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  Table of Contents  
Year : 2017  |  Volume : 54  |  Issue : 1  |  Page : 209-213

Anaplastic lymphoma kinase immunohistochemistry in lung adenocarcinomas: Evaluation of performance of standard manual method using D5F3 antibody

1 Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
2 Department of Medical Oncology, All India Institute of Medical Sciences, New Delhi, India
3 Department of Laboratory Medicine, Jai Prakash Narayan Apex Trauma Center, All India Institute of Medical Sciences, New Delhi, India
4 Department of Pulmonary Medicine and Sleep Disorder, All India Institute of Medical Sciences, New Delhi, India

Date of Web Publication1-Dec-2017

Correspondence Address:
Dr. D Jain
Department of Pathology, All India Institute of Medical Sciences, New Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0019-509X.219588

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 » Abstract 

OBJECTIVE: Immunohistochemistry (IHC) with anaplastic lymphoma kinase (ALK) antibodies is considered as an economical screening method in lung adenocarcinomas. Automated Ventana D5F3-IHC is approved by US Food and Drug Administration for targeted therapy; however, the automated IHC apparatus are not widely used in most laboratories. We evaluated the performance of ALK IHC using the manual semiquantitative method to assess the concordance with Ventana ALK IHC assay. MATERIALS AND METHODS: We tested 156 cases of primary lung adenocarcinomas for ALK protein expression by D5F3-IHC. The intensity of cytoplasmic staining was classified as 0 or 1+/2+/3+ (weak/medium/strong). Binary score of positive and negative was used for Ventana assay. A comparison analysis and clinicopathological features were recorded. RESULTS: ALK IHC was positive in 25 (16.02%) cases, of which 18 were men and mostly nonsmokers. The mean age for all patients was 55 years, and for ALK IHC-positive cases was 48 years. Nine of 25 (36%) ALK IHC-positive cases showed signet ring cell and mucinous morphology. On comparison, all, but one, cases positive by manual method showed positive results by automated assay. IHC negative cases by manual method were negative by Ventana assay. CONCLUSION: Manual IHC is equally effective in the detection of ALK-rearranged cases as automated methods. It can be easily integrated as a screening method into routine practice thus reducing the cost of automated systems. However, equivocal cases should be tested by approved methods.

Keywords: Anaplastic lymphoma kinase, immunohistochemistry, lung adenocarcinoma, Ventana D5F3-IHC

How to cite this article:
Jain D, Jangra K, Malik P S, Arulselvi S, Madan K, Mathur S, Sharma M C. Anaplastic lymphoma kinase immunohistochemistry in lung adenocarcinomas: Evaluation of performance of standard manual method using D5F3 antibody. Indian J Cancer 2017;54:209-13

How to cite this URL:
Jain D, Jangra K, Malik P S, Arulselvi S, Madan K, Mathur S, Sharma M C. Anaplastic lymphoma kinase immunohistochemistry in lung adenocarcinomas: Evaluation of performance of standard manual method using D5F3 antibody. Indian J Cancer [serial online] 2017 [cited 2021 Sep 22];54:209-13. Available from: https://www.indianjcancer.com/text.asp?2017/54/1/209/219588

 » Introduction Top

Since the advent of targeted therapy, molecular testing for common mutations such as epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) has become vital in the diagnostic algorithm of nonsmall cell lung cancer (NSCLC).[1],[2] Currently, detection methods for identifying ALK rearrangements include fluorescence in situ hybridization (FISH), real-time reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemistry (IHC).[3],[4] Although FISH is filed as a companion diagnostic assay by Food and Drug Administration (FDA), it is expensive, time-consuming, needs specialized equipment, and expertise for procedure and interpretation.[5] Thus, it is not appropriate to use FISH as a screening method. ALK RT-PCR is not recommended as a first-line test due to risk of false negativity.[6] IHC, in comparison, is rapid, cheap, and easily integrated into most diagnostic laboratories. Several types of antibody clones are available for detection of ALK rearrangement including the one which has already been used (ALK1 Dako) for the diagnosis of anaplastic large-cell lymphoma and inflammatory myofibroblastic tumor. Other antibody clones include 5A4 (Novocastra), D5F3 (Cell Signaling and Roche) SP8 (Abcam), and 1A4 (Origene). Among them, 5A4, D5F3, and 1A4 are high-affinity clones and have 100% sensitivity for detection of ALK rearrangement.[7],[8] Recently, automated Ventana ALK IHC assay using D5F3 clone of ALK protein has been approved by US FDA for ALK-tyrosine kinase inhibitor crizotinib due to its high concordance with FISH assay.[9] However, the automated IHC apparatus and their proprietary kits and exclusive reagents are not cost effective in low-resource settings and therefore not widely used in most general laboratories. In the present study, we have examined the performance of the manual semiquantitative method of IHC using a D5F3 clone of the ALK antibody by comparing the results with automated Ventana IHC assay.

 » Materials and Methods Top

Patients and samples

We retrospectively analyzed 156 lung biopsies diagnosed as adenocarcinoma based on morphology and IHC (thyroid transcription factor-1 positive, p40 negative) from July 2014 to April 2016 at the Department of Pathology, All India Institute of Medical Sciences, New Delhi. Approval from ethics board of All India Institute of Medical Sciences, New Delhi has been taken before the study (No. IESC/T-245/2015). The diagnosis was made according to the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification for lung adenocarcinomas.[10] Automated Ventana IHC assay was performed in the Department of Laboratory Medicine at Jai Prakash Narayan Apex Trauma Center, All India Institute of Medical Sciences, New Delhi.


Manual method

All cases were independently detected for ALK expression by IHC, using the consecutive 4 μ thick formalin fixed paraffin embedded tissue whole section mounted onto poly-L-lysine coated slides. ALK IHC was performed using primary rabbit monoclonal anti-ALK antibody D5F3 (Cell Signaling Technology, MA, USA). In brief, slides were dried overnight at 65°C first, and then deparaffinized in xylene and dehydrated through a series of graded alcohols. Antigen retrieval was performed using citrate buffer (pH 6.0) for 30 min. Endogenous peroxidase activity was inhibited by incubating the sections in 4% H2O2 for 30 min at room temperature. The slides were then washed in Tris-Cl (pH 7.4–7.6). Nonspecific binding sites were blocked by 10% normal goat serum for 10 min. Again slides were washed in Tris-Cl (pH 7.4–7.6) for 5 min. Sections were then incubated with ALK D5F3 antibody (1:250) in moist chambers for overnight at 4°C. Slides were then analyzed by a detection kit (EnVision Plus HRP, Dako Cytomation, Glostrup, Denmark) according to the manufacturer's instructions, developing peroxidase activity with 3,3'-diaminobenzidine. Finally, slides were counterstained with hematoxylin, dehydrated, and mounted.

Ventana automated immunohistochemistry method

In 66 cases (including all manual IHC-positive cases and few IHC-negative cases), ALK IHC staining was performed on a Ventana BenchMark XT automated slide processing system at Ventana Medical Systems, Inc (VMSI). In brief, slides were subjected to deparaffinization using EZ-Prep (VMSI) and extended cell conditioning 1. Tissue sections were then incubated with anti-ALK antibody (D5F3; clone D5F3, VMSI) for 20 min. OptiView DAB IHC Detection Kit (VMSI) and OptiView Amplification Kit (VMSI) were used according to the manufacturer's recommendations for the visualization of the bound primary antibody. Tissue slides were counterstained with hematoxylin II (VMSI) and bluing reagent (VMSI). Slides were dehydrated and cleared before coverslipping. All cases were paired with negative control run according to the manufacturer's recommendations.[11]

Immunohistochemistry interpretation

The predominant intensity of staining was recorded on a scale of 0–3 (0 = negative, 1 = weak, 2 = moderate, 3 = strong). ALK immunoreactivity was evaluated in modified semiquantitative four-tiered criteria for manual method. IHC score 3+ for strong and diffuse granular cytoplasmic staining in >75% tumor cells, score 2+ for moderate, smooth cytoplasmic staining in >50% tumor cells, and score 1+ for faint, focal cytoplasmic staining in <50% of cells was given. Score 0 was given for completely no staining of the tumor cells.[12]

In addition, binary score of positive (strong granular cytoplasmic staining in any percentage of tumor cells) and negative (absence of strong granular cytoplasmic staining) was used for Ventana IHC.

Control tissues: Lung adenocarcinoma with ALK rearrangement (proved by FISH) and appendix were used as positive tissue control. Lung adenocarcinoma without ALK rearrangement was taken as a negative control.

 » Results Top


Of the 156 patients, 106 were male and 50 female (male:female 2.1:1), with a mean age of 55 years (range, 21–95 years). Ninety-eight percent patients were in Stage IIIb and IV based on the 7th tumor node and metastasis lung cancer staging system [13] and none had received chemotherapy or radiotherapy before biopsy. EGFR mutation information was available in 137 cases; 25 cases were EGFR mutation positive.


A total of 25 cases (25/156; 16.02%) were positive for ALK IHC by both methods. These comprised 18 males and 7 females (male:female = 2.5:1) with a mean age of 48 (range, 30–65) years [Table 1]. The remaining 131 patients were classified as ALK-negative, with scores of 0 (125 patients) and 1+ (6 patients). One ALK-positive case was positive for EGFR also.
Table 1: Clinicopathological features of anaplastic lymphoma kinase positive and negative patients

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Manual method

Staining intensity was strong (score 3+) [Figure 1]a in twenty cases. In six cases, the staining intensity was moderate cytoplasmic in all tumor cells (score 2+) [Figure 1]b.
Figure 1: (a) Photomicrograph shows strong cytoplasmic positivity of anaplastic lymphoma kinase protein (manual method) in a case of lung adenocarcinoma with acinar predominant pattern (×400). (b) Moderate intensity of cytoplasmic positivity of anaplastic lymphoma kinase immunohistochemistry by manual method in solid pattern of adenocarcinoma (×400). (c) Same case shown in 1a shows strong granular cytoplasmic positivity of anaplastic lymphoma kinase protein by automated Ventana immunohistochemistry assay (×200). (d) Same case shown in 1b shows strong granular cytoplasmic positivity of anaplastic lymphoma kinase immunohistochemistry by Ventana assay (×200)

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Automated Ventana immunohistochemistry

Binary score: Strong granular cytoplasmic staining was seen in 25 cases [Figure 1]c and [Figure 1]d; one score 2+ case by manual method was negative.

Forty-one cases which were negative by manual method stained by automated Ventana IHC kit and these were found negative.

When the results of both scoring systems were compared 0 and 1+ corresponded to negative and 2+ and 3+ in any percentage of tumor cells to positive. One score 2+ case on review showed weak positivity in poorly preserved tumor cells.

No background positivity was seen in any of the methods except for granular positivity in alveolar macrophages and brown staining in mucin pools in mucin pools in automated method.

Control specimens: Lung adenocarcinoma with ALK rearrangement showed strong cytoplasmic granular and 3+ positivity in Ventana and manual methods, respectively. Ganglion cells and nerves of the appendix displayed weak to moderate cytoplasmic staining by manual method whereas automated IHC showed strong positivity. Lung adenocarcinoma without ALK rearrangement did not show any positivity by both methods.


Nine of 25 (36%) ALK-positive tumors showed intra- and extra-cellular mucin. Three tumors showed predominantly extracellular mucin (adenocarcinoma with colloid pattern) whereas six tumors showed signet ring cells with extracellular mucin in two cases. The predominant patterns were acinar (12), solid (9), micropapillary (3), and papillary (1).

 » Discussion Top

ALK rearrangements act as the oncogenic drivers in approximately 3%–7% of patients with lung adenocarcinoma in which crizotinib has been shown to be a potent small-molecule inhibitor of ALK tyrosine kinase.[14] Similar to EGFR mutations, frequency of ALK rearrangements varies by ethnicity with a higher range in East Asians (3%–13%) in comparison to non-Asians (2%–7%).[15] To the best of our knowledge, there are only two previous studies from India related to the epidemiology of ALK rearrangement.[16],[17] We have a slightly higher frequency of ALK-positive tumors in nonselected (16.02%) and EGFR negative (25/131 = 19.08%) adenocarcinomas. The reason of higher positivity may be due to the fact that we have tested only by highly sensitive IHC method and taken automated Ventana IHC assay as a gold standard for validation. There was a selection bias as we have taken only those cases where enough tissue was available on the block for analysis. Second, advanced stage of the disease in this cohort may be responsible for the higher frequency of ALK rearrangement. However, our study does not reflect ALK rearrangement epidemiology due to less number of patients; the aims of this study were only to evaluate the performance of standard manual IHC method used for ALK rearrangement detection by comparing the results with approved automated Ventana IHC assay.

Clinically, ALK-rearranged tumors tend to occur in younger age people, nonsmokers, and without any significant sex predilection.[18] In our cohort relatively younger male patients predominated in ALK-positive group and most of them were nonsmokers. Histopathologically tumors typically show acinar, solid, micropapillary, and cribriform growth patterns with signet ring cell cytomorphology and extracellular mucin.[19] We confirm the findings from the literature regarding the histological features of ALK-positive adenocarcinomas. Approximately, 36% of the tumors in our series show signet ring cell morphology with and without extracellular mucin.

Various other studies indicate that IHC for detection of ALK rearrangement is a sensitive and specific method for determination of ALK protein expression.[7],[12],[20] In fact, a significant clinical improvement from crizotinib has been noted in ALK IHC-positive and FISH-negative patients.[21] In addition, IHC can be successfully used on limited or suboptimal samples where FISH analysis would give false negative results.[22],[23] Ultrasensitive automated Ventana D5F3-IHC revealed a very high correlation with FISH in assessing ALK status. The results of Ventana D5F3 staining amplified with OptiView showed 100% correlation with the results of staining using 5A4 and D5F3 without amplification.[24],[25] We have used D5F3 of cell signaling technology and found similar results except for staining intensity which was strong in approved kit-based assay. One of our positive cases (score 2+) was negative on automation which was on review found out to be weak positive (specificity 97.6%). However, none of the negative cases by manual method showed positive pick-up by an automated method (sensitivity100%).

The automated IHC apparatus are not freely available in most routine laboratories. Moreover, these assays are relatively expensive due to mandatory use of patented kits and reagents for signal amplification and high antibody titers in each procedure. Hence, we compared the results of manual D5F3 ALK IHC with automated IHC assay. Accurate detection of ALK rearrangement with manual method and correlation with FISH has been achieved earlier.[8],[12]

The ALK tyrosine kinase inhibitor crizotinib has been developed as a treatment for ALK-rearranged tumors. Data from the clinical trials show promising progression-free survival in advanced ALK-positive NSCLC.[26] One of the ALK IHC-positive patients from the present study is on crizotinib treatment and showing symptomatic relief however the duration is early for response assessment. Remaining patients did not receive crizotinib due mostly to unaffordability of the drug.

 » Conclusion Top

This study suggests that IHC prescreening by a standard manual semiquantitative method without signal amplification is equally effective as automated Ventana IHC assay and it can be easily integrated into routine practice. Mucinous variants and signet ring histology can be used as a predictor for ALK rearrangement and subject to ALK IHC on a priority basis. The risk of missing treatable cases using manual IHC method appears very small. In equivocal cases, automated approved IHC or FISH may be helpful.

The results of this study will help clinicians and pathologists, working at peripheral centers where expensive automated IHC stainer facilities are not freely available, to practice a reliable and cost-effective screening approach for establishing the optimal strategy of clinical ALK detection. However, a prospective validation in a larger cohort along with correlation with treatment response of TKIs is warranted.


The work has been supported by extramural fund given by Indian Council of Medical Research (ICMR), India

Financial support and sponsorship

The financial support has been provided by Indian Council of Medical Research (ICMR), India.

Conflicts of interest

There are no conflicts of interest.

 » References Top

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Shaw AT, Kim DW, Nakagawa K, Seto T, Crinó L, Ahn MJ, et al. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N Engl J Med 2013;368:2385-94.  Back to cited text no. 2
Wu YC, Chang IC, Wang CL, Chen TD, Chen YT, Liu HP, et al. Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan. PLoS One 2013;8:e70839.  Back to cited text no. 3
Teixidó C, Karachaliou N, Peg V, Gimenez-Capitan A, Rosell R. Concordance of IHC, FISH and RT-PCR for EML4-ALK rearrangements. Transl Lung Cancer Res 2014;3:70-4.  Back to cited text no. 4
Camidge DR, Kono SA, Flacco A, Tan AC, Doebele RC, Zhou Q, et al. Optimizing the detection of lung cancer patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements potentially suitable for ALK inhibitor treatment. Clin Cancer Res 2010;16:5581-90.  Back to cited text no. 5
Lindeman NI, Cagle PT, Beasley MB, Chitale DA, Dacic S, Giaccone G, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. J Thorac Oncol 2013;8:823-59.  Back to cited text no. 6
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Goldstraw P, Crowley J, Chansky K, Giroux DJ, Groome PA, Rami-Porta R, et al. The IASLC Lung Cancer Staging Project: Proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours. J Thorac Oncol 2007;2:706-14.  Back to cited text no. 13
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