Clin Exp Metastasis (2009) 26:789–796 DOI 10.1007/s10585-009-9278-3
RESEARCH PAPER
Allelic loss at TP53 in metastatic human endometrial carcinomas Wiktor Szewczuk Æ Danuta Skomra Æ Marek Cybulski Æ Dorota Prza˛dka-Rabaniuk Æ Agata Filip Æ Maciej Jo´z´wik Æ Piotr Olcha Æ Albert Roessner Æ Andrzej Semczuk
Received: 27 December 2008 / Accepted: 15 June 2009 / Published online: 30 June 2009 Ó Springer Science+Business Media B.V. 2009
Abstract Loss of heterozygosity (LOH) is implicated in the initiation and progression of various human neoplasia, and is observed in both early or in advanced-stage human cancers. The current study was aimed at investigating the frequency of LOH TP53 in human endometrial carcinoma (EC) metastases. LOH was analyzed using 3 intragenic polymorphisms in 38 primary ECs and corresponding metastatic lesions. Allelic loss at intron 1 was detected in 14 out of 38 (37%) primary carcinomas and in 11 out of 38 (29%) metastatic lesions. LOH at intron 1 in primary and metastatic tumors was concomitantly noted in 8 out of 38 (21%) cases. LOH at intron 4 was seen in 46% (17 out of
W. Szewczuk D. Prza˛dka-Rabaniuk P. Olcha A. Semczuk (&) IInd Department of Gynecology, Medical University of Lublin, 8 Jaczewski street, 20-954 Lublin, Poland e-mail:
[email protected] D. Skomra Department of Pathology, Medical University of Lublin, Lublin, Poland M. Cybulski Department of Biochemistry and Molecular Biology, Medical University of Lublin, Lublin, Poland A. Filip Department of Human Genetics, Medical University of Lublin, Lublin, Poland M. Jo´z´wik Department of Gynecology, Bialystok Medical University, Bialystok, Poland A. Roessner Department of Pathology, Otto-von-Guericke University, Magdeburg, Germany
37) primary ECs and in 35% (13 out of 37) metastatic lesions. LOH at intron 4 in primary tumor/metastasis was concomitantly demonstrated in 27% (10 out of 33) cases. Allelic loss at exon 4 was detected in 5 out of 33 (15%) primary ECs and in one out of 33 (3%) corresponding metastases. Coexistence of LOH TP53 in primary ECs with metastases at intron 1 and intron 4 was observed in three out of 33 (9%) cases. Correlation between allelic loss at intron 1 in primary ECs and corresponding metastases was found (R = 0.475, p = 0.003). Moreover, there was correlation between LOH at intron 1 in metastastic ECs and allelic imbalance at intron 4 in primary uterine tumors (R = 0.416, p = 0.01). There was a relationship between LOH at intron 4 in primary ECs and corresponding metastatic lesions (R = 0.457, p = 0.004). LOH TP53 at intron 4 correlated with the presence of the neoplasm in the uterine cervix (R = 0.319, p = 0.049), and with the nonendometrioid type of primary tumor (R = 0.371, p = 0.024). There was a significant correlation between exon 4 LOH and patient age (less or equal to 50 years and above this age; R = -0.375, p = 0.032). p53 overexpression was present in thirteen out of 38 (34%) cases, both in primary ECs and in metastatic lesions. Overexpression of p53 was higher in non-endometrioid ECs (three out of 5; 60%) than in endometrioid-type uterine tumors (ten out of 33; 30.3%; p = 0.315). p53 overexpression correlated with the presence of cancer in the lumen of fallopian tube(s) (R = 0.032, p = 0.046), and with allelic loss at intron 1 in primary ECs (R = 0.599, p = 0.0001). In conclusion, LOH occurs not only in primary uterine tumors but also in corresponding metastases, with the higher incidence being reported at intron 4 of the TP53. A significant link existed between LOH TP53 at intron 1 and p53 overexpression in primary ECs, but not in the corresponding metastatic lesions.
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Keywords TP53 LOH Endometrial cancer Metastasis Immunohistochemistry
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Materials and methods Clinical and pathological data
Abbreviations LOH Loss of heterozygosity EC Endometrial cancer G1 Well-differentiated cancer G2 Moderately-differentiated cancer G3 Poorly-differentiated cancer IHC Immunohistochemistry RFLP Restriction fragment length polymorphism VNTR Variable number of tandem repeats VSI Vascular space invasion
Introduction TP53 tumor suppressor gene, known as ‘‘the guardian of the genome’’, is located at human chromosome 17p13.1 [1, 2]. It encodes a nuclear phosphoprotein (p53), which controls the decision of the cell to replicate at G1/S checkpoint of the cell cycle [3, 4]. Activated p53 has been implicated in various human processes, including apoptosis, induction of differentiation, as well as cellular senescence and the repair of DNA damage [4–6]. p53, a member of the well-known p53-pathway (consisting of p14arf/ mdm2/p53), is controlled by various negative regulators and activators [for review see 7]. Various genetic mechanisms of TP53 inactivation have been described in human cancers, including point mutations, homozygous deletions, and/or LOH [8–10]. The short arm of chromosome 17, apart from other chromosomal loci, has been found as one of the regions in which allelic imbalance in primary human ECs is frequently reported [11]. It should be also mentioned that the frequency of LOH in primary uterine tumors at TP53 locus differs in the reports worldwide, ranging from 5.3% up to 62.5% of informative tumors [12, 13; for review see 11]. In the recent study by Graesslin et al. [14], LOH at TP53 was noted in 30.2% of primary human ECs, without any significant relationship between the prevalence of allelic loss and p53 overexpression. Earlier data from our laboratory showed LOH at TP53 in 19% of informative primary human ECs, and we also reported a trend towards an unfavorable outcome in patients affected by uterine corpus cancer with LOH compared to women without allelic imbalance [15]. The aim of the current study was to investigate the frequency of LOH TP53 in human EC metastases, and at comparing the genetic data with the clinical/pathological variables of cancer and p53 overexpression.
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Tissue samples from primary ECs and corresponding metastatic lesions were collected from women having undergone surgery between 1995 and 2008. Altogether, 38 cases were carefully selected, based on the availability of primary and corresponding metastatic tumors on paraffin blocks for the present study. The mean patient age was 61 years (ranged between 36 and 80 years). Chemotherapy, hormonal treatment, or radiation therapy had not been carried out before surgery. The clinicopathological features of EC patients enrolled in this study are presented in Table 1. Histological tumor type was graded on the basis of the WHO classification [16]. Thirty-three cases (87%) were endometrioid-type ECs, and 5 cases (13%) were nonendometrioid carcinomas (clear-cell or papillary serous). Three cases (8%) were well-differentiated (G1) carcinomas, 20 cases (53%) were moderately-differentiated (G2) carcinomas, and 15 cases (39%) were poorly-differentiated (G3) carcinomas. Tumor invasion was present but did not exceed half of the thickness of the myometrial wall in 13 (34%) cases, while cancer infiltrated more than the half of the myometrial wall in 25 (66%) cases. Cancer coexisted with atypical endometrial hyperplasia in one case (2.5%). All patients were staged III/IV according to the FIGO classification [17]. The treatment protocol consisted of total abdominal hysterectomy, bilateral salpingoophorectomy, and pelvic/para-aortic lymph node dissections. Lymph nodes were removed in 22 out of 38 (58%) patients. Lymph node metastases were detected in 12 (58%) cases. Omentectomy was performed in 16 cases, and metastases to the omentum were detected in 9 (56%) cases. In addition, palpation of abdominal organs and collection of peritoneal washings for cytological analysis were performed. In selected cases, the women also underwent appendectomy, tumor cytoreduction, and/or dissection of the distant metastases. At surgery, neoplastic material was immediately fixed in buffered formalin (pH 7.4), embedded in paraffin blocks, and submitted for pathological evaluation at the Department of Pathology, Medical University of Lublin, Lublin, Poland. The material was carefully reviewed by an experienced pathologist (D.S.) who was blinded as to the patients’ clinical records. The pathological variables were assessed with regard to histological type and grade, depth of myometrial infiltration, pattern of ovarian involvement, presence of VSI, presence of cancerous material in the fallopian tube(s), and presence of metastases in the ovary/ ovaries and lymph nodes. The clinical data were evaluated
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Table 1 Patterns of allelic loss at the TP53 among primary (PT) ECs and their corresponding metastases (M) Case
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Patient age
59 51 68 71 36 43 49 73 55 72 45 69 79 64 51 65 67 80 78 61 74 58 48 68 58 41 49 62 64 65 67 55 59 54 57 63 80 59
Clinical stage
IV III III III IV IV IV IV IV III IV IV IV IV IV III IV IV IV IV IV III IV III IV IV IV III III III IV III IV IV IV IV IV III
Histologic type
End End End End End End End End End End End End Non-end End End Non-end End End End End Non-end End End End End End End End End End Non-end end End End End End End Non-end
Histologic grade
G1 G2 G2 G2 G3 G2 G2 G2 G2 G2 G2 G2 G3 G3 G3 G3 G3 G3 G2 G1 G2 G2 G3 G3 G3 G3 G2 G2 G3 G2 G3 G3 G1 G2 G2 G2 G2 G3
Depth of myoinvasion
>1/2 1/2 >1/2 >1/2 1/2 1/2 >1/2 >1/2 >1/2 >1/2 >1/2 1/2 >1/2 1/2 1/2 >1/2 1/2 >1/2 >1/2 1/2
VSI
yes yes no yes yes no no no no no yes yes no yes yes yes yes yes yes yes no no yes yes yes no no yes yes no yes yes no yes yes yes no yes
Presence of the neoplasm in the fallopian tube no yes no yes yes no no yes no yes no no no yes yes no no yes no no no yes no no yes no no no yes no yes no no yes no no yes yes
Presence of the neoplasm in the uterine cervix yes yes no no no yes yes no no yes no yes no yes yes yes yes yes no yes yes yes yes no yes no no yes no no yes yes no yes no yes yes no
Metastases to the ovary/ ovaries
no yes yes yes yes yes yes yes yes yes yes no yes yes yes no yes yes no yes no yes no no yes yes yes no yes no yes no no yes yes yes yes no
Metastases to the lymph nodes
Intron 1
PT
M
Intron 4
PT
M
Exon 4
PT
M p53
no
+ yes
+ no
+ no yes no no
+
no
+
yes
+ +
+ yes yes yes
yes yes yes no yes no yes no no
+
+ + +
+ yes
Patients are indicated by their numbers in the series. Black boxes indicate an allelic loss, empty boxes indicate retained heterozygosity, while grey boxes indicate cases non-informative. Overexpression of p53 is indicated by ‘‘?’’; End, endometrioid type; Non-end, non-endometrioid type
according to patient age, menopausal status and presenting symptoms. The study group was divided into three age categories: the first group consisted of women below 50 years of age (n = 7; 18%), the second group consisted of patients aged between 51 and 60 years (n = 11; 29%), and the third group consisted of women older than 60 years (n = 20; 53%). DNA isolation For DNA isolation, tumor areas were carefully selected based on anatomo-pathological evidence of high-tumor cellularity ([50%). A microdissection technique was applied to further enrich malignant cells. Normal tissues were obtained from the paraffin blocks of the same patient, and consisted of non-malignant myometrium or other nonmalignant reproductive tract organs removed at surgery.
High-molecular weight DNA was extracted using the ‘‘Nucleospin TissueÒ’’ kit (Macheley-Nagel, Duren, Germany). Analysis of LOH at the TP53 We applied three intragenic polymorphic markers located at intron 1 [18], intron 4 [19], and exon 4 [20]. Primer sequences, annealing temperatures, name of restriction endonuclease, and product sizes have been described previously [20]. Briefly, PCR reaction was carried out in a total volume of 50 ll and contained 2.5 ll (500 ng) DNA, 5 ll 109 standard PCR buffer, 6.5 ll dNTP mix (2 lM), 2.5 ll of each primer (50 pmol/ll), 2.5 ll MgCl2 (25 lM), and 1 ll (5 U) Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania). During each cycle, the amplification mixture was denaturated at 95°C for 30 s, annealed for 30 s, and extended at 72°C for 30 s. This procedure was
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repeated in 30–35 cycles. Extension during the final cycle was 7 min. The PCR-products were then purified, digested with appropriate restriction endonuclease (PCR–RFLP), and identified after 2% agarose gel electrophoresis and ethidium bromide staining. For PCR–VNTR, purified PCRproducts were directly electrophorezed and visualized. LOH-positive cases were also analyzed on 8% polyacrylamide gels and silver-stained. Detection of LOH A patient was defined as non-informative when only one allelic band was detected in the normal DNA line. A case was considered informative when two major allelic bands occurred in the normal DNA line. Allelic loss was defined when one allelic band in tumor DNA disappeared completely or when [50% decreased in the relative intensity was noted, compared with the normal DNA pattern. In ambiguous cases, the bands were scanned, and their intensities were compared using densitometry (VDS, Pharmacia Biotech, Uppsala, Sweden). Immunohistochemistry Paraffin-embedded sections (4 lm) were cut and prepared on adhesive slides (Poly-PrepTM slides, SIGMA, St. Louis, MO, USA). We used mouse anti-human p53 monoclonal antibody (clone DO-7, isotype IgG2b; NOVOCASTRA, Newcastle upon Tyne, UK; diluted 1:100) overnight at 4°C. We applied the antigen retrieval technique with microwave pretreatment, consisted of three rounds of heating at 750 W for 5 min., each in a 10 mM citrate buffer (pH 6.0). Immunostaining was performed using Vectastain kit (Vector Laboratories, Burlingame, CA, USA), and the visualization was done using DAB (3,300 diaminobenzidine tetrahydrochloride) chromogen containing 0.05% hydrogen peroxide for 5 min. at room temperature. The sections were finally counterstained with Mayer’s hematoxylin for 30 s at room temperature. Positive controls (primary EC overexpressing p53) and negative controls (the omission of the primary antibody during otherwise identical incubation) were simultaneously incorporated in each experiment.
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graded according to Alkushi et al. [21]: 0, less than 10% of tumor cells expressed p53; 1, 10–50% of cancer cells expressed p53; and 2, more than 50% of the tumor cells expressed p53. The staining score of 2 was considered to indicate p53 overexpression. Statistical analysis Statistical analysis was done using statistical package SPSS 14.0 PL for Windows. Fisher’s exact test, the v2-test and Spearman’s rank correlation test were used for statistical analysis. p \ 0.05 was considered statistically significant.
Results LOH TP53 PCR–LOH data revealed heterozygosity of the TP53 gene in 100% at intron 1, in 97% at intron 4, and in 87% at exon 4. Allelic loss at intron 1 was detected in 14 out of 38 (37%) primary human ECs and in 11 out of 38 (29%) metastatic lesions. LOH TP53 at intron 1 in primary and metastatic tumors was concomitantly noted in 8 out of 38 (21%) cases. LOH at intron 4 was seen in 17 out of 37 (46%) primary ECs and in 13 out of 37 (35%) metastatic lesions. Concomitant allelic loss at intron 4 in primary tumors and metastatic lesions was demonstrated in 10 out of 37 (27%) cases. Allelic loss at exon 4 was detected in 5 out of 33 (15%) primary ECs and in one out of 33 (3%) metastatic lesions (Fig. 1). None of the LOH-positive cases concomitantly revealed exon 4 allelic loss in primary and metastatic tumors. Coexistence of allelic loss at TP53 in primary ECs with metastatic tumors at intron 1 and intron 4 was observed in 3 out of 33 (9%) cases (numbers 12, 31 and 32 from Table 1).
m
N
T
M
Interpretation of immunostaining All slides were analyzed independently by two researchers (D.S., A.S.) without prior knowledge of clinicopathological variables of cancer. The representative areas of nearly 500 tumor cells were counted. The subcellular localization (nuclear or cytoplasmic) and the percentage of positively stained tumor cells were recorded. Only cases with nuclear staining were considered positive. Staining patterns were
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Fig. 1 Representative picture of exon 4 allelic loss at the TP53 in metastatic EC (case number 5 from Table 2). m indicates molecular weight marker; N normal tissue; T tumor tissue; M metastatic tissue
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Analyzing separately type II (non-endometroid) ECs, allelic loss at intron 1, intron 4 and exon 4 was reported in two, three and once cases in primary tumors and in two, four and one metastatic lesions, respectively (Table 1). Interestingly, in a sole case (number 31 from Table 1) allelic imbalance was noted in all TP53 polymorphic markers concomitantly with p53 overexpression. There was no significant relationship between the frequency of allelic loss at intron 1 and the clinicoprognostic variables of cancer. Correlation between allelic loss at intron 1 in primary uterine tumors and corresponding metastases was found (R = 0.475, p = 0.003). Moreover, there was significantly correlation between LOH at intron 1 in metastastic ECs and LOH at intron 4 in primary uterine neoplasms (R = 0.416, p = 0.01). A significant correlation between LOH at intron 4 in primary ECs and corresponding metastatic lesions was noted (R = 0.457, p = 0.004). Moreover, allelic loss at intron 4 correlated with LOH at exon 4 of the TP53 (R = 0.488, p = 0.005). LOH TP53 at intron 4 correlated with the presence of the neoplasm in the uterine cervix (R = 0.319, p = 0.049). Allelic loss at intron 4 in metastatic lesions correlated with the histological type of primary EC, and was observed more frequently in nonendometrioid type compared to endometrioid type uterine carcinomas (R = 0.371, p = 0.024). There was a significant correlation between exon 4 LOH TP53 and patient age (less or equal to 50 years and above this age; R = -0.375, p = 0.032). However, allelic loss at exon 4 in primary ECs was not related to LOH reported in corresponding metastatic lesions (R = -0.075, p = 0.697). p53 overexpression p53 was overexpressed in thirteen out of 38 (34%) cases, either in primary ECs or in metastatic lesions (Fig. 2). Overexpression of p53 was detected in three out of 18 (17%) women aged less than or equal to 60 years, and in 10 out of 20 (50%) women above this age (p = 0.043). A significant correlation existed between p53 overexpression and patient age in different categories (less than 50 years, 50–60 years, and above 60 years; R = 0.343, p = 0.035); (less than or equal to 60 years and above 60 years; R = 0.351, p = 0.031). Interestingly, p53 overexpression correlated with the presence of cancer in the lumen of the fallopian tube(s) (R = 0.032, p = 0.046). A tendency towards p53 overexpression in the cases showing cervical involvement was also observed (R = 0.278, p = 0.091). Overexpression of p53 was higher in non-endometrioid ECs (three out of 5; 60%) than in endometrioid-type uterine tumors (10 out of 33; 30.3%), but this difference was not of significant value (p = 0.315).
Fig. 2 Oveexpression of p53 in primary EC a and corresponding metastatic lesion b (case number 3 from Table 2) (9100)
Correlation between LOH TP53 and p53 overexpression Correlation between p53 overexpression and allelic loss at the TP53 locus in primary ECs and corresponding metastases is presented in Table 1. A significant correlation between p53 overexpression and allelic loss at intron 1 in primary ECs was noted (R = 0.599, p = 0.0001). There was also a tendency towards p53 overexpression and LOH at intron 1 in metastatic lesions and LOH at intron 4 in metastatic tumors, but these differences were not significant.
Discussion LOH of the TP53 is implicated in the initiation and progression of various human malignancies, and is observed in
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both early or in advanced-stage neoplasms [22–24]. Allelic loss at 17p13.1 represents one of two ‘‘hits’’ of tumor suppressor gene inactivation, based on Knutson’s hypothesis [25]. Although there are several reports on allelic loss at TP53 locus in primary human ECs (for review see [11]), data assessing the role of allelic imbalance at TP53 in EC metastases are scarce. Previously, Jones et al. [26] showed no significant correlation between EC lymph node metasteses (n = 5) and LOH at TP53 locus. In the current research, a significant frequency of LOH at the TP53 locus (above 20% of informative cases as suggested by Li et al. [27]) was reported in metastatic lesions at introns 1 and 4 (29% and 35% of informative cases, respectively). Interestingly, allelic loss at intron 1 and intron 4 in primary and metastatic tumors was concomitantly reported in 21% and 27% of informative cases, respectively. These data suggest that LOH at TP53 occurs not only in primary ECs but also in the corresponding metastases. There was significantly higher frequency of TP53 allelic imbalance at intron 4 compared to LOH at exon 4 either at primary tumors or in metastatic lesions. As an explanation, we speculate that allelic loss at intron 4 of the TP53 may be involved with the development of metastases in uterine corpus cancer. Moreover, it is worth pointing out that human TP53 contains an internal promoter located in intron 4 [28]. The alternative promoter leads to the expression of an N-terminally truncated p53 protein, named D133p53. As suggested by Bourdon et al. [28], the failure of appropriate regulation of various p53 isoforms may have a role in cancer formation since attenuation of the wild-type p53 response would render the cells more susceptible to neoplastic development and dissemination. In the literature, Heide et al. [29] showed that most of the colon cancer metastases (72%; 9 out of 12) revealed LOH at TP53 locus. Allelic loss was detectable only in those metastatic lesions, with one exception, where a mutation in the TP53 was found. In another report, Baergen
et al. [30] demonstrated that extrauterine tumors had the same TP53 point mutations as the primary, early-staged uterine serous carcinomas. Their study confirmed the monoclonal origin of extrauterine metastases originating from primary uterine malignancies; however, their study was limited due to the low (n = 4) number of cases included. Using the genetic analysis of TP53, Kupryjanczyk et al. [31] demonstrated that disseminated serous carcinomas (originated from endometrium, ovary or peritoneum) are of monoclonal rather that multicentric origin. Based on the above mentioned results, point mutations, concomitantly with LOH at TP53 locus, may answer the question of whether metastatic tumors originate from the primary ECs. It will be essential to extend our future study to evaluate the prevalence of TP53 alterations (point mutations/deletions) in LOH-positive metastatic lesions originated from primary human ECs. There are several reports on the role of LOH TP53 alterations in relation to clinical/pathological features of primary ECs. For example, Kihana et al. [32] demonstrated significantly higher incidence of TP53 allelic loss in poorly-differentiated than in well- and moderately-differentiated tumors. In another report, LOH TP53 correlated with the histological type and the histological grade of ECs [14]. Moreover, Niederacher et al. [33] correlated LOH TP53 with younger (less than 50 years old) patient age, high histological grading, positive progesterone receptor status, and with uterine tumors from patients not receiving hormonal replacement therapy. Saegusa and Okayasu [34], (using the same polymorphic markers as in the current study) showed a significant link between allelic loss and all the unfavorable clinical/pathological parameters, with the exception of the depth of myometrial invasion. In the current study, LOH TP53 at intron 4 correlated with the invasion of the neoplasm into the uterine cervix (R = 0.319, p = 0.049). It is worth pointing out that allelic loss at intron 4 in EC metastases correlated with the nonendometrioid histological type of primary uterine tumors,
Table 2 Correlation between allelic loss at the TP53 and p53 overexpression
Intron 1
LOH n (%)
p53 overexpression n (%)
Spearman rank correlation test
Primary tumor
14 (37)
10 (26)
R = 0.559 p = 0.0001
Metastasis
11 (29)
6 (16)
R = 0.274 p = 0.097
8 (21)
5 (13)
R = 0.308 p = 0.06
Primary tumor Metastasis
17 (46) 13 (35)
7 (18) 7 (18)
R = 0.117 p = 0.492 R = 0.288 p = 0.083
Primary tumor/metastasis
Primary tumor/metastasis Intron 4
Exon 4
10 (26)
5 (14)
R = 0.189 p = 0.261
Primary tumor
5 (14)
2 (5)
R = 0.089 p = 0.261
Metastasis
1 (3)
1 (3)
R = 0.268 p = 0.131
Primary tumor/metastasis
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–
–
–
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suggesting that this genetic phenomenon may be associated with unfavorable uterine tumor subtype. At present, one can recognize tendencies towards a higher frequency of TP53 allelic loss at intron 1 in primary ECs in relation to p53 overexpression in advanced-stage uterine tumors (Table 2). Interestingly, a tendency towards p53 overexpression and LOH at introns 1 and 4 in metastatic tumors was also observed, but these differences were not of significant value probably due to limited number of LOH-positive cases. Our data are at variance to that of Saegusa and Okayasu [34] and Semczuk et al. [15], who described no relationship between p53 immunopositivity and the presence of TP53 LOH. An explanation could be that clinical studies utilizing the combination of early- and advanced-staged uterine tumors differ in the frequency of allelic imbalances within TP53. Moreover, application of different methods of signal detection and antigen retrieval techniques, choice and the dilution of primary antibodies may complicate the comparison between the studies, even if clinical/pathological features were selected. Finally, the actual level of p53 may be also determined by other factors apart from TP53 dys-regulation, for example MDM2 overexpression, p14ARF alterations or overexpression of hsp family member—mot2 [35, 36]. The prognostic relevance of various genetic alterations in ECs has been reviewed previously [37–40], suggesting that TP53 alterations (base substitution, deletion, or insertional mutations) and p53 overexpression has been consistently associated with unfavorable prognosis, particularly in papillary serous and clear-cell uterine tumors. Kihana et al. [32] were the first to show that EC patients with TP53 LOH had a significantly shorter survival time compared to those with an intact gene, both in stages I– IV and in stage I alone. They found that loss of heterozygosity of the TP53 was an indicator of poor prognosis, that was independent of tumor stage, histologic grade or myometrial invasion [32]. Recently, a tendency towards an unfavorable outcome was reported for EC women displaying allelic loss at TP53 during short-term followup [15]. By contrast, however, LOH at the TP53 locus did not significantly correlate with overall survival and disease-free survival in a large study of 113 EC patients during a median follow-up of 98 months [33]. Presently, a cohort study is in progress in our laboratory and in collaboration with other academic centers from Poland, to assess whether allelic imbalance at TP53 may be an unfavorable prognosticator in patients affected by advanced-stage ECs. Acknowledgments The authors are grateful to the staff of the Department of Pathology, Medial University of Lublin, Lublin, Poland, for the pathological assessment of the material. This research was granted by Medical University of Lublin, Lublin, Poland (Dz. St. 326/07 and 326/08 to A.S.).
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