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Mutations of the TP53 gene in adenocarcinoma and squamous cell carcinoma of the cervix: A systematic review
Gynecologic Oncology 128 (2013) 442–448
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Mutations of the TP53 gene in adenocarcinoma and squamous cell carcinoma of the cervix: A systematic review Maria Lina Tornesello ⁎, Luigi Buonaguro, Franco M. Buonaguro Molecular Biology and Viral Oncology Unit, and AIDS Reference Centre, National Cancer Institute, “Fondazione Pascale”, Naples, Italy
H I G H L I G H T S ► TP53 mutations are significantly more prevalent in adenocarcinoma than in squamous cell carcinoma of the cervix. ► TP53 mutational profile depends from the geographical origin of the tumors. ► Mutated codons show several differences in the two types of tumor.
a r t i c l e
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Available online 17 November 2012 Keywords: TP53 mutation Human papillomavirus Cervical squamous cell carcinoma Cervical adenocarcinoma
a b s t r a c t Objective. Mutations of the tumor suppressor gene TP53 are the most significant events in several human cancers. Few studies have analyzed the frequency of TP53 alterations in squamous cell carcinoma and adenocarcinoma of the cervix with controversial results. This study provides a detailed analysis of TP53 mutation spectra in cervical squamous cell carcinoma and adenocarcinoma from different geographical regions. Methods. The analysis of TP53 mutational profiles was performed in 1353 cervical cancers retrieved from the IARC p53 mutation database (R15, 2010) and the COSMIC data along with the literature review of related studies identified by PubMed searching. Results. This analysis showed a significant higher mutation frequency of TP53 gene in cervical adenocarcinoma (32 of 241; 13.3%) compared to squamous cell carcinoma (39 of 657; 5.9%; P = 0.0003, χ2 test). The proportion of adenocarcinoma with mutated TP53 varied from 4% in North America to 19% in Asia. Among the six hot-spot codons of TP53 gene, three codons (175, 248 and 273) were the most commonly mutated in both types of cervical cancer, one codon (249) mainly in squamous cell carcinoma and one codon (282) only in adenocarcinoma. The G to A and C to T transitions were the prevalent type of mutations in both squamous cell carcinoma and adenocarcinoma (48.7% and 53.5% of all mutations, respectively). The frequency of C to A transversion was relatively high only in adenocarcinoma (25%), while the mirror mutation G to T was comparatively frequent in squamous cell carcinoma (14.6%). Conclusions. Different patterns of TP53 mutations occur in squamous cell carcinoma and adenocarcinoma of the cervix in different regions of the world. The highest frequency of mutated TP53 has been observed in cervical adenocarcinoma from Asia. Further studies are needed to better define the role of TP53 alterations in cervical cancer and possibly to understand the impact of mutations on cancer prognosis and outcomes. © 2012 Elsevier Inc. All rights reserved.
Introduction The two major histopathologic types of cervical cancer are squamous cell carcinoma and adenocarcinoma. Together these tumors represent the third most common cancer in women worldwide with
Abbreviations: HPV, Human papillomavirus; ICC, Invasive cervical carcinoma; SCC, Squamous cell carcinoma; ADC, Adenocarcinoma. ⁎ Corresponding author at: Molecular Biology and Viral Oncology, National Cancer Institute “Fondazione Pascale”, Cappella Cangiani, I-80131 Naples, Italy. Fax: + 39 081 5451 276. E-mail addresses: [email protected], [email protected] (M.L. Tornesello). 0090-8258/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygyno.2012.11.017
an estimated 529,800 new cases and 275,100 deaths in 2008 [1]. The squamous cell carcinoma generally arises at the squamocolumnar junction, between the squamous epithelium of the ectocervix and the columnar epithelium of the endocervix, according to a classical progressive sequence from mild to severe dysplasia, carcinoma in situ and invasive carcinoma [2]. Cervical adenocarcinoma derives from glandular precursor lesions of the endocervical mucosa, designated adenocarcinoma in situ, and comprises several histological subtypes such as the mucinous adenocarcinoma, which may be intestinal, endocervical or signet-ring, and the endometrioid adenocarcinoma [3]. The analysis of the SEER database (National Cancer Institute's Surveillance, Epidemiology, and End Results), including 18,979 women with squamous cell carcinomas and 5583 adenosquamous
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and adenocarcinoma, showed that at diagnosis 26.7% of women with adenocarcinoma had stage IB1 tumors compared to 16.9% of those with squamous cell carcinoma [4]. Women with early stage adenocarcinoma, however, were nearly 40% more likely to die from their disease than patients with squamous cell carcinoma suggesting that distinct molecular mechanisms underlie the pathogenesis of the two types of cervical cancer. Squamous cell carcinoma and adenocarcinoma have been strongly linked to human papillomavirus (HPV) infection, mainly HPV 16 and 18 genotypes [5]. The oncogenic potential of these viruses has been attributed to E6 and E7 early genes which are consistently expressed in HPV-related cancers and derived tumor cell lines and shown to be the main mediators of multistep carcinogenesis for their multiple interactions with several cellular targets [6]. However, highly variable levels of viral oncogene expression have been detected in both cervical intraepithelial neoplasia and invasive cancer, independently from histological grading and physical state of the viral genome [7]. These observations indicate that viral early gene expression is not in itself sufficient to induce cervical cancer and that additional genetic and/or epigenetic events are required [8]. Mutations or functional alterations of cellular genes such as PIK3CA [9], c-Myc (Myc), ErbB2 [10], cIAP1 [11], Ras [12], PTEN [13] and LKB1 [14] have all been reported in variable proportions of cervical cancers. A more extensive although not conclusive mutational analysis has been performed on TP53 tumor suppressor gene [15]. Mutations of the TP53 oncosuppressor gene are among the most common genetic alterations in many human malignancies [16–20]. Epidemiological and molecular studies have demonstrated a link between exposure to several mutagenic compounds and specific TP53 nucleotide changes [21,22]. Among these the well-documented associations are: 1) exposure to ultraviolet irradiation with tandem CC to TT transitions in non melanoma skin cancers [23]; 2) tobacco smoking and polycyclic aromatic hydrocarbons' exposure with G to T transversions at specific G:C base pairs [24,25]; 3) exposure to dietary aflatoxin B1 with G to T transversions at codon 249 in HBV-associated hepatocellular carcinoma [26,27]; and 4) dietary exposure to aristolochic acid with high frequency of A to T transversions in Balkan endemic nephropathy [28–30]. In addition, about 25% of all TP53 mutations are represented by G:C to A:T transitions at CpG sites potentially originating from spontaneous deamination of DNA bases [31]. However, the majority of factors associated with the origin of TP53 mutations along with the heterogeneous distribution by tumor type and geographical location remain largely unknown. The status of TP53 gene was first analyzed in human cervical carcinoma cell lines that were either positive or negative for HPV DNA sequences. The identification of TP53 mutations exclusively in HPVnegative C33-A and HT-3 cell lines led to the initial hypothesis that TP53 somatic mutations are a frequent event in HPV-negative anogenital cancers [32]. This preliminary observation was further confirmed by the analysis of primary cervical cancer presenting TP53 mutations in 60% of HPV-negative and in none of HPV-positive cancers [33]. Later on, TP53 nucleotide changes were identified in metastasis arising from HPVpositive cervical carcinomas, suggesting that p53 mutations could play a major role in the acquisition of aggressive behavior of some primary cancers associated with HPV infection [34]. Conversely, several other studies reported that inactivation of the TP53 gene by allelic loss or by point mutation is an infrequent event in clinical samples of cervical carcinomas, irrespective of the HPV infection [35–37]. Nevertheless, no systematic study has been performed to evaluate the TP53 mutational spectra in the two major histological types of cervical carcinoma from different geographic regions. In this article a systematic review of published studies has been performed to investigate the mutational pattern of TP53 gene in cervical adenocarcinoma and squamous cell carcinoma and possibly to correlate the type of mutations to the presence of specific mutagenic factors in different regions of the world.
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Materials and methods Review of the literature The study was based on the review of available data on the distribution of TP53 mutations in cervical cancer from previous English language literature. The TP53 mutation spectra have been independently analyzed in squamous cell carcinoma and adenocarcinoma of the cervix searching the International Agency for Research on Cancer (IARC) TP53 (http://www-p53.iarc.fr/) and COSMIC databases (http://www.sanger. ac.uk/genetics/CGP/cosmic/) and retrieving articles from Medline using the terms “p53” AND “mutation” AND “cervical” AND “carcinoma” (Table 1). All records evaluating cell lines and tumors other than primary cervical cancer were excluded from the analysis. Ten studies reporting no distinction between adenocarcinoma and squamous cell carcinoma were listed separately in Table 1 and mutations excluded from the comparative analysis between the two histological types of cervical carcinoma. To be included in the systematic review, a study had to present the total number of tumors included in the analysis and individual information on TP53 mutations obtained by several techniques and confirmed by DNA sequencing analysis. The majority of studies focused on exons 5–8, and used SSCP or DDGE as screening method for mutations and direct nucleotide sequencing analysis as confirmatory test, and did not search for mutations in the rest of the gene. Studies were stratified into four geographical regions (Africa, America, Asia and Europe). Analysis The following analyses were planned in the study design: frequency of TP53 mutations in cancer cases stratified by histological grade (squamous cell carcinoma and adenocarcinoma histological subtypes); frequency of TP53 mutations in adenocarcinoma and squamous cell carcinoma stratified by four broad geographical regions. All mutations were verified by MUT-TP53 2.0, a matrix developed by Soussi et al., which using functional and statistical information derived from the IARC p53 database is able to predict the biological activity and likelihood of each TP53 mutant [38]. In particular, the MUT-TP53 2.0 spreadsheet consists of tables in which the functional activity of 2314 p53 mutants, evaluated in terms of loss of transactivation on p21Waf1 promoter by a yeast-based functional assay, has been recorded [39]. The combination of the universal mutation p53 database with the p53 mutant activity database has been shown to be useful to perform functional analysis of thousands of reported p53 alterations in human cancers [38,40]. Results Twenty-seven studies, performed in Asia, Europe, America and Africa, were included in the systematic review comprising a total of 1353 cervical cancers analyzed for TP53 mutations, the majority focused in exons 5 to 8. In Table 1, mutation frequencies in each study were grouped by histological subtype and geography. The proportion of tumors carrying any mutation in TP53 gene showed a broad range across reports. The overall rates of TP53 mutated cases were significantly higher (P = 0.0003) among adenocarcinoma (32 of 241; 13.3%) compared to squamous cell carcinoma (39 of 657; 5.9%). TP53 mutations in adenocarcinoma were relatively frequent in Asia (19.05%) and Europe (12.16%) but rarer in North America (4%). Among squamous cell carcinoma cases, on the other hand, TP53 mutations were rarely identified in Asia (4.31%) and North America (3.64%), while more frequently in Europe (9.22%). Regarding spatial distribution of TP53 mutations, the most commonly mutated codons were 175, 248 and 273 in both types of cervical cancer, the codon 249 mainly in squamous cell carcinoma and the codons 161 and 282 only in adenocarcinoma (Fig. 1). Fig. 2 shows codon mutation patterns stratified by geographic origin of tumors.
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Table 1 List of published data on TP53 mutations' frequencies in invasive squamous cell carcinoma and adenocarcinoma of the cervix in different regions of the world. Continent
Location
Year
Reference
Exons
Method
Tumor*
TP53 Mut
All samples
Europe
UK Norway USA Brazil China Korea Japan
1992 1993 1994 2001 1993 1995 2001
Borresen et al., Lancet. 1992 (339):1350–1 Helland J Pathol. 1993 (171):105–14 Park et al., Oncogene. 1994 (9):205–10 Pinheiro et al., Braz J Med Biol Res. 2001 Jun;34(6):727–33 Choo and Chong, Virology. 1993 (193):1042–1046 Kim & Kim, Yonsei Med J. 1995 Nov;36(5):412–25 Harima et al.,Int J Cancer. 2001 Oct 20;96(5):286–96.
5–8 5–8 5–8 5–8 1–11 5–9 5–8
CDGE CDGE SSCP SSCP SSCP SSCP SSCP
UK UK Germany Germany Norway USA USA, Spain, Colombia China Japan Japan Japan Japan Japan Korea Thailand Egypt
1992 1994 1995 1995 1998 1993 1993 1997 1992 1992 1995 1999 2007 1997 2000 2007
Crook et al., Lancet. 1992 (339):1070–3 Busby-Earle et al., Br J Cancer. 1994 (69):732–7 Milde-Langosch et al., Int J Cancer. 1995 (63):639–45 Ikenberg et al., Cancer. 1995 (76):57–66 Helland et al., Br J Cancer. 1998 Jul;78(1):69–72 Paquette et al., Cancer. 1993 (72):1272–80. Kessis et al., Am J Pathol. 1993 (143):1398–405 Ngan et a., Genitourin Med. 1997 (73):54–8. Fujita et al., Cancer Res 1992 (52):5323–8 Tsuda et al., Jpn J Cancer Res. 1992 (83):1184–1191 Miwa et al., Br J Cancer. 1995 (71):219–26 Nakagawa et al., Br J Cancer. 1999 Mar;79(7–8):1139–44 Iwakawa et al., Cancer Biol Ther. 2007 (6):905–11 Kim et al., Acta Oncol. 1997 (36):295–300 Limpaiboon et al., Southeast Asian J Trop Med. 2000 (31):66–71 Bahnassy et al., BMC Clin Pathol. 2007 May 24;7:4
1–11 5–8 5–8 5–8 5–8 5–8 5–8 2–11 5–8 4–9 2–11 5–8 5–8 4–9 5–8 5–9
Seq DGGE TGGE SSCP CDGE + seq SSCP Seq SSCP SSCP-C-Seq SSCP-C-Seq SSCP SSCP SSCP SSCP SSCP SSCP
UK Germany Germany Italy Norway Germany Sweden USA USA, Spain, Colombia Japan Japan Japan Japan Korea Japan
1992 1995 1995 1998 1998 2001 2006 1993 1993 1992 1992 1994 1995 1997 1999
Crook et al., Lancet. 1992 (339):1070–3 Milde-Langosch et al., Int J Cancer. 1995 (63):639–45 Ikenberg et al., Cancer. 1995 (76):57–66 Tenti et al., Am J Pathol. 1998 (152):1057–63 Helland et al., Br J Cancer. 1998 Jul;78(1):69–72 Denk et al., J Mol Med (Berl). 2001 (79):283–8 Andersson et al., Med Oncol. 2006 (23):113–9. Paquette et al., Cancer. 1993 (72):1272–80. Kessis et al., Am J Pathol. 1993 (143):1398–405 Fujita et al., Cancer Res. 1992 (52):5323–8 Tsuda et al., Jpn J Cancer Res. 1992 (83):1184–1191 Jiko et al., Int J Cancer. 1994 (59):601–6. Miwa et al., Br J Cancer. 1995 (71):219–26 Kim et al., Acta Oncol. 1997 (36):295–300 Nakagawa et al., Br J Cancer. 1999 Mar;79(7–8):1139–44
1–11 5–8 5–8 5–8 5–8 4–10 5–8 5–8 5–8 5–8 4–9 5–8 2–11 4–9 5–8
Seq TGGE SSCP DGGE CDGE + seq Seq SSCP SSCP Seq SSCP-C-Seq SSCP-C-Seq SSCP SSCP SSCP SSCP
ICC ICC ICC ICC ICC ICC ICC Total ICC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC Total SCC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC Total ADC
2 2 2 4 0 6 7 23 3 1 2 2 6 1 1 2 1 0 3 5 3 2 2 5 39 0 2 0 9 1 1 5 1 0 1 3 8 0 0 0 31
92 92 21 122 38 30 65 460 20 47 25 38 11 28 27 103 30 33 42 41 25 127 17 43 657 4 26 5 74 4 5 30 17 8 6 13 25 5 9 5 236
North America South America Asia
Europe
North America Asia
Africa Europe
North America Asia
% 2.17% 2.17% 9.52% 3.28% 20.00% 10.77% 5.00% 15.00% 2.13% 8.00% 5.26% 54.55% 3.57% 3.70% 1.94% 3.33% 7.14% 12.20% 12.00% 1.57% 11.76% 11.63% 5.94% 7.69% 12.16% 25.00% 20.00% 16.67% 5.88% 16.67% 23.08% 32.00%
13.14%
*Abbreviations: ICC, invasive carcinoma (comprising adeno/adenosquamous carcinoma, squamous cell carcinoma and cervical cancer of unspecified histology); ADC, adenocarcinoma; SCC, squamous cell carcinoma.
Interestingly, codon 249 was mainly mutated in Africa, probably due to Aflatoxin exposure, less in Europe and never mutated in Asia and North America. Codons 175 and 273 were found often mutated in Europe and rarely or never in Asia, America and Africa. Codon 248 was the most mutated site in Asian cases. These codons (175, 248 and 273) are the most frequently mutated sites in many human tumors and are associated with loss of p53 function. The TP53 mutations identified in adenocarcinoma and squamous cell carcinoma of the cervix have been compared, using the MUT-TP53 2.0 verification tool, to those of several other tumors present in the p53 database in relation to their effect on p21Waf1 activity [38]. The functional activity of mutated proteins detected in adenocarcinoma and squamous cell carcinoma cases showed a mean value of −1.295 and −0.827, respectively, suggesting major differences in the type of mutations between the two cancers (Fig. 3). Analysis of mutated nucleotides, showed that G to A and the mirror mutations C to T transitions were by far the most abundant, representing 21.4% and 32.1% of changes, respectively, in adenocarcinoma and 26.8% and 21.9%, respectively, in squamous cell carcinoma. The C to A transitions were highly frequent in adenocarcinoma (25%) and rarer in squamous cell carcinoma (2.4%). Conversely G to T transversions were more frequent in squamous cell carcinoma (14.6%) than in adenocarcinoma (3.6%), suggesting that a strand bias occurred during the mutation
process probably due to the preferential repair of damage on the transcribed DNA strand during transcription-coupled repair. In Fig. 4 the 12 types of possible base substitutions were grouped into 6 pairs, each consisting of two complementary substitutions: (G:C, C:G) (A:T, T:A), etc. The overall TP53 pattern then is shown as two complementary sub-patterns in which mutations on the nontranscribed strand are reported above the line in Fig. 1A, and the complementary substitutions on transcribed strand are represented under the line. Discussion The systematic review of twenty-seven studies analyzing TP53 mutational status in cervical cancer showed that nucleotide changes are significantly more frequent in cervical adenocarcinoma (13.3%) than squamous cell carcinoma (5.9%). The highest incidence of TP53 mutations was observed in adenocarcinoma from Asia (19%) and Europe (12.2%). The first evidence of high TP53 mutation rate in cervical adenocarcinoma was reported by Jiko et al. who described alterations in 22.2% of tumors from Japanese women with higher incidence in tumors at advanced clinical stage and with high grade nuclear and structural atypia [41]. Tenti et al. described a TP53 mutation rate of 13.5% among 74 primary cervical adenocarcinoma from Italian women. The majority of
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445
SCC AdC 4
Number of Mutations
3
2
1
0 1 1 3 3 1 1 3 3 5 9 1 9 2 2 3 4 6 4 7 0 1 2 8 9 4 9 0 9 3 0 1 2 3 5 0 2 10 13 13 14 15 16 16 17 17 17 18 18 19 20 21 21 22 23 23 24 24 24 24 24 25 25 26 26 27 28 28 28 28 28 29 29
TP53 Codons Fig. 1. Distribution of mutated codons of the TP53 tumor suppressor gene in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of the cervix.
nucleotide changes were G:C to A:T transitions and were more frequent in cancer cases of high grade, high stage, and large size, suggesting that TP53 mutations play a major role in the progression rather than in the
initiation of cervical adenocarcinoma [42]. Accordingly, Andersson et al. observed that single nucleotide changes occurred in six out of 30 (20%) primary cervical adenocarcinoma independently from HPV status [43].
Fig. 2. Distribution of mutated codons of the TP53 tumor suppressor gene in cervical cancer from different geographical regions.
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SCC 1 0,8 0,6 0,4
WAF1 ACTIVITY
0,2 0 -0,2 -0,4 -0,6 -0,8 -1 -1,2 -1,4 -1,6 -1,8 -2
1
ADC
0,8 0,6 0,4
WAF1 ACTIVITY
0,2 0 -0,2 -0,4 -0,6 -0,8 -1 -1,2 -1,4 -1,6 -1,8 -2
Fig. 3. Meta-analysis of p53 loss of function as measured by transactivation of the WAF1 promoter [38]. On the x-axis is indicated the publication code, the first letter corresponds to the cancer type and the second number is an anonymous ID for the publication. Data from the analysis of squamous cell carcinoma or adenocarcinoma of the cervix are displayed at the right side of the graph (arrow).
Unfortunately no studies to date addressed the issue of TP53 mutation distribution across the different subtypes of adenocarcinoma such as endocervical, endometrioid, intestinal, and mixed adenocarcinoma. The majority of human cancers (including skin, lung, liver, breast, gastrointestinal tract, bladder, etc.) display mutations in six hotspots (codons 175, 245, 248, 249, 273, and 282) within the DNA-binding domain of p53 protein. Accordingly, the most commonly mutated sites in both types of cervical cancer were codons 175, 248 and 273. However, frequencies of codon changes observed in different geographical locations were dissimilar probably due to different environmental or genetic factors. Furthermore, the evaluation of all mutations identified in this study with MUT-TP53 2.0 tool showed that the loss of function of p53 activity was significantly higher in adenocarcinoma (−1.3) than in squamous cell carcinoma (−0.8). The biological significance of this phenomenon, however, remains to be uncovered.
The pattern of p53 mutations was similar in adenocarcinoma and squamous cell carcinoma of the cervix and showed an excess of G:C to A:T transitions. These types of mutations, which are the most common in all human cancers, may originate from spontaneous nucleotide changes deriving from deamination of methylcytosine [4]. However, it is unclear whether their high frequency in cervical cancer may be related to a specific carcinogen or to the excess of oxygen and nitrogen radicals produced by oxidant-generating enzymes during chronic inflammation [44–48]. A major limitation of this pooled analysis of previously published data relies on the fact that several studies included limited sample size and most importantly they have been performed with different screening methods (i.e. SSCP, DGGE, direct sequencing, etc.) with variable sensitivity and specificity. However, despite these limitations, the pooled data provide some interesting insights indicative of future
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A
SCC
ADC
G:C>A:T G:C>T:A G:C>C:G A:T>G:C A:T>C:G A:T>T:A 30% 25% 20% 15% 10% 5% 0% 5% 10% 15% 20% 25% 30%
C:G>T:A C:G>A:T C:G>G:C T:A>C:G T:A>G:C T:A>A:T
B SCC Type of mutations
No. of mutations (%)
NTS
ADC TS
NTS
TS
G:C>A:T
35
50,7%
11
9
6
G:C>T:A
15
21,7%
6
1
1
9 7
G:C>C:G
9
13,0%
4
2
2
1
A:T>G:C
5
7,2%
3
0
2
0
A:T>C:G
3
4,3%
2
1
0
0
2
2,9%
A:T>T:A Total
69
2
0
0
0
28
13
11
17
Fig. 4. A. TP53 mutational patterns represented as pairs of complementary transitions and transversions in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of the cervix; the upper part represents non-transcribed strand (NTS), and the lower part, transcribed strand (TS). B. Summary of TP53 mutations detected in adenocarcinoma and squamous cell carcinoma.
investigations. In particular, more refined genetic epidemiologic studies with more sensitive techniques, such as next-generation sequencing, are needed to better define the prevalence and the type of mutations in the TP53 gene in cervical adenocarcinoma and squamous cell carcinoma from different regions of the world. Moreover, the clinical significance of TP53 status in terms of overall survival, metastasis-free survival and outcome to chemotherapy and/or radiotherapy in cervical cancer is still unknown and needs to be assessed in prospective longitudinal studies. Conflict of interest statement The authors have no conflict of interests.
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Contents lists available at SciVerse ScienceDirect
Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
Mutations of the TP53 gene in adenocarcinoma and squamous cell carcinoma of the cervix: A systematic review Maria Lina Tornesello ⁎, Luigi Buonaguro, Franco M. Buonaguro Molecular Biology and Viral Oncology Unit, and AIDS Reference Centre, National Cancer Institute, “Fondazione Pascale”, Naples, Italy
H I G H L I G H T S ► TP53 mutations are significantly more prevalent in adenocarcinoma than in squamous cell carcinoma of the cervix. ► TP53 mutational profile depends from the geographical origin of the tumors. ► Mutated codons show several differences in the two types of tumor.
a r t i c l e
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Available online 17 November 2012 Keywords: TP53 mutation Human papillomavirus Cervical squamous cell carcinoma Cervical adenocarcinoma
a b s t r a c t Objective. Mutations of the tumor suppressor gene TP53 are the most significant events in several human cancers. Few studies have analyzed the frequency of TP53 alterations in squamous cell carcinoma and adenocarcinoma of the cervix with controversial results. This study provides a detailed analysis of TP53 mutation spectra in cervical squamous cell carcinoma and adenocarcinoma from different geographical regions. Methods. The analysis of TP53 mutational profiles was performed in 1353 cervical cancers retrieved from the IARC p53 mutation database (R15, 2010) and the COSMIC data along with the literature review of related studies identified by PubMed searching. Results. This analysis showed a significant higher mutation frequency of TP53 gene in cervical adenocarcinoma (32 of 241; 13.3%) compared to squamous cell carcinoma (39 of 657; 5.9%; P = 0.0003, χ2 test). The proportion of adenocarcinoma with mutated TP53 varied from 4% in North America to 19% in Asia. Among the six hot-spot codons of TP53 gene, three codons (175, 248 and 273) were the most commonly mutated in both types of cervical cancer, one codon (249) mainly in squamous cell carcinoma and one codon (282) only in adenocarcinoma. The G to A and C to T transitions were the prevalent type of mutations in both squamous cell carcinoma and adenocarcinoma (48.7% and 53.5% of all mutations, respectively). The frequency of C to A transversion was relatively high only in adenocarcinoma (25%), while the mirror mutation G to T was comparatively frequent in squamous cell carcinoma (14.6%). Conclusions. Different patterns of TP53 mutations occur in squamous cell carcinoma and adenocarcinoma of the cervix in different regions of the world. The highest frequency of mutated TP53 has been observed in cervical adenocarcinoma from Asia. Further studies are needed to better define the role of TP53 alterations in cervical cancer and possibly to understand the impact of mutations on cancer prognosis and outcomes. © 2012 Elsevier Inc. All rights reserved.
Introduction The two major histopathologic types of cervical cancer are squamous cell carcinoma and adenocarcinoma. Together these tumors represent the third most common cancer in women worldwide with
Abbreviations: HPV, Human papillomavirus; ICC, Invasive cervical carcinoma; SCC, Squamous cell carcinoma; ADC, Adenocarcinoma. ⁎ Corresponding author at: Molecular Biology and Viral Oncology, National Cancer Institute “Fondazione Pascale”, Cappella Cangiani, I-80131 Naples, Italy. Fax: + 39 081 5451 276. E-mail addresses: [email protected], [email protected] (M.L. Tornesello). 0090-8258/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygyno.2012.11.017
an estimated 529,800 new cases and 275,100 deaths in 2008 [1]. The squamous cell carcinoma generally arises at the squamocolumnar junction, between the squamous epithelium of the ectocervix and the columnar epithelium of the endocervix, according to a classical progressive sequence from mild to severe dysplasia, carcinoma in situ and invasive carcinoma [2]. Cervical adenocarcinoma derives from glandular precursor lesions of the endocervical mucosa, designated adenocarcinoma in situ, and comprises several histological subtypes such as the mucinous adenocarcinoma, which may be intestinal, endocervical or signet-ring, and the endometrioid adenocarcinoma [3]. The analysis of the SEER database (National Cancer Institute's Surveillance, Epidemiology, and End Results), including 18,979 women with squamous cell carcinomas and 5583 adenosquamous
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and adenocarcinoma, showed that at diagnosis 26.7% of women with adenocarcinoma had stage IB1 tumors compared to 16.9% of those with squamous cell carcinoma [4]. Women with early stage adenocarcinoma, however, were nearly 40% more likely to die from their disease than patients with squamous cell carcinoma suggesting that distinct molecular mechanisms underlie the pathogenesis of the two types of cervical cancer. Squamous cell carcinoma and adenocarcinoma have been strongly linked to human papillomavirus (HPV) infection, mainly HPV 16 and 18 genotypes [5]. The oncogenic potential of these viruses has been attributed to E6 and E7 early genes which are consistently expressed in HPV-related cancers and derived tumor cell lines and shown to be the main mediators of multistep carcinogenesis for their multiple interactions with several cellular targets [6]. However, highly variable levels of viral oncogene expression have been detected in both cervical intraepithelial neoplasia and invasive cancer, independently from histological grading and physical state of the viral genome [7]. These observations indicate that viral early gene expression is not in itself sufficient to induce cervical cancer and that additional genetic and/or epigenetic events are required [8]. Mutations or functional alterations of cellular genes such as PIK3CA [9], c-Myc (Myc), ErbB2 [10], cIAP1 [11], Ras [12], PTEN [13] and LKB1 [14] have all been reported in variable proportions of cervical cancers. A more extensive although not conclusive mutational analysis has been performed on TP53 tumor suppressor gene [15]. Mutations of the TP53 oncosuppressor gene are among the most common genetic alterations in many human malignancies [16–20]. Epidemiological and molecular studies have demonstrated a link between exposure to several mutagenic compounds and specific TP53 nucleotide changes [21,22]. Among these the well-documented associations are: 1) exposure to ultraviolet irradiation with tandem CC to TT transitions in non melanoma skin cancers [23]; 2) tobacco smoking and polycyclic aromatic hydrocarbons' exposure with G to T transversions at specific G:C base pairs [24,25]; 3) exposure to dietary aflatoxin B1 with G to T transversions at codon 249 in HBV-associated hepatocellular carcinoma [26,27]; and 4) dietary exposure to aristolochic acid with high frequency of A to T transversions in Balkan endemic nephropathy [28–30]. In addition, about 25% of all TP53 mutations are represented by G:C to A:T transitions at CpG sites potentially originating from spontaneous deamination of DNA bases [31]. However, the majority of factors associated with the origin of TP53 mutations along with the heterogeneous distribution by tumor type and geographical location remain largely unknown. The status of TP53 gene was first analyzed in human cervical carcinoma cell lines that were either positive or negative for HPV DNA sequences. The identification of TP53 mutations exclusively in HPVnegative C33-A and HT-3 cell lines led to the initial hypothesis that TP53 somatic mutations are a frequent event in HPV-negative anogenital cancers [32]. This preliminary observation was further confirmed by the analysis of primary cervical cancer presenting TP53 mutations in 60% of HPV-negative and in none of HPV-positive cancers [33]. Later on, TP53 nucleotide changes were identified in metastasis arising from HPVpositive cervical carcinomas, suggesting that p53 mutations could play a major role in the acquisition of aggressive behavior of some primary cancers associated with HPV infection [34]. Conversely, several other studies reported that inactivation of the TP53 gene by allelic loss or by point mutation is an infrequent event in clinical samples of cervical carcinomas, irrespective of the HPV infection [35–37]. Nevertheless, no systematic study has been performed to evaluate the TP53 mutational spectra in the two major histological types of cervical carcinoma from different geographic regions. In this article a systematic review of published studies has been performed to investigate the mutational pattern of TP53 gene in cervical adenocarcinoma and squamous cell carcinoma and possibly to correlate the type of mutations to the presence of specific mutagenic factors in different regions of the world.
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Materials and methods Review of the literature The study was based on the review of available data on the distribution of TP53 mutations in cervical cancer from previous English language literature. The TP53 mutation spectra have been independently analyzed in squamous cell carcinoma and adenocarcinoma of the cervix searching the International Agency for Research on Cancer (IARC) TP53 (http://www-p53.iarc.fr/) and COSMIC databases (http://www.sanger. ac.uk/genetics/CGP/cosmic/) and retrieving articles from Medline using the terms “p53” AND “mutation” AND “cervical” AND “carcinoma” (Table 1). All records evaluating cell lines and tumors other than primary cervical cancer were excluded from the analysis. Ten studies reporting no distinction between adenocarcinoma and squamous cell carcinoma were listed separately in Table 1 and mutations excluded from the comparative analysis between the two histological types of cervical carcinoma. To be included in the systematic review, a study had to present the total number of tumors included in the analysis and individual information on TP53 mutations obtained by several techniques and confirmed by DNA sequencing analysis. The majority of studies focused on exons 5–8, and used SSCP or DDGE as screening method for mutations and direct nucleotide sequencing analysis as confirmatory test, and did not search for mutations in the rest of the gene. Studies were stratified into four geographical regions (Africa, America, Asia and Europe). Analysis The following analyses were planned in the study design: frequency of TP53 mutations in cancer cases stratified by histological grade (squamous cell carcinoma and adenocarcinoma histological subtypes); frequency of TP53 mutations in adenocarcinoma and squamous cell carcinoma stratified by four broad geographical regions. All mutations were verified by MUT-TP53 2.0, a matrix developed by Soussi et al., which using functional and statistical information derived from the IARC p53 database is able to predict the biological activity and likelihood of each TP53 mutant [38]. In particular, the MUT-TP53 2.0 spreadsheet consists of tables in which the functional activity of 2314 p53 mutants, evaluated in terms of loss of transactivation on p21Waf1 promoter by a yeast-based functional assay, has been recorded [39]. The combination of the universal mutation p53 database with the p53 mutant activity database has been shown to be useful to perform functional analysis of thousands of reported p53 alterations in human cancers [38,40]. Results Twenty-seven studies, performed in Asia, Europe, America and Africa, were included in the systematic review comprising a total of 1353 cervical cancers analyzed for TP53 mutations, the majority focused in exons 5 to 8. In Table 1, mutation frequencies in each study were grouped by histological subtype and geography. The proportion of tumors carrying any mutation in TP53 gene showed a broad range across reports. The overall rates of TP53 mutated cases were significantly higher (P = 0.0003) among adenocarcinoma (32 of 241; 13.3%) compared to squamous cell carcinoma (39 of 657; 5.9%). TP53 mutations in adenocarcinoma were relatively frequent in Asia (19.05%) and Europe (12.16%) but rarer in North America (4%). Among squamous cell carcinoma cases, on the other hand, TP53 mutations were rarely identified in Asia (4.31%) and North America (3.64%), while more frequently in Europe (9.22%). Regarding spatial distribution of TP53 mutations, the most commonly mutated codons were 175, 248 and 273 in both types of cervical cancer, the codon 249 mainly in squamous cell carcinoma and the codons 161 and 282 only in adenocarcinoma (Fig. 1). Fig. 2 shows codon mutation patterns stratified by geographic origin of tumors.
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Table 1 List of published data on TP53 mutations' frequencies in invasive squamous cell carcinoma and adenocarcinoma of the cervix in different regions of the world. Continent
Location
Year
Reference
Exons
Method
Tumor*
TP53 Mut
All samples
Europe
UK Norway USA Brazil China Korea Japan
1992 1993 1994 2001 1993 1995 2001
Borresen et al., Lancet. 1992 (339):1350–1 Helland J Pathol. 1993 (171):105–14 Park et al., Oncogene. 1994 (9):205–10 Pinheiro et al., Braz J Med Biol Res. 2001 Jun;34(6):727–33 Choo and Chong, Virology. 1993 (193):1042–1046 Kim & Kim, Yonsei Med J. 1995 Nov;36(5):412–25 Harima et al.,Int J Cancer. 2001 Oct 20;96(5):286–96.
5–8 5–8 5–8 5–8 1–11 5–9 5–8
CDGE CDGE SSCP SSCP SSCP SSCP SSCP
UK UK Germany Germany Norway USA USA, Spain, Colombia China Japan Japan Japan Japan Japan Korea Thailand Egypt
1992 1994 1995 1995 1998 1993 1993 1997 1992 1992 1995 1999 2007 1997 2000 2007
Crook et al., Lancet. 1992 (339):1070–3 Busby-Earle et al., Br J Cancer. 1994 (69):732–7 Milde-Langosch et al., Int J Cancer. 1995 (63):639–45 Ikenberg et al., Cancer. 1995 (76):57–66 Helland et al., Br J Cancer. 1998 Jul;78(1):69–72 Paquette et al., Cancer. 1993 (72):1272–80. Kessis et al., Am J Pathol. 1993 (143):1398–405 Ngan et a., Genitourin Med. 1997 (73):54–8. Fujita et al., Cancer Res 1992 (52):5323–8 Tsuda et al., Jpn J Cancer Res. 1992 (83):1184–1191 Miwa et al., Br J Cancer. 1995 (71):219–26 Nakagawa et al., Br J Cancer. 1999 Mar;79(7–8):1139–44 Iwakawa et al., Cancer Biol Ther. 2007 (6):905–11 Kim et al., Acta Oncol. 1997 (36):295–300 Limpaiboon et al., Southeast Asian J Trop Med. 2000 (31):66–71 Bahnassy et al., BMC Clin Pathol. 2007 May 24;7:4
1–11 5–8 5–8 5–8 5–8 5–8 5–8 2–11 5–8 4–9 2–11 5–8 5–8 4–9 5–8 5–9
Seq DGGE TGGE SSCP CDGE + seq SSCP Seq SSCP SSCP-C-Seq SSCP-C-Seq SSCP SSCP SSCP SSCP SSCP SSCP
UK Germany Germany Italy Norway Germany Sweden USA USA, Spain, Colombia Japan Japan Japan Japan Korea Japan
1992 1995 1995 1998 1998 2001 2006 1993 1993 1992 1992 1994 1995 1997 1999
Crook et al., Lancet. 1992 (339):1070–3 Milde-Langosch et al., Int J Cancer. 1995 (63):639–45 Ikenberg et al., Cancer. 1995 (76):57–66 Tenti et al., Am J Pathol. 1998 (152):1057–63 Helland et al., Br J Cancer. 1998 Jul;78(1):69–72 Denk et al., J Mol Med (Berl). 2001 (79):283–8 Andersson et al., Med Oncol. 2006 (23):113–9. Paquette et al., Cancer. 1993 (72):1272–80. Kessis et al., Am J Pathol. 1993 (143):1398–405 Fujita et al., Cancer Res. 1992 (52):5323–8 Tsuda et al., Jpn J Cancer Res. 1992 (83):1184–1191 Jiko et al., Int J Cancer. 1994 (59):601–6. Miwa et al., Br J Cancer. 1995 (71):219–26 Kim et al., Acta Oncol. 1997 (36):295–300 Nakagawa et al., Br J Cancer. 1999 Mar;79(7–8):1139–44
1–11 5–8 5–8 5–8 5–8 4–10 5–8 5–8 5–8 5–8 4–9 5–8 2–11 4–9 5–8
Seq TGGE SSCP DGGE CDGE + seq Seq SSCP SSCP Seq SSCP-C-Seq SSCP-C-Seq SSCP SSCP SSCP SSCP
ICC ICC ICC ICC ICC ICC ICC Total ICC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC Total SCC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC ADC Total ADC
2 2 2 4 0 6 7 23 3 1 2 2 6 1 1 2 1 0 3 5 3 2 2 5 39 0 2 0 9 1 1 5 1 0 1 3 8 0 0 0 31
92 92 21 122 38 30 65 460 20 47 25 38 11 28 27 103 30 33 42 41 25 127 17 43 657 4 26 5 74 4 5 30 17 8 6 13 25 5 9 5 236
North America South America Asia
Europe
North America Asia
Africa Europe
North America Asia
% 2.17% 2.17% 9.52% 3.28% 20.00% 10.77% 5.00% 15.00% 2.13% 8.00% 5.26% 54.55% 3.57% 3.70% 1.94% 3.33% 7.14% 12.20% 12.00% 1.57% 11.76% 11.63% 5.94% 7.69% 12.16% 25.00% 20.00% 16.67% 5.88% 16.67% 23.08% 32.00%
13.14%
*Abbreviations: ICC, invasive carcinoma (comprising adeno/adenosquamous carcinoma, squamous cell carcinoma and cervical cancer of unspecified histology); ADC, adenocarcinoma; SCC, squamous cell carcinoma.
Interestingly, codon 249 was mainly mutated in Africa, probably due to Aflatoxin exposure, less in Europe and never mutated in Asia and North America. Codons 175 and 273 were found often mutated in Europe and rarely or never in Asia, America and Africa. Codon 248 was the most mutated site in Asian cases. These codons (175, 248 and 273) are the most frequently mutated sites in many human tumors and are associated with loss of p53 function. The TP53 mutations identified in adenocarcinoma and squamous cell carcinoma of the cervix have been compared, using the MUT-TP53 2.0 verification tool, to those of several other tumors present in the p53 database in relation to their effect on p21Waf1 activity [38]. The functional activity of mutated proteins detected in adenocarcinoma and squamous cell carcinoma cases showed a mean value of −1.295 and −0.827, respectively, suggesting major differences in the type of mutations between the two cancers (Fig. 3). Analysis of mutated nucleotides, showed that G to A and the mirror mutations C to T transitions were by far the most abundant, representing 21.4% and 32.1% of changes, respectively, in adenocarcinoma and 26.8% and 21.9%, respectively, in squamous cell carcinoma. The C to A transitions were highly frequent in adenocarcinoma (25%) and rarer in squamous cell carcinoma (2.4%). Conversely G to T transversions were more frequent in squamous cell carcinoma (14.6%) than in adenocarcinoma (3.6%), suggesting that a strand bias occurred during the mutation
process probably due to the preferential repair of damage on the transcribed DNA strand during transcription-coupled repair. In Fig. 4 the 12 types of possible base substitutions were grouped into 6 pairs, each consisting of two complementary substitutions: (G:C, C:G) (A:T, T:A), etc. The overall TP53 pattern then is shown as two complementary sub-patterns in which mutations on the nontranscribed strand are reported above the line in Fig. 1A, and the complementary substitutions on transcribed strand are represented under the line. Discussion The systematic review of twenty-seven studies analyzing TP53 mutational status in cervical cancer showed that nucleotide changes are significantly more frequent in cervical adenocarcinoma (13.3%) than squamous cell carcinoma (5.9%). The highest incidence of TP53 mutations was observed in adenocarcinoma from Asia (19%) and Europe (12.2%). The first evidence of high TP53 mutation rate in cervical adenocarcinoma was reported by Jiko et al. who described alterations in 22.2% of tumors from Japanese women with higher incidence in tumors at advanced clinical stage and with high grade nuclear and structural atypia [41]. Tenti et al. described a TP53 mutation rate of 13.5% among 74 primary cervical adenocarcinoma from Italian women. The majority of
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445
SCC AdC 4
Number of Mutations
3
2
1
0 1 1 3 3 1 1 3 3 5 9 1 9 2 2 3 4 6 4 7 0 1 2 8 9 4 9 0 9 3 0 1 2 3 5 0 2 10 13 13 14 15 16 16 17 17 17 18 18 19 20 21 21 22 23 23 24 24 24 24 24 25 25 26 26 27 28 28 28 28 28 29 29
TP53 Codons Fig. 1. Distribution of mutated codons of the TP53 tumor suppressor gene in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of the cervix.
nucleotide changes were G:C to A:T transitions and were more frequent in cancer cases of high grade, high stage, and large size, suggesting that TP53 mutations play a major role in the progression rather than in the
initiation of cervical adenocarcinoma [42]. Accordingly, Andersson et al. observed that single nucleotide changes occurred in six out of 30 (20%) primary cervical adenocarcinoma independently from HPV status [43].
Fig. 2. Distribution of mutated codons of the TP53 tumor suppressor gene in cervical cancer from different geographical regions.
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SCC 1 0,8 0,6 0,4
WAF1 ACTIVITY
0,2 0 -0,2 -0,4 -0,6 -0,8 -1 -1,2 -1,4 -1,6 -1,8 -2
1
ADC
0,8 0,6 0,4
WAF1 ACTIVITY
0,2 0 -0,2 -0,4 -0,6 -0,8 -1 -1,2 -1,4 -1,6 -1,8 -2
Fig. 3. Meta-analysis of p53 loss of function as measured by transactivation of the WAF1 promoter [38]. On the x-axis is indicated the publication code, the first letter corresponds to the cancer type and the second number is an anonymous ID for the publication. Data from the analysis of squamous cell carcinoma or adenocarcinoma of the cervix are displayed at the right side of the graph (arrow).
Unfortunately no studies to date addressed the issue of TP53 mutation distribution across the different subtypes of adenocarcinoma such as endocervical, endometrioid, intestinal, and mixed adenocarcinoma. The majority of human cancers (including skin, lung, liver, breast, gastrointestinal tract, bladder, etc.) display mutations in six hotspots (codons 175, 245, 248, 249, 273, and 282) within the DNA-binding domain of p53 protein. Accordingly, the most commonly mutated sites in both types of cervical cancer were codons 175, 248 and 273. However, frequencies of codon changes observed in different geographical locations were dissimilar probably due to different environmental or genetic factors. Furthermore, the evaluation of all mutations identified in this study with MUT-TP53 2.0 tool showed that the loss of function of p53 activity was significantly higher in adenocarcinoma (−1.3) than in squamous cell carcinoma (−0.8). The biological significance of this phenomenon, however, remains to be uncovered.
The pattern of p53 mutations was similar in adenocarcinoma and squamous cell carcinoma of the cervix and showed an excess of G:C to A:T transitions. These types of mutations, which are the most common in all human cancers, may originate from spontaneous nucleotide changes deriving from deamination of methylcytosine [4]. However, it is unclear whether their high frequency in cervical cancer may be related to a specific carcinogen or to the excess of oxygen and nitrogen radicals produced by oxidant-generating enzymes during chronic inflammation [44–48]. A major limitation of this pooled analysis of previously published data relies on the fact that several studies included limited sample size and most importantly they have been performed with different screening methods (i.e. SSCP, DGGE, direct sequencing, etc.) with variable sensitivity and specificity. However, despite these limitations, the pooled data provide some interesting insights indicative of future
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A
SCC
ADC
G:C>A:T G:C>T:A G:C>C:G A:T>G:C A:T>C:G A:T>T:A 30% 25% 20% 15% 10% 5% 0% 5% 10% 15% 20% 25% 30%
C:G>T:A C:G>A:T C:G>G:C T:A>C:G T:A>G:C T:A>A:T
B SCC Type of mutations
No. of mutations (%)
NTS
ADC TS
NTS
TS
G:C>A:T
35
50,7%
11
9
6
G:C>T:A
15
21,7%
6
1
1
9 7
G:C>C:G
9
13,0%
4
2
2
1
A:T>G:C
5
7,2%
3
0
2
0
A:T>C:G
3
4,3%
2
1
0
0
2
2,9%
A:T>T:A Total
69
2
0
0
0
28
13
11
17
Fig. 4. A. TP53 mutational patterns represented as pairs of complementary transitions and transversions in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of the cervix; the upper part represents non-transcribed strand (NTS), and the lower part, transcribed strand (TS). B. Summary of TP53 mutations detected in adenocarcinoma and squamous cell carcinoma.
investigations. In particular, more refined genetic epidemiologic studies with more sensitive techniques, such as next-generation sequencing, are needed to better define the prevalence and the type of mutations in the TP53 gene in cervical adenocarcinoma and squamous cell carcinoma from different regions of the world. Moreover, the clinical significance of TP53 status in terms of overall survival, metastasis-free survival and outcome to chemotherapy and/or radiotherapy in cervical cancer is still unknown and needs to be assessed in prospective longitudinal studies. Conflict of interest statement The authors have no conflict of interests.
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