Virchows Arch (2015) 467:295–301 DOI 10.1007/s00428-015-1804-x
ORIGINAL ARTICLE
Dysregulation of the Rb pathway in recurrent pleomorphic adenoma of the salivary glands Ana Amélia de Souza 1 & Albina Altemani 2 & Fabricio Passador-Santos 1 & Cecilia Pedroso Turssi 3 & Ney Soares de Araujo 1 & Vera Cavalcanti de Araújo 1 & Andresa Borges Soares 1
Received: 12 March 2015 / Revised: 5 May 2015 / Accepted: 24 June 2015 / Published online: 9 July 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Pleomorphic adenoma (PA) is the most common salivary gland neoplasm, and while mostly benign, recurrences (RPA) and malignant transformation to carcinoma exPA (CXPA) do occur. Cell cycle proteins important in its tumorigenesis have been studied as markers for PA with a high risk of RPA or CXPA. The aim of the present study was to investigate cell cycle markers p-16, cyclin D1, CDK4, E2F, and retinoblastoma (Rb) in this context. Expression of p16, cyclin D1, E2F, CDK4, and Rb was studied by immunohistochemistry in 24 cases of PA, 21 of RPA, and 2 of CXPA. The presence of HPV was assessed by in situ hybridization. Immunostaining for p16 and cyclin D1 was negative or weakly positive in most cases of PA while strongly positive in the majority of RPA and both CXPA cases. Staining for Rb and CDK4 was either negative or weakly positive in PA, RPA, and CXPA. Expression of E2F was stronger in RPA and CXPA than in PA. Nuclear reactivity for HPV was not observed in any case. In conclusion, the strong staining for p16, cyclinD1, and E2F in RPA and CXPA, while weak or negative in PA, suggests that these proteins might be involved in recurrence and malignant transformation of PA. Keywords Pleomorphic adenoma . Recurrence . Cell cycle * Andresa Borges Soares [email protected] 1
Department of Oral Pathology, São Leopoldo Mandic Institute and Research Center, Rua José Rocha Junqueira13 Ponte Preta, Campinas, SP 13045-755, Brazil
2
Department of Pathology, School of Medicine, State University of Campinas (UNICAMP), Campinas, SP, Brazil
3
Department of Statistics, São Leopoldo Mandic Institute and Research Center, Campinas, SP, Brazil
Introduction Pleomorphic adenoma (PA) is the most common salivary gland tumor [1–4]. Despite its benign character, PA can recur following surgical excision [5, 6]. Recurrent PA (RPA) has been associated with an increased risk of malignant transformation to carcinoma ex-PA (CXPA) [7–9]. The retinoblastoma (Rb) protein, transcription factor 2 (E2F), p16, cyclin D1, and cyclin-dependent kinases (CDKs) are all components of the Rb cell cycle control pathway. The active hyperphosphorylated form of the Rb protein (pRb) binds to E2F, inhibits its action, and prevents the transition from G1 to S phase [10–12]. p16 binds to CDK4 and 6, which blocks their binding to D-type cyclins. Dysregulation of the Rb pathway leads to unrestricted proliferation and plays a role in carcinogenesis in a variety of human tumors, including those of the salivary glands [10–13]. The aim of the present study was to evaluate expression of Rb, p16, cyclin D1, CDK4, and E2F1 in PA without recurrence (PA) in comparison with their expression in RPA and RPA with malignant transformation (CXPA). In addition, correlations between expression patterns and clinical characteristics as well as between the different proteins were studied.
Materials and methods Twenty-four cases of PA, 20 cases of RPA, and 2 cases of CXPA were obtained from the Department of Pathology, University of Campinas, Brazil. Five-micrometer sections were cut from formalin-fixed paraffin-embedded tissue blocks and subsequently stained with hematoxylin-eosin to confirm the diagnosis. PA cases were chosen according to site (parotid or submandibular) and histological features, including the absence of cytological atypia, low mitotic index, and absence
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Virchows Arch (2015) 467:295–301
of extracapsular extension. Patients with RPA had previously undergone surgery, resulting in a histopathological diagnosis of PA. A diagnosis of CXPA was based upon histopathological characteristics, notably intracapsular CXPA with extraductular expansion [14] and immunohistochemical expression of epithelial or luminal rather than myoepithelial cell markers [15]. Demographical and clinical information was obtained from the medical records. Patient characteristics The PA group was composed of 18 patients with a parotid (78 %) and 5 with a submandibular gland (22 %) tumor (mean age 44 years), of which 14 were women (61 %) and 9 men (39 %).The RPA group consisted of 19 parotid (95 %) and 1 submandibular gland (5 %) tumor from 9 women (45 %) and 11 men (55 %), with a median age of 43 years at the time of initial surgery. The CXPA group included one woman and one man, mean age of 42 years, both of whom had tumors located in the parotid gland. The median time interval between initial operation and recurrence (RPA) was 10 years (range 1 to 25 years), with five (25 %) cases with a first recurrence within 5 years, six (30 %) after 5 years, and four (20 %) after more than 10 years. Information was not available for five (25 %) patients. Immunohistochemistry The tumors had been routinely fixed in 10 % buffered formalin, processed, and embedded in paraffin. From each b l o ck , 3 -μ m se ct i o ns w e r e cu t an d m o u nt ed o n a m i n o p r o p y l t r i et h o x y s i l an e - c o a t e d sl i d e s . A f t e r deparaffinization in xylene and rehydration through decreasing concentrations of ethanol, endogenous peroxidases were inhibited in 3 % H2O2 in methanol at room temperature. Immunohistochemistry was subsequently performed using antibodies and detection methods as specified in Table 1. Immunoreactivity was visualized with 3.3′-diaminobenzidine tetrahydrochloride (DAB, 5 min at 37 °C) and the slides were Table 1 Specificity p-16 cyclin D1 Rb CDK4 E2F1
Details of the antibodies used for immunohistochemistry Clone
EP12 73598 EPR4513 135251
Dilution
Source
Buffer (AR)
1:50 1:500 1:150 1:400
Cintec Kita Dakob Santa Cruzc Abcamd Abcamb
EDTA Citrate EDTA Citrate
a
Cintec Histology Kit, Rocha, Germany
b
Dako Corporation, Glostrup, Denmark
c
Santa Cruz Biotechnology, USA
d
Abcam, Canada
counterstained with Mayer hematoxylin. Negative controls consisted of omission of primary antibody. Evaluation of staining For p16, cyclin D1, Rb, CDK4, and E2F1, only nuclear immunoreactivity was observed. Staining was evaluated qualitatively and semiquantitatively. Qualitative analysis was performed via visual assessment of immunoreactivity in all areas of the section. Semiquantitative assessment of protein expression was based upon the relative number of immunoreactive neoplastic cells as a percentage of all neoplastic cells in a 0–3 scoring system: 0 corresponding to
ORIGINAL ARTICLE
Dysregulation of the Rb pathway in recurrent pleomorphic adenoma of the salivary glands Ana Amélia de Souza 1 & Albina Altemani 2 & Fabricio Passador-Santos 1 & Cecilia Pedroso Turssi 3 & Ney Soares de Araujo 1 & Vera Cavalcanti de Araújo 1 & Andresa Borges Soares 1
Received: 12 March 2015 / Revised: 5 May 2015 / Accepted: 24 June 2015 / Published online: 9 July 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Pleomorphic adenoma (PA) is the most common salivary gland neoplasm, and while mostly benign, recurrences (RPA) and malignant transformation to carcinoma exPA (CXPA) do occur. Cell cycle proteins important in its tumorigenesis have been studied as markers for PA with a high risk of RPA or CXPA. The aim of the present study was to investigate cell cycle markers p-16, cyclin D1, CDK4, E2F, and retinoblastoma (Rb) in this context. Expression of p16, cyclin D1, E2F, CDK4, and Rb was studied by immunohistochemistry in 24 cases of PA, 21 of RPA, and 2 of CXPA. The presence of HPV was assessed by in situ hybridization. Immunostaining for p16 and cyclin D1 was negative or weakly positive in most cases of PA while strongly positive in the majority of RPA and both CXPA cases. Staining for Rb and CDK4 was either negative or weakly positive in PA, RPA, and CXPA. Expression of E2F was stronger in RPA and CXPA than in PA. Nuclear reactivity for HPV was not observed in any case. In conclusion, the strong staining for p16, cyclinD1, and E2F in RPA and CXPA, while weak or negative in PA, suggests that these proteins might be involved in recurrence and malignant transformation of PA. Keywords Pleomorphic adenoma . Recurrence . Cell cycle * Andresa Borges Soares [email protected] 1
Department of Oral Pathology, São Leopoldo Mandic Institute and Research Center, Rua José Rocha Junqueira13 Ponte Preta, Campinas, SP 13045-755, Brazil
2
Department of Pathology, School of Medicine, State University of Campinas (UNICAMP), Campinas, SP, Brazil
3
Department of Statistics, São Leopoldo Mandic Institute and Research Center, Campinas, SP, Brazil
Introduction Pleomorphic adenoma (PA) is the most common salivary gland tumor [1–4]. Despite its benign character, PA can recur following surgical excision [5, 6]. Recurrent PA (RPA) has been associated with an increased risk of malignant transformation to carcinoma ex-PA (CXPA) [7–9]. The retinoblastoma (Rb) protein, transcription factor 2 (E2F), p16, cyclin D1, and cyclin-dependent kinases (CDKs) are all components of the Rb cell cycle control pathway. The active hyperphosphorylated form of the Rb protein (pRb) binds to E2F, inhibits its action, and prevents the transition from G1 to S phase [10–12]. p16 binds to CDK4 and 6, which blocks their binding to D-type cyclins. Dysregulation of the Rb pathway leads to unrestricted proliferation and plays a role in carcinogenesis in a variety of human tumors, including those of the salivary glands [10–13]. The aim of the present study was to evaluate expression of Rb, p16, cyclin D1, CDK4, and E2F1 in PA without recurrence (PA) in comparison with their expression in RPA and RPA with malignant transformation (CXPA). In addition, correlations between expression patterns and clinical characteristics as well as between the different proteins were studied.
Materials and methods Twenty-four cases of PA, 20 cases of RPA, and 2 cases of CXPA were obtained from the Department of Pathology, University of Campinas, Brazil. Five-micrometer sections were cut from formalin-fixed paraffin-embedded tissue blocks and subsequently stained with hematoxylin-eosin to confirm the diagnosis. PA cases were chosen according to site (parotid or submandibular) and histological features, including the absence of cytological atypia, low mitotic index, and absence
296
Virchows Arch (2015) 467:295–301
of extracapsular extension. Patients with RPA had previously undergone surgery, resulting in a histopathological diagnosis of PA. A diagnosis of CXPA was based upon histopathological characteristics, notably intracapsular CXPA with extraductular expansion [14] and immunohistochemical expression of epithelial or luminal rather than myoepithelial cell markers [15]. Demographical and clinical information was obtained from the medical records. Patient characteristics The PA group was composed of 18 patients with a parotid (78 %) and 5 with a submandibular gland (22 %) tumor (mean age 44 years), of which 14 were women (61 %) and 9 men (39 %).The RPA group consisted of 19 parotid (95 %) and 1 submandibular gland (5 %) tumor from 9 women (45 %) and 11 men (55 %), with a median age of 43 years at the time of initial surgery. The CXPA group included one woman and one man, mean age of 42 years, both of whom had tumors located in the parotid gland. The median time interval between initial operation and recurrence (RPA) was 10 years (range 1 to 25 years), with five (25 %) cases with a first recurrence within 5 years, six (30 %) after 5 years, and four (20 %) after more than 10 years. Information was not available for five (25 %) patients. Immunohistochemistry The tumors had been routinely fixed in 10 % buffered formalin, processed, and embedded in paraffin. From each b l o ck , 3 -μ m se ct i o ns w e r e cu t an d m o u nt ed o n a m i n o p r o p y l t r i et h o x y s i l an e - c o a t e d sl i d e s . A f t e r deparaffinization in xylene and rehydration through decreasing concentrations of ethanol, endogenous peroxidases were inhibited in 3 % H2O2 in methanol at room temperature. Immunohistochemistry was subsequently performed using antibodies and detection methods as specified in Table 1. Immunoreactivity was visualized with 3.3′-diaminobenzidine tetrahydrochloride (DAB, 5 min at 37 °C) and the slides were Table 1 Specificity p-16 cyclin D1 Rb CDK4 E2F1
Details of the antibodies used for immunohistochemistry Clone
EP12 73598 EPR4513 135251
Dilution
Source
Buffer (AR)
1:50 1:500 1:150 1:400
Cintec Kita Dakob Santa Cruzc Abcamd Abcamb
EDTA Citrate EDTA Citrate
a
Cintec Histology Kit, Rocha, Germany
b
Dako Corporation, Glostrup, Denmark
c
Santa Cruz Biotechnology, USA
d
Abcam, Canada
counterstained with Mayer hematoxylin. Negative controls consisted of omission of primary antibody. Evaluation of staining For p16, cyclin D1, Rb, CDK4, and E2F1, only nuclear immunoreactivity was observed. Staining was evaluated qualitatively and semiquantitatively. Qualitative analysis was performed via visual assessment of immunoreactivity in all areas of the section. Semiquantitative assessment of protein expression was based upon the relative number of immunoreactive neoplastic cells as a percentage of all neoplastic cells in a 0–3 scoring system: 0 corresponding to
