Neurosurg Rev (2015) 38:89–99 DOI 10.1007/s10143-014-0579-x
ORIGINAL ARTICLE
Immunohistochemical expression of receptor tyrosine kinase PDGFR-α, c-Met, and EGFR in skull base chordoma R. Akhavan-Sigari & M. Abili & M. R. Gaab & V. Rohde & N. Zafar & P. Emami & H. Ostertag
Received: 23 October 2013 / Revised: 15 April 2014 / Accepted: 22 June 2014 / Published online: 17 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Chordomas are rare, locally aggressive malignancies that often exhibit an insidious natural history and are difficult to eradicate. Surgery and radiotherapy are the treatment mainstays of chordoma, but the chance of local recurrence remains high. Reports of receptor tyrosine kinase (RTK) expression in chordoma suggest that these tumors may respond to kinase inhibitor therapy. Currently, there are no effective R. Akhavan-Sigari (*) : V. Rohde : N. Zafar Department of Neurosurgery, University Medical Center Göttingen, Georg-August-University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany e-mail: [email protected] V. Rohde e-mail: [email protected] N. Zafar e-mail: [email protected] M. Abili Department of Neurosurgery, Imam Reza University, Razavi Hospital, Mashhad, Iran e-mail: [email protected] M. Abili Mashhad University of Medical Sciences, Taleghani Hospital, Shahid Kalantari Avenue, after Ghaem Bridge, Mashhad, Iran M. R. Gaab Department of Neurosurgery, Nordstadt Medical Center, Klinikum Hannover, Haltenhoffstr, 41, 30167 Hannover, Germany e-mail: [email protected] P. Emami Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Martinistr, 52, 20246 Hamburg, Germany e-mail: [email protected] H. Ostertag Department of Pathology, Nordstadt Medical Center, Klinikum Hannover, Haltenhoffstr, 41, 30167 Hannover, Germany e-mail: [email protected]
chemotherapeutic protocols for chordoma. A tissue microarray containing 74 tumor specimens from primary chordoma patients and 71 from their recurrent tumors for a total of 145 tumor specimens was immunohistochemically analyzed for expression of a number of proteins involved in signal transduction from RTKs. Platelet-derived growth factor receptor-α (PDGFR-α), epidermal growth factor receptor (EGFR), c-Met, and CD-34 were detected in 100, 92, 100, and 59 % of cases, respectively. PDGFR-α and c-Met staining was of moderate to strong intensity in all cases. In contrast, total EGFR staining was variable; weak staining was detected in 10 cases. Our results contribute to the understanding of the expression of RTKs in skull base chordomas and support the development of targeted therapies that inhibit RTKs, which may have a synergistic effect for chemotherapy in patients. There were statistically significant correlations between the expression of PDGFR-α, c-Met, and EGFR and disease-free survival. The results nonetheless suggest that chordomas may respond to RTK inhibitors or modulators of other downstream signaling. Keywords Chordoma . Tyrosine kinase receptors . EGFR . c-Met . PDGFR-α
Introduction Chordomas are rare tumors with an incidence of about 0.05/ 100,000/year. They arise from embryonic notochordal remnants usually along the axial skeleton, primarily in the sacrococcygeal and petroclival regions, and account for less than 5 % of all primary malignant bone tumors [49]. Mainly adults between 40 and 60 years are affected, but cases of children presenting with chordoma were also rarely reported (5 % of cases) [23]. The median survival time for all patients is 6.29 years, with a 5-year survival rate of 67.6 % [4]. Unfortunately, standard chemotherapy was shown to be
90
basically unsuccessful, which causes serious problems for managing patients with locally recurrent or metastatic disease [11, 15]. Immunohistochemical (IHC) studies have shown that receptor tyrosine kinases (RTKs) such as platelet-derived growth factor receptor-α (PDGFR-α), platelet-derived growth factor receptor-β (PDGFRB), tyrosine protein kinase KIT, epithelial growth factor receptor (EGFR), mesenchymal-epithelial transition factor (MET), and human epithelial growth factor receptor 2 (HER2) are expressed in chordoma by immunohistochemistry [17, 28, 44, 50]. Given that RTKs could prove to be essential for the survival or proliferation of chordoma tumor cells, targeting these RTKs using antibodies or small molecule tyrosine kinase inhibitors (TKI) might offer new treatment options. Interestingly, imatinib was found to have antitumor activity in patients with chordoma [10]. In addition, expression of the MET oncogene has been reported in chordoma [28]. Of note, the MET oncogene is known to be expressed in various chondroid neoplasms, normal articular cartilage, and fetal notochord [9, 34]. Given their possible relationship to notochordal development and chondroid differentiation, further investigation is warranted to clarify the roles of these and other RTKs in chordomagenesis. Therapies aimed at several of these molecules have demonstrated initial success in treating both in vitro models of chordoma and patients with chordoma, supporting the feasibility of this approach [7, 19]. We examined whether selected RTKs are demonstrable in a large series of chordoma tissue, and their clinicopathologic significance with prognosis was clarified, suggesting that RTK inhibitors may be useful for the systemic treatment of chordoma.
Methods Chordomas A series of 74 chordoma patients for a total of 145 tumor specimens (74 primary tumor specimens and 71 recurrent tumors) were treated at the Neurosurgical Departments of the Nordstadt Hospital Hannover and of the Hannover Medical School in a period of 20 years in Germany. Out of 71 recurrent lesions, 42 were from the first, 15 from the second, 8 from the third, 4 from the fourth, 1 from the fifth, and 1 from the sixth recurrences. Tumor recurrence was defined as a return of symptoms and signs, with verification of tumor regrowth radiologically (Table 1). All patients were included; we had no exclusion criteria other than lack of available material for immunohistochemical analysis.
Neurosurg Rev (2015) 38:89–99 Table 1 A total of 145 tumor specimens were analyzed, of which 74 were from primary chordomas and 71 from recurring lesions (i.e., 42 from first recurrences, 15 from second, 8 from third, and 4 from fourth recurrences. One patient had a fifth and one patient a sixth recurrence) Primary tumor Recurrences 1 N=42
N=74 2 N=15
3 N=8
4 N=4
5 N=1
Total
6 N=1
N=71 N=145
Cohort demographics The patients (32 women and 42 men) ranged in age from 16 to 88 years (median age, 58 years). Seventy-one patients experienced recurrence and subsequent resection. Locations of the chordomas were skull base. The cases selected included patients with no history of chemotherapy or radiotherapy prior to surgery and a follow-up time of ≥12 months. The patient records were reviewed to collect the pathological data (tumor location and status of neurological deficits), demographic data (patient age and gender), and recurrence (Table 2). Multitumor tissue microarray construction A multitumor tissue microarray was assembled and used for comparison of the expression of molecular markers of chordoma. Following institutional review board approval, we constructed the tissue microarray as previously described [37]. The tissue microarrays (TMA) were constructed using a tissue arraying instrument (Beecher Instruments, Hackensack, NJ). Three tissue core cylinders with a diameter of 0.6 mm were punched from each donor paraffin block in targeted areas corresponding to previously demarcated neoplastic areas on the parallel slide. These tissue cores were then deposited into a recipient “master” paraffin block. The punches were placed 1 mm apart on the x-axis and 1.5 mm apart on the y-axis. Two microarray blocks, respectively, contained 75 and 70 punches. Sections 5 μm thick were cut from the master block, stained with H&E, and reviewed to ensure the presence of morphologically pure cores of chordoma for each case. Morphologic features of each core were confirmed by reviewing the corresponding whole tissue sections stained with H&E. We obtained tissue cores from paraffin-embedded formalin-fixed tissue blocks from the archives at the Department of Pathology, Nordstadt Medical Center, Klinikum Hannover. A pathologist (H.O.) reviewed the slides from all blocks to select representative areas of invasive tumor or normal tissue to be scored. Immunohistochemistry All slides were processed simultaneously under identical condition using standard methods. Immunohistochemistry was performed on the 145 tumor specimens for the following
Neurosurg Rev (2015) 38:89–99
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Table 2 Correlation between RTK scores and clinicopathological factors in skull base chordomas (n=74); P values (italics for P
ORIGINAL ARTICLE
Immunohistochemical expression of receptor tyrosine kinase PDGFR-α, c-Met, and EGFR in skull base chordoma R. Akhavan-Sigari & M. Abili & M. R. Gaab & V. Rohde & N. Zafar & P. Emami & H. Ostertag
Received: 23 October 2013 / Revised: 15 April 2014 / Accepted: 22 June 2014 / Published online: 17 October 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Chordomas are rare, locally aggressive malignancies that often exhibit an insidious natural history and are difficult to eradicate. Surgery and radiotherapy are the treatment mainstays of chordoma, but the chance of local recurrence remains high. Reports of receptor tyrosine kinase (RTK) expression in chordoma suggest that these tumors may respond to kinase inhibitor therapy. Currently, there are no effective R. Akhavan-Sigari (*) : V. Rohde : N. Zafar Department of Neurosurgery, University Medical Center Göttingen, Georg-August-University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany e-mail: [email protected] V. Rohde e-mail: [email protected] N. Zafar e-mail: [email protected] M. Abili Department of Neurosurgery, Imam Reza University, Razavi Hospital, Mashhad, Iran e-mail: [email protected] M. Abili Mashhad University of Medical Sciences, Taleghani Hospital, Shahid Kalantari Avenue, after Ghaem Bridge, Mashhad, Iran M. R. Gaab Department of Neurosurgery, Nordstadt Medical Center, Klinikum Hannover, Haltenhoffstr, 41, 30167 Hannover, Germany e-mail: [email protected] P. Emami Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Martinistr, 52, 20246 Hamburg, Germany e-mail: [email protected] H. Ostertag Department of Pathology, Nordstadt Medical Center, Klinikum Hannover, Haltenhoffstr, 41, 30167 Hannover, Germany e-mail: [email protected]
chemotherapeutic protocols for chordoma. A tissue microarray containing 74 tumor specimens from primary chordoma patients and 71 from their recurrent tumors for a total of 145 tumor specimens was immunohistochemically analyzed for expression of a number of proteins involved in signal transduction from RTKs. Platelet-derived growth factor receptor-α (PDGFR-α), epidermal growth factor receptor (EGFR), c-Met, and CD-34 were detected in 100, 92, 100, and 59 % of cases, respectively. PDGFR-α and c-Met staining was of moderate to strong intensity in all cases. In contrast, total EGFR staining was variable; weak staining was detected in 10 cases. Our results contribute to the understanding of the expression of RTKs in skull base chordomas and support the development of targeted therapies that inhibit RTKs, which may have a synergistic effect for chemotherapy in patients. There were statistically significant correlations between the expression of PDGFR-α, c-Met, and EGFR and disease-free survival. The results nonetheless suggest that chordomas may respond to RTK inhibitors or modulators of other downstream signaling. Keywords Chordoma . Tyrosine kinase receptors . EGFR . c-Met . PDGFR-α
Introduction Chordomas are rare tumors with an incidence of about 0.05/ 100,000/year. They arise from embryonic notochordal remnants usually along the axial skeleton, primarily in the sacrococcygeal and petroclival regions, and account for less than 5 % of all primary malignant bone tumors [49]. Mainly adults between 40 and 60 years are affected, but cases of children presenting with chordoma were also rarely reported (5 % of cases) [23]. The median survival time for all patients is 6.29 years, with a 5-year survival rate of 67.6 % [4]. Unfortunately, standard chemotherapy was shown to be
90
basically unsuccessful, which causes serious problems for managing patients with locally recurrent or metastatic disease [11, 15]. Immunohistochemical (IHC) studies have shown that receptor tyrosine kinases (RTKs) such as platelet-derived growth factor receptor-α (PDGFR-α), platelet-derived growth factor receptor-β (PDGFRB), tyrosine protein kinase KIT, epithelial growth factor receptor (EGFR), mesenchymal-epithelial transition factor (MET), and human epithelial growth factor receptor 2 (HER2) are expressed in chordoma by immunohistochemistry [17, 28, 44, 50]. Given that RTKs could prove to be essential for the survival or proliferation of chordoma tumor cells, targeting these RTKs using antibodies or small molecule tyrosine kinase inhibitors (TKI) might offer new treatment options. Interestingly, imatinib was found to have antitumor activity in patients with chordoma [10]. In addition, expression of the MET oncogene has been reported in chordoma [28]. Of note, the MET oncogene is known to be expressed in various chondroid neoplasms, normal articular cartilage, and fetal notochord [9, 34]. Given their possible relationship to notochordal development and chondroid differentiation, further investigation is warranted to clarify the roles of these and other RTKs in chordomagenesis. Therapies aimed at several of these molecules have demonstrated initial success in treating both in vitro models of chordoma and patients with chordoma, supporting the feasibility of this approach [7, 19]. We examined whether selected RTKs are demonstrable in a large series of chordoma tissue, and their clinicopathologic significance with prognosis was clarified, suggesting that RTK inhibitors may be useful for the systemic treatment of chordoma.
Methods Chordomas A series of 74 chordoma patients for a total of 145 tumor specimens (74 primary tumor specimens and 71 recurrent tumors) were treated at the Neurosurgical Departments of the Nordstadt Hospital Hannover and of the Hannover Medical School in a period of 20 years in Germany. Out of 71 recurrent lesions, 42 were from the first, 15 from the second, 8 from the third, 4 from the fourth, 1 from the fifth, and 1 from the sixth recurrences. Tumor recurrence was defined as a return of symptoms and signs, with verification of tumor regrowth radiologically (Table 1). All patients were included; we had no exclusion criteria other than lack of available material for immunohistochemical analysis.
Neurosurg Rev (2015) 38:89–99 Table 1 A total of 145 tumor specimens were analyzed, of which 74 were from primary chordomas and 71 from recurring lesions (i.e., 42 from first recurrences, 15 from second, 8 from third, and 4 from fourth recurrences. One patient had a fifth and one patient a sixth recurrence) Primary tumor Recurrences 1 N=42
N=74 2 N=15
3 N=8
4 N=4
5 N=1
Total
6 N=1
N=71 N=145
Cohort demographics The patients (32 women and 42 men) ranged in age from 16 to 88 years (median age, 58 years). Seventy-one patients experienced recurrence and subsequent resection. Locations of the chordomas were skull base. The cases selected included patients with no history of chemotherapy or radiotherapy prior to surgery and a follow-up time of ≥12 months. The patient records were reviewed to collect the pathological data (tumor location and status of neurological deficits), demographic data (patient age and gender), and recurrence (Table 2). Multitumor tissue microarray construction A multitumor tissue microarray was assembled and used for comparison of the expression of molecular markers of chordoma. Following institutional review board approval, we constructed the tissue microarray as previously described [37]. The tissue microarrays (TMA) were constructed using a tissue arraying instrument (Beecher Instruments, Hackensack, NJ). Three tissue core cylinders with a diameter of 0.6 mm were punched from each donor paraffin block in targeted areas corresponding to previously demarcated neoplastic areas on the parallel slide. These tissue cores were then deposited into a recipient “master” paraffin block. The punches were placed 1 mm apart on the x-axis and 1.5 mm apart on the y-axis. Two microarray blocks, respectively, contained 75 and 70 punches. Sections 5 μm thick were cut from the master block, stained with H&E, and reviewed to ensure the presence of morphologically pure cores of chordoma for each case. Morphologic features of each core were confirmed by reviewing the corresponding whole tissue sections stained with H&E. We obtained tissue cores from paraffin-embedded formalin-fixed tissue blocks from the archives at the Department of Pathology, Nordstadt Medical Center, Klinikum Hannover. A pathologist (H.O.) reviewed the slides from all blocks to select representative areas of invasive tumor or normal tissue to be scored. Immunohistochemistry All slides were processed simultaneously under identical condition using standard methods. Immunohistochemistry was performed on the 145 tumor specimens for the following
Neurosurg Rev (2015) 38:89–99
91
Table 2 Correlation between RTK scores and clinicopathological factors in skull base chordomas (n=74); P values (italics for P
