Frequent β-catenin gene mutations in atypical polypoid adenomyoma of the uterus,

Human Pathology (2014) 45, 33 40 www.elsevier.com/locate/humpath Original contribution Frequent β-catenin gene mutations in atypical polypoid adenomyoma of the uterus, Hiroyuki Takahashi MD, PhD a, Tsutomu Yoshida MD, PhD a, Toshihide Matsumoto PhD a, Yoichi Kameda MD, PhD b, Yasuo Takano MD, PhD b, Yuki Tazo MD a, Hisako Inoue MD a, Makoto Saegusa MD, PhD a, a Department of Pathology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan b Pathology Section, Kanagawa Cancer Center Research Institute, 1-1-2, Nakao, Asahi-ku, Yokohama 241-0815, Japan Received 22 April 2013; revised 3 June 2013; accepted 6 June 2013 Keywords: APA; β-catenin; α-sma; CD10; Cyclin D1; p21 waf1 Summary Atypical polypoid adenomyoma (APA) is an uncommon polypoid lesion of the uterus. To clarify the mechanism of its histogenesis, we examined the functional role of β-catenin, with reference to expression of p21 waf1, cyclin D1, cyclin E, CD10, and α smooth muscle actin (SMA), as well as cell proliferation, in 7 lesions. In the epithelial components, expression of nuclear β-catenin, p21 waf1, and cyclin D1 was increased in a stepwise fashion from normal tissue through complex atypical hyperplasia and adenomyoma to APA lesions, particularly in squamous morular areas, whereas cell proliferation, as well as cyclin E expression, was significantly decreased in the latter. Similar findings were evident in the stromal lesions, with the exception of a case of nuclear β-catenin. In addition, coexpression of CD10 and α-sma markers was observed in the stromal components in 3 APA cases, in line with the results of normal secretory endometrial and adenomyoma samples, suggesting that cells progress to myofibromatous cells in response to differentiation-promoting events. Finally, β-catenin gene (CTNNB1) mutations were detected in all APA cases, the single nucleotide substitutions being in the epithelial but not the stromal components. These findings suggest that activation of β-catenin signaling, probably secondary to the gene abnormalities, plays an important role in the formation of the complex epithelial architecture in APAs, leading to inhibition of cell proliferation through overexpression of p21 waf1. In contrast, changes in the stromal cell phenotype may occur through a shift from CD10 to α- SMA immunopositivity, independent of CTNNB1 status. 2014 Elsevier Inc. All rights reserved. Funding: This study was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Tokyo) (20590352). Conflict of interest: The authors declare no conflicts of interest. Corresponding author. Department of Pathology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan. E-mail address: msaegusa@med.kitasato-u.ac.jp (M. Saegusa). 1. Introduction Atypical polypoid adenomyoma (APA), an uncommon focal and polypoid lesion of the uterus, is characterized by irregular structures of cytologically atypical endometrial epithelial components with squamous morula formation and prominent cellular smooth muscle stromal components [1,2]. Most patients with APAs are of reproductive age, nulliparous, 0046-8177/$ see front matter 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.06.020

34 H. Takahashi et al. Table Clinicopathlogical findings in 7 APAs, 2 AMs, 10 CAHs, and 11 normal endometrium samples Treatment Morule CTNNB1 Nu-β-catenin LIs p21 waf1 LIs Cyclin D1 LIs Cycin E LIs Ki-67 LIs CD10 + Case Age (y) Epithelium Stroma Epithelium Stroma Epithelium Stroma Epithelium Stroma Epithelium Stroma Stroma Gla Mo Gla Mo Gla Mo Gla Mo Gla Mo Nucleotides Amino acid APA-1 34 Curettage + TCT37TTT Ser Phe 8 39.4 5.4 1.1 41.5 36 42.7 35.9 36.2 1.7 2.8 0 0.8 1.1 1.5 + APA-2 40 Curettage + TCT37TGT Ser Cys 10.4 48.8 5.8 1.5 28.8 17.5 30 66.9 35.3 0.8 0.3 0 17.5 1.2 2.4 APA-3 43 Hysterectomy + TCT33TGT Ser Cys 5 47.9 4.9 0.7 2.5 32 36.6 76.1 9.1 1 0 0.3 20 1.8 1.5 + APA-4 44 Curettage + TCT37GCT Ser Ala 55.3 61.3 13.2 1.9 1.3 7.6 19.9 36.7 30.3 6.1 * 0 9.5 0.8 1.6 + APA-5 38 Curettage + TCT33TTT Ser Phe 12.2 57.5 3.8 19 40.3 51.4 81.8 62.5 38.1 * * * 29.7 1.1 3.1 APA-6 37 Curettage + GAC32CAC Asp His 13 19 5.8 18.8 * 25 50 36.4 18.1 2.5 * 0.9 30 2.8 1.5 APA-7 46 Hysterectomy + GAC32TAC Asp Tyr 10.5 16.1 1.4 9.5 55.6 0.6 17.2 54.4 1.7 9.6 40.2 0 10.7 5 2.6 AM-1 42 Polypectomy Wild 0 * 1.1 2.8 * 8.1 15.2 * 8.6 22.1 * 0.3 3.2 * 0 + AM-2 34 Polypectomy Wild 8.3 * 0 0.8 * 4.1 10.7 * 17.2 6.5 * 0 1.8 * 0 + + * 10.8 ± 12.6 31.7 ± 23.9 * 0.5 ± 0.6 27.8 ± 27.9 * 5.2 ± 5.9 14.6 ± 10.8 * 1.6 ± 1.8 7.4 ± 7.7 * 4.9 ± 3.5 Curettage Wild 8.5 ± 5.9 44.6 ± 7.0 CAH (n = 10) + * 34.2 ± 19.1 36.8 ± 13.6 * 3.7 ± 3.2 11.8 ± 12.1 * 10.4 ± 8.8 20.1 ± 8.5 * 1.6 ± 1.0 6.1 ± 8.3 * 3.3 ± 2.2 Biopsy NE 5.8 ± 1.9 41 ± 9.1 Normal (P) (n = 11) Abbreviations: CAH, complex atypical hyperplasia; NE, not evaluable; Nu, nuclear; *, more than 50% CD10-positive cells in stromal lesion; normal (P), normal proliferative endothelium. and premenopausal, with an age range of 21 to 53 years (median, 39.7 years) [2,3]. The lesions frequently develop in the lower uterine segments [2,4]. In some cases, curettage samples obtained from the lesions may be responsible for misidentification as an invasive carcinoma because of the presence of dense proliferation of smooth muscle cells [2]. Previous studies have demonstrated conflicting results regarding the biological behavior of APAs. For example, a review of 136 APA cases indicated that 30% had residual or recurrent tumors after local excision by curettage and that the risk of coexistent endometrial hyperplasia or carcinoma was approximately 9%, indicating a potentially high risk of malignant disease [5]. In contrast, others have found that the lesions appeared to be histologically and biologically benign, as suggested by DNA ploidy studies and Ki-67 immunohistochemical staining [4,6]. Interestingly, several risk factors for APAs, including obesity, hormone replacement therapy, and prolonged estrogenic stimulation, are common to patients with endometrial hyperplasia and carcinoma [2,7], indicating the possibility of coexistence of these lesions [8-10]. β-catenin is a multifunctional protein involved in E-cadherin mediated cell-cell adhesion, acting as a downstream effector in the Wnt signaling pathway [11-13]. We previously demonstrated that mutations in exon 3 of the gene were frequent in endometrial carcinomas with squamous morular differentiation, suggesting that the nuclear accumulation is an initial signal for an induction of the process, leading to inhibition of cell proliferation through overexpression of p21 waf1 [14-16]. Given that the trans-differentiation toward the squamous morular phenotype is common in APAs [2], we hypothesized a role for β- catenin signaling in its histogenesis. To test this idea, we investigated alterations in β-catenin status with reference to expression of several cell cycle and stromal-related molecules and cell proliferation in APAs, as well as normal, complex atypical hyperplasia (CAH) and adenomyoma (AM) lesions. 2. Materials and methods 2.1. Cases We reviewed uterine tumor samples obtained by curettage or hysterectomy from the patient records of Kitasato University Hospital and Kanagawa Cancer Center Hospital between 1990 and 2012. According to the criteria described by Longacre et al [2], tumors were designated as APAs if they featured localized polypoid proliferation of irregular glands with various degrees of squamous morular differentiation and cellular smooth muscle or hybrid smooth muscle/ fibrous stroma. In curettage samples, we used CD10 immunoreactivity patterns to distinguish APAs from

CTNNB1 mutations in APA 35 Fig. 1 Serial sections of APA. Hematoxylin and eosin (HE) staining and IHC for β-catenin, p21 waf1, cyclin D1, cyclin E, Ki-67, CD10, and α-sma. Boxes with solid outlines enclose both squamous morules (arrows in H&E panel) and stromal lesions and are magnified in lower panels. Note focal Ki-67 immunoreactivity in glandular components (arrow). Original magnifications 100 (upper panels); 400 (lower panels). myoinvasive endometrioid carcinomas, as described by Ohishi and colleagues [17], referred for clinical examinations such as imaging studies. Briefly, CD10-positive stromal cells immediately surrounding the irregular glands (fringelike staining pattern) were frequently evident in myoinvasive endometrioid carcinoma; but such findings were relatively rare in APAs. Finally, a total of 7 APA cases were selected for this study. In addition, 19 biopsy samples of normal endometrial tissue (11 in the proliferative and 8 in the late secretory phase), 10 curettage samples of CAH without squamous morules, and 2 polypectomy specimens of AMs were investigated (Table 1). All tissues were routinely fixed in 10% formalin and embedded in paraffin. This study was approved by the Kitasato University Medical Ethics Committee (B12-95). 2.2. Immunohistochemistry staining Immunohistochemistry (IHC) examination was performed using a combination of microwave-oven heating and polymer immunocomplex (Envision; DAKO, Copenhagen, Denmark) methods, as described previously [14-16]. Antibodies to β-catenin (clone 14; BD Bioscience, San Jose, CA), α-sma (clone 1A4; DAKO), CD10 (clone 56C6; Leica Microsystems, Wetzlar, Germany), Ki-67 (MIB-1; DAKO), cyclin D1 (SP4; DAKO), cyclin E (HE12; BD Bioscience), and p21 waf1 (SX118; DAKO) were used. For evaluation of IHC results, tumor lesions were subdivided into glandular and stromal components. To determine labeling indices (LIs) for nuclear β-catenin, p21 waf1, cyclin D1, cyclin E, and Ki-67, immunopositive nuclei were counted in at least 500 cells in 3 randomly selected fields in each of the 2 phenotypes, as described previously [15]. The LI values for cytoplasmic α-sma immunopositivity in normal endometrial stromal cells were examined in a similar manner. In addition, cases were defined as positive for CD10 immunostaining when 50% of the stroma lesions in APAs were stained in each section. 2.3. Mutation analysis of CTNNB1 gene Six serial 10-μm paraffin-wax sections were reviewed microscopically; and areas with tumor lesions were microdissected, carefully avoiding contamination with non-apa tissue. Tumor areas containing epithelial and stromal components were microdissected separately in 3 APA cases. Genomic DNA was extracted using a QIAamp DNA FFPE Tissue Kit (Qiagen, Tokyo, Japan). A 148 base pair fragment of exon 3 of the β- catenin gene (CTNNB1) was amplified by heminested polymerase chain reactions using the following primers: common forward, 5 -ATTTGATGGAGTTGGACATGG- 3 ; outer reverse, 5 -TGTTCTTGAGTGAAGGACTGA-3 ; and inner reverse, 5 -TCTTCCTCAGGATTGCCTT-3. Water was used instead of template DNA as a negative control for each examination. The polymerase chain reaction products were sequenced using BigDye Terminators v1.1 Cycle Sequencing Kit (Life Technologies Japan, Tokyo, Japan) and analyzed with an ABI PRISM

36 H. Takahashi et al. Fig. 2 Nuclear LIs for several markers investigated in epithelial (A) and stromal (B) components of APAs. Data are shown as means ± SDs. Nu, nuclear; P, normal proliferative endometrium; H, complex atypical hyperplasia; Gla, glandular components; Mo, squamous morular lesions. 3130 genetic analyzer (Perkin-Elmer Japan, Yokohama, Japan) according to the manufacturers protocols. 2.4. Statistical analysis Comparative data were analyzed using the Mann- Whitney U test. The cutoff for statistical significance was P b.05. 3. Results 3.1. Immunohistochemical findings Examples of immunoreactivity for β-catenin, p21 waf1, cyclin D1, cyclin E, Ki-67, CD10, and α-sma in both epithelial and stromal components of APAs are illustrated in Fig. 1. Nuclear accumulations of β-catenin, p21 waf1, and cyclin D1, as well as cytoplasmic CD10 immunoreactivity, were frequently observed in epithelial components, including squamous morular lesions, in contrast to the less intense immunoreactivity for cyclin E and Ki-67. Distinct immunopositivities for p21 waf1 and cyclin D1, but not for nuclear β-catenin, cyclin E, and Ki-67, were evident in the myofibromatous stromal components. In addition to diffuse and strong immunoreaction for α-sma, focal and weak CD10 immunopositivity was observed in the stroma of 3 cases (Table 1). In the epithelial components, the average LI values for nuclear β-catenin, as well as p21 waf1 and cyclin D1, showed stepwise increases from normal proliferative endometrium through CAH and AM to APA lesions, particularly in squamous morular areas, whereas the LIs of both Ki-67 and cyclin E were significantly decreased in APAs, with the exception of the squamous morular APA lesions (Fig. 2A). In the stromal components, significantly higher LI values for p21 waf1 and cyclin D1 were evident in APAs than in normal, CAH, or AM lesions, whereas the LIs of Ki-67, as well as cyclin E, were decreased in stepwise fashion from normal tissue to APA lesions. There was no significant difference in nuclear β-catenin LIs (Fig. 2B). In normal endometrial stromal components with strong CD10 immunoreactivity, significantly higher α-sma LIs were observed in the secretory than in the proliferative phase (Fig. 3A and B). In 2 AM cases, strong α-sma and weak CD10 immunopositivity were common in stromal components (Fig. 3C), supporting an earlier report that such lesions consist of endometrial-type stroma and smooth muscles, with the latter predominant [18]. 3.2. Mutation analysis of CTNNB1 Sequence analysis of exon 3 of CTNNB1 revealed heterozygous substitution mutations in all 7 APA cases (Table 1). Of these cases, 3 also demonstrated singlenucleotide substitutions in the epithelial but not the stromal components (Fig. 4A), in contrast to absence of the gene mutations in AM and CAH (Fig. 4B and Table 1). 4. Discussion The present study provides evidence for frequent mutations of CTNNB1 in epithelial but not stromal components of APAs, in line with results showing high nuclear β-catenin accumulation, particularly in squamous

CTNNB1 mutations in APA 37 Fig. 3 Expression of α-sma and CD10 in stroma of normal endometrium and AM. A, Normal proliferative (left) and late secretory (right) endometrial tissues; H&E staining (upper panels) and IHC staining for α-sma (lower). Note apparent immunoreactivity for α-sma immunoreactivity in stromal cells in late secretory phase (stromal lesions indicated by arrow are magnified in the inset), in contrast to the immunoreaction in vessel walls only (arrow) in the proliferative phase (original magnifications 200; inset, 400). B, α-sma LI values in proliferative (Proli) and late secretory (Late Sec) stromal components. Data are shown as means ± SDs. C, Serial sections of AM case. H&E staining (left) and IHC staining for CD10 (middle) and α-sma (right). Original magnification 100. morular lesions. Considering the absence of mutations in AM and CAH cases, it is suggested that activation of β- catenin signaling, probably by the gene abnormalities, is essential for establishment of the architecturally characteristic glandular components. This conclusion may be supported by our previous findings of a functional role of the signaling during trans-differentiation toward the morular phenotype of endometrial carcinoma cells [14-16]. In contrast to our present data, an earlier report revealed cytoplasmic and nuclear accumulation of β-catenin in 5 of 6 APAs, despite the absence of gene mutations [19]. Although we are unable to provide an explanation for the discrepant results, it is possible that there were differences in the microdissection of the paraffin-embedded tissues because the presence of stromal components not having the mutations appeared to be false-negative results. In addition, it is possible that CTNNB1-mutated tumor cells escaped detection because of smaller numbers or heterogeneous distribution in the lesions. An important finding was that p21 waf1 was overexpressed in both the epithelial and stromal components of APAs, in line with the significant decrease in cell proliferation. In general, p21 waf1 is considered to inhibit cell-cycle progression primarily through inhibition of cyclin-dependent kinase 2 [20], allowing us to speculate that its overexpression may be key in the modulation of cell proliferation in APA lesions. In addition, it appears that CTNNB1 abnormalities serve as a regulator of the cell kinetics in epithelial but not stromal components through induction of p21 waf1 expression, as the nuclear accumulation was demonstrated to be secondary to activation of the p14 ARF /p53/p21 waf1 pathway [15]. As unexpected results in this study, overexpression of the cyclin D1 gene (CCND1), a target of β-catenin signaling, was observed in both components, with a tendency to demonstrate an inverse relationship to cell proliferation. In general, G1 cyclins, including cyclin D1, Cdk4, and p16 INK4A, are considered nonessential for completion of most normal cell division cycles, as overexpressed G1 cyclins do not as a rule increase net cell proliferation, despite accelerating G 1 to S progression [21]. In fact, frequent coexpression of nuclear β-catenin, cyclin D1, and p16 INK4A has been demonstrated at invasive fronts with low proliferation rates in colorectal adenocarcinomas [22]. Together with

38 H. Takahashi et al. Fig. 4 Sequence analysis of exon 3 of CTNNB1 in APA and AM. A, Case APA-3. Note heterozygous substitution mutation (arrows) in total and epithelial samples (upper panel), in line with nuclear β-catenin accumulation, whereas such mutations are not evident in stromal samples (lower left, dotted arrow). B, Case AM-2. No mutations in hot spots (indicated by C33, C37, C41, and C45) in exon 3 of CTNNB1 (upper panel). IHC staining for β-catenin (lower). Note absence of nuclear β-catenin immunoreactivity in glandular components, in contrast to strong membranous immunoreaction. Boxes with solid outlines that enclose a gland are magnified in the insets; original magnifications 100; inset 400. the evidence of cyclin D1 expression in squamous morular lesions lacking cell proliferative activity within endometrial carcinomas [15], its overexpression may contribute to cell differentiation rather than to proliferation. Second, the observed overexpression of cyclin E in the squamous morular lesions was unexpected, as E-type cyclins induce progression through the G 1 phase of the cell cycle [23]. Given the evidence that β-catenin coactivates liver receptor homolog 1 effects on the cyclin E promoter [24], it is likely that β-catenin signaling also contributes to cyclin E expression through interaction with other transcription factors. There is a rapidly growing body of evidence that APAs have a hybrid myofibromatous stroma with admixtures of smooth muscle, collagenous fibrous tissue, and normal endometrial stroma, indicating the histogenesis as myofibromatous metaplasia of normal endometrial stromal cells [2,17,25]. In this study, coexpression of CD10 and α-sma was evident in stromal components in 3 of the 7 APAs. Similar findings were observed in 2 AM cases. Together with the evidence of changes in α-sma immunopositivity in normal endometrial stroma during the menstrual cycle, it appears that established cells progress to myofibromatous cells in response to differentiation-promoting events, probably hormonal effects, leading to a shift from CD10 to α-sma immunophenotypes. Previously, a concept of APA with low malignant potential (APA-LMP), which shows a high risk for local recurrence and infiltration into myometrium, has been proposed [2]. Although the number of cases in our series was small, it was impressive that both components showed significantly decreased cell proliferation, implying that most APAs can be categorized as benign or at least lowgrade malignant lesions. Further studies on this point clearly are warranted. Together, our observations suggest a model for establishment and maintenance of epithelial and stromal parts of APAs (Fig. 5). A combination of β-catenin signaling and overexpression of p21 waf1 may be necessary for formation of the architectural complexity of the glandular components, leading to inhibition of cell proliferation. In contrast, changes in the cell phenotype through a shift from CD10

CTNNB1 mutations in APA 39 Fig. 5 Schematic representation of possible histogenesis of epithelial and stromal components of APA. to α-sma immunopositivity may occur during myofibromatous formation in the stromal components, independent of CTNNB1 status. In conclusion, our data clearly demonstrate different mechanisms for the histogenesis of the epithelial and stromal components of APAs. References [1] Mazur MT. Atypical polypoid adenomyomas of the endometrium. Am J Surg Pathol 1981;5:473-82. [2] Longacre T, Chung MH, Rouse RV, et al. Atypical polypoid adenomyofibromas (atypical polypoid adenomyomas) of the uterus. Am J Surg Pathol 1996;20:1-20. [3] Young RH, Treger T, Scully RE. Atypical polypoid adenomyoma of the uterus: a report of 27 cases. Am J Clin Pathol 1986;86:139-45. [4] Fukunaga M, Endo Y, Ushigome S, et al. Atypical polypoid adenomyomas of the uterus. Histopathology 1995;27:35-42. [5] Heatley MK. Atypical polypoid adenomyoma: a systematic review of the English literature. Histopathology 2006;48:609-10. [6] Kuwashima Y, Uehara T, Kurosumi M, et al. Atypical polypoid adenomyomas of the uterus in a very old woman: report of a case with immunohistochemical characterization of its stromal components and proliferative status. Eur J Gynaecol Oncol 1995;16:115-9. [7] Clement PB, Young RH. Atypical polypoid adenomyomas of the uterus associated with Turner s syndrome: a report of three cases, including a review of estrogen-associated endometrial neoplasm and neoplasms associated with Turner s syndrome. Int J Gynecol Pathol 1987;71:141-4. [8] Horita A, Kurata A, Komatsu K, et al. Coexistent atypical polypoid adenomyoma and complex atypical endometrial hyperplasia in the uterus. Diagn Cytopathol 2010;38:527-32. [9] Sugiyama T, Ohta S, Nishida T, et al. Two cases of endometrial adenocarcinoma arising from atypical polypoid adenomyoma. Gynecol Oncol 1998;71:141-4. [10] Fukuda M, Sakurai N, Yamamoto Y, et al. Case of atypical adenomyoma that possibly underwent a serial progression from endometrial hyperplasia to carcinoma. J Obstet Gynecol Res 2011;5:468-71. [11] Ozawa M, Baribault H, Kemler R. The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J 1989;8:1711-7. [12] Kemler R. From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. Trends Genet 1993;9: 317-21. [13] Gumbiner B. Signal transduction by β-catenin. Curr Opin Cell Biol 1997;7:634-40. [14] Saegusa M, Okayasu I. Frequent nuclear β-catenin accumulation and associated mutations in endometrioid-type endometrial and ovarian carcinomas with squamous differentiation. J Pathol 2001; 194:59-67. [15] Saegusa M, Hashimura M, Kuwata T, et al. β-catenin simultaneously induces activation of the p53-p21waf1 pathway and overexpression of cyclin D1 during squamous differentiation of endometrial carcinoma cells. Am J Pathol 2004;164:1739-49. [16] Saegusa M, Hashimura M, Kuwata T, et al. A functional role of Cdx in β-catenin signaling during transdifferentiation in endometrial carcinomas. Carcinogenesis 2007;28:1885-92. [17] Ohishi Y, Kaku T, Kibayashi H, et al. CD10 immunostaining distinguishes atypical polypoid adenomyofibroma (atypical polypoid adenomyoma) from endometrial carcinoma invading the myometrium. HUM PATHOL 2008;39:1446-53. [18] Gilks CB, Clement PB, Hart WR, et al. Uterine adenomyomas excluding atypical polypoid adenomyomas and adenomyomas of endocervical type: a clinicopathologic study of 30 cases of an underemphasized lesion that may cause diagnostic problems with brief consideration of adenomyomas of other female genital tract sites. Int J Gynecol Pathol 2000;19:195-205. [19] Ota S, Catasus L, Matias-Guiu X, et al. Molecular pathology of atypical polypoid adenomyoma of the uterus. HUM PATHOL 2003;34:784-8. [20] Abbas T, Dutta A. p21 in cancer: intricate networks and multiple activities. Nat Rev Cancer 2009;9:400-14.

40 H. Takahashi et al. [21] Quelle DE, Ashmun RA, Shurtleff SA, et al. Overexpression of mouse D-type cyclins accelerates G 1 phase in rodent fibroblasts. Genes Dev 1993;7:1559-71. [22] Jung A, Schrauder M, Oswald U, et al. The invasion front of human colorectal adenocarcinomas shows co-localization of nuclear β-catenin, cyclin D1, and p16 INK4A and is a region of low proliferation. Am J Pathol 2001;159:1613-7. [23] Sherr CJ. Cancer cell cycles. Cell 1996;274:1672-7. [24] Botrugno OA, Fayard E, Annicotte J-S, et al. Synergy between LRH-1 and β-catenin induces G 1 cyclin-mediated cell proliferation. Mol Cell 2004;15:499-509. [25] Horita A, Kurata A, Maeda D, et al. Immunohistochemical characteristics of atypical polypoid adenomyoma with special reference to h-caldesmon. Int J Gynecol Pathol 2010;30:64-70.