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Pathology International 2014; 64: 133–141
doi:10.1111/pin.12142
Case Report
Renal epithelioid angiomyolipoma with malignant features: Histological evaluation and novel immunohistochemical findings
Sachiko Konosu-Fukaya,1 Yasuhiro Nakamura,1 Fumiyoshi Fujishima,1 Atsuko Kasajima,1 Keely M McNamara,1 Yayoi Takahashi,1 Kensuke Joh,3 Hideo Saito,2 Naomasa Ioritani,4 Yoshihiro Ikeda,4 Yoichi Arai,2 Mika Watanabe1 and Hironobu Sasano1 Departments of 1Pathology and 2Urology, Tohoku University Graduate School of Medicine, and Divisions of 3 Pathology and 4Urology, Sendai Shakai Hoken Hospital, Sendai, Japan
Renal epithelioid angiomyolipoma (EAML) is a potentially malignant tumor type whose characteristics and biomarkers predictive of malignant behavior have not been elucidated. Here, we report three cases of renal EAML with malignant features but without histories of tuberous sclerosis complex. Case 1 involved a 29-year-old man with a 12-cm solid mass in the right kidney who underwent radical right nephrectomy. Case 2 involved a 22-year-old woman with a retroperitoneal mass who underwent radical right nephrectomy and retroperitoneal tumorectomy. Local recurrence was detected 7 years post-surgery. Case 3 involved a 23-year-old man with a 14-cm solid mass in the left kidney who underwent radical left nephrectomy. Microscopically, the tumors in all cases demonstrated proliferation of epithelioid cells with atypia, mitotic activity, necrosis, hemorrhage, and vascular invasion. Epithelioid cells in all cases were immunohistochemically positive for melanocytic and myoid markers and weakly positive for E-cadherin and β-catenin. Immunohistochemistry revealed activation of the mammalian target of rapamycin pathway. Here, we report the morphological and immunohistochemical features of clinically or histologically malignant renal EAML.
TSC1/TSC2 complex function can cause over-activation of the mammalian target of rapamycin (mTOR) signaling pathway, reported to contribute to disease progression in EAML patients.2 It is considered a potentially malignant tumor type because approximately one-third of EAML patients are reported to develop metastasis, resulting in poor clinical outcomes.3 Currently, no markers exist for clinical differentiation of malignant and non-malignant AML at initial presentation; diagnosis is made after metastasis or local recurrence.4 Previous studies have proposed several clinicopathological parameters to predict the malignant potential of EAML, but definitive predictive biomarkers have not been determined.5 Therefore, we report three cases of sporadic renal EAML with a malignant presentation, analyzed by immunohistochemistry (IHC) for markers associated with tumorigenesis and disease progression in AML, including E-cadherin, β-catenin, and molecules involved in the mTOR pathway, to explore potential biomarkers for identification of malignant renal EAML.
Key words: β-catenin, E-cadherin, epithelioid angiomyolipoma, malignant angiomyolipoma, mTOR
CLINICAL SUMMARY
Epithelioid angiomyolipoma (EAML) was first reported by Pea et al. in 1991 as a rare angiomyolipoma (AML) variant associated with tuberous sclerosis complex (TSC).1 Loss of Correspondence: Yasuhiro Nakamura, MD, PhD, Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan. Email: yasu-naka @patholo2.med.tohoku.ac.jp Disclosure: None declared. Received 15 April 2013. Accepted for publication 27 January 2014. © 2014 The Authors Pathology International © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Case 1 A 29-year-old man with an unremarkable medical history visited the hospital with an abdominal mass. A computed tomography (CT) scan revealed bilateral masses: a 12-cm solid lesion in the upper pole of the right kidney and an approximately 1-cm mass in the ventral cortex of the left kidney (Fig. 1a). No metastatic lesions were detected. Subsequent imaging studies suggested renal cell carcinoma (RCC) in the right kidney and AML in the left kidney, and radical right nephrectomy was performed. No signs of TSC were identified. Macroscopically, the lesion appeared white on the cut surface, measured 12 cm at the widest part, and
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Figure 1 (a) Contrast-enhanced computed tomography scan of Case 1 showing a 12-cm mass in the right kidney. Microscopic findings from Case 1 revealed (b) a sheet of epithelioid cells and (c) extensive necrosis and hemorrhage. (d) Immunohistochemical examination of Case 1 showed CD34-positive vascular endothelium with neoplastic cells and vascular invasion (arrow). The epithelial cells were positive for (e) HMB-45, (f) α-smooth muscle actin, and had (g) focal membranous E-cadherin expression and (h) diffuse membranous β-catenin expression.
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had scattered intratumoral hemorrhage. It extended into extrarenal tissues and to the pelvic wall. Neither recurrence nor metastasis was clinically identified at the last follow-up visit, 10 months post-surgery.
Case 2 A 22-year-old woman was admitted to the hospital with a growing retroperitoneal mass in the upper part of her right kidney (Fig. 2a). Her medical and family histories were unremarkable. Signs of TSC were not clinically detected. Retroperitoneal tumor resection was subsequently performed. The tumor measured 21 cm at its largest diameter, and protruded from the organ exterior. Seven years after surgery, follow-up CT revealed a mass mainly located on the para-aortic retroperitoneum, focally expanding into the posterior mediastinum. Radiographic examination revealed partial fat density in the tumor, suggestive of AML. Additionally, multicystic lesions were detected bilaterally in the lungs. A 2-cm tumor was also identified in the liver. The cystic pulmonary lesions were clinically and radiologically diagnosed as lymphangiomyomatosis (LAM). Radical nephrectomy of the right kidney and retroperitoneal tumor resection were performed. However, an underlying respiratory disorder and broad expansion of the tumor made the retroperitoneal tumor clinically unresectable. Ten years after the initial surgery, the patient is alive with the disease.
Case 3 A 23-year-old man visited the hospital for evaluation of macrohematuria and flank pain. His medical and family histories were unremarkable. CT revealed a 14-cm solid mass in
the lower pole of the left kidney and hydronephrosis (Fig. 3a). No metastatic lesions were detected. Clinically, RCC was suggested, and radical left nephrectomy was subsequently performed. Macroscopically, the lesion appeared gray to white on the cut surface and was 14 cm along its largest diameter, with no evidence of extrarenal extension. No significant signs of TSC were clinically detected. Details on Cases 1 and 3 were retrieved from the Department of Pathology, Tohoku University. Details on Case 2 were retrieved from the Division of Pathology, Sendai Shakai Hoken Hospital. Hematoxylin and eosin (H&E) staining and IHC were performed on 10% formalin-fixed and paraffinembedded tissue specimens. Table 1 summarizes relevant IHC marker information. TSC was diagnosed based on criteria adapted from Roach et al.6 Patients received no form of therapy before or after surgery. As a control, we obtained information from the Department of Pathology, Tohoku University files on three classic AML cases between 2005 and 2012. All control cases were right kidney tumors; control case 1 involved a 75-year-old man (5-cm tumor); control case 2, a 77-year-old man (1.2-cm tumor); and control case 3, a 62-year-old man (4-cm tumor). All three cases were histologically diagnosed as classic AML comprising thick-walled blood vessels, mature adipose cells, and smooth muscle-like spindle cells without foci of atypical epithelial cells. Additionally, on IHC, tumor cells in all cases were positive for melanocytic and myoid markers. To assess the mTOR pathway activation status in these three cases, we examined various phosphorylated or activated forms of factors involved in this pathway by IHC using an immunoreaction score (IRS), obtained by multiplying the proportion of positive cells and staining intensity.7–9 Values for the proportion of positive cells were assigned as follows: negative, 0; 1–10%, 1; 11–50%, 2; 51–80%, 3; and
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Figure 2 (a) Contrast-enhanced computed tomography of Case 2 showing a 21-cm mass in the right kidney. (b) Histological examination of case 2 revealed a sheet of epithelioid cells. Immunohistochemical examination of Case 2 showed that the epithelial cells were positive for (c) HMB-45, (d) α-smooth muscle actin, (e) E-cadherin (diffuse membranous expression), and (f) β-catenin (diffuse membranous expression). The epithelioid cells of Case 2 were also positive for (g) the phosphorylated form of mammalian target of rapamycin (p-mTOR), (h) cytoplasmic phosphorylated form of S6, and (i) 4E-binding protein-1. (j) The spindle cells of control 1 were positive for p-mTOR.
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81–100%, 4. Staining intensity was qualitatively evaluated as 0, 1+, 2+, or 3+. The total value range was 0–12.
PATHOLOGICAL FINDINGS Microscopically, all tumors were composed of an atypical polygonal cell sheet mixed with classic AML components such as blood vessels with markedly thick vascular walls, adipocytes, and smooth muscle-like cells. Case 1 and 3 tumors predominantly comprised atypical polygonal cells; however, classic AML components were predominant in the Case 2 tumor. All tumors had areas of medium- to large-sized polygonal cells with eosinophilic or clear cytoplasm, prominent nuclear atypia, and conspicuous nucleoli and bizarre or polynuclear cells (Figs 1b, 2b, and 3b). Mitotic figures were encountered at frequencies of 3/10 high-power fields (HPF), 1/10 HPF, and 4/10 HPF in Cases 1, 2, and 3, respectively. Extensive intratumoral coagulative necrosis, marked hemorrhage (Figs 1c,3c), and
lymphovascular invasion were detected in Cases 1 and 3 (Figs 1d,3d). Extrarenal tumor invasion was detected in Case 1. No lymphovascular invasion, necrosis, or extrarenal invasion was detected in Case 2. On IHC, epithelioid cells were focally positive for vimentin, melanocytic markers (HMB-45 and Melan A; Figs 1e, 2c and 3e), myoid markers (α-smooth muscle actin and desmin; Figs 1f, 2d and 3f), and CD68; negative for cytokeratin, epithelial membrane antigen, CD10, and S-100; and weakly and focally immunopositive for transcription factor E3, in all cases. The Ki67 labeling index (LI) in epithelioid cells was approximately 11%, 13%, and 20% in focal ‘hot spots’ for Cases 1, 2, and 3, respectively, but less than 1% in the spindle cells of classic components in each case. Accordingly, these tumors were diagnosed as renal EAML.10 In addition to the classic EAML markers described above, membranous immunoreactivity for both E-cadherin and β-catenin was detected in these three cases. In Cases 1 and 3, approximately one-third of epithelioid cells were weakly positive for E-cadherin, with an incomplete,
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Figure 3 (a) Contrast-enhanced computed tomography of Case 3 showing a 14-cm mass in the left kidney. Histological examination of Case 3 revealed (b) a sheet of epithelioid cells and (c) extensive necrosis and hemorrhage. (d) Immunohistochemical examination of case 3 showed CD31-positive vascular endothelium with neoplastic cells and vascular invasion (arrow). The epithelial cells were positive for (e) HMB-45, (f) α-smooth muscle actin, (g) E-cadherin (focal membranous expression), and (h) β-catenin (diffuse membranous expression). © 2014 The Authors Pathology International © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
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Table 1 Antibodies and their dilutions used for immunohistochemical studies Antibody
Clone
HMB45
HMB45
Melan A α-smooth muscle actin desmin CD68 vimentin AE1/AE3 E-cadherin
A103 1A4
β-catenin
Ki67 p53 EMA CD10 S-100 P-mTOR
P-S6 eIF4E P-4EBP1
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D33 KP-1 V9 AEandAE3 4A2C7
Source
Dilution
Immunotech (Marseille, 1:100 France) Biogenesis (Poole, UK) 1:50 DAKO (Glostrup, (1:40) × 20 Denmark)
DAKO DAKO DAKO DAKO Life Technologies Corporation (Carlsbad, CA, USA) β-catenin Becton, Dickinson and Company (Franklin Lakes, NJ, USA) MIB-1 DAKO D0-7 Novocastra (Welzlar, Germany) E29 Nichirei (Tokyo, Japan) 56C6 Novocastra 15EZE12 BioGenex (Fremont, CA, USA) P-mTOR Cell Signaling Technology (Beverly, MA, USA) P-S6 ribosomal Cell Signaling Technology eIF4E Cell Signaling Technology P-4EBP1 Cell Signaling Technology
1:800 1:100 1:500 1:300 1:400
1:200
1:300 1:100 1:2 1:40 1:10
β-catenin staining was detected in all cases (Figs 1h, 2f, and 3h). However, E-cadherin and β-catenin were only focally detected in spindle cell cytoplasm in areas of classic components. Nuclear immunoreactivity was not detected for either component in these three cases. IHC findings for each case are summarized in Table 2. IHC scoring for phosphorylated phosphatidylinositol-3 kinase (p-PI3K), AKT (p-AKT), mTOR (p-mTOR), S6 (p-S6), and 4E-binding protein-1 (p-4EBP1), as well as for eukaryotic translation initiation factor 4E (eIF4E), is summarized in Table 2. In all three cases, p-mTOR and its downstream effectors, p-S6, p-4EBP-1, and eIF4E, were detected by IHC. IRS values were 3–4 for p-mTOR, 2–6 for p-S6, 1–4 for nuclear p-4EBP-1, 6–8 for cytoplasmic p-4EBP-1, and 2–4 for eIF4E. These markers were also detected in the three classic AMLs, with IRS values of 4–6, 2–4, 1–2, 4, and 2–3 for p-mTOR, p-S6, nuclear p-4EBP-1, cytoplasmic p-4EBP-1, and eIF4E, respectively, with no significant differences in IHC scores between our present cases and the control cases (spindle cells in AML).
1:50
1:100 1:50 1:50
circumscribed staining line (Figs 1g,3g). In Case 2, epithelioid cells demonstrated diffuse immunoreactivity for E-cadherin, but circumscribed membrane immunoreactivity was weak and incomplete (Fig. 2e). Diffuse membranous
DISCUSSION Aydin et al. defined EAML as an AML variant where epithelioid cells constitute more than 10% of tumor cells.10 Renal AML is the main manifestation of TSC.10 LAM is a rare neoplasm affecting the lungs, and is also considered to be closely related to TSC.11 However, TSC was not identified in any of the current cases. Brimo et al. suggested that the presence of at least three of four specific histological findings could predict malignant EAML progression, defined by an epithelioid cell composition greater than 5%. These histological findings are: (i) cellular atypia in ≥70% of the epithelioid cell population; (ii) mitotic
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Table 2 The immunohistochemical findings of E-cadherin and β-catenin in the present cases and the immunohistochemical scoring of the factors involving mammalian target of rapamycin (mTOR) pathway in the present cases and the controls
E-cadherin
β-catenin P-mTOR P-S6 P-4EBP1(n) P-4EBP1 (cy) eIF4E
Case 1
Case 2
Case 3
Control 1
Control 2
Control 3
focal, incomplete circumscribe weak diffuse, membranous 4 6 2 6 4
diffuse, incomplete circumscribe weak diffuse, membranous 4 6 4 8 4
focal, incomplete circumscribe weak diffuse, membranous 3 2 1 6 2
–
–
–
– 4 4 2 4 3
– 6 3 1 4 3
– 4 2 2 4 2
cy, cytoplasmic; eIF4E, eukaryotic translation initiation factor 4E; n, nuclear; p-4EBP1, phosphorylated form of 4E-binding protein-1; p-mTOR, phosphorylated form of mTOR; p-S6, phosphorylated form of S6.
count greater than 2/10 HPF; (iii) presence of atypical mitotic figures; and (iv) necrosis.5 Cases 1 and 3 met criteria (i), (ii), and (iv), whereas Case 2 only met (i). Therefore, Case 2 was clinically malignant EAML based on the presence of massive retroperitoneal invasion, whereas Cases 1 and 3 were possibly histologically malignant EAML based on the previously reported criterion. Recently, Wang et al. reported that epithelioid cells in EAML markedly express membranous E-cadherin.12 E-cadherin, a molecule in the cadherin superfamily, is expressed in cell membranes of various epithelial and mesenchymal neoplastic cells.13,14 β-catenin is an undercoat protein linked to membranous E-cadherin and acts as an intracellular signaling factor.15 In this study, we examined E-cadherin and β-catenin expression. Epithelioid cells were positive for membranous E-cadherin and β-catenin, consistent with their proposed function as cell adhesion molecules. E-cadherin is also a potent inhibitor of aggressive biological and clinical features such as invasion and metastasis.16 E-cadherin downregulation was also reported to correspond to poor prognosis in various types of neoplasms because of its involvement in epithelial-mesenchymal transition (EMT).16 E-cadherin membranous localization is regulated by an Akt/ mTORC1/CLIP-170-dependent pathway associated with microtubule transport.17 Therefore, mTORC1 activation due to loss of TSC2 can result in E-cadherin retention in the Golgi apparatus and significant reduction in membranous E-cadherin, leading to reduced cell adhesion and EMT induction.17 In our cases, the intensity of E-cadherin immunoreactivity in epithelioid cells was weak and focal. Further investigation is necessary to clarify whether these IHC findings are associated with EMT and subsequent malignant behavior. TSC is caused by dysfunction of the TSC1/TSC2 complex, which negatively regulates the mTOR pathway and promotes tumor progression and growth.2 Inappropriate mTOR pathway upregulation leads to tumorigenesis of TSCassociated AML, as well as sporadic AML and EAML.18 Recently, mTOR inhibitors have been proposed as treatment
for malignant AML based on reports demonstrating the efficacy of temsirolimus and everolimus in malignant AML.19,20 Our IHC findings show that the mTOR pathway is active in both clinically and histologically malignant EAML, suggesting that mTOR inhibitors may be a therapeutic option. IHC revealed that the epithelioid cells were diffusely positive for the above markers in the current EAML cases, whereas spindle cells in the control cases showed marked but scattered immunoreactivity. Further investigation is required to understand the significance of these findings in relation to mTOR inhibitor therapy.
REFERENCES 1 Pea M, Bonetti F, Martignoni G et al. Apparent renal cell carcinomas in tuberous sclerosis are heterogeneous: The identification of malignant epithelioid angiomyolipoma. Am J Surg Pathol 1998; 22: 180–87. 2 Inoki K, Corradetti MN, Guan KL. Dysregulation of the TSCmTOR pathway in human disease. Nat Genet 2005; 37: 19–24. 3 Cibas ES, Goss GA, Kulke MH, Demetri GD, Fletcher CD. Malignant epithelioid angiomyolipoma (‘sarcoma ex angiomyolipoma’) of the kidney: A case report and review of the literature. Am J Surg Pathol 2011; 25: 121–6. 4 L’Hostis H, Deminiere C, Ferriere JM, Coindre JM. Renal angiomyolipoma: A clinicopathologic, immunohistochemical, and follow-up study of 46 cases. Am J Surg Pathol 1999; 23: 1011–20. 5 Brimo F, Robinson B, Guo C, Zhou M, Latour M, Epstein JI. Renal epithelioid angiomyolipoma with atypia: A series of 40 cases with emphasis on clinicopathologic prognostic indicators of malignancy. Am J Surg Pathol 2010; 34: 715–22. 6 Roach ES, Sparagana SP. Diagnosis of ruberous sclerosis complex. J Child Neurol 2004; 19: 643–9. 7 Nur S, Chuang L, Ramaswamy G. Immunohistochemical characterization of cancer antigen in uterine cancers. Int J Gynecol Cancer 2006; 16: 1903–10. 8 Alì G, Boldrini L, Capodanno A et al. Expression of p-AKT and p-mTOR in a large series of bronchopulmonary neuroendocrine tumors. Exp Ther Med 2011; 2: 787–92. 9 Li N, Zhong M, Song M. Expression of phosphorylated mTOR and its regulatory protein is related to biological behaviors of ameloblastoma. Int J Clin Exp Pathol 2012; 5: 660–67.
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10 Aydin H, Magi-Galluzzi C, Lane BR et al. Renal Angiomyolipoma Clinicopathologic Study of 194 cases: With emphasis in the epithelioid histology and tuberous sclerosis association. Am J Surg Pathol 2009; 33: 289–97. 11 McCormack FX. Lyphangioleiomyomatosis: A clinical update. Chest 2008; 133: 507–16. 12 Wang Z, Gong Q, Fan Q. Expression of E-cadherin in angiomyolipoma. Hum Pathol 2012; 43: 2348–53. 13 Subramaniam MM, Navarro S, Llombart-Bosch A. Immunohistochemical study of correlation between histologic subtype and expression of epithelial-mesenchymal transition-related proteins in synovial sarcomas. Arch Pathol Lab Med 2011; 135: 1001–9. 14 Saito T, Oda Y, Itakura E et al. Expression of intercellular adhesion molecules in epithelioid sarcoma and malignant rhabdoid tumor. Pathol Int 2001; 51: 532–42. 15 Mak BC, Kenerson HL, Aicher LD, Barnes EA, Yeung RS. Aberrant beta-catenin signaling in tuberous sclerosis. Am J Pathol 2005; 167: 107–16.
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16 Berx G, van Roy F. Involvement of members of the cadherin superfamily in cancer. Cold Spring Harb Perspect Biol 2009; 1: a003129. 17 Barnes EA, Kenerson HL, Jiang X, Yeung RS. Tuberin regulates E-cadherin localization: Implications in epithelial-mesenchymal transition. Am J Pathol 2010; 177: 1765–78. 18 Kenerson H, Folpe AL, Takayama TK, Yeung RS. Activation of the mTOR pathway in sporadic angiomyolipomas and other perivascular epithelioid cell neoplasms. Hum Pathol 2007; 38: 1361–71. 19 Italiano A, Delcambre C, Hostein I et al. Treatment with the mTOR inhibitor temsirolimus in patients with malignant PEComa. Ann Oncol 2010; 21: 1135–7. 20 Shitara K, Yatabe Y, Mizota A, Sano T, Nimura Y, Muro K. Dramatic tumor response to everolimus for malignant epithelioid angiomyolipoma. Jpn J Clin Oncol 2011; 41: 814–16.
© 2014 The Authors Pathology International © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Pathology International 2014; 64: 133–141
doi:10.1111/pin.12142
Case Report
Renal epithelioid angiomyolipoma with malignant features: Histological evaluation and novel immunohistochemical findings
Sachiko Konosu-Fukaya,1 Yasuhiro Nakamura,1 Fumiyoshi Fujishima,1 Atsuko Kasajima,1 Keely M McNamara,1 Yayoi Takahashi,1 Kensuke Joh,3 Hideo Saito,2 Naomasa Ioritani,4 Yoshihiro Ikeda,4 Yoichi Arai,2 Mika Watanabe1 and Hironobu Sasano1 Departments of 1Pathology and 2Urology, Tohoku University Graduate School of Medicine, and Divisions of 3 Pathology and 4Urology, Sendai Shakai Hoken Hospital, Sendai, Japan
Renal epithelioid angiomyolipoma (EAML) is a potentially malignant tumor type whose characteristics and biomarkers predictive of malignant behavior have not been elucidated. Here, we report three cases of renal EAML with malignant features but without histories of tuberous sclerosis complex. Case 1 involved a 29-year-old man with a 12-cm solid mass in the right kidney who underwent radical right nephrectomy. Case 2 involved a 22-year-old woman with a retroperitoneal mass who underwent radical right nephrectomy and retroperitoneal tumorectomy. Local recurrence was detected 7 years post-surgery. Case 3 involved a 23-year-old man with a 14-cm solid mass in the left kidney who underwent radical left nephrectomy. Microscopically, the tumors in all cases demonstrated proliferation of epithelioid cells with atypia, mitotic activity, necrosis, hemorrhage, and vascular invasion. Epithelioid cells in all cases were immunohistochemically positive for melanocytic and myoid markers and weakly positive for E-cadherin and β-catenin. Immunohistochemistry revealed activation of the mammalian target of rapamycin pathway. Here, we report the morphological and immunohistochemical features of clinically or histologically malignant renal EAML.
TSC1/TSC2 complex function can cause over-activation of the mammalian target of rapamycin (mTOR) signaling pathway, reported to contribute to disease progression in EAML patients.2 It is considered a potentially malignant tumor type because approximately one-third of EAML patients are reported to develop metastasis, resulting in poor clinical outcomes.3 Currently, no markers exist for clinical differentiation of malignant and non-malignant AML at initial presentation; diagnosis is made after metastasis or local recurrence.4 Previous studies have proposed several clinicopathological parameters to predict the malignant potential of EAML, but definitive predictive biomarkers have not been determined.5 Therefore, we report three cases of sporadic renal EAML with a malignant presentation, analyzed by immunohistochemistry (IHC) for markers associated with tumorigenesis and disease progression in AML, including E-cadherin, β-catenin, and molecules involved in the mTOR pathway, to explore potential biomarkers for identification of malignant renal EAML.
Key words: β-catenin, E-cadherin, epithelioid angiomyolipoma, malignant angiomyolipoma, mTOR
CLINICAL SUMMARY
Epithelioid angiomyolipoma (EAML) was first reported by Pea et al. in 1991 as a rare angiomyolipoma (AML) variant associated with tuberous sclerosis complex (TSC).1 Loss of Correspondence: Yasuhiro Nakamura, MD, PhD, Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan. Email: yasu-naka @patholo2.med.tohoku.ac.jp Disclosure: None declared. Received 15 April 2013. Accepted for publication 27 January 2014. © 2014 The Authors Pathology International © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Case 1 A 29-year-old man with an unremarkable medical history visited the hospital with an abdominal mass. A computed tomography (CT) scan revealed bilateral masses: a 12-cm solid lesion in the upper pole of the right kidney and an approximately 1-cm mass in the ventral cortex of the left kidney (Fig. 1a). No metastatic lesions were detected. Subsequent imaging studies suggested renal cell carcinoma (RCC) in the right kidney and AML in the left kidney, and radical right nephrectomy was performed. No signs of TSC were identified. Macroscopically, the lesion appeared white on the cut surface, measured 12 cm at the widest part, and
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Figure 1 (a) Contrast-enhanced computed tomography scan of Case 1 showing a 12-cm mass in the right kidney. Microscopic findings from Case 1 revealed (b) a sheet of epithelioid cells and (c) extensive necrosis and hemorrhage. (d) Immunohistochemical examination of Case 1 showed CD34-positive vascular endothelium with neoplastic cells and vascular invasion (arrow). The epithelial cells were positive for (e) HMB-45, (f) α-smooth muscle actin, and had (g) focal membranous E-cadherin expression and (h) diffuse membranous β-catenin expression.
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had scattered intratumoral hemorrhage. It extended into extrarenal tissues and to the pelvic wall. Neither recurrence nor metastasis was clinically identified at the last follow-up visit, 10 months post-surgery.
Case 2 A 22-year-old woman was admitted to the hospital with a growing retroperitoneal mass in the upper part of her right kidney (Fig. 2a). Her medical and family histories were unremarkable. Signs of TSC were not clinically detected. Retroperitoneal tumor resection was subsequently performed. The tumor measured 21 cm at its largest diameter, and protruded from the organ exterior. Seven years after surgery, follow-up CT revealed a mass mainly located on the para-aortic retroperitoneum, focally expanding into the posterior mediastinum. Radiographic examination revealed partial fat density in the tumor, suggestive of AML. Additionally, multicystic lesions were detected bilaterally in the lungs. A 2-cm tumor was also identified in the liver. The cystic pulmonary lesions were clinically and radiologically diagnosed as lymphangiomyomatosis (LAM). Radical nephrectomy of the right kidney and retroperitoneal tumor resection were performed. However, an underlying respiratory disorder and broad expansion of the tumor made the retroperitoneal tumor clinically unresectable. Ten years after the initial surgery, the patient is alive with the disease.
Case 3 A 23-year-old man visited the hospital for evaluation of macrohematuria and flank pain. His medical and family histories were unremarkable. CT revealed a 14-cm solid mass in
the lower pole of the left kidney and hydronephrosis (Fig. 3a). No metastatic lesions were detected. Clinically, RCC was suggested, and radical left nephrectomy was subsequently performed. Macroscopically, the lesion appeared gray to white on the cut surface and was 14 cm along its largest diameter, with no evidence of extrarenal extension. No significant signs of TSC were clinically detected. Details on Cases 1 and 3 were retrieved from the Department of Pathology, Tohoku University. Details on Case 2 were retrieved from the Division of Pathology, Sendai Shakai Hoken Hospital. Hematoxylin and eosin (H&E) staining and IHC were performed on 10% formalin-fixed and paraffinembedded tissue specimens. Table 1 summarizes relevant IHC marker information. TSC was diagnosed based on criteria adapted from Roach et al.6 Patients received no form of therapy before or after surgery. As a control, we obtained information from the Department of Pathology, Tohoku University files on three classic AML cases between 2005 and 2012. All control cases were right kidney tumors; control case 1 involved a 75-year-old man (5-cm tumor); control case 2, a 77-year-old man (1.2-cm tumor); and control case 3, a 62-year-old man (4-cm tumor). All three cases were histologically diagnosed as classic AML comprising thick-walled blood vessels, mature adipose cells, and smooth muscle-like spindle cells without foci of atypical epithelial cells. Additionally, on IHC, tumor cells in all cases were positive for melanocytic and myoid markers. To assess the mTOR pathway activation status in these three cases, we examined various phosphorylated or activated forms of factors involved in this pathway by IHC using an immunoreaction score (IRS), obtained by multiplying the proportion of positive cells and staining intensity.7–9 Values for the proportion of positive cells were assigned as follows: negative, 0; 1–10%, 1; 11–50%, 2; 51–80%, 3; and
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Figure 2 (a) Contrast-enhanced computed tomography of Case 2 showing a 21-cm mass in the right kidney. (b) Histological examination of case 2 revealed a sheet of epithelioid cells. Immunohistochemical examination of Case 2 showed that the epithelial cells were positive for (c) HMB-45, (d) α-smooth muscle actin, (e) E-cadherin (diffuse membranous expression), and (f) β-catenin (diffuse membranous expression). The epithelioid cells of Case 2 were also positive for (g) the phosphorylated form of mammalian target of rapamycin (p-mTOR), (h) cytoplasmic phosphorylated form of S6, and (i) 4E-binding protein-1. (j) The spindle cells of control 1 were positive for p-mTOR.
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81–100%, 4. Staining intensity was qualitatively evaluated as 0, 1+, 2+, or 3+. The total value range was 0–12.
PATHOLOGICAL FINDINGS Microscopically, all tumors were composed of an atypical polygonal cell sheet mixed with classic AML components such as blood vessels with markedly thick vascular walls, adipocytes, and smooth muscle-like cells. Case 1 and 3 tumors predominantly comprised atypical polygonal cells; however, classic AML components were predominant in the Case 2 tumor. All tumors had areas of medium- to large-sized polygonal cells with eosinophilic or clear cytoplasm, prominent nuclear atypia, and conspicuous nucleoli and bizarre or polynuclear cells (Figs 1b, 2b, and 3b). Mitotic figures were encountered at frequencies of 3/10 high-power fields (HPF), 1/10 HPF, and 4/10 HPF in Cases 1, 2, and 3, respectively. Extensive intratumoral coagulative necrosis, marked hemorrhage (Figs 1c,3c), and
lymphovascular invasion were detected in Cases 1 and 3 (Figs 1d,3d). Extrarenal tumor invasion was detected in Case 1. No lymphovascular invasion, necrosis, or extrarenal invasion was detected in Case 2. On IHC, epithelioid cells were focally positive for vimentin, melanocytic markers (HMB-45 and Melan A; Figs 1e, 2c and 3e), myoid markers (α-smooth muscle actin and desmin; Figs 1f, 2d and 3f), and CD68; negative for cytokeratin, epithelial membrane antigen, CD10, and S-100; and weakly and focally immunopositive for transcription factor E3, in all cases. The Ki67 labeling index (LI) in epithelioid cells was approximately 11%, 13%, and 20% in focal ‘hot spots’ for Cases 1, 2, and 3, respectively, but less than 1% in the spindle cells of classic components in each case. Accordingly, these tumors were diagnosed as renal EAML.10 In addition to the classic EAML markers described above, membranous immunoreactivity for both E-cadherin and β-catenin was detected in these three cases. In Cases 1 and 3, approximately one-third of epithelioid cells were weakly positive for E-cadherin, with an incomplete,
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a
b
c
d
e
f
Figure 3 (a) Contrast-enhanced computed tomography of Case 3 showing a 14-cm mass in the left kidney. Histological examination of Case 3 revealed (b) a sheet of epithelioid cells and (c) extensive necrosis and hemorrhage. (d) Immunohistochemical examination of case 3 showed CD31-positive vascular endothelium with neoplastic cells and vascular invasion (arrow). The epithelial cells were positive for (e) HMB-45, (f) α-smooth muscle actin, (g) E-cadherin (focal membranous expression), and (h) β-catenin (diffuse membranous expression). © 2014 The Authors Pathology International © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd
Malignant epithelioid angiomyolipoma
g
Figure 3
h
Continued
Table 1 Antibodies and their dilutions used for immunohistochemical studies Antibody
Clone
HMB45
HMB45
Melan A α-smooth muscle actin desmin CD68 vimentin AE1/AE3 E-cadherin
A103 1A4
β-catenin
Ki67 p53 EMA CD10 S-100 P-mTOR
P-S6 eIF4E P-4EBP1
139
D33 KP-1 V9 AEandAE3 4A2C7
Source
Dilution
Immunotech (Marseille, 1:100 France) Biogenesis (Poole, UK) 1:50 DAKO (Glostrup, (1:40) × 20 Denmark)
DAKO DAKO DAKO DAKO Life Technologies Corporation (Carlsbad, CA, USA) β-catenin Becton, Dickinson and Company (Franklin Lakes, NJ, USA) MIB-1 DAKO D0-7 Novocastra (Welzlar, Germany) E29 Nichirei (Tokyo, Japan) 56C6 Novocastra 15EZE12 BioGenex (Fremont, CA, USA) P-mTOR Cell Signaling Technology (Beverly, MA, USA) P-S6 ribosomal Cell Signaling Technology eIF4E Cell Signaling Technology P-4EBP1 Cell Signaling Technology
1:800 1:100 1:500 1:300 1:400
1:200
1:300 1:100 1:2 1:40 1:10
β-catenin staining was detected in all cases (Figs 1h, 2f, and 3h). However, E-cadherin and β-catenin were only focally detected in spindle cell cytoplasm in areas of classic components. Nuclear immunoreactivity was not detected for either component in these three cases. IHC findings for each case are summarized in Table 2. IHC scoring for phosphorylated phosphatidylinositol-3 kinase (p-PI3K), AKT (p-AKT), mTOR (p-mTOR), S6 (p-S6), and 4E-binding protein-1 (p-4EBP1), as well as for eukaryotic translation initiation factor 4E (eIF4E), is summarized in Table 2. In all three cases, p-mTOR and its downstream effectors, p-S6, p-4EBP-1, and eIF4E, were detected by IHC. IRS values were 3–4 for p-mTOR, 2–6 for p-S6, 1–4 for nuclear p-4EBP-1, 6–8 for cytoplasmic p-4EBP-1, and 2–4 for eIF4E. These markers were also detected in the three classic AMLs, with IRS values of 4–6, 2–4, 1–2, 4, and 2–3 for p-mTOR, p-S6, nuclear p-4EBP-1, cytoplasmic p-4EBP-1, and eIF4E, respectively, with no significant differences in IHC scores between our present cases and the control cases (spindle cells in AML).
1:50
1:100 1:50 1:50
circumscribed staining line (Figs 1g,3g). In Case 2, epithelioid cells demonstrated diffuse immunoreactivity for E-cadherin, but circumscribed membrane immunoreactivity was weak and incomplete (Fig. 2e). Diffuse membranous
DISCUSSION Aydin et al. defined EAML as an AML variant where epithelioid cells constitute more than 10% of tumor cells.10 Renal AML is the main manifestation of TSC.10 LAM is a rare neoplasm affecting the lungs, and is also considered to be closely related to TSC.11 However, TSC was not identified in any of the current cases. Brimo et al. suggested that the presence of at least three of four specific histological findings could predict malignant EAML progression, defined by an epithelioid cell composition greater than 5%. These histological findings are: (i) cellular atypia in ≥70% of the epithelioid cell population; (ii) mitotic
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Table 2 The immunohistochemical findings of E-cadherin and β-catenin in the present cases and the immunohistochemical scoring of the factors involving mammalian target of rapamycin (mTOR) pathway in the present cases and the controls
E-cadherin
β-catenin P-mTOR P-S6 P-4EBP1(n) P-4EBP1 (cy) eIF4E
Case 1
Case 2
Case 3
Control 1
Control 2
Control 3
focal, incomplete circumscribe weak diffuse, membranous 4 6 2 6 4
diffuse, incomplete circumscribe weak diffuse, membranous 4 6 4 8 4
focal, incomplete circumscribe weak diffuse, membranous 3 2 1 6 2
–
–
–
– 4 4 2 4 3
– 6 3 1 4 3
– 4 2 2 4 2
cy, cytoplasmic; eIF4E, eukaryotic translation initiation factor 4E; n, nuclear; p-4EBP1, phosphorylated form of 4E-binding protein-1; p-mTOR, phosphorylated form of mTOR; p-S6, phosphorylated form of S6.
count greater than 2/10 HPF; (iii) presence of atypical mitotic figures; and (iv) necrosis.5 Cases 1 and 3 met criteria (i), (ii), and (iv), whereas Case 2 only met (i). Therefore, Case 2 was clinically malignant EAML based on the presence of massive retroperitoneal invasion, whereas Cases 1 and 3 were possibly histologically malignant EAML based on the previously reported criterion. Recently, Wang et al. reported that epithelioid cells in EAML markedly express membranous E-cadherin.12 E-cadherin, a molecule in the cadherin superfamily, is expressed in cell membranes of various epithelial and mesenchymal neoplastic cells.13,14 β-catenin is an undercoat protein linked to membranous E-cadherin and acts as an intracellular signaling factor.15 In this study, we examined E-cadherin and β-catenin expression. Epithelioid cells were positive for membranous E-cadherin and β-catenin, consistent with their proposed function as cell adhesion molecules. E-cadherin is also a potent inhibitor of aggressive biological and clinical features such as invasion and metastasis.16 E-cadherin downregulation was also reported to correspond to poor prognosis in various types of neoplasms because of its involvement in epithelial-mesenchymal transition (EMT).16 E-cadherin membranous localization is regulated by an Akt/ mTORC1/CLIP-170-dependent pathway associated with microtubule transport.17 Therefore, mTORC1 activation due to loss of TSC2 can result in E-cadherin retention in the Golgi apparatus and significant reduction in membranous E-cadherin, leading to reduced cell adhesion and EMT induction.17 In our cases, the intensity of E-cadherin immunoreactivity in epithelioid cells was weak and focal. Further investigation is necessary to clarify whether these IHC findings are associated with EMT and subsequent malignant behavior. TSC is caused by dysfunction of the TSC1/TSC2 complex, which negatively regulates the mTOR pathway and promotes tumor progression and growth.2 Inappropriate mTOR pathway upregulation leads to tumorigenesis of TSCassociated AML, as well as sporadic AML and EAML.18 Recently, mTOR inhibitors have been proposed as treatment
for malignant AML based on reports demonstrating the efficacy of temsirolimus and everolimus in malignant AML.19,20 Our IHC findings show that the mTOR pathway is active in both clinically and histologically malignant EAML, suggesting that mTOR inhibitors may be a therapeutic option. IHC revealed that the epithelioid cells were diffusely positive for the above markers in the current EAML cases, whereas spindle cells in the control cases showed marked but scattered immunoreactivity. Further investigation is required to understand the significance of these findings in relation to mTOR inhibitor therapy.
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