|Year : 2021 | Volume
| Issue : 1 | Page : 28
Expression of anaplastic lymphoma kinase in uveal melanoma
Jacqueline Coblentz, Ana Beatriz Dias, Jose Joao Mansure, Miguel Noel Burnier
The MUHC-McGill University Ocular Pathology and Translational Research Laboratory, Montreal, QC, Canada
|Date of Submission||25-Mar-2021|
|Date of Acceptance||12-Jul-2021|
|Date of Web Publication||24-Aug-2021|
Dr. Jacqueline Coblentz
4800 Boulevard de Maisonneuve W, Montreal, QC
Source of Support: None, Conflict of Interest: None
Purpose: The purpose of this study was to determine the expression of anaplastic lymphoma kinase (ALK) in uveal melanoma (UM) to evaluate its potential utility as a therapeutic target.
Materials and Methods: A total of 80 formalin-fixed paraffin-embedded enucleated eyes of UM patients, as well as 11 eyes and 11 pulmonary metastases from a rabbit model of human UM, were collected. All samples were stained for ALK using a fully automated immunohistochemical procedure. Human UM cases were classified according to cell type in spindle or epithelioid. Differences in ALK positivity according to cell type were determined using Pearson's Chi-square test.
Results: In human UM specimens, ALK was positive in 2 of 39 spindle cell type cases (2.5%) and in 13 of 41 (16.25%) epithelioid cell type cases. The difference in ALK expression between cell types was statistically significant (P = 0.002). In the animal model of human UM cells, all cases (100%) were positive for ALK in both ocular and pulmonary lesions.
Conclusion: ALK is expressed in a small proportion of UM, with statistically significant more expression in the more aggressive epithelioid cell type. Furthermore, when ocular tumors and corresponding lung metastasis from a highly metastatic animal model of UM were examined, ALK was positive in all samples. Collectively, our data suggest that ALK expression may be associated with more aggressive tumors. To the best of our knowledge, this is the first demonstration of the potential of ALK as a therapeutic target in human UM, particularly in aggressive tumors.
Keywords: Anaplastic lymphoma kinase, animal model, immunohistochemistry, uveal melanoma
|How to cite this article:|
Coblentz J, Dias AB, Mansure JJ, Burnier MN. Expression of anaplastic lymphoma kinase in uveal melanoma. Pan Am J Ophthalmol 2021;3:28
|How to cite this URL:|
Coblentz J, Dias AB, Mansure JJ, Burnier MN. Expression of anaplastic lymphoma kinase in uveal melanoma. Pan Am J Ophthalmol [serial online] 2021 [cited 2021 Oct 20];3:28. Available from: https://www.thepajo.org/text.asp?2021/3/1/28/324523
| Introduction|| |
While uveal melanoma (UM) is effectively controlled by enucleation or plaque radiation, approximately 50% of patients will develop metastasis, at which point the median survival is <1 year., As such, new therapeutic targets related to the cellular mechanisms involved in the processes of tumor progression and metastasis are needed. To this end, better understanding of the biological behavior of the tumor will allow us to uncover target molecules for the systemic treatment of UM.,
Over the last decades, considerable effort has been directed toward the research field, with a common goal of developing suitable experimental models for the study of UM. Models of UM using mice and rabbits have undergone many improvements, leading to experimental systems that better represent the natural progression of UM in patients. Significant advance has come from the use of human UM cell lines that, once injected in animals, are capable of inducing tumor growth and metastatic disease in immunodeficient hosts.
The protein anaplastic lymphoma kinase (ALK) is expressed physiologically in peripheral neuronal tissues and in some neuroendocrine cells and tumors.
The ALK receptor tyrosine kinase was initially discovered as a component of the fusion protein nucleophosmin (NPM)-ALK in anaplastic large cell lymphoma. Genomic alterations in ALK have been detected, such as rearrangements, point mutations, and genomic amplification. Those genomic alterations have been found in several malignancies, including lymphoma, non-small cell lung cancer (NSCLC), neuroblastoma, and inflammatory myofibroblastic tumor. Importantly, ALK serves as a validated therapeutic target in these diseases.
The objectives of this study are to characterize the immunohistochemical expression of ALK in enucleated eyes of UM patients and in the eyes and pulmonary metastasis of an animal model of human UM and to correlate ALK expression with prognostic factors such as cell type.
| Materials and Methods|| |
A total of 80 formalin-fixed paraffin-embedded enucleated eyes from patients with UM, as well as 11 eyes from a rabbit model of UM and their corresponding 11 pulmonary metastases from the same rabbits, were collected.
Human UM samples were classified according to cell type, as either spindle or epithelioid. Rabbit samples were obtained from a well-established human UM cell animal model in our laboratory. Briefly, human UM epithelioid cells were inoculated into the suprachoroidal space of rabbits that were immunosuppressed using daily injections of cyclosporine A. Fundoscopy and ultrasound were performed weekly in order to verify tumor growth. Lungs with metastasis from the same rabbits were also collected.
All samples were stained for ALK using a fully automated immunohistochemical procedure using Ventana Medical Systems, Inc., Tucson, AZ, USA. Samples were incubated with anti-ALK monoclonal antibody at a dilution of 1:100. Human normal lung samples were used as a positive control. For the negative control, the antibody was omitted.
Staining was scored as negative (0) or positive (1) according to the presence of any positivity in the tumor. Differences in ALK expression according to cell type in human samples were determined using Pearson's Chi-square test. In the animal model samples, the statistical analysis was descriptive, due to the fact that all rabbits had the same epithelioid cell type.
| Results|| |
In the 80 clinical samples, 39 were spindle and 41 were epithelioid cell type tumors. In the spindle cell type group, 2 cases were positive and 37 were negative for ALK. In the epithelioid cell type group, 13 cases were positive and 28 were negative for ALK [Figure 1]. Statistical analysis using Pearson's Chi-square test demonstrated a statistically significant difference in ALK staining between the two groups (P = 0.002).
|Figure 1: Histopathology of a human uveal melanoma, showing positive expression for anaplastic lymphoma kinase (red)|
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All samples from rabbit primary UMs were positive for ALK (n = 11; 100%) [Figure 2]. Furthermore, all pulmonary metastases from the animal model were positive for ALK (n = 11; 100%) [Figure 3].
|Figure 2: Histopathology of a rabbit uveal melanoma, showing positive expression for anaplastic lymphoma kinase (red)|
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|Figure 3: Histopathology of a rabbit pulmonary metastasis, showing positive expression for anaplastic lymphoma kinase (red)|
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| Discussion|| |
ALK is a receptor tyrosine kinase. It is normally expressed in several parts of the body such as brain, eye, skin, esophagus, stomach, midgut, testis, and ovary. The presence of ALK-mRNA in both central and peripheral nervous systems, as well as other organs, plays an important physiologic role in the normal development and normal functioning of these systems.
However, while ALK is important for normal function, it was first described as the fusion product oncogene for a subtype of non-Hodgkin's lymphoma. Later, translocations involving ALK were found to be useful to characterize specific types of lymphoma. The discovery of NPM-ALK as the specific lymphoma gene mutation involved in anaplastic large cell lymphoma allowed for this subtype to be redefined on the molecular level. This finding led to a better understanding of the role of ALK.
Advances in cancer management can occur through the discovery of key oncogenic drivers of the malignant process, understanding their detailed molecular mechanisms, and exploiting this transdisciplinary knowledge therapeutically. A variety of human malignancies have ALK translocations, amplifications, or oncogenic mutations, including ALCL, inflammatory myofibroblastic tumors, NSCLC, and neuroblastoma. These findings have driven intense interest in targeting the ALK signaling pathway as a therapeutic strategy.
In the present study, we investigated the expression of ALK in enucleated eyes of UM patients as well as in the ocular tumors and pulmonary metastasis from the same rabbits in a human UM animal model. Our results show that, in human eyes, ALK expression is higher in epithelioid cell type tumors when compared to spindle cell type tumors. Previous studies have shown that cell type is an important prognostic factor in patients with UM. Other prognostic factors, such as size, extraocular extension, lymphocytic infiltrate, and vascular loops, are closely related to UM cell type. Regarding the animal model, high cytoplasmic ALK expression was found in all ocular tumors, possibly because the injected cells were epithelioid. Furthermore, metastatic pulmonary cells revealed higher positivity with regard to the extent of staining compared to the primary tumor. This suggested that ALK positivity may be associated with a more malignant/metastatic phenotype.
This study in both human and experimental eyes is based on the immunohistochemical results using a monoclonal antibody to detect the presence of ALK in the primary and the metastasis from the animal model. As a general rule, monoclonal antibodies are more specific; therefore, the results are reliable. On the other hand, even considering the score that adds intensity and extension of staining, the final grading of the IHC marker is still somewhat subjective.
Another important point is the difficulty of assessing the staining in spindle cells in a given tumor, due to the fact that ALK is a cytoplasmic staining.
Currently, several anti-ALK drugs have been approved for clinical use by the FDA, including the first to market, crizotinib, which is an orally available dual inhibitor of ALK and the hepatocyte growth factor receptor (c-Met). Yamazaki et al. previously verified the relationship between ALK inhibition and antitumor efficacy in human tumor xenograft models. In the mentioned study, crizotinib was orally administered to athymic mice implanted with non-small cell lung carcinomas. This resulted in inhibition of tumor growth. Furthermore, results obtained from the present nonclinical xenograft mouse model projected >70% ALK inhibition in patients with NSCLC who were administered the clinically recommended dosage of crizotinib. The results suggest that crizotinib may effectively inhibit ALK phosphorylation, thereby exerting antitumor effects in patients.
However, Surriga et al. showed that crizotinib inhibited the phosphorylation of the c-Met receptor but not of ALK or ROS1 in UM cells. Consequently, migration of UM cells was suppressed in vitro at a concentration associated with the specific inhibition of c-Met phosphorylation. This effect on cell migration could be achieved with siRNA specific to c-Met but not to ALK or ROS1. In the model developed by Surriga et al., there was development of melanoma in the eye with subsequent metastasis to the liver and lung at 7 weeks after the initial transplantation. When mice were treated with crizotinib starting 1 week after the transplantation, there was a significant reduction in the development of metastasis as compared to untreated control sets. These results suggest crizotinib as a potential adjuvant therapy for patients with primary UM who are at high risk for the development of metastatic disease.
Our study supports the use of anti-ALK drugs for the treatment of UM. While Surriga et al. used animal cell lines for their study, our current study on ALK expression was conducted on tissue from rabbits inoculated with human cell lines, in particular with epithelioid cells. As such, the results may more accurately correlate to clinical UM, especially in regard to the use of anti-ALK drugs.
More recently, studies involving the use of crizotinib in patients with UM have been performed, although the results were not very promising.
Finally, our research shows that ALK is expressed in a small proportion of human eyes with UM; ALK is expressed to a greater extent in human UM of epithelioid cell type; ALK is strongly expressed in all primary UMs from this animal model; ALK is strongly expressed in all metastases from this animal model. High ALK expression in epithelioid cells and in metastasis from the animal model corroborates the hypothesis of a relationship between ALK expression and a more morphologically aggressive phenotype in human UM.
This study is based on human enucleated eyes which usually represent large malignant melanomas. For this reason, we could not draw any conclusion in regard to the expression of ALK in small-to-medium-sized melanomas. However, there is a link between the intensity of the expression of ALK and epithelioid cells. This finding may indicate that the expression of ALK correlates with cell type, therefore, with size. In the animal model, only epithelioid cells were injected. It is interesting that the metastatic sites in the animal model showed a much stronger positivity for ALK than the primary (ocular) site.
Further studies on the expression of ALK in UM are ongoing and its potential value as a therapeutic target needs yet to be determined.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Singh AD, Turell ME, Topham AK. Uveal melanoma: Trends in incidence, treatment, and survival. Ophthalmology 2011;118:1881-5.
Singh AD, Topham A. Incidence of uveal melanoma in the United States: 1973-1997. Ophthalmology 2003;110:956-61.
McLean IW, Saraiva VS, Burnier MN Jr. Pathological and prognostic features of uveal melanomas. Can J Ophthalmol 2004;39:343-50.
Mooy CM, De Jong PT. Prognostic parameters in uveal melanoma: A review. Surv Ophthalmol 1996;41:215-28.
Blanco PL, Caissie AL, Burnier MN Jr. Experimental models of uveal melanoma. Can J Ophthalmol 2004;39:441-7.
Kerr KM, López-Ríos F. Precision medicine in NSCLC and pathology: How does ALK fit in the pathway? Ann Oncol 2016;27 Suppl 3:i16-24.
Katayama R, Lovly CM, Shaw AT. Therapeutic targeting of anaplastic lymphoma kinase in lung cancer: A paradigm for precision cancer medicine. Clin Cancer Res 2015;21:2227-35.
Vernersson E, Khoo NK, Henriksson ML, Roos G, Palmer RH, Hallberg B. Characterization of the expression of the ALK receptor tyrosine kinase in mice. Gene Expr Patterns 2006;6:448-61.
Duyster J, Bai RY, Morris SW. Translocations involving anaplastic lymphoma kinase (ALK). Oncogene 2001;20:5623-37.
Mossé YP, Wood A, Maris JM. Inhibition of ALK signaling for cancer therapy. Clin Cancer Res 2009;15:5609-14.
McLean IW, Keefe KS, Burnier MN. Uveal melanoma. Comparison of the prognostic value of fibrovascular loops, mean of the ten largest nucleoli, cell type, and tumor size. Ophthalmology 1997;104:777-80.
Yamazaki S, Vicini P, Shen Z, Zou HY, Lee J, Li Q, et al.
Pharmacokinetic/pharmacodynamic modeling of crizotinib for anaplastic lymphoma kinase inhibition and antitumor efficacy in human tumor xenograft mouse models. J Pharmacol Exp Ther 2012;340:549-57.
Surriga O, Rajasekhar VK, Ambrosini G, Dogan Y, Huang R, Schwartz GK. Crizotinib, a c-Met inhibitor, prevents metastasis in a metastatic uveal melanoma model. Mol Cancer Ther 2013;12:2817-26.
Khan S, Lutzky J, Shoushtari AN, Jeter JM, Chiuzan C, Sender N, et al.
Adjuvant crizotinib in high-risk uveal melanoma following definitive therapy. Carvajal Journal of Clinical Oncology 2020;38:15_suppl, 10075.
Proença RP, Fonseca C, Goyeneche AA, Ito H, Dias A, Marques-Neves C, et al.
The expression of ALK, RET, ROS1, c-MET, EGFR and IGF-R1 in uveal melanoma and its association to clinicopathological characteristics and prognosis. Investig Ophthalmol Vis Sci 2020;61:2833.
[Figure 1], [Figure 2], [Figure 3]