K-ras mutation in tamoxifen-related endometrial polyps

Abstract

BACKGROUND

K-ras mutation is thought to occur at an early stage of neoplastic progression in the endometrium. The authors investigated mutations in codon 12 of K-ras in tamoxifen (TAM)-related endometrial polyps.

METHODS

DNA was extracted from 11 frozen endometrial polyps from TAM-treated patients with breast carcinoma. Mutations were detected using the mutant allele–specific amplification method. The results subsequently were analyzed for correlations with immunohistochemical data that were obtained using antibodies against estrogen receptors (ERs; α and β forms), progesterone receptors (PRs; A and B forms), and Ki-67.

RESULTS

Mutations in codon 12 of K-ras were observed in 7 of 11 TAM-related endometrial polyps. Expression levels of ER-α and PR-B were high in the glandular epithelium and low in the stroma. PR-A expression was high in both the glandular epithelium and the stroma. In the glandular epithelium, expression of ER-β appeared to be lower than expression of ER-α. The Ki-67 index in the glandular epithelium ranged from 2 to 38, whereas the index ranged from 0 to 4 in the stroma (P < 0.01).

CONCLUSIONS

The incidence of mutations in codon 12 of K-ras in TAM-related endometrial polyps (64%) was greater than the incidence of these same mutations in sporadic endometrial hyperplasias (4.5–23%). High expression levels of ER-α, PR-A, and PR-B in the glandular epithelium were observed in all polyps, regardless of K-ras codon 12 mutation status and Ki-67 index. The authors' findings may support the hypothesis that the polyp-carcinoma sequence partly indicates the development of endometrial carcinoma in postmenopausal women who have been treated with TAM. Cancer 2003. © 2003 American Cancer Society.

Tamoxifen (TAM) is a nonsteroidal triphenylethylene derivative that is widely used for adjuvant treatment and chemoprevention of breast carcinoma. TAM possesses estrogen-agonistic and estrogen-antagonistic effects in various types of tissue, depending on the ambient estradiol concentration.1 Since the publication of the report by Fornander et al.2 in 1989, an increased incidence of endometrial carcinoma has been observed among postmenopausal women with breast carcinoma who have been treated with TAM. In larger epidemiologic studies,3–5 the relative risk of endometrial carcinoma has been estimated to range from two to three, with risk increasing along with the duration and cumulative dose of TAM treatment.

Endometrial polyps and hyperplastic endometrial changes are among the most common pathologic changes in women who have been treated with TAM,6 and the presence of these changes typically is interpreted as evidence of the estrogenic effects of TAM.7–9 Silva et al.10 observed that 10 of 13 (77%) postmenopausal patients who developed endometrial carcinoma after treatment with TAM had endometrial polyps, whereas 16 of 47 (34%) patients in a comparable group who did not receive TAM had endometrial polyps. Deligdisch et al.6 reported that 15 of 33 endometrial carcinomas that developed in postmenopausal patients with breast carcinoma after adjuvant treatment with TAM were found in endometrial polyps. These findings may suggest that the polyp-carcinoma sequence plays a role in the development of endometrial carcinoma in postmenopausal patients with breast carcinoma who are treated with TAM.11

Estrogen receptor (ER)-α is a transcription factor that regulates the expression of target genes in a ligand-dependent manner. ER-β is thought to play a role in the modulation of estrogenic activity, either in combination with ER-α or on its own.12, 13 Paech et al.14 reported that as a ligand of ER-β, TAM, but not estradiol, functions as a potent transcriptional activator at an AP1 site. Kato et al.15 demonstrated that the activity of the amino-terminal activation function–1 of ER-α is modulated by the phosphorylation of serine 118 via the Ras/MAP kinase cascade of the growth factor signaling pathways. Serine 118 phosphorylation was found to be induced by 4-hydroxytamoxifen.16 TAM was found to activate the MAP kinase cascade in Ishikawa cells (an endometrial carcinoma cell line) but not in MCF-7 cells (a breast carcinoma cell line).17

TAM adducts to DNA were detected in endometrial tissue obtained from women who were treated with TAM. Site-specific deoxyguanosine-N²-TAM adducts induced primary GC→TA transversions in COS-7 cells.18 Codons 12 and 14 of the K-ras gene were identified as hotspots for the formation of carcinogenic adducts to DNA in human bronchial epithelial cells.19 In pancreatic tumors, patients with G→T mutations had a significantly higher level of aromatic DNA adducts than did those with G→A mutations or wild-type K-ras.20 K-ras mutations are thought to be correlated with phenotypic progression from complex atypic hyperplasia to endometrial carcinoma. K-ras mutations have been identified in 4.5–23% of endometrial hyperplasias and in 18–26% of endometrial carcinomas.21–25

Although it is not known whether the induction of endometrial carcinoma occurs via DNA adducts and/or because of the estrogenic nature of TAM, the ras gene may or may not play a major role in the development of endometrial carcinoma in women who have been treated with TAM. In the current study, we were able to demonstrate the high incidence of mutations in codon 12 of K-ras in TAM-related endometrial polyps.

MATERIALS AND METHODS

Patients

Between October 1993 and September 2002, 136 patients were recruited from the group of women who received TAM as adjuvant hormonal therapy after surgery for breast carcinoma and who regularly attended the Fukuoka University Hospital (Fukuoka, Japan) outpatient gynecologic clinic. Hysteroscopy was performed for 26 patients who exhibited an endometrial thickness of > 0.5 cm on ultrasound and who reported vaginal bleeding and/or were thought to have either premalignant or malignant lesions based on endometrial cytology studies. Hysteroscopic findings included single polyps (n = 16), multiple polyps (n = 4), cystically atrophic changes in the endometrium (n = 4), and endometrial carcinoma (n = 2). Eleven frozen polypectomy specimens were available for analysis.

Immunohistochemical Methods

Sections were deparaffinized, rehydrated, and incubated for 30 minutes with 3% H₂O₂ in methanol to block endogenous peroxidase activity. After briefly being rinsed in Tris-buffered saline, sections (in 0.01 M citrate buffer) were irradiated in a microwave oven (power, 800 watts). After the sections were cooled and rinsed in Tris-buffered saline, they were incubated for 6 hours at room temperature with monoclonal antibodies directed against ER-α (clone 6F11 [1:40 dilution]; Novocastra, Newcastle, UK), ER-β (clone H-150 [1:100 dilution]; Santa Cruz Biotechnology, Santa Cruz, CA), progesterone receptor (PR)-A (clone 16 [1:100 dilution]; Novocastra), PR-B (clone SAN27 [1:100 dilution]; Novocastra), and Ki-67 (clone MIB-1 [1:100 dilution]; Dako, Glostrup, Denmark). The thoroughly washed sections were subjected to immunostaining for 30 minutes using an alkaline phosphatase–mediated system (EnvisionAP; Dako). Sections were counterstained with hematoxylin. For each specimen, the glandular epithelium and stroma were evaluated separately. Two investigators blindly and independently assessed staining intensity as negative (−), positive (+), or intensely positive (++). The Ki-67 labeling index was determined using 500 cells in the most active area of a given specimen. The primary antibody was omitted in negative control specimens.

Preparation of DNA from Tissue Samples

High–molecular weight DNA was extracted from the available frozen tissue samples. Each sample was placed in liquid nitrogen and then pulverized in a blender. The resultant powder was dissolved in lysis buffer (0.1% sodium dodecyl sulfate with 200 mg/mL proteinase K). DNA was isolated after phenol/chloroform extraction and precipitation with ethanol.

Detection of K-ras Codon 12 Mutations

As is described elsewhere,26 the mutant allele–specific amplification (MASA) method was used to detect mutations in codon 12 of K-ras. Wu et al.27 reported on the allele-specific amplification method and described the significance of the 3′ nucleotide of one of the primers to PCR efficiency. In the current study, the MASA method called for the use of a mixture of three synthetic oligonucleotides corresponding to the possible variants of the first nucleotide or three oligonucleotides corresponding to the possible variants of the second nucleotide of codon 12 of K-ras as one of the PCR primers. The primer mixtures, K1203 (5'-GTACTGGTGGAGTATTTGATAGT-3') and K1204 (5'-CATGAAAATGGTCAGAGAAACC-3'), were used to amplify exon 1 and its flanking intronic sequences. The MASA primer mixtures were 5′-ACTTGTGGTAGTTGGAGCTC-3′, 5′-ACTTGTGGTAGTTGGAGCTT-3′, and 5′-ACTTGTGGTAGTTGGAGCTA-3′ (corresponding to variants of the first nucleotide of codon 12) and 5′-CTTGTGGTAGTTGGAGCTGC-3′, 5′-CTTGTGGTAGTTGGAGCTGT-3′, and 5′-CTTGTGGTAGTTGGAGCTGA-3′ (corresponding to variants of the second nucleotide of codon 12). Polymerase chain reaction (PCR) amplification involved 40 cycles of 0.5 minutes at 95 °C, 2 minutes at 53 °C, and 2 minutes at 70 °C. MASA-PCR amplification involved 32 cycles of 0.5 minutes at 95 °C, 2 minutes at 60 °C, and 2 minutes at 70 °C. Synthetic DNA with a single-codon mutation was used as a positive control. On gel electrophoresis, a band representing more than 50 copies of the amplified fragment was considered to be a positive finding, because bands representing fewer than 50 copies were unstable. For cases that were found by MASA analysis to be positive for mutations in codon 12 of K-ras, DNA isolated from paraffin-embedded tissue samples was further analyzed using an enriched PCR–enzyme-linked minisequence assay (ELMA; Sumitomo Metal Industry, Tokyo, Japan).28 As is described elsewhere,29 the PCR-ELMA assay was based on the enrichment of mutant K-ras followed by the incorporation of a biotin-labeled nucleotide specific for the mutant gene, which then was quantified enzymatically using a chromogenic substance. The PCR-amplified K-ras gene was bound by probes designed to detect wild-type K-ras codon 12 (GGT) and its six mutant forms (GAT, GCT, GTT, AGT, CGT, and TGT), and a microtiter plate reader was used for detection and for quantification of results. The results of the semiquantitative analysis were scored as (3+), (2+), (1+), (+−), or (−), according to the percentage of mutant K-ras. According to the manufacturer, the scores (3+), (2+), (1+), (+−), and (−) correspond to > 20%, 2–20%, 0.2–2%, < 0.2%, and 0% (undetected) mutant K-ras, respectively.

Statistical Analysis

Statistical analysis was performed using StatView software (Version 4.57; Abacus Concepts, Berkeley, CA). The student t test was used to assess associations between categoric variables. Statistical significance was considered to exist if P was less than 0.05.

RESULTS

Clinical Findings

The clinicopathologic characteristics of the 11 TAM-related endometrial polyps are summarized in Table 1. Patient age ranged from 43 to 73 years. One patient (Patient 10) was premenopausal, two patients (Patients 3 and 6) had TAM-induced amenorrhea, and seven patients were postmenopausal. Patients had been treated with 20 mg TAM daily for 6–99 months (mean, 32.2 months; dose range, 3.6–59.4 g) and had received no hormone therapy except for TAM. After polypectomy, 3 patients (Patients 1, 3, and 5) stopped receiving TAM and 7 patients, including a patient (Patient 7) who underwent a transvaginal hysterectomy for prolapse of the uterus 1 year after polypectomy, continued to receive TAM. One patient (Patient 4) had a recurrent TAM-related endometrial polyp hysteroscopically removed at age 73 years, whereas the remaining patients exhibited no abnormal ultrasonographic findings, with follow-up periods ranging from 12 to 52 months.

Table 1. Clinical Data on Women with Tamoxifen-Related Endometrial Polyps

Patient no.	Age (yrs)	Age at menopause (yrs)	Vaginal bleeding	Endometrial cytology	Tamoxifen dose (g)	Diameter of EMa (cm)
EM: endometrium; Pre: premenopausal. a Measured by transvaginal ultrasonography.
1	57	54	Yes	Benign	17.4	2.8
2	64	45	No	Suspicious	3.6	2.3
3	53	50	Yes	Benign	16.8	1.8
4 (Specimen a)	71	53	Yes	Benign	12.0	2.2
4 (Specimen b)	73	53	Yes	Benign	24.0	1.8
5	60	54	Yes	Suspicious	59.4	1.5
6	52	50	Yes	Benign	18.6	1.8
7	69	45	Yes	Benign	15.0	1.6
8	62	49	No	Suspicious	15.6	1.3
9	62	55	Yes	Benign	28.8	1.6
10	43	Pre	Yes	Benign	13.2	2.1

Histologic Findings

On microscopic examination, all polyps exhibited cystically dilated atrophic and proliferative glands embedded in abundant fibrotic stroma. Focal endometrial hyperplasias also were found in all polyps (Fig. 1). Five polyps exhibited ciliated metaplasias, and one exhibited a mucinous metaplasia.

Figure 1. Tamoxifen-related endometrial polyp. Note the aggregation of proliferative glands in the fibrotic stroma (hematoxylin and eosin, original magnification ×100).

Immunohistochemical Findings

Immunohistochemical data are summarized in Table 2. ER-α expression levels were high in the glandular epithelium and low in the stroma (Fig. 2A). In the glandular epithelium, ER-β appeared to be expressed at lower levels compared with ER-α (Fig. 2B). PR-A expression was high in both the glandular epithelium and the stroma (Fig. 2C). PR-B expression was high in the glandular epithelium and low in the stroma (Fig. 2D). In the glandular epithelium, the Ki-67 index ranged from 2 to 38 (mean, 13.6) (Fig. 3), compared with 0 to 4 (mean, 1.3; P < 0.01) in the stroma.

Table 2. Immunohistochemical Data and K-ras Mutation Data

Patient no.	ER-α		ER-β		PR-A		PR-B		Ki-67 index		K-ras mutation
	G	S	G	S	G	S	G	S	G	S	MASA	PCR-ELMA
ER: estrogen receptor; PR: progesterone receptor; G: gland; S: stroma; MASA: mutant allele–specific amplification; PCR-ELMA: enriched polymerase chain reaction–enzyme-linked minisequence assay; ++: intensely positive staining; +: positive staining; −: negative staining; ND: not detected.
1	++	+	+	−	++	++	++	−	11	0	No	—
2	++	+	+	+	++	++	++	+	8	1	No	—
3	++	+	+	+	++	++	++	+	16	0	Yes	GCT
4 (Specimen a)	++	+	+	+	++	++	++	+	18	0	Yes	GAT
4 (Specimen b)	++	+	+	+	++	++	++	+	38	4	Yes	GAT
5	++	+	+	+	++	++	++	+	17	3	Yes	GAT
6	++	+	+	−	++	++	++	+	18	2	Yes	GAT
7	++	−	+	+	++	+	++	−	7	2	Yes	ND
8	++	−	+	−	++	++	++	−	22	0	No	—
9	++	+	−	−	++	++	++	+	2	0	Yes	GCT
10	++	+	+	+	++	++	++	++	3	2	No	—

Figure 2. (A) Estrogen receptor (ER)-α, (B) ER-β, (C) progesterone receptor (PR)-A, and (D) PR-B immunostaining in tamoxifen-related endometrial polyps (EnvisionAP [Dako, Glostrup, Denmark], original magnification ×100).

Figure 3. MIB-1 (Dako, Glostrup, Denmark) immunostaining of a tamoxifen-related endometrial polyp specimen (EnvisionAP [Dako], original magnification ×100).

K-ras Mutations

Mutations in codon 12 of K-ras were found using the MASA method in 7 of 11 TAM-related endometrial polyps (Fig. 4). All observed mutations appeared to occur at the second base of codon 12 (i.e., codon 12 was mutated to GCT, GTT, or GAT). Enriched PCR-ELMA was performed on frozen and paraffin-embedded tissue samples from the seven TAM-related endometrial polyps in which K-ras mutations were detected using MASA. The mutation types of the frozen tissue samples as determined by PCR-ELMA were GAT (+−) (Patient 6), GAT (1+) (Patients 4 [Specimen b] and 5), GAT (3+) (Patient 4 [Specimen a]), and GCT (3+) (Patients 3 and 9) (Fig. 5). The mutation types of the paraffin-embedded tissue samples as determined by PCR-ELMA were GAT (+−) (Patient 6), GAT (1+) (Patients 4 [Specimens a and b] and 5), GCT (1+) (Patient 9), and GCT (3+) (Patient 3). No mutation in codon 12 of K-ras was detected in either the frozen tissue sample or the paraffin-embedded tissue sample from Patient 7.

Figure 4. Detection of mutations in codon 12 of K-ras in tamoxifen (TAM)-related endometrial polyps. Lane 1: marker (pUC/MspI9); Lanes 2–4: positive controls (5 × 10⁵ copies, 5 × 10² copies, and 5 × 10¹ copies, respectively); Lanes 5–7: negative controls (normal white blood cells from 3 individuals). Lanes 8–13 and 15–17: TAM-related endometrial polyps (Patients 9, 4 [Specimen a], 4 [Specimen b], 1, 2, 7, 8, 6, and 10, respectively); Lane 14: hysterectomy specimen from Patient 7.

Figure 5. Semiquantitative analysis of mutations in codon 12 of K-ras using the enriched polymerase chain reaction (PCR)–enzyme-linked minisequence assay (ELMA; Sumitomo Metal Industry, Tokyo, Japan). Lane 4: pancreatic carcinoma with a known GTT mutant version of codon 12; Lanes 1–3, 5, and 6: tamoxifen-related endometrial polyps (Patients 6, 5, 7, 4 [Specimen a], and 4 [Specimen b], respectively). The mutation types as determined by PCR-ELMA for Lanes 1–3, 5, and 6 were GAT (+−), GAT (1+), GGT (wild-type), GAT (3+), and GAT (1+), respectively. BACK: background.

DISCUSSION

Several immunohistochemical studies have reported that compared with endometrial tissue from postmenopausal women who have not received TAM, endometrial tissue from postmenopausal women treated with TAM exhibited a high proliferative index8, 9 and increased expression of PR in the glandular epithelium7 or stroma.9 In the current study, stromal PR-A expression was high, whereas stromal PR-B expression was low in all specimens except for a premenopausal TAM-related endometrial polyp. In all polyps, high expression levels of ER-α, PR-A, and PR-B were observed in the glandular epithelium. These findings were consistent with the findings for hormone receptors in glandular epithelium in the proliferative phase.13, 30 The presence of ER-β has been noted in the epithelial, vascular, and stromal compartments of the endometrium. Menstrual-cyclic changes in expression are less evident in ER-β than in ER-α.13 We confirmed these results using an antibody (clone H150, Santa Cruz Biotechnology) against ER-β. In the glandular epithelium, ER-β expression levels consistently are lower than ER-α expression levels. ER-α may be a key receptor in the development of TAM-related endometrial polyps.

Although ras mutations are the most common oncogenic mutations in human malignancies, the incidence of K-ras activation varies widely among carcinomas. K-ras mutations are rare in human primary breast carcinomas but occur in approximately half of all colorectal carcinomas.31 Transformations in ras oncogenes occur after single point mutations arise within the coding sequences of these genes. Mutations in naturally occurring ras oncogenes have been localized to codons 12, 13, and 61. Alterations in codon 12, GGT, are the most common mutations. In the current study, mutations in codon 12 of K-ras were found in 7 of 11 TAM-related endometrial polyps. This incidence rate is higher than that of K-ras codon 12 mutations in ordinary endometrial hyperplasia (range, 4.5–23%).21–25 The K-ras codon 12 mutation status of the specimen from Patient 7 was positive based on MASA findings but negative according to enriched PCR-ELMA findings. This discrepancy is thought to have resulted from the different sensitivities of the two methods. The enriched PCR-ELMA data obtained from frozen and paraffin-embedded tissue samples may support the idea that paraffin-embedded tissue samples can be used in the analysis of the mutation status of K-ras codon 12.

Mutter et al.23 reported that GGT→GTT mutations in codon 12 of K-ras were observed in 6 of 9 nonmalignant endometrial tissue samples from women with K-ras-mutated endometrial carcinoma. Sasaki et al.22 found GGT→GTT mutations in codon 12 of K-ras in 5 of 11 K-ras-mutated endometrial hyperplasias in Japanese women; in the current study, this GGT→GTT mutation was not observed. Therefore, K-ras mutations in TAM-related endometrial polyps and sporadic endometrial hyperplasias may be induced via different mechanisms. In rat liver DNA, TAM was found to induce adducts and to cause a significant (approximately threefold) increase in the mutation frequency at the lacI gene32; these mutations were characterized by an increased proportion of G:C→T:A transversions. In a study of the mutagenicity of two model reactive intermediates of TAM, α-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide, the majority of mutations in plasmid DNA treated with the former compound were GC→TA transversions, whereas GC→AT transitions were the most common mutations in plasmid DNA treated with the latter agent.33 Because microsatellite instability (MI) sometimes is correlated with altered methylation status, several authors have suggested that MI-positive tumors should be associated with a higher frequency of methylation-related GC→AT transitions. Inactivation of the DNA repair gene MGMT (O⁶-methylguanine-DNA methyltransferase) by promoter hypermethylation is associated with G→A mutations in K-ras in colorectal tumorigenesis.34 Lagarda et al.24 demonstrated the close association between K-ras mutations and MI in endometrial carcinoma, as methylation-related transitions were found to occur frequently in MI-positive tumors.

In their study, Rutqvist et al.4 reported that the cumulative endometrial carcinoma incidence curves for the TAM-treated and control groups diverged over the entire course of the 15-year follow-up period. The authors suggested that such an early divergence might indicate that TAM promoted the growth of endometrial lesions that were present before the initiation of treatment with TAM. The continued divergence several years after the cessation of treatment suggested that TAM also might have produced some of the observed lesions.

In summary, mutations in codon 12 of K-ras were found in 7 of 11 endometrial polyps from women treated with TAM. The incidence of these mutations was much higher than that of K-ras codon 12 mutations in sporadic endometrial hyperplasia (4.5–23%). Among the observed mutations in codon 12 of K-ras, four were identified as GGT→GAT mutations and two were identified as GGT→GCT mutations; in contrast, in previous studies, GGT→GTT was found to be the most common mutation in sporadic endometrial hyperplasias.22, 23 High expression levels of ER-α, PR-A, and PR-B in the glandular epithelium were observed in all polyps, regardless of K-ras codon 12 mutation status and Ki-67 index. In the glandular epithelium, ER-β expression levels appeared to be lower than ER-α expression levels. Thus, the authors' findings may lend further support to the hypothesis that the polyp-carcinoma sequence partly indicates the development of TAM-induced endometrial carcinoma.