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Uterine leiomyosarcoma with osteoclast-like giant cells associated with high expression of receptor activator of nuclear factor κB ligand
Human Pathology (2015) 46, 1679–1684
www.elsevier.com/locate/humpath
Original contribution
Uterine leiomyosarcoma with osteoclast-like giant cells associated with high expression of receptor activator of nuclear factor κB ligand☆ Mika Terasaki MD, PhD a,⁎, Yasuhiro Terasaki MD, PhD a , Koichi Yoneyama MD, PhD b , Naomi Kuwahara MT a , Kyoko Wakamatsu MT a , Kiyotaka Nagahama MD, PhD a , Shinobu Kunugi MD, PhD a , Toshiyuki Takeshita MD, PhD b , Akira Shimizu MD, PhD a a
Department of Analytic Human Pathology, Nippon Medical School, Tokyo 113-8602, Japan Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo 113-8602, Japan
b
Received 28 January 2015; revised 20 March 2015; accepted 10 April 2015
Keywords: Leiomyosarcoma; Osteoclast-like giant cells; Receptor activator of nuclear factor κB ligand; RANKL; Leiomyoma; Uterus; Immunohistochemistry
Summary The occurrence of osteoclast-like giant cells (OLGCs) in uterine leiomyosarcomas (LMSs) is a rare phenomenon. The nature of OLGCs and the significance of their accumulation in these tumors are poorly understood. Recent studies revealed that the formation of osteoclasts requires a specific cytokine, receptor activator of nuclear factor κB ligand (RANKL), in bone. In this study, we investigated the expression of RANKL in 2 cases of uterine LMS with OLGCs by means of immunohistochemistry and compared the extent of RANKL expression with that in conventional uterine LMSs and leiomyomas by using real-time reversetranscription quantitative polymerase chain reaction. Our cases of uterine LMS with OLGCs showed markedly high expression of RANKL messenger RNA with clear RANKL immunoreactivity compared with messenger RNA expression and immunoreactivity of conventional uterine LMSs and leiomyomas. These findings suggest that the tumors producing RANKL may account for accumulation of OLGCs in tumor tissue because of RANKL-related osteoclastogenesis. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Uterine leiomyosarcomas (LMSs) comprise approximately 1% to 2% of uterine malignancies [1]. Osteoclast-like giant Abbreviations LMS, leiomyosarcoma; OLGC, osteoclast-like giant cell; RANKL, receptor activator of nuclear factor κB; RT-qPCR, reverse transcription–quantitative polymerase chain reaction; SMA, α-smooth muscle actin ☆
Disclosures: The authors have no disclosures or financial support to report. ⁎ Corresponding author at: Department of Analytic Human Pathology, Nippon Medical School, 1-25-16 Nezu, Bunkyo-ku, Tokyo 113-0031, Japan. E-mail address: [email protected] (M. Terasaki). http://dx.doi.org/10.1016/j.humpath.2015.04.018 0046-8177/© 2015 Elsevier Inc. All rights reserved.
cells (OLGCs) are rarely seen in uterine LMSs [1], and this type of LMS is named uterine LMS with OLGCs [2-13], which has a poor prognosis [7]. This phenomenon of infiltrating OLGCs in tumor tissues was reported not only for uterine LMS but also for several other malignant tumors from various organs including carcinomas and sarcomas [14-21]. Certain studies revealed that the regulation of osteoclast formation requires a specific cytokine, receptor activator of nuclear factor κB ligand (RANKL), in bone [22,23]. RANKL expressed by osteoblasts or osteocytes interacts with its receptor, receptor activator of nuclear factor κB (RANK), expressed by the monocyte/macrophage lineage precursor cells of osteoclasts, and this RANK-RANKL
1680 interaction induces precursor cells that transform into osteoclasts [22,23]. This evidence led previous studies to report finding expression of RANKL in certain bone tumor tissues with OLGCs [24-27]. However, few studies reporting RANKL expression in tumors with OLGCs arising from organ or tissue sites other than bone exist [18,28], and no report of uterine LMS with OLGCs has appeared. The nature of these OLGCs and the significance of their accumulation in uterine LMSs are still unknown. We therefore studied the expression of RANKL in uterine LMS with OLGCs and compared it with RANKL expression in conventional uterine LMS and leiomyomas using immunohistochemistry and real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR).
2. Materials and methods 2.1. Case 1 The patient was an 81-year-old Japanese woman who presented with a 30-year history of leiomyoma. She reported abdominal bloating. Pelvic examinations revealed an enlarged uterus, and magnetic resonance imaging showed an irregularly shaped mass, with a diameter of approximately 12 cm, in the uterus. A chest radiograph and a computed tomographic image suggested multiple metastases to the lungs. A total hysterectomy and bilateral oophorectomy were performed. No significant peripheral lymphadenopathy was observed. The patient was treated with the chemotherapeutic agents gemcitabine and docetaxel. However, the metastatic lung lesions grew larger, and new metastatic bone lesions appeared. She died 11 months after the operation.
2.2. Case 2 The patient was a 60-year-old Japanese woman who presented with an 8-year history of leiomyoma. She complained of abdominal bloating and dysuria. Magnetic resonance imaging revealed an irregularly shaped mass with a diameter of approximately 13 cm on the right side of the uterine wall. Several enlarged peripheral lymph nodes were noted. A total hysterectomy, bilateral oophorectomy, omentectomy, and lymph node biopsy were performed. Despite chemotherapy with gemcitabine and docetaxel, the patient had recurrence and died 21 months after the operation.
2.3. Comparative cases To compare RANKL expression in other cases by means of immunohistochemistry (IHC) and RT-qPCR, we collected and evaluated 3 cases of uterine leiomyomas and 3 cases of conventional uterine LMSs. The diagnosis of uterine LMS was based on the presence of nuclear atypia, a high mitotic index, coagulative tumor cell necrosis, and immunoreactivity for smooth muscle markers of tumor cells [29].
M. Terasaki et al.
2.4. Histopathology and IHC Surgical specimens of uterine tumors were obtained during routine clinical investigations. The study protocol was approved by the Human Ethics Review Committee of Nippon Medical School; a signed consent form was obtained from each patient. Formalin-fixed tissues from uterine tumors were embedded in paraffin blocks according to standard histopathologic laboratory methods. Sections were cut 4 μm thick and stained with hematoxylin and eosin. Immunohistochemical studies were performed by using autoclave heating and polymer immunocomplex (N-Histofine Simple Stain MAX PO; Nichirei Biosciences, Tokyo, Japan) methods with appropriately reacting positive and negative controls. We used the following primary antibodies for diagnosis: SMA (Dako, Carpinteria CA), h-caldesmon (Dako), desmin (Dako), Ki-67 (Dako), CD68 (Dako), and cytokeratin AE1/ AE3 (Dako). To detect RANKL expression, we used anti-RANKL antibody (rabbit polyclonal antibody; Abcam plc, Cambridge, UK).
2.5. Real-time RT-qPCR amplification We analyzed RANKL messenger RNA (mRNA) expression by performing real-time RT-qPCR. We collected 6 serial 10-μm paraffin sections from each case and extracted total RNA with TRIzol reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. To remove contaminating DNA, poly A+ RNA was treated with DNase I (RNase-Free DNase Set; Qiagen). All samples had a ratio of the optic density at 260 and 280 nm (OD260/OD280) of greater than 1.8. To generate first-strand complementary DNA, we reverse transcribed total RNA (100 ng) with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) in a total volume of 20 μL. For each PCR, we used 5 μL of cDNA (total volume 20 μL). To detect RANKL mRNA, we used ready-to-use primer and probe sets from Applied Biosystems (Assays-on-Demand Gene Expression Catalog nos. Hs00243522_m1 for RANKL and Hs03929097_g1 for GAPDH). We optimized primer and probe concentrations for each target gene according to the manufacturer's procedure. PCR (2 minutes at 50°C, 10 minutes at 95°C, and 45 cycles of 15 seconds of denaturation at 95°C and 60 seconds of annealing at 60°C) was performed with the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) and the fluorescent TaqMan methodology. For all experiments, we quantified RANKL mRNA and GAPDH mRNA in duplicate and normalized RANKL mRNA against GAPDH mRNA and calculated relative gene expression data using the 2−ΔΔCt method [30].
3. Results 3.1. Pathologic findings: histopathology and IHC The gross examination of case 1 showed that a tumor measuring 12 × 10 cm occupied the whole uterine corpus,
RANKL in uterine leiomyosarcoma with OLGCs and that of case 2 showed that a tumor measuring 13 × 10 cm was located in the right side of the uterine wall. Both cases manifested focal necrosis and degenerative changes. In both cases, we also found benign-appearing leiomyomatous nodules on gross examination. The histologic study of case 1 showed that the lesion consisted mostly of proliferating tumor cells that differed in size and shape,
1681 such as atypical, pleomorphic, and short spindle-shaped cells, with numerous OLGCs (Fig. 1A). Mitotic activity was 20/10 high-power fields (HPFs). Some foci of geographic coagulative tumor cell necrosis were seen. In case 2, the mass consisted of atypical short spindle or round proliferating cells of different sizes, with numerous OLGCs (Fig. 1B). Mitotic activity was 15/10 HPFs, and focal coagulative tumor cell necrosis was seen.
Fig. 1 Light microscopic and immunohistochemical findings in case 1 (A, D, and G), case 2 (B, E, and H), and one of the cases of conventional LMS (C, F, and I). A and B, The tumor tissue in both cases of uterine LMS with OLGCs consisted of atypical short spindle cells with numerous OLGCs (arrowheads). C, The conventional uterine LMS shows proliferation of atypical spindle cells. D to F, Positive SMA staining results are seen in tumor cells in cases of uterine LMS with OLGCs (D and E) and conventional uterine LMS (F). D and E, No SMA staining is noted in OLGCs (arrowheads). G to I, The OLGCs show strong CD68 staining (arrowheads), as do infiltrating macrophages. Tumor cells are negative for CD68. A to C, Hematoxylin and eosin stain.
1682 All 3 cases of conventional uterine LMSs demonstrated proliferation of atypical spindle cells (Fig. 1C), and mitotic activities in those cases were 12/10 HPFs, 25/10 HPFs, and 10/10 HPFs. All cases of conventional uterine LMSs showed some foci of coagulative tumor cell necrosis. By immunohistochemistry, tumor cells in all cases of uterine LMSs with OLGCs and conventional LMSs showed expression of SMA (Fig. 1D-F) and h-caldesmon but no expression of CD68 (Fig. 1G-I) and AE1/AE3. The OLGCs were positive for CD68 (Fig. 1G and H) but negative for SMA (Fig. 1D and E). In addition, numerous CD68+ mononuclear macrophages had infiltrated the tumor tissue in both LMS with OLGCs cases (Fig. 1G and H), but not many macrophages had infiltrated the tumor tissue in conventional uterine LMS cases (Fig. 1I).
3.2. Comparative study of RANKL expression via IHC and real-time RT-qPCR Immunohistochemical analyses for RANKL showed clear immunoreactivity in the cytoplasm of the tumor cells in both cases of uterine LMS with OLGCs (Fig. 2A and B). Immunoreactivity for RANKL was not detected (Fig. 2C) in 2 cases of conventional uterine LMS, was weakly positive in 1 case of conventional uterine LMS, and was not detected in any leiomyomas (data not shown). Real-time RT-qPCR assays revealed markedly high expression of RANKL mRNA in both cases of uterine LMS with OLGCs compared with cases of conventional uterine LMSs and leiomyomas (Fig. 3).
4. Discussion We found that our cases of uterine LMS with OLGCs manifested markedly high expression of RANKL mRNA and
M. Terasaki et al. clear RANKL immunoreactivity in tumor cells compared with cases of conventional uterine LMSs and leiomyomas. Osteoclasts are members of the monocyte/macrophage lineage that differentiates under the influence of the critical cytokine RANKL [22,23]. Any RANKL expressed by osteoblasts or osteocytes interacts with its receptor, RANK, which is expressed by monocyte/macrophage precursor cells of osteoclasts, and this RANK-RANKL interaction leads to transformation of precursor cells into osteoclasts [22,23]. These data prompted previous studies of certain bone tumors with OLGCs to report that a RANK-RANKL–related mechanism also has an important role in tumorigenesis because tumors with OLGCs, such as giant cell tumor of bone, chondroblastoma, Ewing sarcoma, and osteosarcoma, have high RANKL expression [24-27]. For other bone sites, a few studies reported on the relationship between RANKL expression and tumors with OLGCs. Ikeda et al [28] reported on a case of sarcomatoid hepatocellular carcinoma with OLGCs in which the tumor cells expressed RANKL mRNA, as determined by in situ hybridization. Those investigators speculated that a similar mechanism of osteoclastogenesis in bone occurred in this tumor [28]. Gibbons et al [18] found, by means of RT-PCR, expression of RANKL and other related cytokines in 2 cases of soft tissue LMS with OLGCs; the isolated tumor cells induced monocytes to transform into OLGCs. In our study, we observed markedly higher expression of RANKL mRNA in uterine LMS with OLGCs compared with expression in conventional uterine LMS and leiomyomas, with clear RANKL immunoreactivity in tumor cells and numerous CD68+ mononuclear macrophages infiltrating the tumor tissues. However, RANKL immunoreactivity was weakly positive or not detected in conventional uterine LMSs and was not detected in leiomyomas without notable CD68+ mononuclear macrophage infiltration. We thus suggest that the highly expressed RANKL of
Fig. 2 Immunohistochemical findings for RANKL. Case 1 (A) and case 2 (B) of uterine LMS with OLGCs and a case of conventional LMS (C). A and B, The tumors with OLGCs show RANKL staining in the cytoplasm. However, the OLGCs (arrowheads) demonstrate no RANKL staining. C, Tumor cells in the conventional uterine LMS are almost all negative for RANKL.
RANKL in uterine leiomyosarcoma with OLGCs
Fig. 3 Expression of RANKL mRNA in cases 1 and 2 compared with that in 3 cases of uterine leiomyoma and 3 cases of conventional uterine LMS, as determined via real-time RT-qPCR. Both cases 1 and 2 have markedly high expression of RANKL mRNA.
our uterine LMS tumor cells may induce accumulation of OLGCs from numerous macrophages in the tumor tissues. Both our patients had a disease with an aggressive course, and chemotherapy with gemcitabine and docetaxel was ineffective. These cases are similar to others in reports that described uterine LMSs with OLGCs as aggressive tumors [7]. Some studies showed that carcinomas with OLGCs tended to be undifferentiated or anaplastic cancers with a poor prognosis [19-21]. Denosumab, a fully humanized monoclonal antibody against RANKL, inhibits osteoclastogenesis and is widely used to treat not only osteoporosis [31] but also certain bone tumors [32] and to prevent bone metastases of some other cancers [33,34]. Whether highly expressed RANKL affects tumor progression and aggressiveness in those specific cases is still unclear. Continued investigation of the relationship between RANKL expression and progression of uterine LMS with OLGCs is warranted. In summary, our report is the first to show markedly high RANKL expression in tumor cells in uterine LMSs with OLGCs compared with tumor cells in conventional uterine LMSs and leiomyomas. We suggest that tumors producing RANKL may account for OLGC accumulation in tumor tissues, which may result from RANKL-related osteoclastogenesis.
Acknowledgments The authors thank Ms A. Ishikawa, Ms M. Kataoka, and Mr T. Arai for expert technical assistance.
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1683 [2] Darby AJ, Papadaki L, Beilby JO. An unusual leiomyosarcoma of the uterus containing osteoclast-like giant cells. Cancer 1975;36:495-504. [3] Pilon VA, Parikh N, Maccera J. Malignant osteoclast-like giant cell tumor associated with a uterine leiomyosarcoma. Gynecol Oncol 1986; 23:381-6. [4] Marshall RJ, Braye SG, Jones DB. Leiomyosarcoma of the uterus with giant cells resembling osteoclasts. Int J Gynecol Pathol 1986;5:260-8. [5] Sieiński W. Malignant giant cell tumor associated with leiomyosarcoma of the uterus. Cancer 1990;65:1838-42. [6] Watanabe K, Hiraki H, Ohishi M, Mashiko K, Saginoya H, Suzuki T. Uterine leiomyosarcoma with osteoclast-like giant cells: histopathological and cytological observations. Pathol Int 1996;46:656-60. [7] Aru A, Norup P, Bjerregaard B, Andreasson B, Horn T. Osteoclast-like giant cells in leiomyomatous tumors of the uterus: a case report and review of the literature. Acta Obstet Gynecol Scand 2001;80:371-4. [8] Sukpan K, Khunamornpong S, Suprasert P, Siriaunkgul S. Leiomyosarcoma with osteoclast-like giant cells of the uterus: a case report and literature review. J Med Assoc Thai 2010;93:510-5. [9] van Meurs HS, Dieles JJ, Stel HV. A uterine leiomyoma in which a leiomyosarcoma with osteoclast-like giant cells and a metastasis of a ductal breast carcinoma are present. Ann Diagn Pathol 2012;16:67-70. [10] Ben Salah R, Mekni A, Oueslati H, Zitouna M, Bouchoucha S. Leiomyosarcoma of the cervix uteri with osteoclast-like giant cells. Tunis Med 2012;90:896-7. [11] Chen KT. Leiomyosarcoma with osteoclast-like giant cells. Am J Surg Pathol 1995;19:487-8. [12] Patai K, Illyes G, Varbiro S, Gidai J, Kosa L, Vajo Z. Uterine leiomyosarcoma with osteoclast like giant cells and long standing systemic symptoms. Gynecol Oncol 2006;102:403-5. [13] Anh Tran T, Holloway RW. Metastatic leiomyosarcoma of the uterus with heterologous differentiation to malignant mesenchymoma. Int J Gynecol Pathol 2012;31:453-7. [14] Holland R, van Haelst UJ. Mammary carcinoma with osteoclast-like giant cells: additional observations on six cases. Cancer 1984;53: 1963-73. [15] Kuwano H, Sonoda T, Hashimoto H, Enjoji M. Hepatocellular carcinoma with osteoclast-like giant cells. Cancer 1984;54:837-42. [16] Gaffey MJ, Lack EE, Christ ML, Weiss LM. Anaplastic thyroid carcinoma with osteoclast-like giant cells. A clinicopathologic, immunohistochemical, and ultrastructural study. Am J Surg Pathol 1991;15:160-8. [17] Molberg KH, Heffess C, Delgado R, Albores-Saavedra J. Undifferentiated carcinoma with osteoclast-like giant cells of the pancreas and periampullary region. Cancer 1998;82:1279-87. [18] Gibbons CL, Sun SG, Vlychou M, et al. Osteoclast-like cells in soft tissue leiomyosarcomas. Virchows Arch 2010;456:317-23. [19] Rosenkrantz AB, Melamed J, Stifelman M. Osteoclast-like giant cell tumor of the renal pelvis associated with urothelial carcinoma: computed tomography, gross, and histologic appearance. Urology 2011;78:1310-2. [20] Singhal A, Shrago SS, Li SF, Huang Y, Kohli V. Giant cell tumor of the pancreas: a pathological diagnosis with poor prognosis. Hepatobiliary Pancreat Dis Int 2010;9:433-7. [21] Gaumann A, Hansen T, Köhler HH, et al. The expression of cathepsins in osteoclast-like giant cells of an anaplastic thyroid carcinoma with tracheal perforation. Pathol Res Pract 2001;197:257-62. [22] Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 1998;95:3597-602. [23] Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;93:165-76. [24] Taylor R, Knowles HJ, Athanasou NA. Ewing sarcoma cells express RANKL and support osteoclastogenesis. J Pathol 2011;225:195-202. [25] Huang L, Cheng YY, Chow LT, Zheng MH, Kumta SM. Receptor activator of NF-κB ligand (RANKL) is expressed in chondroblastoma:
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www.elsevier.com/locate/humpath
Original contribution
Uterine leiomyosarcoma with osteoclast-like giant cells associated with high expression of receptor activator of nuclear factor κB ligand☆ Mika Terasaki MD, PhD a,⁎, Yasuhiro Terasaki MD, PhD a , Koichi Yoneyama MD, PhD b , Naomi Kuwahara MT a , Kyoko Wakamatsu MT a , Kiyotaka Nagahama MD, PhD a , Shinobu Kunugi MD, PhD a , Toshiyuki Takeshita MD, PhD b , Akira Shimizu MD, PhD a a
Department of Analytic Human Pathology, Nippon Medical School, Tokyo 113-8602, Japan Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo 113-8602, Japan
b
Received 28 January 2015; revised 20 March 2015; accepted 10 April 2015
Keywords: Leiomyosarcoma; Osteoclast-like giant cells; Receptor activator of nuclear factor κB ligand; RANKL; Leiomyoma; Uterus; Immunohistochemistry
Summary The occurrence of osteoclast-like giant cells (OLGCs) in uterine leiomyosarcomas (LMSs) is a rare phenomenon. The nature of OLGCs and the significance of their accumulation in these tumors are poorly understood. Recent studies revealed that the formation of osteoclasts requires a specific cytokine, receptor activator of nuclear factor κB ligand (RANKL), in bone. In this study, we investigated the expression of RANKL in 2 cases of uterine LMS with OLGCs by means of immunohistochemistry and compared the extent of RANKL expression with that in conventional uterine LMSs and leiomyomas by using real-time reversetranscription quantitative polymerase chain reaction. Our cases of uterine LMS with OLGCs showed markedly high expression of RANKL messenger RNA with clear RANKL immunoreactivity compared with messenger RNA expression and immunoreactivity of conventional uterine LMSs and leiomyomas. These findings suggest that the tumors producing RANKL may account for accumulation of OLGCs in tumor tissue because of RANKL-related osteoclastogenesis. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Uterine leiomyosarcomas (LMSs) comprise approximately 1% to 2% of uterine malignancies [1]. Osteoclast-like giant Abbreviations LMS, leiomyosarcoma; OLGC, osteoclast-like giant cell; RANKL, receptor activator of nuclear factor κB; RT-qPCR, reverse transcription–quantitative polymerase chain reaction; SMA, α-smooth muscle actin ☆
Disclosures: The authors have no disclosures or financial support to report. ⁎ Corresponding author at: Department of Analytic Human Pathology, Nippon Medical School, 1-25-16 Nezu, Bunkyo-ku, Tokyo 113-0031, Japan. E-mail address: [email protected] (M. Terasaki). http://dx.doi.org/10.1016/j.humpath.2015.04.018 0046-8177/© 2015 Elsevier Inc. All rights reserved.
cells (OLGCs) are rarely seen in uterine LMSs [1], and this type of LMS is named uterine LMS with OLGCs [2-13], which has a poor prognosis [7]. This phenomenon of infiltrating OLGCs in tumor tissues was reported not only for uterine LMS but also for several other malignant tumors from various organs including carcinomas and sarcomas [14-21]. Certain studies revealed that the regulation of osteoclast formation requires a specific cytokine, receptor activator of nuclear factor κB ligand (RANKL), in bone [22,23]. RANKL expressed by osteoblasts or osteocytes interacts with its receptor, receptor activator of nuclear factor κB (RANK), expressed by the monocyte/macrophage lineage precursor cells of osteoclasts, and this RANK-RANKL
1680 interaction induces precursor cells that transform into osteoclasts [22,23]. This evidence led previous studies to report finding expression of RANKL in certain bone tumor tissues with OLGCs [24-27]. However, few studies reporting RANKL expression in tumors with OLGCs arising from organ or tissue sites other than bone exist [18,28], and no report of uterine LMS with OLGCs has appeared. The nature of these OLGCs and the significance of their accumulation in uterine LMSs are still unknown. We therefore studied the expression of RANKL in uterine LMS with OLGCs and compared it with RANKL expression in conventional uterine LMS and leiomyomas using immunohistochemistry and real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR).
2. Materials and methods 2.1. Case 1 The patient was an 81-year-old Japanese woman who presented with a 30-year history of leiomyoma. She reported abdominal bloating. Pelvic examinations revealed an enlarged uterus, and magnetic resonance imaging showed an irregularly shaped mass, with a diameter of approximately 12 cm, in the uterus. A chest radiograph and a computed tomographic image suggested multiple metastases to the lungs. A total hysterectomy and bilateral oophorectomy were performed. No significant peripheral lymphadenopathy was observed. The patient was treated with the chemotherapeutic agents gemcitabine and docetaxel. However, the metastatic lung lesions grew larger, and new metastatic bone lesions appeared. She died 11 months after the operation.
2.2. Case 2 The patient was a 60-year-old Japanese woman who presented with an 8-year history of leiomyoma. She complained of abdominal bloating and dysuria. Magnetic resonance imaging revealed an irregularly shaped mass with a diameter of approximately 13 cm on the right side of the uterine wall. Several enlarged peripheral lymph nodes were noted. A total hysterectomy, bilateral oophorectomy, omentectomy, and lymph node biopsy were performed. Despite chemotherapy with gemcitabine and docetaxel, the patient had recurrence and died 21 months after the operation.
2.3. Comparative cases To compare RANKL expression in other cases by means of immunohistochemistry (IHC) and RT-qPCR, we collected and evaluated 3 cases of uterine leiomyomas and 3 cases of conventional uterine LMSs. The diagnosis of uterine LMS was based on the presence of nuclear atypia, a high mitotic index, coagulative tumor cell necrosis, and immunoreactivity for smooth muscle markers of tumor cells [29].
M. Terasaki et al.
2.4. Histopathology and IHC Surgical specimens of uterine tumors were obtained during routine clinical investigations. The study protocol was approved by the Human Ethics Review Committee of Nippon Medical School; a signed consent form was obtained from each patient. Formalin-fixed tissues from uterine tumors were embedded in paraffin blocks according to standard histopathologic laboratory methods. Sections were cut 4 μm thick and stained with hematoxylin and eosin. Immunohistochemical studies were performed by using autoclave heating and polymer immunocomplex (N-Histofine Simple Stain MAX PO; Nichirei Biosciences, Tokyo, Japan) methods with appropriately reacting positive and negative controls. We used the following primary antibodies for diagnosis: SMA (Dako, Carpinteria CA), h-caldesmon (Dako), desmin (Dako), Ki-67 (Dako), CD68 (Dako), and cytokeratin AE1/ AE3 (Dako). To detect RANKL expression, we used anti-RANKL antibody (rabbit polyclonal antibody; Abcam plc, Cambridge, UK).
2.5. Real-time RT-qPCR amplification We analyzed RANKL messenger RNA (mRNA) expression by performing real-time RT-qPCR. We collected 6 serial 10-μm paraffin sections from each case and extracted total RNA with TRIzol reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. To remove contaminating DNA, poly A+ RNA was treated with DNase I (RNase-Free DNase Set; Qiagen). All samples had a ratio of the optic density at 260 and 280 nm (OD260/OD280) of greater than 1.8. To generate first-strand complementary DNA, we reverse transcribed total RNA (100 ng) with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) in a total volume of 20 μL. For each PCR, we used 5 μL of cDNA (total volume 20 μL). To detect RANKL mRNA, we used ready-to-use primer and probe sets from Applied Biosystems (Assays-on-Demand Gene Expression Catalog nos. Hs00243522_m1 for RANKL and Hs03929097_g1 for GAPDH). We optimized primer and probe concentrations for each target gene according to the manufacturer's procedure. PCR (2 minutes at 50°C, 10 minutes at 95°C, and 45 cycles of 15 seconds of denaturation at 95°C and 60 seconds of annealing at 60°C) was performed with the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) and the fluorescent TaqMan methodology. For all experiments, we quantified RANKL mRNA and GAPDH mRNA in duplicate and normalized RANKL mRNA against GAPDH mRNA and calculated relative gene expression data using the 2−ΔΔCt method [30].
3. Results 3.1. Pathologic findings: histopathology and IHC The gross examination of case 1 showed that a tumor measuring 12 × 10 cm occupied the whole uterine corpus,
RANKL in uterine leiomyosarcoma with OLGCs and that of case 2 showed that a tumor measuring 13 × 10 cm was located in the right side of the uterine wall. Both cases manifested focal necrosis and degenerative changes. In both cases, we also found benign-appearing leiomyomatous nodules on gross examination. The histologic study of case 1 showed that the lesion consisted mostly of proliferating tumor cells that differed in size and shape,
1681 such as atypical, pleomorphic, and short spindle-shaped cells, with numerous OLGCs (Fig. 1A). Mitotic activity was 20/10 high-power fields (HPFs). Some foci of geographic coagulative tumor cell necrosis were seen. In case 2, the mass consisted of atypical short spindle or round proliferating cells of different sizes, with numerous OLGCs (Fig. 1B). Mitotic activity was 15/10 HPFs, and focal coagulative tumor cell necrosis was seen.
Fig. 1 Light microscopic and immunohistochemical findings in case 1 (A, D, and G), case 2 (B, E, and H), and one of the cases of conventional LMS (C, F, and I). A and B, The tumor tissue in both cases of uterine LMS with OLGCs consisted of atypical short spindle cells with numerous OLGCs (arrowheads). C, The conventional uterine LMS shows proliferation of atypical spindle cells. D to F, Positive SMA staining results are seen in tumor cells in cases of uterine LMS with OLGCs (D and E) and conventional uterine LMS (F). D and E, No SMA staining is noted in OLGCs (arrowheads). G to I, The OLGCs show strong CD68 staining (arrowheads), as do infiltrating macrophages. Tumor cells are negative for CD68. A to C, Hematoxylin and eosin stain.
1682 All 3 cases of conventional uterine LMSs demonstrated proliferation of atypical spindle cells (Fig. 1C), and mitotic activities in those cases were 12/10 HPFs, 25/10 HPFs, and 10/10 HPFs. All cases of conventional uterine LMSs showed some foci of coagulative tumor cell necrosis. By immunohistochemistry, tumor cells in all cases of uterine LMSs with OLGCs and conventional LMSs showed expression of SMA (Fig. 1D-F) and h-caldesmon but no expression of CD68 (Fig. 1G-I) and AE1/AE3. The OLGCs were positive for CD68 (Fig. 1G and H) but negative for SMA (Fig. 1D and E). In addition, numerous CD68+ mononuclear macrophages had infiltrated the tumor tissue in both LMS with OLGCs cases (Fig. 1G and H), but not many macrophages had infiltrated the tumor tissue in conventional uterine LMS cases (Fig. 1I).
3.2. Comparative study of RANKL expression via IHC and real-time RT-qPCR Immunohistochemical analyses for RANKL showed clear immunoreactivity in the cytoplasm of the tumor cells in both cases of uterine LMS with OLGCs (Fig. 2A and B). Immunoreactivity for RANKL was not detected (Fig. 2C) in 2 cases of conventional uterine LMS, was weakly positive in 1 case of conventional uterine LMS, and was not detected in any leiomyomas (data not shown). Real-time RT-qPCR assays revealed markedly high expression of RANKL mRNA in both cases of uterine LMS with OLGCs compared with cases of conventional uterine LMSs and leiomyomas (Fig. 3).
4. Discussion We found that our cases of uterine LMS with OLGCs manifested markedly high expression of RANKL mRNA and
M. Terasaki et al. clear RANKL immunoreactivity in tumor cells compared with cases of conventional uterine LMSs and leiomyomas. Osteoclasts are members of the monocyte/macrophage lineage that differentiates under the influence of the critical cytokine RANKL [22,23]. Any RANKL expressed by osteoblasts or osteocytes interacts with its receptor, RANK, which is expressed by monocyte/macrophage precursor cells of osteoclasts, and this RANK-RANKL interaction leads to transformation of precursor cells into osteoclasts [22,23]. These data prompted previous studies of certain bone tumors with OLGCs to report that a RANK-RANKL–related mechanism also has an important role in tumorigenesis because tumors with OLGCs, such as giant cell tumor of bone, chondroblastoma, Ewing sarcoma, and osteosarcoma, have high RANKL expression [24-27]. For other bone sites, a few studies reported on the relationship between RANKL expression and tumors with OLGCs. Ikeda et al [28] reported on a case of sarcomatoid hepatocellular carcinoma with OLGCs in which the tumor cells expressed RANKL mRNA, as determined by in situ hybridization. Those investigators speculated that a similar mechanism of osteoclastogenesis in bone occurred in this tumor [28]. Gibbons et al [18] found, by means of RT-PCR, expression of RANKL and other related cytokines in 2 cases of soft tissue LMS with OLGCs; the isolated tumor cells induced monocytes to transform into OLGCs. In our study, we observed markedly higher expression of RANKL mRNA in uterine LMS with OLGCs compared with expression in conventional uterine LMS and leiomyomas, with clear RANKL immunoreactivity in tumor cells and numerous CD68+ mononuclear macrophages infiltrating the tumor tissues. However, RANKL immunoreactivity was weakly positive or not detected in conventional uterine LMSs and was not detected in leiomyomas without notable CD68+ mononuclear macrophage infiltration. We thus suggest that the highly expressed RANKL of
Fig. 2 Immunohistochemical findings for RANKL. Case 1 (A) and case 2 (B) of uterine LMS with OLGCs and a case of conventional LMS (C). A and B, The tumors with OLGCs show RANKL staining in the cytoplasm. However, the OLGCs (arrowheads) demonstrate no RANKL staining. C, Tumor cells in the conventional uterine LMS are almost all negative for RANKL.
RANKL in uterine leiomyosarcoma with OLGCs
Fig. 3 Expression of RANKL mRNA in cases 1 and 2 compared with that in 3 cases of uterine leiomyoma and 3 cases of conventional uterine LMS, as determined via real-time RT-qPCR. Both cases 1 and 2 have markedly high expression of RANKL mRNA.
our uterine LMS tumor cells may induce accumulation of OLGCs from numerous macrophages in the tumor tissues. Both our patients had a disease with an aggressive course, and chemotherapy with gemcitabine and docetaxel was ineffective. These cases are similar to others in reports that described uterine LMSs with OLGCs as aggressive tumors [7]. Some studies showed that carcinomas with OLGCs tended to be undifferentiated or anaplastic cancers with a poor prognosis [19-21]. Denosumab, a fully humanized monoclonal antibody against RANKL, inhibits osteoclastogenesis and is widely used to treat not only osteoporosis [31] but also certain bone tumors [32] and to prevent bone metastases of some other cancers [33,34]. Whether highly expressed RANKL affects tumor progression and aggressiveness in those specific cases is still unclear. Continued investigation of the relationship between RANKL expression and progression of uterine LMS with OLGCs is warranted. In summary, our report is the first to show markedly high RANKL expression in tumor cells in uterine LMSs with OLGCs compared with tumor cells in conventional uterine LMSs and leiomyomas. We suggest that tumors producing RANKL may account for OLGC accumulation in tumor tissues, which may result from RANKL-related osteoclastogenesis.
Acknowledgments The authors thank Ms A. Ishikawa, Ms M. Kataoka, and Mr T. Arai for expert technical assistance.
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