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Anaplastic lymphoma kinase gene expression in small round cell tumors of childhood—a comparative ımmunohistochemical study
Annals of Diagnostic Pathology 19 (2015) 239–242
Contents lists available at ScienceDirect
Annals of Diagnostic Pathology
Anaplastic lymphoma kinase gene expression in small round cell tumors of childhood—a comparative ımmunohistochemical study☆,☆☆ Esra Karakuş, MD a,⁎, Suna Emir, MD, PhD b, Ayper Kaçar, MD, PhD c, Resul Karakuş, MD, PhD d, Hacı Ahmet Demir, MD, PhD e, Derya Özyörük, MD b a
Pathology Department, Ankara Children's Hematology and Oncology Research and Training Hospital, Ankara, Turkey Pediatric Oncology Department, Ankara Children's Hematology and Oncology Research and Training Hospital, Ankara, Turkey Department of Pathology, Ordu University Faculty of Medicine, Ordu, Turkey d Department of Immunology, Gazi University Faculty of Medicine, Ankara, Turkey e Departments of Pediatrics and Pediatric Oncology, Private Memorial Ankara Hospital, Ankara, Turkey b c
a r t i c l e
i n f o
Keywords: Anaplastic lymphoma kinase Neuroblastoma Wilms tumor Rhabdomyosarcoma Immunohistochemistry Ewing sarcoma
a b s t r a c t The focus of this study was to investigate anaplastic lymphoma kinase (ALK) expression by immunohistochemistry using a highly specific antibody. Distribution and frequency of ALK expression may provide a clue for ALK inhibitor use in small round cell tumors of childhood. The study group involved 76 small round cell tumors of childhood, which composed of 11 rhabdomyosarcomas, 13 Wilms tumors, 7 Ewing sarcoma/primitive neuroectodermal tumors, 34 peripheral neuroblastic tumors, and 11 acute lymphoblastic lymphoma. Anaplastic lymphoma kinase protein expression in small round cell tumors of childhood is poorly described in the literature. The findings of our study highlight a potential and possible role of targeting ALK in pediatric solid tumors by using ALK immunohistochemistry. Anaplastic lymphoma kinase may also have an oncogenic role in rhabdomyosarcomas and peripheral neuroblastic tumors, and they may possibly be treated with ALK inhibitors. Anaplastic lymphoma kinase expression in Wilms tumors is not reported in the literature, previously. Our study evaluated ALK expression in Wilms tumor samples. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The anaplastic lymphoma kinase (ALK) gene is a member of the insulin receptor family involved in the development of nervous system during embryogenesis [1]. Anaplastic lymphoma kinase is also known as ALK tyrosine receptor kinase, or CD246. Anaplastic lymphoma kinase gene was first shown to have a role in anaplastic large cell lymphomas, and studies performed later revealed that inhibition of the kinase by specific ALK targeting drugs results in tumor growth arrest and cell death in patients with non–small cell lung cancer (NSCLC) [2]. The receptor ALK was found to be mutated in many cases of lymphomas, inflammatory myofibroblastic tumors, esophageal cancers, breast cancers, colorectal cancers, and NSCLC and was targeted in biological therapies [3]. Anaplastic lymphoma kinase rearrangement–positive patients may be treated with crizotinib, a novel ALK inhibitor that was approved by the Food and Drug Administration in 2011 for the treatment for patients with locally advanced or metastatic NSCLC that is ALK positive. If additional pediatric solid tumor types would be identified by
☆ The authors declare that they have no competing interests. ☆☆ The article was financed by the authors. ⁎ Corresponding author at: Pathology Department, Ankara Child Diseases Hematology and Oncology Education and Research Hospital, Ziraat mah, İrfan Basbug cad. Kurtdereli sok, No:4 Diskapi, Ankara, Turkey. Tel.: +90 5071636748; fax: +90 312 347 23 30. E-mail addresses: [email protected], [email protected] (E. Karakuş). http://dx.doi.org/10.1016/j.anndiagpath.2015.04.007 1092-9134/© 2015 Elsevier Inc. All rights reserved.
ALK fusion expression, such cases could possibly benefit from new ALK inhibitor therapies. The focus of this study was to investigate ALK presence by immunohistochemistry (IHC) using a highly specific ALK antibody in order to define the frequency and distribution of ALK expression. Distribution and frequency of ALK expression may provide a clue for ALK inhibitor use in small round cell tumors of childhood. Among the small round cell tumors of childhood, we included rhabdomyosarcomas (RMSs), Wilms tumors, Ewing sarcoma/primitive neuroectodermal tumors (ES/ PNETs), peripheral neuroblastic tumors (pNTs), acute lymphoblastic lymphoma (ALL). To our knowledge, immunohistochemical comparison studies based on ALK expression on such a heterogenous collection of pediatric tumors are quite rare.
2. Materials and methods 2.1. Tissue specimens The study group consisted of 76 small round cell tumors of childhood sampled from patients treated at Ankara Children's Hematology and Oncology Research and Training Hospital. Ethical approval for the study was given by the ethical committee (2014-078) at the Ankara Children's Hematology and Oncology Research and Training Hospital, Ankara, Turkey.
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E. Karakuş et al. / Annals of Diagnostic Pathology 19 (2015) 239–242
2.2. Immunohistochemistry Tissue samples were processed for routine histologic examination with standard formalin fixation and paraffin embedding, and 5-μm thin sections were stained with hematoxylin-eosin. Samples were immunohistochemically stained for ALK expression. For IHC, 3-μmthick sections were tested with Ventana Benchmark GX immunostainer (Ventana, Tucson, Arizona). Diaminobenzidine was used as chromogen. Immunohistochemistry was performed with modified tissue microarray blocks, constructed for each case. The individual cases were represented with 4 different 0.5-cm cores in the blocks. Anti-ALK antibody (anti-CD246, clone ALK1; DAKO, Glostrup, Denmark) was used. We used sample of anaplastic large cell lymphomas as positive controls. For negative controls, the primary antibodies were omitted. A pathologist evaluated immunohistochemical staining of ALK in a blinded fashion. Immunoreactivity was scored as follows: 0, no staining; 1, faint cytoplasmic staining without any background staining; 2, moderate cytoplasmic staining; and 3, strong granular cytoplasmic staining in at least 10% of tumor cells [4].
3. Results The study group involved 76 small round cell tumors of childhood, which composed of 11 RMSs, 13 Wilms tumors, 7 ES/PNETs, 34 pNTs, and 11 ALLs. These samples were obtained from patients treated at the Ankara Children's Hematology and Oncology Research and Training Hospital between 2006 and 2014. Staining characteristics of the tumors are detailed in Table 1.
Fig. 1. Moderately ALK staining in ERMS. Original magnifications ×400.
3.4. Peripheral neuroblastic tumors Twenty-seven (84%) of 34 pNTs showed positivity for ALK, all with cytoplasmic staining pattern. Eighteen (52%) of 34 showed moderate to strong cytoplasmic, dot-like, and membranous staining (Fig. 3A-C). Peripheral neuroblastic tumors are a generic term including neuroblastoma (NB), ganglioneuroblastoma (GNB), and ganglioneuroma (GN). The distribution ALK expression in the pNTs subtypes is detailed in Table 2. It was observed that higher frequency of ALK expression in pNTs correlated with advanced tumor types. 3.5. Acute lymphoblastic lymphoma
3.1. Rhabdomyosarcomas
None of the 11 ALL showed ALK positivity.
Nine (81%) of 11 RMSs showed positivity for ALK, all with cytoplasmic staining pattern. Four (36%) of 11 showed moderate to strong cytoplasmic, dot-like, and membranous staining. Among the histologic parameters, pure solid growth pattern, pleomorphic and spindle cell morphologies were significantly associated with ALK status (Fig. 1).
3.2. Wilms tumors Five (38%) of 13 Wilms tumors showed positivity for ALK in the epithelial component, all with cytoplasmic staining pattern. One (7%) of 13 showed moderate to strong cytoplasmic, dot-like, and membranous staining (Fig. 2). Renal tubules also showed strong cytoplasmic and membranous staining.
4. Discussion This study evaluates ALK expression in a group of pediatric solid tumors by IHC. Studies in this field and around ALK expression may contribute to identification of possible new targets for new agents. There is still a need to develop new treatments for patients with refractory diseases. Multiple malignancies such as ALCL, NSCLC, inflammatory myofibroblastic tumor, colorectal cancer, breast cancer, and NB are known to show ALK expressions. A study performed by Tennstedt et al [3] reported a comprehensive analysis of solid tumor types for ALK fusion and demonstrated that ALK was also expressed in thyroid cancers, seminomas, and lung and ovarian cancer. Limited data exist regarding expression and genetic alterations and the potential effect of ALK inhibitors in RMSs. van Gaal et al [5] reported that ALK expression was increased in alveolar RMS (ARMS) as
3.3. Ewing sarcoma/primitive neuroectodermal tumors Two (28%) of 7 ES/PNETs showed positivity for ALK, all with cytoplasmic staining pattern. None of the them showed moderate to strong ALK positivity. Table 1 Results of IHC staining for ALK in small round cell tumors of childhood
RMSs Wilms tumors ES/PNET pNTs ALL
Pediatric solid tumors
Tumors with any cells positive for ALK, n (%)
Tumors with moderate-strong staining for ALK, n (%)
11 13 7 34 11
9 (81) 5 (38) 2 (28) 27 (79) 0 (0)
4 (36) 1 (7) 0 (0) 18 (52) 0 (0)
Moderate-strong, 2-3+ positivity for ALK.
Fig. 2. Immunohistochemical staining of Wilms tumor specimen with ALK. Original magnifications ×200.
E. Karakuş et al. / Annals of Diagnostic Pathology 19 (2015) 239–242
Fig. 3. Anaplastic lymphoma kinase IHC staining patterns in different tumor types. (A) NB sample with membrane staining of ALK. (B) GNB biopsy specimen with moderate staining of the neuroblastic component. (C) GN sample with cytoplasmic staining of only ganglion cells. A-C, original magnifications ×200.
compared with embryonal RMS (ERMS; 81% vs 32%, respectively). They also reported that ALK gene copy number was detected more frequently in ARMS (88%), compared with 52% in ERMS. One study investigated 116 RMSs using a polymer-based ALK immunostaining method, dualcolor fluorescence in situ hybridization, polymerase chain reaction, Table 2 The distribution ALK expression in the pNT subtypes Subtypes
n = 34
Tumors with any cells positive for ALK, n (%)
Tumors with moderate-strong staining for ALK, n (%)
NB GNB GN
24 6 4
17 (70) 6 (100) 4 (100)
14 (58) 4 (66) 0 (0)
241
and sequencing and showed a striking difference in ALK reactivity between ARMS (69%) and other (4%) subtypes [6]. The same study also found that ALK immunoreactivity correlated with ALK gene copy number. We found that 9 (81%) of 11 RMSs showed positivity for ALK, and pleomorphic and spindle cell morphologies were significantly associated with ALK status. However, because the number of our RMS cases is small, our results should be interpreted with caution. Beside RMSs, the expression of ALK receptor has been identified in some other mesenchymal malignancies such as ES/PNET. Li et al [7] showed that ALK protein was detected in ES/PNET cases 6 of 10. Fleuren et al [8] found that ALK expression was detected in most ES/PNET cases, 51 of 59. The same study also reported that ALK expression was lower in postchemotherapy resections compared with paired untreated primary tumors. In line with the results of previous studies, ALK expression was demonstrated by immunostaining in 2 ES/PNET cases in our series. Among several reports that have documented ALK expression in pNTs, Duijkers et al [9] defined that ALK tumor positivity by IHC is an independent poor prognostic marker for patients with NB and GNB and that ALK IHC is an easily applicable technique for routine patient risk stratification, particularly in NB patient trials in which ALK inhibitors were used. Wang et al [10] evaluated MYCN/ALK gene copy number by fluorescence in situ hybridization and detected ALK expression by IHC in 188 NB, 52 GNB, and 6 GN samples. Anaplastic lymphoma kinase positivity in NB (50.5%; 51/101) was significantly higher than that in GNB (22.6%; 7/31) and in GN (0.0%; 0/4). In the same study, it was also demonstrated that high ALK protein expression has been linked to inferior survival in NB. Although the number of patients in our study is low, we showed that ALK IHC is an easily applicable technique which may be also used for routine patient risk stratification, which is in agreement with previous reports. To our knowledge, ALK expression in Wilms tumors is not reported in the literature, previously. Our study evaluated ALK expression in Wilms tumor samples. Five (38%) of 13 Wilms tumor samples showed cytoplasmic staining, with 1 (7%) of 13 demonstrating a moderate and patchy pattern. Renal tubules also showed strong cytoplasmic and membranous staining. Further studies are needed to evaluate the function of ALK and its possible role in the pathogenesis and evaluation of Wilms tumor. Anaplastic lymphoma kinase protein expression in small round cell tumors of childhood is poorly described in the literature. The findings of our study highlight a potential and possible role of targeting ALK in pediatric solid tumors by using ALK IHC. Anaplastic lymphoma kinase may also have an oncogenic role in RMSs and pNTs, and they may possibly be treated with ALK inhibitors. However, it should also be noted that immunohistochemical expression of ALK does not itself confer sensitivity to ALK inhibitors and that ALK immunohistochemical overexpression may not be linked to ALK translocation or copy number increase. Although these results are preliminary and further research is required, these findings may have a major impact on future pediatric solid tumors to target pathways for potential treatment strategies. Also, further clinical studies should investigate whether ALK inhibitors are beneficial for treating ALK-immunopositive pediatric tumors.
References [1] Bellmunt J, Selvarajah S, Rodig S, et al. Identification of ALK gene alterations in urothelial carcinoma. PLoS One 2014;9:e103325. [2] Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non–small-cell lung cancer who harbor EML4-ALK. J Clin Oncol 2009; 27:4247–53. [3] Tennstedt P, Strobel G, Bölch C, et al. Patterns of ALK expression in different human cancer types. J Clin Pathol 2014;67:477–81. [4] Park HS, Lee JK, Kim DW, et al. Immunohistochemical screening for anaplastic lymphoma kinase (ALK) rearrangement in advanced non–small cell lung cancer patients. Lung Cancer 2012;77:288–92. [5] van Gaal JC, Flucke UE, Roeffen MH, et al. Anaplastic lymphoma kinase aberrations in rhabdomyosarcoma: clinical and prognostic implications. J Clin Oncol 2012;20: 308–15. [6] Yoshida A, Shibata T, Wakai S, et al. Anaplastic lymphoma kinase status in rhabdomyosarcomas. Mod Pathol 2013;26:772–81.
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E. Karakuş et al. / Annals of Diagnostic Pathology 19 (2015) 239–242
[7] Li XQ, Hisaoka M, Shi DR, Zhu XZ, Hashimoto H. Expression of anaplastic lymphoma kinase in soft tissue tumors: an immunohistochemical and molecular study of 249 cases. Hum Pathol 2004;35:711–21. [8] Fleuren ED, Roeffen MH, Leenders WP, et al. Expression and clinical relevance of MET and ALK in Ewing sarcomas. Int J Cancer 2013;15:427–36.
[9] Duijkers FA, Gaal J, Meijerink JP, et al. High anaplastic lymphoma kinase immunohistochemical staining in neuroblastoma and ganglioneuroblastoma is an independent predictor of poor outcome. Am J Pathol 2012;180:1223–31. [10] Wang M, Zhou C, Sun Q, et al. ALK amplification and protein expression predict inferior prognosis in neuroblastomas. Exp Mol Pathol 2013;95:124–30.
Contents lists available at ScienceDirect
Annals of Diagnostic Pathology
Anaplastic lymphoma kinase gene expression in small round cell tumors of childhood—a comparative ımmunohistochemical study☆,☆☆ Esra Karakuş, MD a,⁎, Suna Emir, MD, PhD b, Ayper Kaçar, MD, PhD c, Resul Karakuş, MD, PhD d, Hacı Ahmet Demir, MD, PhD e, Derya Özyörük, MD b a
Pathology Department, Ankara Children's Hematology and Oncology Research and Training Hospital, Ankara, Turkey Pediatric Oncology Department, Ankara Children's Hematology and Oncology Research and Training Hospital, Ankara, Turkey Department of Pathology, Ordu University Faculty of Medicine, Ordu, Turkey d Department of Immunology, Gazi University Faculty of Medicine, Ankara, Turkey e Departments of Pediatrics and Pediatric Oncology, Private Memorial Ankara Hospital, Ankara, Turkey b c
a r t i c l e
i n f o
Keywords: Anaplastic lymphoma kinase Neuroblastoma Wilms tumor Rhabdomyosarcoma Immunohistochemistry Ewing sarcoma
a b s t r a c t The focus of this study was to investigate anaplastic lymphoma kinase (ALK) expression by immunohistochemistry using a highly specific antibody. Distribution and frequency of ALK expression may provide a clue for ALK inhibitor use in small round cell tumors of childhood. The study group involved 76 small round cell tumors of childhood, which composed of 11 rhabdomyosarcomas, 13 Wilms tumors, 7 Ewing sarcoma/primitive neuroectodermal tumors, 34 peripheral neuroblastic tumors, and 11 acute lymphoblastic lymphoma. Anaplastic lymphoma kinase protein expression in small round cell tumors of childhood is poorly described in the literature. The findings of our study highlight a potential and possible role of targeting ALK in pediatric solid tumors by using ALK immunohistochemistry. Anaplastic lymphoma kinase may also have an oncogenic role in rhabdomyosarcomas and peripheral neuroblastic tumors, and they may possibly be treated with ALK inhibitors. Anaplastic lymphoma kinase expression in Wilms tumors is not reported in the literature, previously. Our study evaluated ALK expression in Wilms tumor samples. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The anaplastic lymphoma kinase (ALK) gene is a member of the insulin receptor family involved in the development of nervous system during embryogenesis [1]. Anaplastic lymphoma kinase is also known as ALK tyrosine receptor kinase, or CD246. Anaplastic lymphoma kinase gene was first shown to have a role in anaplastic large cell lymphomas, and studies performed later revealed that inhibition of the kinase by specific ALK targeting drugs results in tumor growth arrest and cell death in patients with non–small cell lung cancer (NSCLC) [2]. The receptor ALK was found to be mutated in many cases of lymphomas, inflammatory myofibroblastic tumors, esophageal cancers, breast cancers, colorectal cancers, and NSCLC and was targeted in biological therapies [3]. Anaplastic lymphoma kinase rearrangement–positive patients may be treated with crizotinib, a novel ALK inhibitor that was approved by the Food and Drug Administration in 2011 for the treatment for patients with locally advanced or metastatic NSCLC that is ALK positive. If additional pediatric solid tumor types would be identified by
☆ The authors declare that they have no competing interests. ☆☆ The article was financed by the authors. ⁎ Corresponding author at: Pathology Department, Ankara Child Diseases Hematology and Oncology Education and Research Hospital, Ziraat mah, İrfan Basbug cad. Kurtdereli sok, No:4 Diskapi, Ankara, Turkey. Tel.: +90 5071636748; fax: +90 312 347 23 30. E-mail addresses: [email protected], [email protected] (E. Karakuş). http://dx.doi.org/10.1016/j.anndiagpath.2015.04.007 1092-9134/© 2015 Elsevier Inc. All rights reserved.
ALK fusion expression, such cases could possibly benefit from new ALK inhibitor therapies. The focus of this study was to investigate ALK presence by immunohistochemistry (IHC) using a highly specific ALK antibody in order to define the frequency and distribution of ALK expression. Distribution and frequency of ALK expression may provide a clue for ALK inhibitor use in small round cell tumors of childhood. Among the small round cell tumors of childhood, we included rhabdomyosarcomas (RMSs), Wilms tumors, Ewing sarcoma/primitive neuroectodermal tumors (ES/ PNETs), peripheral neuroblastic tumors (pNTs), acute lymphoblastic lymphoma (ALL). To our knowledge, immunohistochemical comparison studies based on ALK expression on such a heterogenous collection of pediatric tumors are quite rare.
2. Materials and methods 2.1. Tissue specimens The study group consisted of 76 small round cell tumors of childhood sampled from patients treated at Ankara Children's Hematology and Oncology Research and Training Hospital. Ethical approval for the study was given by the ethical committee (2014-078) at the Ankara Children's Hematology and Oncology Research and Training Hospital, Ankara, Turkey.
240
E. Karakuş et al. / Annals of Diagnostic Pathology 19 (2015) 239–242
2.2. Immunohistochemistry Tissue samples were processed for routine histologic examination with standard formalin fixation and paraffin embedding, and 5-μm thin sections were stained with hematoxylin-eosin. Samples were immunohistochemically stained for ALK expression. For IHC, 3-μmthick sections were tested with Ventana Benchmark GX immunostainer (Ventana, Tucson, Arizona). Diaminobenzidine was used as chromogen. Immunohistochemistry was performed with modified tissue microarray blocks, constructed for each case. The individual cases were represented with 4 different 0.5-cm cores in the blocks. Anti-ALK antibody (anti-CD246, clone ALK1; DAKO, Glostrup, Denmark) was used. We used sample of anaplastic large cell lymphomas as positive controls. For negative controls, the primary antibodies were omitted. A pathologist evaluated immunohistochemical staining of ALK in a blinded fashion. Immunoreactivity was scored as follows: 0, no staining; 1, faint cytoplasmic staining without any background staining; 2, moderate cytoplasmic staining; and 3, strong granular cytoplasmic staining in at least 10% of tumor cells [4].
3. Results The study group involved 76 small round cell tumors of childhood, which composed of 11 RMSs, 13 Wilms tumors, 7 ES/PNETs, 34 pNTs, and 11 ALLs. These samples were obtained from patients treated at the Ankara Children's Hematology and Oncology Research and Training Hospital between 2006 and 2014. Staining characteristics of the tumors are detailed in Table 1.
Fig. 1. Moderately ALK staining in ERMS. Original magnifications ×400.
3.4. Peripheral neuroblastic tumors Twenty-seven (84%) of 34 pNTs showed positivity for ALK, all with cytoplasmic staining pattern. Eighteen (52%) of 34 showed moderate to strong cytoplasmic, dot-like, and membranous staining (Fig. 3A-C). Peripheral neuroblastic tumors are a generic term including neuroblastoma (NB), ganglioneuroblastoma (GNB), and ganglioneuroma (GN). The distribution ALK expression in the pNTs subtypes is detailed in Table 2. It was observed that higher frequency of ALK expression in pNTs correlated with advanced tumor types. 3.5. Acute lymphoblastic lymphoma
3.1. Rhabdomyosarcomas
None of the 11 ALL showed ALK positivity.
Nine (81%) of 11 RMSs showed positivity for ALK, all with cytoplasmic staining pattern. Four (36%) of 11 showed moderate to strong cytoplasmic, dot-like, and membranous staining. Among the histologic parameters, pure solid growth pattern, pleomorphic and spindle cell morphologies were significantly associated with ALK status (Fig. 1).
3.2. Wilms tumors Five (38%) of 13 Wilms tumors showed positivity for ALK in the epithelial component, all with cytoplasmic staining pattern. One (7%) of 13 showed moderate to strong cytoplasmic, dot-like, and membranous staining (Fig. 2). Renal tubules also showed strong cytoplasmic and membranous staining.
4. Discussion This study evaluates ALK expression in a group of pediatric solid tumors by IHC. Studies in this field and around ALK expression may contribute to identification of possible new targets for new agents. There is still a need to develop new treatments for patients with refractory diseases. Multiple malignancies such as ALCL, NSCLC, inflammatory myofibroblastic tumor, colorectal cancer, breast cancer, and NB are known to show ALK expressions. A study performed by Tennstedt et al [3] reported a comprehensive analysis of solid tumor types for ALK fusion and demonstrated that ALK was also expressed in thyroid cancers, seminomas, and lung and ovarian cancer. Limited data exist regarding expression and genetic alterations and the potential effect of ALK inhibitors in RMSs. van Gaal et al [5] reported that ALK expression was increased in alveolar RMS (ARMS) as
3.3. Ewing sarcoma/primitive neuroectodermal tumors Two (28%) of 7 ES/PNETs showed positivity for ALK, all with cytoplasmic staining pattern. None of the them showed moderate to strong ALK positivity. Table 1 Results of IHC staining for ALK in small round cell tumors of childhood
RMSs Wilms tumors ES/PNET pNTs ALL
Pediatric solid tumors
Tumors with any cells positive for ALK, n (%)
Tumors with moderate-strong staining for ALK, n (%)
11 13 7 34 11
9 (81) 5 (38) 2 (28) 27 (79) 0 (0)
4 (36) 1 (7) 0 (0) 18 (52) 0 (0)
Moderate-strong, 2-3+ positivity for ALK.
Fig. 2. Immunohistochemical staining of Wilms tumor specimen with ALK. Original magnifications ×200.
E. Karakuş et al. / Annals of Diagnostic Pathology 19 (2015) 239–242
Fig. 3. Anaplastic lymphoma kinase IHC staining patterns in different tumor types. (A) NB sample with membrane staining of ALK. (B) GNB biopsy specimen with moderate staining of the neuroblastic component. (C) GN sample with cytoplasmic staining of only ganglion cells. A-C, original magnifications ×200.
compared with embryonal RMS (ERMS; 81% vs 32%, respectively). They also reported that ALK gene copy number was detected more frequently in ARMS (88%), compared with 52% in ERMS. One study investigated 116 RMSs using a polymer-based ALK immunostaining method, dualcolor fluorescence in situ hybridization, polymerase chain reaction, Table 2 The distribution ALK expression in the pNT subtypes Subtypes
n = 34
Tumors with any cells positive for ALK, n (%)
Tumors with moderate-strong staining for ALK, n (%)
NB GNB GN
24 6 4
17 (70) 6 (100) 4 (100)
14 (58) 4 (66) 0 (0)
241
and sequencing and showed a striking difference in ALK reactivity between ARMS (69%) and other (4%) subtypes [6]. The same study also found that ALK immunoreactivity correlated with ALK gene copy number. We found that 9 (81%) of 11 RMSs showed positivity for ALK, and pleomorphic and spindle cell morphologies were significantly associated with ALK status. However, because the number of our RMS cases is small, our results should be interpreted with caution. Beside RMSs, the expression of ALK receptor has been identified in some other mesenchymal malignancies such as ES/PNET. Li et al [7] showed that ALK protein was detected in ES/PNET cases 6 of 10. Fleuren et al [8] found that ALK expression was detected in most ES/PNET cases, 51 of 59. The same study also reported that ALK expression was lower in postchemotherapy resections compared with paired untreated primary tumors. In line with the results of previous studies, ALK expression was demonstrated by immunostaining in 2 ES/PNET cases in our series. Among several reports that have documented ALK expression in pNTs, Duijkers et al [9] defined that ALK tumor positivity by IHC is an independent poor prognostic marker for patients with NB and GNB and that ALK IHC is an easily applicable technique for routine patient risk stratification, particularly in NB patient trials in which ALK inhibitors were used. Wang et al [10] evaluated MYCN/ALK gene copy number by fluorescence in situ hybridization and detected ALK expression by IHC in 188 NB, 52 GNB, and 6 GN samples. Anaplastic lymphoma kinase positivity in NB (50.5%; 51/101) was significantly higher than that in GNB (22.6%; 7/31) and in GN (0.0%; 0/4). In the same study, it was also demonstrated that high ALK protein expression has been linked to inferior survival in NB. Although the number of patients in our study is low, we showed that ALK IHC is an easily applicable technique which may be also used for routine patient risk stratification, which is in agreement with previous reports. To our knowledge, ALK expression in Wilms tumors is not reported in the literature, previously. Our study evaluated ALK expression in Wilms tumor samples. Five (38%) of 13 Wilms tumor samples showed cytoplasmic staining, with 1 (7%) of 13 demonstrating a moderate and patchy pattern. Renal tubules also showed strong cytoplasmic and membranous staining. Further studies are needed to evaluate the function of ALK and its possible role in the pathogenesis and evaluation of Wilms tumor. Anaplastic lymphoma kinase protein expression in small round cell tumors of childhood is poorly described in the literature. The findings of our study highlight a potential and possible role of targeting ALK in pediatric solid tumors by using ALK IHC. Anaplastic lymphoma kinase may also have an oncogenic role in RMSs and pNTs, and they may possibly be treated with ALK inhibitors. However, it should also be noted that immunohistochemical expression of ALK does not itself confer sensitivity to ALK inhibitors and that ALK immunohistochemical overexpression may not be linked to ALK translocation or copy number increase. Although these results are preliminary and further research is required, these findings may have a major impact on future pediatric solid tumors to target pathways for potential treatment strategies. Also, further clinical studies should investigate whether ALK inhibitors are beneficial for treating ALK-immunopositive pediatric tumors.
References [1] Bellmunt J, Selvarajah S, Rodig S, et al. Identification of ALK gene alterations in urothelial carcinoma. PLoS One 2014;9:e103325. [2] Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non–small-cell lung cancer who harbor EML4-ALK. J Clin Oncol 2009; 27:4247–53. [3] Tennstedt P, Strobel G, Bölch C, et al. Patterns of ALK expression in different human cancer types. J Clin Pathol 2014;67:477–81. [4] Park HS, Lee JK, Kim DW, et al. Immunohistochemical screening for anaplastic lymphoma kinase (ALK) rearrangement in advanced non–small cell lung cancer patients. Lung Cancer 2012;77:288–92. [5] van Gaal JC, Flucke UE, Roeffen MH, et al. Anaplastic lymphoma kinase aberrations in rhabdomyosarcoma: clinical and prognostic implications. J Clin Oncol 2012;20: 308–15. [6] Yoshida A, Shibata T, Wakai S, et al. Anaplastic lymphoma kinase status in rhabdomyosarcomas. Mod Pathol 2013;26:772–81.
242
E. Karakuş et al. / Annals of Diagnostic Pathology 19 (2015) 239–242
[7] Li XQ, Hisaoka M, Shi DR, Zhu XZ, Hashimoto H. Expression of anaplastic lymphoma kinase in soft tissue tumors: an immunohistochemical and molecular study of 249 cases. Hum Pathol 2004;35:711–21. [8] Fleuren ED, Roeffen MH, Leenders WP, et al. Expression and clinical relevance of MET and ALK in Ewing sarcomas. Int J Cancer 2013;15:427–36.
[9] Duijkers FA, Gaal J, Meijerink JP, et al. High anaplastic lymphoma kinase immunohistochemical staining in neuroblastoma and ganglioneuroblastoma is an independent predictor of poor outcome. Am J Pathol 2012;180:1223–31. [10] Wang M, Zhou C, Sun Q, et al. ALK amplification and protein expression predict inferior prognosis in neuroblastomas. Exp Mol Pathol 2013;95:124–30.