
those values need to be carefully interpreted (Figure 1).
The suppressor gene TP53 has been investigated in
many human neoplasias and is altered in about half of it
[18]. Many different alterations have been reported in-
cluding breaks, punctual mutations and deletions.
4. Discussion The TP53 SSCP analysis after PCR amplification re-
vealed conformational changes in 5 patients and se-
quencing analysis confirmed the presence of mutations in
Many molecular alterations have been described in os-
teosarcoma; nevertheless few researchers could estab-
TP53 Mutations and Chemotherapy Response to Neoadjuvant Metotrexate, Cisplatin and Adryamicin
Chemotherapy in Resected Osteosarcoma
4 out 5. The SSCP is an efficient method for screening
genetic variations, including single mutations. The sensi-
bility allows detecting between 80% to 90% of the muta-
tions in 200 - 400 bp length DNA fragments [19]. It is an
affordable method for institutions that do not dispose of a
high throughput sequencer.
Our analysis of TP53 mutations extended from exons
5 to 9, a region that encompasses about 90% of the muta-
tions [8]. We found mutations in 23.5% of our patients.
In osteosarcoma, there are no consensual data concerning
the TP53 mutations rate and the response to chemother-
apy. In a study published in 1998, Goto and coworkers
(1998) observed TP53 mutations in 41% of the samples,
and among them, 15% were good responders while 64%
of patients without TP53 mutations presented a good
response, with a significant correlation (p < 0.05). They
considered as good responders who presented tumoral
necrosis rate >90% [20]. This criterion is different from
those suggested by Rosen and coworkers (1993) and
adopted by us in this study, in which the good responders
present >95% of tumor necrosis rate [21].
Radig and coworkers (1996) analyzed 40 osteosarco-
ma patients and found TP53 mutations in 19%. In an-
other study, published in 1998, the same group found
15.7% of mutated osteosarcomas which do not correlate
with tumor progression [22]. In a large study containing
272 primary osteosarcomas, TP53 mutation was obser-
ved in 22% of patients without the correlation of tumor
progression [23]. The mutation rate varying from 13.3%
[24] (associated with disease free survival), 18% [25],
and up to 26.5% [26] has been reported. In a study with a
large cohort of 196 osteosarcoma patients, TP53 mu t a-
tions were observed in 19.4% of osteosarcoma patients,
and no correlation with p53 mutations and prognosis
were observed, nevertheless patient age was the only
factor that varied with TP53 gene status (p < 0.05) [27].
We confirmed this data. In our study, TP53 mutation
status was not predictive of chemoresistance; suggesting
that chemotherapy response is independent of the TP53
gene. Nevertheless, the mutation rate could be associated
with patient age (p<0.05), as already described [26,27].
A very surprisingly result was observed when we
compared the overall survival rate between osteosarcoma
patients with TP53 wild type and mutated TP53. In our
cohort, mutated patients have the same live span with
patients harboring wild type TP53. It is interesting to
note that the follow-up over 15 years, as we have des-
cribed, is very rare to see in the literature. Nevertheless,
the sample size of the current study was too small to
draw definitive conclusions regarding the relationship
between clinicopathologic features and p53 changes.
Studying the expression of IDH1 and TP53 in os-
teosarcoma, Hu and coworkers (2010), detected higher
levels of IDH1 in the wild type than in TP53 mutant cells.
IDH1 correlates with histological Rosen grade and me-
tastasis negatively. TP53 correlates with histological
Rosen grade, metastasis and overall survival in clinical
osteosarcoma biopsies. Osteosarcoma patients with high
IDH1 expression have a very high p53 expression, so
IDH1 may correlate with p53 and is a candidate bio-
marker for osteosarcoma [28].
Finally, a recent whole-genome sequencing study on
osteosarcomas and chordomas describes a new process of
cancer genome evolution termed chromothripsis. Rather
than a multistep accumulation of unbalanced rearrange-
ments, there is a single catastrophic genomic instability
event that primarily affects a single chromosome [29];
maybe this could explain why TP53 mutations alone do
not correlate to tumor progression. Moreover, to establish
clinical implications of p53, it is also necessary to con-
sider miRNA expression. They are generally deleted
and/or disregulated in cancer. Certain microRNAs, (mir-
34a, b, and c) can be transcriptionally transactivated by
p53 [30,31]. On the other hand, mir-125b, negatively re-
gulates TP53 expression [32]. So, it seems that not only
TP53 mutations do affect its downstream functions, but
also change in the related miRNAs need to be considered.
Furthermore, p53 isoform variants originated from alter-
native splicing and the promoter usage can also have
clinical implications [33]. In conclusion, the analysis of
TP53 mutations status is the first step in the understand-
ing of this important tumor suppressor gene in osteosar-
comatumorigenesis. Further analysis, at the protein level
and miRNA expression, needs to be performed aiming to
establish a correlation between p53 and osteosarcoma
clinical features.
5. Author’s Contribution
LR: carried out the molecular genetic studies and par-
ticipated in the design of the study and drafted the
manuscript. CDB: participated in the design of the study,
participated in the sequence alignment, performed the
statistical analysis and coordination and draft the manu-
script. MB conceived the study, and participated in its
design and coordination. All authors read and approved
the final manuscript.
6. Acknowledgments and Funding
We are grateful to Dr Ricardo Karan Kalil for technical
support and Dr Andréa Carla de Souza Góes for critical
reading. The Sarah Network of Hospitals for Rehabilita-
tion financed this work.
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Chemotherapy in Resected Osteosarcoma
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List of Abbreviations
TP53: tumor protein p53;
IARC International Agency for Research on Cancer;
DNA: Deoxyribonucleic acid;
HNSCC: head and neck squamous cell carcinoma;
PCR: polymerase chain reaction;
SSCP: Single Strand Conformation Polymorphism;
Avh: accumulated volts per hour;
IDH1: isocitrate dehydrogenase.