Correlation of DNA Mismatch Repair Protein Hmsh2 Immunohistochemistry with P53 and Apoptosis in Cervical Carcinoma

ANTICANCER RESEARCH 27: 63-68 (2007)

Correlation of DNA Mismatch Repair Protein hMSH2 Immunohistochemistry with p53 and Apoptosis in Cervical Carcinoma

FRANK KÖSTER1, ANDREAS SCHRÖER1, DOROTHEA FISCHER1, THOMAS GREWELDINGER2, KLAUS DIEDRICH1 and MICHAEL FRIEDRICH1

1Department of Obstetrics and Gynaecology, UK-SH, Campus Lübeck, D-23538 Lübeck; 2Department of Obstetrics and Gynaecology, University of Saarland, D-66421 Homburg/Saar, Germany

Abstract. Background: Mutations in genes of the DNA heterodimer with hMSH3 (MutS‚) (4). The MutL· mismatch repair system (MMR) are linked to hereditary non- heterodimer comprising the Mut-L-homologon-1 (hMLH1) polyposis colorectal cancer and also play a role in sporadic and postmeiotic-segregation-increased-2 (PMS2) binds to cancer. Besides its repair function, the MMR is the linkage of the MutS heterodimer. The replication processivity factor DNA mismatch recognition to the cell cycle control. Materials proliferating-cell-nuclear-antigen (PCNA) links the MMR and Methods: The correlation between the immunoreactivity complex to an enzyme complex which exchanges the of the MMR protein hMSH2 and p53, apoptosis, clinical mispaired part of the mutated strand (5, 6). prognosis factors and the survival rate in 102 samples of Defects or altered expression of MMR-proteins were not cervical carcinoma was determined. Results: hMSH2 only found in HNPCC, but also in a number of sporadic immunoreactivity was correlated with p53 and weakly cancers, where they were not necessarily linked to the correlated with apoptosis. hMSH2 immunoreactivity could not appearance of MSI (7-10). In gynaecological cancer, be correlated to any tumour markers, while apoptosis hMSH2 alterations were found, e.g. in endometrial cancer correlated significantly with T stage, FIGO classification and (11, 12), ovarian cancer (13, 14), malignancies of the uterine the relative risk of death from cervical cancer. Conclusion: In corpus (15) and breast cancer (16-18). Only a few studies cervical cancer, the processes of DNA mismatch repair, cell focused on hMSH2 and cervical cancer. Two describe an cycle control and apoptosis seemingly act in concert. Decreased intensified expression of hMSH2 in malignant tissues expression of the hMSH2 mismatch repair protein might lead compared with non-neoplastic samples (19, 20). An to a failure in the induction of apoptosis. association between cancer and MMR is not necessarily linked to the loss of immunoreactivity for one of the MMR An association between the DNA mismatch repair (MMR) proteins since a mutation of hMSH2, with the subsequent protein, Mut-S-homologon-2 (hMSH2) and cancer was first loss of MMR-function, was identified in breast cancer to be described in 1993 in hereditary non-polyposis colorectal still immunoreactive (21). cancer (HNPCC) (1-3). The loss of MMR function in On chromosomal damage the MMR proteins can activate HNPCC results in a high frequency of microsatellite proteins of cell cycle checkpoints and apoptosis by an as yet instabilities (MSI), the impairment of chromosomal unclear mechanism (22). An interaction of PMS2 with p73, integrity and promotes tumorigenesis. an associate of the tumour suppressor p53, is necessary for As part of the MMR complex, hMSH2 binds to DNA the induction of apoptosis after cisplatin administration (23). base-to-base mismatches as a heterodimer with hMSH6 Point mutations in hMSH2 resulted in partially functional (MutS·), or to insertion- and deletion-loop mismatches as a proteins that made it possible to distinguish between the mismatch repair function of hMSH2 and its ability to induce cell death after cisplatin exposure (24, 25). The tumour suppressor protein p53 inhibits tumorigenesis by cell cycle Correspondence to: Dr. Frank Köster, Department of Obstetrics arrest and induction of apoptosis. Mutations in this central and Gynaecology, UK-SH, Campus Lübeck, D-23538 Lübeck, gatekeeper of the cell cycle are involved in a high percentage Germany. Tel: +49 451 500 3604, Fax: +49 451 500 4760, e-mail: [email protected] of cancer. Scherer et al. (26) identified a response element for p53 in the promoter region of the hMSH2 gene and Key Words: Cervical cancer, DNA mismatch repair, hMSH2, p53, Warnick et al. showed that p53 actively regulates the hMSH2 apoptosis, immunohistochemistry. expression in ovarian cancer cells (27).

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The relation between hMSH2 and p53 has been Table I. Percentages of samples with positive immunostaining and investigated in various studies with diverse outcomes in medium IRS for hMSH2, p53, Ki-67 and apoptosis after TUNEL-assay. different types of cancer. Rass et al. (28) found increased Positive Negative IRS immunoreactive scores in malignant melanoma for both hMSH2 and p53, whereas Spagnoletti et al. found hMSH2- hMSH2 80.4% 19.6% 5.47±3.2 positive tumour cells tended to be negative for the p53 71.6% 28.4% 4.74±3.45 expression of p53 in breast cancer (10). Apoptosis 77.4% 22.6% 3.8±2.4 Ki-67 93% 7% 2.59±0.86 In this study the relationship between the expression of the MMR protein hMSH2, p53 and apoptosis was investigated in samples of cervical carcinoma. Further, the correlation of these results with prognostic factors in cervical carcinoma, such as steroid receptor status, TNM deparaffinized and rehydrated. Proteinase-K digestion was stage, FIGO classification and tumour grading, as well as performed for 25 min at room temperature. The TUNEL reaction with the survival rate, was determined. was undertaken at 37ÆC for 1 h under a cover glass. For the detection of fluorescein-dUTP-labelled cells, an alkaline Materials and Methods phosphatase-linked antifluorescein antibody was used. The immunoreaction was visualised with the New-Fuchsin-Substrate- Tissue specimens. Tumour tissue from 102 patients with cervical Kit (DAKO, Hamburg, Germany). carcinoma was collected. The investigations on human material were approved by the ethics-committee of the University of Interpretation of immunoreactivity. The immunoreactive score (IRS) Homburg/Saar. Each patient consented to participate in this study. described by Remmele and Stegner (29) was used for the Steroid receptor expression and the tumour staging (T interpretation of the immunoreactivity in tumour sections. The IRS stage=tumour size and character; N stage=nodal status; M is the product of the staining intensity (SI: negative=0; weak=1; stage=distant metastases), histopathological staging and FIGO moderate=2; strong=3) and the percentage score of grading were taken from the reports of the pathologists. immunopositive cells (PP: 0=no positive cells; 1=1-10%; 2=11- 50%; 3=51-80%; 4=>80% positive cells). Immunoreactivity for Immunohistochemistry. Freshly excised tissue samples from cervical Ki-67 was evaluated and compared with other immuno-reactivities carcinoma were fixed in 4% phosphate-buffered paraformaldehyde with a scoring after Friedrich et al. (15) that creates a ranking of for 24 h at 4ÆC and embedded in paraffin. Serial sections of 6-Ìm the percentages of immunopositive cells (PP) (rank 0=0%; 1=1- thickness were cut on a rotation microtome and mounted on 10%; 2=11-25%; 3=>25%). microscope slides previously silanised and treated with 0.5% ovalbumin. After drying, slides were deparaffinated in xylol and Microscopy and photography. The stained sections were viewed rehydrated in descending concentrations of alcohol, submerged in under a Leica DM4000 microscope and photographed using an SIS 3% hydrogen peroxide to prevent endogenous peroxidase activity Colorview 12 digital camera. and demasked in a microwave oven at 500 W for 10 min in 0.1 M citrate-buffer. Slides were then washed with Tris-buffer. Non- Statistical analysis of correlation. The statistical correlations among specific protein binding was blocked with normal rabbit sera hMSH2, p53, Ki-67 and apoptosis immunoreactivity, as well as the (DAKO, Hamburg, Germany; 1:50 in Tris-buffer) for 15 min. correlations with pathological data, were analysed using Spearman’s As primary antibody for hMSH2, the monoclonal mouse rank correlation co-efficient in SPSS version 10.0 (SPSS Inc., antibody clone FE-11 was used at a dilution of 1:20 (Calbiochem, Chicago, IL, USA). Statistical significance was set at p<0.05. Schwalbach, Germany), for p53 the monoclonal mouse antibody clone DO-7, at a 1:30 dilution and for Ki-67 the polyclonal rabbit Results antibody A-047 was used at a 1:50 dilution (both DAKO, Hamburg, Germany). The primary antibodies were incubated on We examined cervical carcinoma samples from 102 patients the tumour sections overnight at 4ÆC. using immunohistochemistry for hMSH2, p53, Ki-67 and Immunodetection was performed using the ABC method TUNEL-reactivity for apoptosis detection. Examples of (DAKO, Hamburg, Germany). The secondary biotinylated antibody was incubated for 15 min at 37ÆC followed by the streptavidin- sections after immunostaining are shown in Figure 1. biotin-complex for 15 min at 37ÆC and an amplification reaction with streptavidin-peroxidase and biotinyl-tyramine for another 15 Immunoreactivity for hMSH2, p53, Ki-67 proteins and for min at 37ÆC. Three wash steps were performed after all incubations. apoptosis. The results from immunodetection with the Bound peroxidase activity was visualised with 3,3’-diaminobenzidine antibodies for hMSH, p53 apoptosis and Ki-67 are (DAB) (DAKO, Hamburg, Germany). presented in Table I. The IRS state the mean of Immunoreactive detection of apoptosis after TdT-mediated-dUTP- immunoreactivity scores of all samples. For Ki-67 only the nick-end-labelling-(TUNEL) assay. For the detection of apoptotic PP-score representing the number of positive cells is shown. cells in tissue sections the In-Situ-Cell-Death-Detection-Kit, AP All antibodies reacted positively with the majority of the was used (Roche, Mannheim, Germany). The slides were samples with the highest IRS for hMSH2.

64 Köster et al: hMSH2 in Cervical Cancer

Figure 1. Immunohistochemistry of samples from cervical carcinoma. Magnification: x400. A) hMSH2 detection. Left: sample 89 with 20% immunoreactive tumour cells and right: sample 86 with 70% immunoreactive tumour cells. B) p53 detection. Left: sample 86 with 10% immunoreactive tumour cells and right: sample 87 with 30% immunoreactive tumour cells. C) Detection of apoptosis. Left: sample 88 with 90% immunoreactive tumour cells and right: sample 85 with 65% immunoreactive tumour cells.

Correlations of the immunoreactivity scores by the spearman’s immunoreactivity positively correlated with p53 rank correlation coefficient. Table II summarises the immunoreactivity (p≤0.001) with a medium strong correlations between the IRS of hMSH2, p53 and apoptosis. Spearman’s rank correlation coefficient (rs) of 0.449 and Correlations of Ki-67 with hMSH2, p53 and apoptosis are with apoptosis (p=0.005) with a low rs of 0.279. p53 represented only with regard to the PP. hMSH2 immunoreactivity and apoptosis were not significantly

65 ANTICANCER RESEARCH 27: 63-68 (2007)

Table II. Spearman’s rank correlation coefficient between hMSH2 and and rarely detectable. High levels of p53 immunostaining p53 expressions and the immunostaining of apoptosis following TUNEL are generally interpreted as abnormal nuclear assay. accumulation of mutated p53 (30), but in cervical IRS PP-score carcinoma the appearance of p53 mutation are reportedly rare (31). A further complication for the interpretation of hMSH2 – p53 0.449* (p<0.001) 0.507* (p<0.001) p53 immunoreactivity is the fact that cervical carcinoma is hMSH2 – apoptosis 0.279* (p=0.005) 0.190 (p=0.055) strongly linked to infection with the human papilloma virus p53 – apoptosis 0.178 (p=0.074) 0.105 (p=0.294) (HPV). The viral oncoprotein E6 interacts with cellular hMSH2 – Ki-67 0.045 (p=0.658) p53 – Ki-67 0.086 (p=0.392) proteins and induces degradation of p53 (32). Apoptosis – Ki-67 –0.027 (p=0.792) Nevertheless, we found 74% of the tumour tissues stained positively for p53, so we expect that our data reveal in *Statistically significant. most of the cases the detection of wild-type p53. While many studies presume a p53 mutation frequency of less than 20% in cervical cancer reflected in immunodiagnostic detection, other studies report higher percentages of correlated (p=0.074). There were no significant correlations immunodetectable p53. Spagnoletti et al. report of 48% of between the Ki-67 PP-score in tumour cells and the PP- tissues from breast cancer expressing p53 (10), Cheah et al. scores for hMSH2 (p=0.658), p53 (p=0.392) or apoptosis detected over 70% in cervical carcinoma (31) and (p=0.792). Haensgen et al. found immunodetectable p53 in 85% in cervical carcinoma cases using the same antibody against Correlation of the immunoreactivity for hMSH2 and p53 p53 that was used in this study (33). proteins, and for apoptosis with clinical data of the patients. The correlation between immunoreactivities for hMSH2 Table III shows the correlation of patient data with the IRS and p53, probably wild-type p53, seems to be reasonable of hMSH2, p53 and apoptosis. No correlations were found because hMSH2 is regulated by p53. Scherer et al. detected with ER and PR status. hMSH2 and p53 immunoreactivity a response element in the promoter region of the hmsh2 did not correlate with TNM stage, tumour grading or FIGO gene (34) and Warnick et al. showed that it is active (27). stage. For apoptosis, there were weak correlations between Moreover, in cancer tissue, where high proliferative activity the IRS and T stage (rs=0.297; p=0.003) and FIGO stage with frequent replicative errors is presumed, the mismatch (rs=0.262; p=0.01). repair system with hMSH2 activity is comprehensibly linked to apoptosis induction through p53 (35). Correlation of the immunoreactivity for hMSH2 and p53 In contrast to our results, Giarnieri et al. found a proteins and for apoptosis, with the survival rate of cervical correlation between a high histochemical grade of p53 and carcinoma patients. The survival rates were analysed using the absence of hMSH2 and hMLH1 expression in non- the Cox’s regression model. The relative risk of death from invasive squamous cell carcinoma of the uterine cervix cervical cancer was significantly increased to 1.335 for (NISCC) (19). Positive staining for hMSH2 was found in patients with increasing immunoreactivity for apoptosis 92.9% of NISCC, where only 56.5% of invasive squamous (p=0.0102). The IRS for hMSH2 or p53 did not correlate cell carcinoma of the uterine cervix (ISCC) expressed with the rate of survival (Table IV). hMSH2. For ISCC the correlation of p53 overexpression and hMLH1 expression was positive. Discussion Correlation of hMSH2 immunoreactivity with predictive Correlation of the immunoreactivity for hMSH2 with p53 tumour markers. No correlations between hMSH2 expression and apoptosis. Immunoreactivity for hMSH2 was immunoreactivity and tumour grading could be detected in significantly correlated with immunoreactivity for p53 and the 102 samples of cervical carcinoma tissues. Chung et al. apoptosis. The correlation between the high expression of (36) investigated the association of the absence of the hMSH2 and apoptosis reflects the expected relation MMR-proteins hMSH2 or hMLH1 with clinico- between the mismatch repair system and the induction of pathological features of cervical cancer. In accordance with apoptosis, as demonstrated by Zhang et al., who showed our study, they could not relate the MMR protein that hMSH2-deficient cells do not undergo apoptosis and expression to any of the investigated tumour gradings, develop tumours after the accumulation of mutations (22). including the frequency of MSI. Giarnieri et al. report a It is difficult to comment on p53 immunoreactivity, correlation between the absence of these MMR proteins especially in the case of cervical cancer. Because of its with the invasiveness of the squamous cell carcinoma of the short half life, the immunoreactivity of wild-type p53 is low uterine cervix. No further correlations could be found

66 Köster et al: hMSH2 in Cervical Cancer

Table III. Spearman’s rank correlation coefficients between hMSH2-expression, p53-expression and apoptosis with clinical data.

ER PR T stage N stage M stage Histopathological FIGO grading stage hMSH2 –0.029 0.019 0.126 0.033 0.025 0.098 0.115 p-value (0.77) (0.847) (0.218) (0.753) (0.81) (0.328) (0.263) p53 0.145 –0.087 0.02 0.007 –0.1 0.109 –0.035 p-value (0.146) (0.387) (0.845) (0.944) (0.35) (0.273) (0.732)

Apoptosis 0.019 –0.086 0.297* 0.167 0.18 0.066 0.262* p-value (0.85) (0.393) (0.003) (0.113) (0.09) (0.512) (0.01)

*Statistically significant.

between MMR protein expression and histological tumour Table IV. Correlations of hMSH2 and p53-expression and apoptosis with grading (19). Ciavattini et al. found the highest levels of the relative risk of death from cervical cancer, calculated using the Cox’s hMSH2 and hMLH1 in preinvasive lesions compared to regression model. normal tissue and invasive squamous cervical carcinoma. Relative risk p-value The MMR is probably highly active in early cancer stages. For breast cancer, Bock et al. report a correlation between hMSH2 0.9423 0.4106 the loss of hMSH2 expression with lymph node appearance, p53 0.9823 0.795 higher degrees of malignancy and elevated proliferative Apoptosis 1.3352* 0.0102 activity (17). *Statistically significant.

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