Abstract

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The Expression of Progesterone Receptor Isoforms

in Endometrial Cancer*

Ekspresja izoform receptora progesteronowego w raku endometrium

1_{1st Department of Gynaecology and Obstetrics, Silesian Piasts University of Medicine in Wrocław, Poland} 2_{2nd Department of Gynaecology, Obstetrics and Neonatology Silesian Piasts University of Medicine}

in Wrocław, Poland

3_{Department of Pathomorphology, Silesian Piasts University of Medicine in Wrocław, Poland} 4_{Lower Silesian Oncology Centre, Institute of Pathomorhology, Wrocław, Poland}

5_{Departament of Animal Physiology Institute of Zoology, Jagiellonian University, Kraków, Poland} Adv Clin Exp Med 2008, 17, 1, 45–52

ISSN 1230−025X

ORIGINAL PAPERS

© Copyright by Silesian Piasts University of Medicine in Wrocław

Abstract

Background. Endometrial cancer is one of the most common malignant tumors affecting women. It has also been established that hormones, estrogens and progesterone, play an unquestionable role in its etiology. Progesterone acts in all tissues through the progesterone receptor (PR), which belongs to the steroid receptor superfamily. PR exists as two isoforms, PRA and PRB, which differ in structure and function and their role in the pathogenesis of endometrial cancer is still unknown.

Objectives. The goal of this study was to evaluate PRA and PRB progesterone receptor expression in endometrial cancer in relation to histological grade and the relationship between patient survival rate and the isoforms’ expres− sions.

Material and Methods. Analysis of the presence of PRA and PRB in the examined tissues was conducted using immunohistochemical methods.

Results. All immunohistochemical parameters of PRA and PRB isoform expression were lower in moderately dif− ferentiated than in well−differentiated endometrial cancer tissues, but these differences were not statistically signi− ficant. Comparison of PRA and PRB expression in well and poorly differentiated cancer showed that this expres− sion is significantly lower in the latter. Both expressions were also lower in poorly differentiated than in modera− tely differentiated cancer. A slight decrease in five−year survival rate in patients with low expressions of both PRA and PRB was observed.

Conclusions. Disorders of the progesterone receptor isoforms occur in all histological grades of endometrial can− cer, although as a rule this only seems to be significant in cases of poorly differentiated cancer tissues (Adv Clin Exp Med 2008, 17, 1, 45–52).

Key words: endometrial cancer, isoforms, progesterone, receptors, steroid.

Streszczenie

Wprowadzenie. Rak błony śluzowej trzonu macicy należy do najczęściej występujących nowotworów złośliwych u kobiet. Stwierdzono, że hormony, zarówno estrogeny jak i progesteron, odgrywają niekwestionowaną rolę w je− go etiologii. Progesteron wywiera działanie w tkankach przez receptor progesteronowy (PR) należący do nadro− dziny receptorów steroidowych. Receptor progesteronowy występuje w postaci dwóch izoform: PRA i PRB, róż− niących się zarówno strukturalnie, jak i funkcjonalnie, a ich rola w patogenezie raka endometrium pozostaje wciąż niewyjaśniona.

Cel pracy. Ocena ekspresji izoform PRA i PRB w raku endometrium w zależności od stopnia zróżnicowania his− tologicznego nowotworu oraz ocena zależności między ekspresją izoform a przeżyciem pacjentek.

Endometrial cancer is known to be one of the most common malignant tumors affecting women. It has also been established that hormones play an unquestionable role in its etiology. It is highly pro− bable that the presence of estrogen is a significant factor in promoting the development of cancer. However, its influence is more likely to be mitoge− nic than mutagenic [1, 2]. Estrogen activity is in− hibited by the influence of progesterone, which causes a reduction in estrogen receptor expression [1, 3]. The anti−estrogenic action of progesterone implies an essential role in the pathogenesis of en− dometrial cancer. Progesterone acts in all tissues by means of the progesterone receptor, which be− longs to the steroid receptor superfamily. Progeste− rone receptor (PR) synthesis and activity, as well as its expression disorders, could well play a cru− cial role in the pathogenesis of endometrial cancer. In 1983, Horwitz and Alexander described for the first time two sub−units of the progesterone re− ceptor in breast cancer [4]. In 1990, however, Ka− stner observed different transcription paths of two alternative progesterone receptor isoforms, PRA (progesterone receptor A, 94 kDa) and PRB (pro− gesterone receptor B, 114 kDa) [5]. The isoforms are coded by the same gene, but their transcription is carried out under the control of two alternative promoters and translation with the use of two al− ternative initiating AUG codons [5–7]. The lack of conclusive data concerning the role of these two receptor proteins in the pathogenesis of endome− trial cancer as well as the diversified results on this issue presented so far in world literature encoura− ged the authors to conduct further research in this field. The goal of the study was to analyze the PRA and PRB isoforms of progesterone receptor expression in well (n = 38), moderately (n = 22), and poorly differentiated (n = 12) endometrial can− cer. Correlation between the patients’ survival rate and isoform expression was also evaluated.

Material and Methods

Analysis of the presence of both proteins, PRA and PRB, in the examined tissues was con−

ducted using immunohistochemical methods. En− dometrial cancer tissues embedded in paraffin were used. These were prepared in the histopatho− logical laboratory of the 2nd_{Department of Gyne−}

cology and Obstetrics and in the histopathological laboratory of the Department of Oncology, Wroc− law Medical University, Poland, between 1995 and 2004. The expressions of the progesterone re− ceptor isoforms PRA and PRB were examined using computer analysis of the microscopic image (AnalySIS DOCU software package, ver. 3.2 for Windows 95/98/NT, Soft Imaging System GmbH, license no. 100 7557). Immunoreactivity was me− asured in epithelial cells, as endometrial cancer is known to originate in these cells.

The tissues embedded in paraffin were cut in− to 6−µm−thick slices using microtomes. Then the sections were repeatedly washed in the series xy− lene I, xylene II, absolute alcohol I, absolute alco− hol II, 96% ethanol I, 96% ethanol II, 70% etha− nol, 50% ethanol, and distilled water. The prepa− red sections were immersed in each alcohol for a period of five minutes. The dewaxed tissues we− re then heated with a 0.01 M citrate buffer, pH 6.0, three times for three minutes using an 800−W mi− crowave oven. Following cooling to room tempe− rature, the sections were washed three times in TBS (tris−buffered saline) and incubated in 0.3% H2O2in TBS to quench endogenous peroxidase ac−

tivity. Then the tissues were incubated with 3% horse serum for 30 minutes and with a monoclonal antibody specific to PRB (NCL−PGR−B clone, No− vocastra) using a dilution of 1:100. The A form was detected by immunohistochemical procedures using the specific monoclonal antibody NCL− PRG−312 clone (Novocastra).

Incubation with monoclonal antibodies was carried for 12 hours at 4°C. The specimens were then washed three times for five minutes with TBST (tris−buffered saline with 0.05% Tween) and incubated with biotinylated anti−mouse IgG anti− body for 90 minutes at a dilution of 1:400. Then the specimens were washed again three times for 5 minutes with TBST. In the next phase, ABC com− plex (Avidin−Biotin−Peroxidase Complex, DAKO)

immunohistochemicznej.

Wyniki.Wszystkie parametry reakcji immunohistochemicznej były niższe w rakach średnio zróżnicowanych niż w rakach dobrze zróżnicowanych, choć otrzymane różnice nie były istotne statystycznie. Porównanie ekspresji PRA i PRB w dobrze i nisko zróżnicowanych rakach endometrium wykazało, że jest ona istotnie niższa w nisko zróżnicowanych nowotworach niż w dobrze zróżnicowanych. Ekspresja obu izoform była również niższa w rakach nisko zróżnicowanych niż w średnio zróżnicowanych. Zaobserwowano także nieznaczne skrócenie przeżycia pac− jentek z niską ekspresją zarówno PRA, jak i PRB.

Wnioski. Zaburzenia ekspresji izoform receptora progesteronowego stwierdzono we wszystkich stopniach zróżni− cowania histologicznego raka endometrium, najwyraźniej jednak są zaznaczone w rakach nisko zróżnicowanych. (Adv Clin Exp Med 2008, 17, 1, 45–52).

was used at a dilution of 1:100 for 40 minutes at room temperature. The tissues were then washed twice with TBST for five minutes and once with TBS for five minutes. Then a color reaction was conducted in a solution containing TBS (pH 7.4) with the addition of imidazole (68 mg/100 ml), DAB (50 mg/100 ml), and 30% H2O2(70 µl/100

ml). When the color reaction was present, the spe− cimens were washed in distilled water in order to restrain the reaction. As a final step the specimens were dehydrated in a set of alcohols and xylene.

Three parameters of immunohistochemical re− action were examined in the tissue samples: locali− zation of the color reaction, intensity of the color re− action, and the percentage of positive cells. Five fields were randomly selected from each specimen and examined at a magnification of ×400 using an Olympus light microscope. The percentage of posi− tive cells was calculated using the Touch Count function of AnalySIS software (Soft Imaging Sy− stem GmbH). All cells were counted in the exami− ned fields and the percentage of color−positive cells was calculated. The mean value was then calculated from the percentages of positive cells. The expres− sions of PRA and PRB were evaluated employing the modified semiquantitive IRS scale according to Remmelle [8]. Points for three immunohistochemi− cal parameters of PRA and PRB isoform expression were calculated: the percentage of positive cells, the intensity of the color reaction, and the rIRS product (the product of the points for intensity of the color reaction and the percentage of positive cells).

The results were statistically analyzed. Each parameter was calculated separately for the mean, median, and standard deviation. The statistical si− gnificance between the means for different groups was calculated by means of one−way analysis of variance (ANOVA) or by the Wilcoxon signed rank test when the variances in the groups were not homogeneous (the homogeneity of variance was determined by Bartlett’s test) or when the number of cases was too small. Statistical signifi− cance between frequencies was calculated using the Mantel−Haenszel test. Survival curves obtai− ned with the Kaplan−Meier method were used to evaluate the statistical association between results. Survival curves were compared using the log−rank test. Apvalue of less than 0.05 was required to re− ject the null hypothesis. Statistical analysis was performed using EPIINFO Ver. 3.2 (4.02.2004) software package.

Results

The ages of the patients examined in the study ranged between 36 and 86 years (average: 61 years). They had had menarche between the ages of 9 and

18 years (average: 13.6 years). Material from 13 pre−menopausal and 59 post−menopausal women with endometrial cancer was studied. Menopause occurred between the ages of 39 and 57 years (average: 51.2 years). Fifteen women of the study group had never been pregnant (21%), 22 patients only once (31%), 25 women 2 or 3 times (35%), and 10 patients (14%) had delivered more than 4 times. In the study group as a whole, 16 (22%) patients had had one or two miscarriages.

The patients’ age at the time of endometrial cancer diagnosis had no statistically significant re− lationship with PRA and PRB expression in the examined cancer tissue. Furthermore, there was no association between the expressions of the iso− forms in the cancer tissues and patients age at me− narche or menopause. There was no evidence that parity had any influence on PRA and PRB expres− sion in the examined endometrial cancer tissues. Similarly, no statistically significant correlation was found between PRA and PRB expression in the patients’ cancer tissues.

The main goal of this experiment was to ana− lyze the expression parameters of progesterone re− ceptor isoforms in the cancer group in relation to histological grade. Although it was found that all immunohistochemical parameters of PRA and PRB isoform expression, such as the rIRS product, percentage of the positive cells, and the intensity of the color reaction, were lower in the moderate− ly differentiated than in the well−differentiated en− dometrial cancer tissues, these differences did not reach statistical significance. Only the percentage of PRA isoform−positive cells was statistically si− gnificantly lower in the moderately differentiated than in the well−differentiated endometrial cancer tissues. These results are presented in Table 1.

The expressions of PRA (p= 0.0000) and PRB (p= 0.0023) were statistically significantly lower in the poorly differentiated than in the well−diffe− rentiated cancer tissue. The pvalue showed stati− stical significance in all the evaluated parameters of the immunohistochemical reaction. The results are presented in Table 2.

The comparison of PRA and PRB expression in moderately and poorly differentiated cancer showed that all the expression parameters of PRA and PRB, i.e. rIRS, the percentage of positive cells, and color reaction intensity, were lower in the poorly differentiated than in the moderately differentiated tissue. The differences between the mean values of all the expression parameters were also statistically significant. These results are pre− sented in Table 3. Also, lack of PRA and PRB expression was confirmed mainly in the poorly differentiated endometrial cancer.

ly differentiated endometrial cancer

Tabela 1. Porównanie parametrów ekspresji PRA i PRB pomiędzy rakami endometrium dobrze i średnio zróżnicowanymi Well−differentiated cancer group Moderately differentiated cancer group p

(Grupa nowotworów dobrze (Grupa nowotworów średnio

zróżnicowanych) zróżnicowanych)

n = 38 n = 22

x M SD x M SD

PRA rIRS 4.58 4.00 2.43 3.59 2.50 2.48 0.0945

% 2.45 2.00 0.78 2.00 2.00 0.69 0.0362*

I 1.82 2.00 0.65 1.68 2.00 0.84 0.482

PRB rIRS 3.74 2.00 2.96 3.64 3.00 2.97 0.882

% 2.08 2.00 1.10 2.00 2.00 0.82 0.827

I 1.61 2.00 0.86 1.59 2.00 0.85 0.909

PRA – progesterone receptor A. PRB – progesterone receptor B. % – percentage of positive cells. I – intensity of the color reaction.

rIRS – product of points for intensity of the color reaction and the percentage of positive cells. x – mean value.

PRA – receptor progesteronowy A. PRB – receptor progesteronowy B. % – procent komórek dodatnich. I – intensywność reakcji barwnej.

rIRS – iloczyn punktów przyznanych za intensywność reakcji i za procent komórek dodatnich. x – wartość średnia.

Table 2.Comparison of the expression parameters of the PRA and PRB isoforms between well−differentiated and poorly differentiated endometrial cancer

Tabela 2. Porównanie parametrów ekspresji PRA i PRB między rakami endometrium dobrze i nisko zróżnicowanymi Well−differentiated cancer group Poorly differentiated cancer group p (Grupa nowotworów dobrze (Grupa nowotworów nisko

zróżnicowanych) zróżnicowanych)

n = 38 n = 12

x M SD x M SD

PRA rIRS 4.58 4.00 2.43 0.833 1.000 0.718 0.0000*

% 2.45 2.00 0.78 1.08 1.00 0.51 0.0000*

I 1.82 2.00 0.65 0.583 1.000 0.515 0.0000*

PRB rIRS 3.74 2.00 2.96 1.08 1.00 0.793 0.0023*

% 2.08 2.00 1.10 1.42 1.00 0.515 0.0446*

I 1.61 2.00 0.86 0.750 1.000 0.452 0.0014*

PRA – progesterone receptor A. PRB – progesterone receptor B. % – percentage of positive cells. I – intensity of the color reaction.

rIRS – product of points for intensity of the color reaction and the percentage of positive cells. x – mean value.

M – median.

SD – standard deviation. p – calculated probability value. PRA – receptor progesteronowy A. PRB – receptor progesteronowy B. % – procent komórek dodatnich. I – intensywność reakcji barwnej.

rIRS – iloczyn punktów przyznanych za intensywność reakcji i za procent komórek dodatnich. x – wartość średnia.

M – mediana.

meters of both isoforms in well, moderately, and poorly differentiated cancer one can conclude that in all histological grades of endometrial cancer, progesterone receptor isoform disorders occur and they are most significant in poorly differentiated cancer tissues. The expressions of PRA and PRB in well, moderately, and poorly differentiated en− dometrial cancer are presented in Figure 1.

The relationship between the two isoforms of the progesterone receptor expression in the endo− metrial cancer tissues and patient survival rate we− re also analyzed. The follow−up lasted from 4 to 106 months, with a mean value of 41.4. Over the entire period it was observed that all the assessed immunohistochemical features (such as the per− centage of positive cells, intensity of the immuno− histochemical reaction, and final results of this re− action marked as rIRS) for PRA and for PRB were lower in the cancer tissues taken from the patients who died during the observation period, although it should be noted that the differences were not sta− tistically significant. Survival curves were drawn up using Kaplan−Meier methods and compared using the log−rank test. The expression of isoforms PRA and PRB in the examined tissues was consi−

dered to be low when the IRS factor was ≤4, i.e. the selected border value. The survival curves show that in the majority of tissues taken from the patients who died during the observation, the expression value of PRA and PRB was lower than 4. The results are presented in Figure 2. Only in one case, in which tissue was taken from a patient who died during the observation period, were the PRA and PRB expressions higher than the border value.

Based on the survival curves it was also obser− ved that the five−year survival rate was 77.7 ± ± 8.1%, but it was shorter when PRA expression was ≤4. In cases where PRA expression was > 4, the survival rate was 95.0 ± 4.9%. An analysis of the five−year survival rate in relation to PRB expression level produced a similar dependency. The survival rate was 79.3 ± 7.4% in cases where PRB expression was ≤4, but 93.8 ± 6.1% when it was > 4. These results are presented in Figure 3. The overall differences observed between survival rates of patients who had cancer tissues with PRA and PRB expression ≤4 as opposed to that of pa− tients who had protein expressions > 4 did not reach a statistically significant level. However, the

Table 3. Comparison of the expression parameters of the PRA and PRB isoforms between moderately differentiated and poorly differentiated endometrial cancer

Tabela 3.Porównanie parametrów ekspresji PRA i PRB między rakami endometrium średnio i nisko zróżnicowanymi Moderately differentiated cancer group Poorly differentiated cancer group p (Grupa nowotworów średnio (Grupa nowotworów nisko

zróżnicowanych) zróżnicowanych)

n = 22 n = 12

x M SD x M SD

PRA rIRS 3.59 2.50 2.48 0.833 1.000 0.718 0.0001*

% 2.00 2.00 0.69 1.08 1.00 0.51 0.0007*

I 1.68 2.00 0.84 0.583 1.000 0.515 0.0005*

PRB rIRS 3.64 3.00 2.97 1.08 1.00 0.793 0.0034*

% 2.00 2.00 0.82 1.42 1.00 0.515 0.0353*

I 1.59 2.00 0.85 0.750 1.000 0.452 0.0034*

PRA – progesterone receptor A. PRB – progesterone receptor B. % – percentage of positive cells. I – intensity of the color reaction.

rIRS – product of points for intensity of the color reaction and the percentage of positive cells. x – mean value.

M – median.

SD – standard deviation. p – calculated probability value. PRA – receptor progesteronowy A. PRB – receptor progesteronowy B. % – procent komórek dodatnich. I – intensywność reakcji barwnej.

rIRS – iloczyn punktów przyznanych za intensywność reakcji i za procent komórek dodatnich. x – wartość średnia.

M – mediana.

differences did show that the five−year survival rate tended to decrease in patients with low expressions of PRB and PRA.

Discussion

In the literature pertaining to this subject it can be observed that researchers generally agree that there is a lack of or at least a decrease in progeste− rone receptor expression in endometrial cancer. The present study was focused on the expression of both isoforms (PRA and PRB) of the progeste− rone receptor in endometrial cancer. Akahira et al. observed that down−regulation of PRA is associa− ted with the development of an ovarian epithelial carcinoma [10]. Disorders in PRA and PRB expression were described by Mote et al. in a stu− dy concerning the role of progesterone receptor isoforms in breast cancer carcinogenesis [11]. The authors observed that PRA and PRB were co− expressed within the same cells in comparable amounts in normal breast tissues and in prolifera− tive disease without atypia (PDWA) [11]. In atypi− cal breast lesions, ductal carcinoma in situ, and in− vasive breast lesions they observed disorders in the occurrence of both proteins which were cha− racterized by a predominance of one of the two proteins’ expressions. PRA predominance was particularly evident in a high proportion in ductal carcinomas in situand invasive breast lesions. Ho−

wever, Balleine et al. analyzed the expressions of PRA and PRB in low−grade endometrial stromal sarcoma [12]. The authors observed the expression of the PRB isoform in all nine analyzed cases. Ei− ght tumors expressed PRA, in seven cases as the predominant isoform [12]. Arnett−Mansfield et al. observed the expression of both isoforms at a simi− lar level in normal endometrial glands. There was an increased predominance of one of the isoforms in atypical hyperplastic areas and in tumors [13]. The present study observed only a few cases whe− re isoform expression disorders were manifested by the total loss of one or both of the isoforms. Both isoforms’ expressions were identified in most of the examined endometrial cancer tissues; however, the isoform expression levels were lower in the poorly and moderately differentiated cancer group than in the well−differentiated one.

The final goal of the present study was an ana− lysis of patient survival in relation to the expres− sions of the two progesterone receptor isoforms. The survival curves showed that five−year survival was slightly lower in cases where the PRA and PRB expression was ≤4 (given by the rIRS factor)

G1

G2

G3

PRA PRB 0,0

0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0

PRA

PRB

rIRS [x]

Fig. 1. Expressions of PRA and PRB in well, moder− ately, and poorly differentiated endometrial cancer. PRA – progesterone receptor A, PRB – progesterone receptor B, G1 – well−differentiated endometrial can− cer, G2 – moderately differentiated endometrial can− cer, G3 – poorly differentiated endometrial cancer

Ryc. 1. Ekspresja PRA i PRB w dobrze, średnio i nisko zróżnicowanych rakach endometrium:

G1 – dobrze zróżnicowane raki endometrium,

G2 – średnio zróżnicowane raki endometrium,

G3 – nisko zróżnicowane raki endometrium

Fig. 2. The five−year survival rate for PRA≤4 (77.7 ± 8.1%) and PRA > 4 (95.0 ± 4.9%), p = 0.214

Ryc. 2. Przeżycie pięcioletnie dla PRA≤4 77,7 ±

±8,1% i dla PRA > 4 95,0 ±4,9%, p= 0,214

Fig. 3. The five−year survival rate PRB ≤4

(79.3 ± 7.4%) and PRB > 4 (93.8 ± 6.1%), p = 0.214

Ryc. 3. Przeżycie pięcioletnie dla PRB ≤4 79,3 ±

than in cases where the expression of both proteins was > 4 (border value). Although this difference did not reach a statistically significant level, it de− monstrated a tendency to shorten the five−year su− rvival rate of patients with low progesterone re− ceptor PRA and PRB isoform expression (≤4 in the examined tissues). Based on the examined ma− terial and the results of analysis one can conclude that the low PRA and PRB expression is connec− ted with a shortened survival rate of patients suffe− ring from endometrial cancer.

Kleine and Steiner described similar relations between patient survival and progesterone receptor expression in endometrial cancer tissues [14, 15]. Kleine et al. stated that in endometrial cancer, the progesterone receptor is a significant prognostic factor, next to the clinical stage. Steiner et al. dec− lared that not only the FIGO stage, tumor grading, tumor type, and the depth of myometrial invasion, but also biochemically measured progesterone re− ceptor status had a significantly association with the overall survival rate. Sakaguchi et al. observed that only a high level of PRB mRNA correlates with the survival of patients with endometrial can− cer. They suggested that measurement of the proge− sterone receptor isoforms could have a prognostic value in endometrial cancer [16]. Akahira came to a similar conclusion based on a study of both iso−

forms’ PRA and PRB expression in ovarian cancer [17]. He went further to suggest that it is important to examine PRB LI (labeling indices) as a progno− stic factor of human epithelial ovarian cancer.

The present authors conclude that the study re− sults are complementary to current knowledge in this field as presented thus far in international lite− rature. The authors would like to underline that this study is one of the few in the literature which analyzed the progesterone receptor isoforms sepa− rately as a prognostic factors in endometrial can− cer. There is no doubt that significant disorders in PRA and PRB expression occur in endometrial cancer tissues and thus have an impact on the pa− thogenesis, development, and progression of endo− metrial cancer. In the present study a decline in the survival rate of patients whose endometrial tissues showed low PRA and PRB expression was ob− served. However, it was not possible to explicitly prove that the isoforms of the progesterone recep− tor could be taken as an independent prognostic factor for endometrial cancer due to the fact that the observed differences failed to reach a statisti− cal significance. Therefore the authors plan further studies on the subject with a larger and heteroge− neous group of patients to determine the possible prognostic meaning of PRA and PRB expression in endometrial cancer.

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Address for correspondence:

Magdalena Tłaczała

1st Department of Gynecology and Obstetrics Silesian Piasts University of Medicine T. Chałubińskiego 3

50−369 Wroclaw Poland

E−mail: [email protected] Conflict of interest: None declared Received: 8.11.2007