|Year : 2016 | Volume
| Issue : 2 | Page : 1065-1069
Evaluation of anaplastic lymphoma kinase expression in nonsmall cell lung cancer; a tissue microarray analysis
Raheleh Roudi1, Gholamreza Haji2, Zahra Madjd3, Ahmad Shariftabrizi4, Mitra Mehrazma5
1 Oncopathology Research Center, FATiM, Iran University of Medical Sciences, Tehran, Iran
2 Department of Pathology, FATiM, Iran University of Medical Sciences, Tehran, Iran
3 Oncopathology Research Center; Department of Pathology; Department of Molecular Medicine, FATiM, Iran University of Medical Sciences, Tehran, Iran
4 Department of Nuclear Medicine and Molecular Imaging, State University of New York, Buffalo, NY 14214, USA
5 Oncopathology Research Center; Department of Pathology, FATiM, Iran University of Medical Sciences, Tehran, Iran
|Date of Web Publication||25-Jul-2016|
Department of Pathology, Ali-Asghar Children Hospital, No: 201, Vahid Dastgerdi Street, Modarres Highway, Tehran
Source of Support: None, Conflict of Interest: None
Introduction: The oncogenic form of anaplastic lymphoma kinase (ALK) gene is an attractive candidate marker for diagnostic and therapeutic purposes in several malignancies, including nonsmall cell lung cancer (NSCLC). This study aimed to examine the expression levels and clinical significance of ALK in a series of NSCLC tumors.
Material and Methods: We retrospectively reviewed 140 samples of NSCLC, including 64 (46%) squamous cell carcinoma (SCC), 62 (44%) adenocarcinoma (ADC), and 14 (10%) large cell carcinoma (LCC) for expression of ALK using immunohistochemistry; and immunostaining patterns were correlated with clinicopathological parameters.
Results: Expression of ALK was significantly different between SCC with ADC (P < 0.001) and LCC samples (P < 0.001). The highest level of ALK expression was found in ADC cases with poor differentiation and high nuclear grade (P = 0.005 and P = 0.005, respectively). Furthermore, high level of ALK expression was more often observed in ADC cases with poor prognosis features (P = 0.013).
Conclusions: These findings suggested that ALK can be considered as a promising target in the targeted therapy in patients with lung ADC.
Keywords: Anaplastic lymphoma kinase, lung cancer, receptor tyrosine kinases, tissue microarray
|How to cite this article:|
Roudi R, Haji G, Madjd Z, Shariftabrizi A, Mehrazma M. Evaluation of anaplastic lymphoma kinase expression in nonsmall cell lung cancer; a tissue microarray analysis. J Can Res Ther 2016;12:1065-9
|How to cite this URL:|
Roudi R, Haji G, Madjd Z, Shariftabrizi A, Mehrazma M. Evaluation of anaplastic lymphoma kinase expression in nonsmall cell lung cancer; a tissue microarray analysis. J Can Res Ther [serial online] 2016 [cited 2020 Sep 24];12:1065-9. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/1065/170940
| > Introduction|| |
Lung cancer remains as leading of cancer-related mortality worldwide. It is reported that at the time of diagnosis 54% of patients harbor advanced stage disease or distant metastasis and 5-year survival rate for lung cancer is only 15%., Nonsmall cell lung cancer (NSCLC) represents nearly 85% of all cases and includes adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC).
Several studies have attempted to screen genetic alterations, including gene amplification, and mutations in lung cancer tissue samples for diagnostic and therapeutic purposes.,, Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that upon ligand binding, autophosphorylate specific tyrosine residues and thus play a critical role in cell proliferation and survival, dedifferentiation of cancer cells and tumor progression, and migration., Anaplastic lymphoma kinase (ALK) is a member of the RTKs superfamily that is encoded by the ALK gene and its expression is can be affected by dysregulation through one or more different mechanisms, including fusion gene formation, mutations or gaining additional copies. Oncogenic forms of ALK have been reported in several malignancies, including glioblastoma, melanoma, breast cancer, and neuroblastoma as well as NSCLC.,,,,,, In a study by Rikova group, evaluation of purified phosphotyrosine superfamily proteins including ALK from NSCLCs cell lines and tumor samples suggested that 4.4% of NSCLCs were positive for ALK fusion protein. In addition, echinoderm microtubule-associated protein-like 4 (EML4) - ALK gene fusion, as the most common genetic alterations in this gene, was examined in NSCLC; which showed that production of this larger protein was found in 6.7% of NSCLC, which can lead to a constitutively active ALK tyrosine kinase. Several subsequent studies investigated ALK rearrangement using fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) based techniques, while limited reports are available regarding the evaluation of ALK rearrangement in NSCLCs using immunohistochemistry (IHC). In a previous report, it has been shown that the sensitivity and specificity of IHC approaches were 100% and 95.8%, respectively; thus this method could be used as a reliable method for detection of ALK rearrangement in NSCLCs. Due to lack of data on IHC expression patterns of ALK in NSCLC, this study was designed to explore the expression level of ALK and its correlation with tumor characteristics in the most prevalent subtypes of NSCLC including ADC, SCC, and LCC.
| > Materials and Methods|| |
Patients and clinicopathological data
The study population consisted of 140 NSCLC patients (64 SCC, 62 ADC, and 14 LCC), who were diagnosed from 2004 to 2012 (Tehran, Iran). Tissue samples were obtained before therapy for patients with primary NSCLC. For each case, hematoxylin and eosin (H and E)-slides and medical records were reviewed to confirm the diagnosis and obtain the clinicopathological parameters, including tumor types, histological grade (in SCC and ADC), nuclear grade (only in ADC) and inflammation (only in SCC), and available patients' information (age and gender). Based on the new classification of ADC, lepidic, and microinvasive tumors were considered as good prognosis, while acinar, papillary, mucinous carcinoma, and solid-acinar tumors were considered as moderate prognosis, and solid, and micropapillary tumors were considered as poor prognosis. This project was approved by Iran University of Medical Sciences (IUMS) Research Ethics Committee. Patients' data were kept fully anonymous.
Construction of tissue microarrays
Tissue microarrays (TMAs) were constructed from formalin-fixed paraffin-embedded NSCLC tissues as described previously., Briefly, representative tumor areas were defined by examination of H and E stained sections from all tissue blocks by a pathologist (GH). The selected area was then excised and re-embedded in recipient blocks using tissue-array instrument (Minicore; ALPHELYS, Plaisir, France). Furthermore, the normal lung tissues adjacent to tumors were included in TMA blocks.
Sections of the TMA blocks were cut at 5 μm onto Superfrost Plus™ positively charged glass slides (Thermo Scientific, Germany) and immunostained. For this purpose, the sections were deparaffinized with xylene and rehydrated through graded ethanol rinses. Endogenous peroxidase activity was blocked by incubation of sections in 3% hydrogen peroxidase in methanol at room temperature (RT) for 20 min. Antigen retrieval was performed by placing the slides in the Tris-ethylenediaminetetraacetic acid buffer (pH = 9.0) and treating with pressure cooker heating at high temperature for 10 min. After cooling at RT and washing in Tris-buffered saline (TBS), the sections were incubated overnight at 4°C with specific mouse monoclonal anti-human ALK antibody (Product code = M 7195, Dako, Denmark) with optimal dilution of 1/100. The immunoreactivity was detected with enVision+/DAB system (Dako, Denmark) and counterstained with hematoxylin (Dako, Denmark). An anaplastic large cell lymphoma tumor sample was used as a positive control sample to confirm the specificity of ALK staining and negative control was made by replacement of the primary antibody with TBS buffer.
Evaluation of immunohistochemical staining and scoring system
The immunostained tissue arrays were evaluated using a semi-quantitative scoring system after a series of TMAs were eye-balled on a multi-headed microscope by two observers (MM and GH) in a coded manner without a previous knowledge of clinical and pathological parameters of patients. In difficult cases, the scoring was confirmed by both observers and an agreement was reached. Immunostaining was scored on a scale of 1+ to 3+ (1 = weak, 2 = moderate and 3 = intense staining) and percentage of positive tumor cells were graded as 1 - <10% of positive tumor cells, 2 - 10-50% of positive tumor cells and 3 - <50% positive tumor cells. Finally, histochemical score (H-score) was calculated by multiplying the intensity of staining and the percentage of positive tumor cells.,
All data were analyzed using SPSS software version 20 (SPSS, Chicago, IL, USA). The correlation between ALK expression and clinicopathological parameters were evaluated using Pearson's χ2 and Pearson's R tests. The level of expression of ALK in various subtypes of NSCLC (including SCC, ADC, and LCC) were compared using Mann-Whitney U test. P ≤ 0.05 was considered to be statistically significant.
| > Results|| |
The study population consisted of 140 NSCLC tumors, including 64 (46%) SCC, 62 (44%) ADC, and 14 (10%) of LCC samples. The median age of patients was 66 (range: 34-89); of whom 80 (57%) cases were younger than 67, and 60 (43%) patients were over 67 years of age. The median age of SCC patients was 68 (range: 34-89), the median age of ADC patients was 66 (range: 34-89) and the median age of LCC patients was 67 (range: 43-75). The study population consisted of 106 (76%) male and 34 (24%) female subjects with a male/female (M/F) ratio of 3.11. The M/F ratio in SCC cases was 20.3 (61/3), whereas in ADC and LCC samples decreased to 1.36 (36/26) and 1.8 (9/5), respectively. Of the SCC samples 27 (42%) were well differentiated, 20 (31%) were moderately differentiated and 17 (27%) were poorly differentiated carcinomas. Twenty-one (33%) of SCC samples showed the presence of inflammation, whereas 43 (67%) cases showed absence of inflammation. Of the ADC samples, 8 (13%), 29 (47%) and 25 (40%) were well, moderately and poorly differentiated, respectively. All patients and tumor characteristics are shown in [Table 1].
|Table 1: Patients and tumor characteristics in NSCLC samples (the significance levels are shown in bold)|
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Analysis of anaplastic lymphoma kinase expression
The staining pattern of anaplastic large cell lymphoma, as a positive control for ALK expression, was strong and uniform without any background staining in the nuclei. IHC staining on TMAs built from various types of NSCLC showed that the expression pattern of ALK was mainly cytoplasmic and rarely membranous.
From a total of 140 NSCLC samples examined for expression of ALK, 101 (72%) cases showed weak intensity of staining, whereas 37 (27%) cases showed moderate intensity of staining and only 2 (1%) cases showed strong intensity of staining [Figure 1]a,[Figure 1]b,[Figure 1]c. Expression of ALK in the SCC cases showed a wide variation in terms of intensity of staining; 56 (88%) of cases showed weak staining, whereas only 8 (12%) of samples showed moderate staining and strong staining was not found in SCC specimens. Thirty-six (58%) of stained ADC cores displayed weak intensity of staining, 24 (39%) of ADC cases showed moderate staining of intensity and 2 (3%) of examined samples displayed strong intensity of staining. In the LCC cases, weak and moderate staining were found in 9 (64%) and 5 (36%) of cases, respectively, whereas strong staining was not found in LCC samples. The staining patterns of NSCLC samples are summarized in [Table 1].
|Figure 1: Immunohistochemical staining of anaplastic lymphoma kinase from nonsmall cell lung cancer patients, (a) +1 = Weak. (b) +2 = Moderate and (c) +3 = Strong intensity of staining (×200)|
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In addition, the level of ALK expression was divided into either lower (≤mean of H-scores) or higher (>mean of H-scores) expression. In the NSCLC cases, 81/140 (58%) of cases expressed low levels of ALK and 59/140 (42%) of samples expressed high levels of ALK. Fifty-one (80%) and 13 (20%) of SCC samples had low and high ALK expression, respectively. Low expression of ALK was found in 26/62 (42%) of ADC cases and high expression was observed in 36/62 (58%) of ADC stained cores. Of 14 LCC samples, 4 (29%) of the cases exhibited low levels of ALK expression and 10 (71%) of cases showed higher levels of expression. The mean of H-score is summarized in [Table 1].
Association of anaplastic lymphoma kinase expression and clinicopathological parameters
The difference in expression of ALK among NSCLCs was analyzed in two groups using the Mann-Whitney U Test. There was a significant association between expression of ALK in SCC cases with ADC cases (all P< 0.001) and LCC (intensity of staining P = 0.036, percentage of positive tumor cells P = 0.001 and H-score P < 0.001), but no significant association was found between expression of ALK in ADC and LCC (intensity of staining P=0.61, percentage of positive tumor cells P = 0.49 and H-score P = 0.35) [Figure 2].
|Figure 2: Box-Plot analysis of ALK expression distribution in NSCLC tumor. In the panel, the vertical axis gives immunostaining score and the horizontal axis showed different subtypes of NSCLC, including SCC, ADC, and LCC.|
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The correlation between the level of ALK expression and clinicopathological parameters was evaluated. Our analysis demonstrated no significant correlation between the expression levels of ALK with histological grade and inflammation in SCC samples (P = 0.11 and P = 0.4, respectively) [Table 2]. ALK immunoreactivity showed a significant association with histological grade (P = 0.005) and nuclear grade (P = 0.005) in ADC cases indicating high levels of ALK expression in ADC cases with poor differentiation and high nuclear grade [Table 3]. Additionally, univariate analysis indicated that ALK expression showed a significant association with tumor differentiation in the ADC samples suggesting high levels of ALK expression was mainly found in moderate (acinar, papillary, mucinous carcinoma, and solid-acinar tumors) and poor prognosis (solid, and micropapillary tumors) compared to good prognosis (lepidic and microinvasive tumors) ADC tumors (P = 0.013) [Table 3].
|Table 2: Association between the expression levels of ALK with pathological factors in SCC samples (P value; Pearson χ2)|
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|Table 3: Association between the expression levels of ALK with pathological factors in ADC samples (P value; Pearson χ2), (the significance levels are shown in bold)|
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| > Discussion|| |
RTKs are a large family of single-pass receptors that activate several signaling pathways, including PI3/AKT, MAPK/ERK, and JAK/STAT and eventually lead to cell proliferation and survival, tumor progression, and metastasis; thus members of RTKs family can provide a unique opportunity for tailored therapies in various cancers, including lung cancer., ALK is an enzyme from RTKs family that is encoded by the ALK gene on chromosome 2 and the most common oncogenic form of ALK is fusion with the gene EML4. The ALK-EML4 gene rearrangement has been widely investigated in NSCLC samples using FISH and PCR-based methods, although evaluation of protein expression of ALK using IHC is a more practical and convenient technique, especially for clinical applications. In addition, sufficient and high-quality DNA or RNA is required for molecular based techniques such as PCR. On the other hand, bronchoscopy is the most commonly used diagnostic procedure for obtaining only very small amount of tissue and thus techniques such as TMA are very useful in this regard. Our results demonstrated that the majority of ADC tumor cells were strongly positive for ALK, whereas SCC cases mainly displayed weak intensity, which was consistent with previous reports.,
We also explored the correlation between the levels of ALK expression and some of the clinicopathological parameters. In ADC cases, the increased expression of ALK was significantly related to poorly differentiated and high nuclear grade. Additionally, tumor cells expressing ALK were significantly more often found in ADC cases with poor prognosis features. These findings reveal that overexpression of ALK confers tumor aggressiveness and progression in ADC cases. ALK immunostaining showed no significant correlation with histological grade or inflammation in SCC cases. It has been shown that overexpression of ALK may be a reliable therapeutic target in NSCLC patients, especially ADC, whose tumors harbor ALK mutations.,
| > Conclusions|| |
Our findings indicated that evaluation of ALK in ADC cases can be considered as a reliable diagnostic and therapeutic target for tailored therapy of lung cancer patients.
This study was part of a residency thesis and supported by a grant from Iran University of Medical Sciences (Grant #17365).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013;63:11-30.
Ettinger DS, Akerley W, Borghaei H, Chang AC, Cheney RT, Chirieac LR, et al.
Non-small cell lung cancer. J Natl Compr Canc Netw 2012;10:1236-71.
Planchard D. Identification of driver mutations in lung cancer:First step in personalized cancer. Target Oncol 2013;8:3-14.
Köhler J, Schuler M. Afatinib, erlotinib and gefitinib in the first-line therapy of EGFR mutation-positive lung adenocarcinoma: A review. Onkologie 2013;36:510-8.
Ohashi K, Sequist LV, Arcila ME, Moran T, Chmielecki J, Lin YL, et al.
Lung cancers with acquired resistance to EGFR inhibitors occasionally harbor BRAF gene mutations but lack mutations in KRAS, NRAS, or MEK1. Proc Natl Acad Sci U S A 2012;109:E2127-33.
Adrain C, Freeman M. Regulation of receptor tyrosine kinase ligand processing. Cold Spring Harb Perspect Biol 2014;6. pii: a008995.
Lemmon MA, Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2010;141:1117-34.
Shackelford RE, Vora M, Mayhall K, Cotelingam J. ALK-rearrangements and testing methods in non-small cell lung cancer: A review. Genes Cancer 2014;5:1-14.
Robertson FM, Petricoin Iii EF, Van Laere SJ, Bertucci F, Chu K, Fernandez SV, et al.
Presence of anaplastic lymphoma kinase in inflammatory breast cancer. Springerplus 2013;2:497.
Wang M, Zhou C, Sun Q, Cai R, Li Y, Wang D, et al.
ALK amplification and protein expression predict inferior prognosis in neuroblastomas. Exp Mol Pathol 2013;95:124-30.
Hurley SP, Clary DO, Copie V, Lefcort F. Anaplastic lymphoma kinase is dynamically expressed on subsets of motor neurons and in the peripheral nervous system. J Comp Neurol 2006;495:202-12.
Niu HT, Zhou QM, Wang F, Shao Q, Guan YX, Wen XZ, et al.
Identification of anaplastic lymphoma kinase break points and oncogenic mutation profiles in acral/mucosal melanomas. Pigment Cell Melanoma Res 2013;26:646-53.
Takeuchi K, Choi YL, Togashi Y, Soda M, Hatano S, Inamura K, et al.
KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer. Clin Cancer Res 2009;15:3143-9.
Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, et al
. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007;448:561-6.
Rikova K, Guo A, Zeng Q, Possemato A, Yu J, Haack H, et al.
Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 2007;131:1190-203.
Paik JH, Choe G, Kim H, Choe JY, Lee HJ, Lee CT, et al.
Screening of anaplastic lymphoma kinase rearrangement by immunohistochemistry in non-small cell lung cancer: Correlation with fluorescence in situ
hybridization. J Thorac Oncol 2011;6:466-72.
Travis WD, Brambilla E, Noguchi M, Nicholson AG, Geisinger K, Yatabe Y, et al
. Diagnosis of lung cancer in small biopsies and cytology: Implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society Classification. Arch Pathol Lab Med 2012;137:668-4.
Roudi R, Madjd Z, Korourian A, Mehrazma M, Molanae S, Sabet MN, et al.
Clinical significance of putative cancer stem cell marker CD44 in different histological subtypes of lung cancer. Cancer Biomark 2014;14:457-67.
Roudi R, Korourian A, Shariftabrizi A, Madjd Z. Differential Expression of Cancer Stem Cell Markers ALDH1 and CD133 in Various Lung Cancer Subtypes. Cancer Invest 2015;33: 294-302.
Zhang YG, Jin ML, Li L, Zhao HY, Zeng X, Jiang L, et al.
Evaluation of ALK rearrangement in Chinese non-small cell lung cancer using fish, immunohistochemistry, and real-time quantitative RT – PCR on paraffin-embedded tissues. PLoS One 2013;8:e64821.
Huo L, Jennifer LH, Mien-Chie H. Receptor tyrosine kinases in the nucleus: Nuclear functions and therapeutic implications in cancers. In: Kumar R, editor. Nuclear Signaling Pathways and Targeting Transcription in Cancer. 1st
ed. New York: Humana Press; 2014. p. 189-229.
Maruyama IN. Mechanisms of activation of receptor tyrosine kinases: Monomers or dimers. Cells 2014;3:304-30.
Wang Y, Wang S, Xu S, Qu J, Liu B. Clinicopathologic features of patients with non-small cell lung cancer harboring the EML4-ALK fusion gene: A meta-analysis. PLoS One 2014;9:e110617.
Cook S, Dodge C, Morgan R, Sandusky GE. DNA/RNA Degradation rate in long term fixed museum specimens. Forensic Med Anat Res 2014;3:1.
Suter MJ, Hariri LP, Miller AJ, Adams DC, Lanuti M, Mino-Kenudson M. Increasing the diagnostic yield and accuracy of bronchial biopsy for the assessment of lung cancer. In: CLEO: Applications and Technology. Optical Society of America; 2014.
Barreca A, Lasorsa E, Riera L, Machiorlatti R, Piva R, Ponzoni M, et al.
Anaplastic lymphoma kinase in human cancer. J Mol Endocrinol 2011;47:R11-23.
Koivunen JP, Mermel C, Zejnullahu K, Murphy C, Lifshits E, Holmes AJ, et al.
EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res 2008;14:4275-83.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]