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  Table of Contents  
Year : 2017  |  Volume : 54  |  Issue : 1  |  Page : 148-154

A comparative analysis of immunohistochemistry and fluorescent in situ hybridization assay to detect anaplastic lymphoma kinase status in lung adenocarcinoma cases: A search for a testing algorithm

1 Department of Pathology, Tata Memorial Hospital, Mumbai, Maharashtra, India
2 Department of Medical Oncology, Tata Memorial Hospital, Mumbai, Maharashtra, India
3 Department of Surgical Oncology, Tata Memorial Hospital, Mumbai, Maharashtra, India
4 Department of Radiation Oncology, Tata Memorial Hospital, Mumbai, Maharashtra, India

Date of Web Publication1-Dec-2017

Correspondence Address:
Dr. P B Wagle
Department of Pathology, Tata Memorial Hospital, Mumbai, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijc.IJC_202_17

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 » Abstract 

INTRODUCTION: Testing for echinoderm microtubule-associated protein-like 4 (EML4) anaplastic lymphoma kinase (ALK) translocation by fluorescence in situ hybridization (FISH) is well established whereas the Food and Drug Administration (FDA) ALK immunohistochemical (IHC) test is relatively new. AIMS AND OBJECTIVE: The aim of this study is to compare FDA-approved ALK IHC test (D5F3 clone) with the standard ALK FISH test. MATERIALS AND METHODS: A validation and a test arm with 100 and 200 cases of Formalin-Fixed, Paraffin-embedded blocks of lung adenocarcinoma, respectively, comprised the material. All cases had ALK IHC test on automated Ventana Benchmark XT IHC slide stainer using anti-ALK D5F3 rabbit monoclonal primary antibody; when positive tumor cells (any percentage) showed strong granular cytoplasmic staining. For the FISH test, Vysis ALK Dual Color Break Apart Rearrangement Probe (Abbott Molecular Inc.,) was used to detect ALK gene 2p23 rearrangements; when positive the red and green signals were split two signal diameter apart and/or isolated 3'red signal were detected in more than 15% tumor cells. The ALK FISH results were available in all 100 validation cases and 64-test arm cases which formed the basis of this analysis. RESULTS: The ALK IHC test was positive in 16% cases; four discordant cases were ALK IHC positive but ALK FISH negative, but no case was ALK IHC negative and ALK FISH positive. There was 100% sensitivity, 90.5% specificity, and 93.75% accuracy. CONCLUSION: A negative ALK IHC result obviates the need for a FISH test barring those with a strong clinical profile, and a positive ALK IHC result is sufficient basis for the initiation of treatment.

Keywords: Anaplastic lymphoma kinase, fluorescence in situ hybridization, immunohistochemistry, lung adenocarcinoma

How to cite this article:
Wagle P B, Jambhekar N A, Kumar R, Prabhash K, Pramesh C S, Desai S B, Noronha V, Karimundackal G, Shah A, Joshi A, Laskar S G, Jiwnani S, Pai T, Agarwal J P. A comparative analysis of immunohistochemistry and fluorescent in situ hybridization assay to detect anaplastic lymphoma kinase status in lung adenocarcinoma cases: A search for a testing algorithm. Indian J Cancer 2017;54:148-54

How to cite this URL:
Wagle P B, Jambhekar N A, Kumar R, Prabhash K, Pramesh C S, Desai S B, Noronha V, Karimundackal G, Shah A, Joshi A, Laskar S G, Jiwnani S, Pai T, Agarwal J P. A comparative analysis of immunohistochemistry and fluorescent in situ hybridization assay to detect anaplastic lymphoma kinase status in lung adenocarcinoma cases: A search for a testing algorithm. Indian J Cancer [serial online] 2017 [cited 2020 Sep 23];54:148-54. Available from:

 » Introduction Top

Transforming rearrangements of the anaplastic lymphoma kinase (ALK1) gene have been well recognized in a subset of human malignancies.[1],[2],[3],[4] In 2007, two investigators Soda et al. and Rikova et al. independently described that some Non small cell lung carcinoma (NSCLCs) harboring ALK gene rearrangement create an in-frame fusion protein between echinoderm microtubule-associated protein-like 4 (EML4) wherein the novel fusion gene arises from an inversion on the short arm of chromosome 2. The ALK-rearranged lung cancer cell lines are dependent on ALK kinase activity for transforming activity, oncogenicity, growth, and survival.[5],[6],[7],[8],[9] The incidence of ALK gene rearrangements in NSCLC is estimated to range from 3% to 13% and even up to 20% in the Western population.[7],[10],[11],[12],[13],[14],[15],[16],[17] Several ALK kinase inhibitors causing apoptosis and tumor shrinkage in vitro have been identified.[5],[6]

In 2011, the US Food and Drug Administration (FDA) approved the use of ALK inhibitor drug crizotinib in ALK-positive lung carcinoma, and concurrently, there was approval of Vysis ALK Break-Apart fluorescence in situ hybridization (FISH) Probe Kit (Abbott Molecular, Inc.).[18] Later in June 2015, US-FDA approved Ventana ALK immunohistochemical (IHC) test as a companion test to detect ALK rearrangement in NSCLC.[19] Since IHC tests are routine in surgical pathology practice, the availability of an antibody to detect the presence of ALK gene rearrangement holds promise of an easily applicable, fast, and cost-effective test and moreover, IHC results are also more stable than results of a FISH test.

The objective of this analysis was to compare the performance of the FDA-approved ALK IHC test using D5F3 clone on Formalin Fixed Paraffin-embedded (FFPE) blocks of lung adenocarcinoma specimens with the standard ALK FISH test and to seek practical insights regarding the testing algorithm for ALK alterations with IHC and FISH.

 » Materials and Methods Top

This is an observational study comprising separate analysis of a validation arm and a test arm with 100 and 200 FFPE blocks, respectively, of lung adenocarcinoma. The histological classification was as per the WHO 2015.[20] The performance and interpretation of the IHC test were as per the vendor kit instructions.

Validation arm

For validation, the ALK IHC test was done on 100 sequential lung adenocarcinoma FFPE blocks of cases whose ALK FISH test results were available from the laboratory electronic records; this included 54 lung biopsies, 44 extrapulmonary metastatic lung carcinoma deposits, and two lung resections. The entire freshly cut section was subjected to IHC.

Test arm

In the test arm, ALK IHC test was done on 200 lung adenocarcinomas FFPE sections from 106 lung biopsies, resections of 90 metastatic lung adenocarcinomas deposits, and four lung resections. A total of 32 out of 200 cases tested for ALK IHC were positive and 26 had a subsequent ALK FISH test, and six cases with inadequate tissue were excluded from the analysis. The ALK IHC test was negative in the remaining 168 cases and 38 of these had ALK FISH test based on the request of treating physician. Thus, out of 200 cases, 64 had both ALK IHC and ALK FISH results, and these form the basis of this analysis.


The ALK IHC assay was performed as per the protocol provided by Ventana diagnostics on automated Ventana Benchmark XT IHC slide stainer. The reagents comprised Ventana anti-ALK D5F3 rabbit monoclonal primary antibody, OptiView DAB IHC kit, and OptiView Amplification ALK IHC kit. Three sections were made from each FFPE block: the first for H and E, the second for ALK IHC test with Ventana anti-ALK (D5F3) antibody, and the third for Rabbit Monoclonal Negative Control Ig antibody. The FISH-validated ALK positive and ALK negative controls were included with each batch and each case to ensure that there was no false negative, or no false positive reaction, respectively.

Neoplastic cells labeled with the ALK IHC assay were evaluated for the presence or absence of DAB signal. The interpretation was as per the Ventana ALK Scoring Interpretation Guide for nonsmall cell lung carcinoma (NSCLC)[21] wherein a positive ALK IHC result is one which shows strong granular cytoplasmic staining in tumor cells (any percentage) [Figure 1]. The matched negative control slide was used to assess the degree of nonspecific background staining known to occur due to tissue elements such as alveolar macrophages, cells of neural origin, cells within lymphocytic infiltrate, glandular epithelial elements, mucin, and necrotic tumor areas.
Figure 1: Lung adenocarcinoma; (a) solid growth pattern (H and E); (b) anaplastic lymphoma kinase immunohistochemistry with DF53 antibody clone showing intense diffuse brown expression in the tumor in the positive control slide (fluorescence in situ hybridization validated); (c) lack of any immuno expression in negative control slide (fluorescence in situ hybridization validated)

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Fluorescent in-situ hybridization test

The FISH test was performed with Vysis ALK Dual Color Break Apart Rearrangement Probe (Abbott Molecular Inc.,) which detects chromosomal rearrangements involving the ALK gene 2p23. The probe kit comprises a combination of spectrum orange-labeled probe on the telomeric 3'side of the ALK gene and a spectrum green-labeled probe on the centromeric 5'side of the gene. A case was considered positive when 15 percent or more of tumor cells revealed either red and green signals split two signal diameter apart and/or isolated 3'red signals were detected cases wherein the total value of split, or loss of green, or sum of both these is <15% were considered negative.[22]

Statistical analysis

Statistical analysis was done on the data in Excel spreadsheet using standard formulae as given below to calculate the accuracy of ALK IHC test and its concordance with ALK FISH test which was the point of reference.

Sensitivity = True positive/true positive + false negative, Specificity = True negative/true negative + false positive, Positive predictive value: True positive/true positive + false positive, Negative predictive value: True negative/true negative + false negative, Accuracy = (true positive + true negative)/(true positive + false positive + true negative + false negative).

 » Results Top

The results of the validation and test arm are as shown in the ensuing text.

Validation arm

A comparison of the ALK IHC expression and ALK FISH test in the 100 cases revealed a positive concordance in 27 and negative concordance in 68; thus there were 95 concordant and five discordant results [Table 1] and [Figure 2]. The five discordant cases included four wherein IHC was positive, but FISH was negative, and in the fifth case, IHC was negative, but FISH was positive. Thus, ALK IHC test detected 3% more positive cases than the ALK FISH test.
Figure 2: Positive concordance ([a-c]: Same case); (a) lung adenocarcinoma solid growth pattern (H and E); (b) intense diffuse cytoplasmic immunoreactivity (anaplastic lymphoma kinase immunohistochemistry positive); (c) numerous cells show split red and green signals (anaplastic lymphoma kinase fluorescence in situ hybridization positive). Negative concordance ([d-f]: Same case); (d) papillary pattern of lung adenocarcinoma (H and E); (e) tumor cells lack immunoreactivity (anaplastic lymphoma kinase immunohistochemistry negative); (f) fused yellow signals (anaplastic lymphoma kinase fluorescence in situ hybridization negative)

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Table 1: Comparative analysis anaplastic lymphoma kinase fluorescent in situ hybridization and anaplastic lymphoma kinase immunohistochemistry: Validation arm (n=100*)

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Statistical analysis revealed sensitivity 96.4%, specificity 94.4%, positive predictive value 87.1%, negative predictive value 98.6%, and accuracy 95%.

On further analysis of the five discordant cases [Table 2], the histological pattern was acinar in two and in one case each, it was solid, signet ring, and micropapillary. The four IHC positive FISH negative cases showed 97%–100% fused signals whereas the IHC negative FISH positive had 17% split signals.
Table 2: Analysis of discordant cases: Validation arm (n=5*)

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A single patient out of the first four (ALK IHC positive/ALK FISH negative group) received crizotinib and revealed partial response so too the fifth patient whose tumor was ALK IHC negative/ALK FISH positive also received crizotinib and too showed partial response.

Test arm

Four out of the 64 cases wherein both ALK IHC and ALK FISH testing were done showed discordant results and all of them were ALK IHC positive but ALK FISH negative [Table 3]; not a single ALK IHC negative case was ALK FISH positive. Thus, overall ALK IHC test detected 6.25% more cases than the ALK FISH test.
Table 3: Comparative analysis anaplastic lymphoma kinase fluorescent in situ hybridization and anaplastic lymphoma kinase immunohistochemistry: Test arm (n=64*)

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Statistical analysis of test arm revealed: Sensitivity 100%, specificity 90.5%, positive predictive value 84.6%, negative predictive value 100%, true positivity 84.6%, true negativity 100%, false positivity 15.4%, false negativity 0%, and accuracy 93.75%.

Further, the analysis of the four discordant cases wherein ALK IHC was positive and ALK FISH was negative revealed solid pattern in two and an acinar and signet ring cell morphology in one case each [Table 4] and [Figure 3].
Figure 3: Discordant cases from test arm: Top row (left to right) shows four adenocarcinoma cases (H and E) showing following patterns: (a) Signet ring cell, (b) solid, (c) acinar, (d) solid. The middle row shows corresponding anaplastic lymphoma kinase immunohistochemical tests with DF53 antibody clone showing intense immunoreactivity in all four cases (e-h). Lowermost row shows corresponding anaplastic lymphoma kinase fluorescence in situ hybridization tests of above cases with fused signals in all four cases (i-l)

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Table 4: Analysis of discordant cases: Test arm (n=4*)

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In the corresponding FISH test, the percentage of fused signals ranged from 88% to 100% [Figure 3]. A single patient who received crizotinib had stable disease thereafter at 14 months.

 » Discussion Top

In 2011, the use of targeted drug crizotinib to treat ALK positive lung adenocarcinoma and a companion diagnostic test to diagnose ALK gene rearrangement by FISH test were approved by the US FDA.[10],[22] However, most surgical pathology laboratories are better equipped to do IHC tests, and hence, a reliable IHC test to detect ALK gene rearrangement would have a wider scope of application. Several investigators have tested primary anti-ALK antibodies such as ALK 1 (Dako), 5A4 (Novacastra and Abcam), and D5F3 (Ventana and Cell Signaling Technology) and compared their performance with FISH assays.[23],[24],[25],[26],[27],[28] The Ventana ALK IHC assay using D5F3 antibody clone received FDA approval as a stand-alone companion diagnostic test (CDx assay) in June 2015.[19] It includes an amplification step which increases the signal difference between the specific immunoreaction and the background signal and thus helps to reduce equivocal results and thus facilitates immediate identification of patients eligible for anti-ALK treatment.

As of now, there is an overall paucity of literature comparing ALK IHC with ALK FISH test, and there is none from India (at the time of going to press).[23],[24],[25],[26],[27],[28] In the present analysis, the ALK IHC test had 100% sensitivity and 90.5% specificity in the test arm. A comparison of the test arm results of the present study with published world literature shows a good concordance [Table 5].
Table 5: Literature review: Anaplastic lymphoma kinase immunohistochemistry versus anaplastic lymphoma kinase fluorescent in situ hybridization with D5F3 antibody

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The 90.6% to 100% range of sensitivity noted by various investigators [Table 5] implies that false negatives are low and hence patients would generally not lose out on the benefit of treatment due to a wrong negative report; the specificity ranged from 90.5% to 99.8%.

Thus, none of the published studies show 100% ALK IHC and ALK FISH concordance; such discordance can be anticipated and explained. The IHC positive FISH negative results could be because ALK IHC is easier to interpret; the positive expression is generally of moderate to strong intensity, and moreover, it is diffuse and stains almost all of the tumor-bearing area. In contrast, a FISH test requires sufficient and better-preserved tissue to visualize the requisite number of 15% split signals. A correct interpretation of ALK FISH needs specialized training and under diagnosis is more likely since the minimum distance between the red and green signals to interpret it as a “split signal” is two signals apart and under diagnoses is more likely as the split signal can be underestimated when the split is relatively narrow. Over diagnosis while interpreting FISH is also known to occur due to nuclear truncation leading to loss of 5' signals [22] and over interpretation of nonspecific signals in the nontumor tissue in the background. This can explain why some ALK FISH test positive patients do not respond to anti-TKI therapy.[22],[23] The single discordant case in the validation arm showing IHC negative but FISH positive result showed a good response to crizotinib. This can also be explained by the fact that rarely ALK rearrangement per se may not result into the translation or transcription of the fusion gene causing lack of protein expression. In addition, technical artifacts such as late fixation or over fixation may compromise the detection of the ALK protein.[23]

A concept of borderline or equivocal FISH results was recently introduced by Ilie et al. wherein the ALK signals, either in the form of split or isolated red signals, hover in the range of 10%–20% bracket; it was emphasized that such borderline results are best tested by another modality.[25] In the present study, the four discordant cases of the test arm were unequivocally ALK IHC positive the corresponding ALK FISH signals in the first three cases were a mere 3%, 8%, and none (0%), and only in the fourth case, there were 12% split signals which would “qualify” for the borderline bracket. The IHC and FISH tests of all these discordant cases were repeated to doubly reconfirm the results.

Discordance between ALK IHC and ALK FISH have been the subject of much discussion in literature, and testing with real-time reverse transcription polymerase chain reaction (RT-PCR) and next generation sequencing (NGS) are additional options; however, we did not have access to these. Some investigators therefore recommend both IHC and FISH test for treatment initiation; however, a few others have shown response to crizotinib when IHC was positive, but FISH was negative. Cabillic et al. did parallel testing with IHC using clone 5A4 and FISH on 3244 cases and found major discordances between the two assays: hence, proposed parallel testing since no assay is 100% specific or 100% sensitive, making multiple testing essential.[27] On similar lines, a recent multicenter study by Von Laffert et al. identified two ALK FISH borderline cases showing an ALK IHC expression positive profile which were confirmed to be positive for ALK gene rearrangement by RT-PCR assay. They hence opined that IHC with D5F3 clone can be a stand-alone diagnostic test, but a rational use of both IHC and FISH is essential in view of discordance.[28] Another recent editorial by Yatabe also stressed the need for multiple modality testing for each patient.[29] In 2016, Marchetti et al. reviewed 13 recently published comparative studies, totally comprising 10388 cases including his own 1031 cases where parallel testing was done; he found 41 IHC positive FISH negative and 82 IHC negative FISH positive cases. However, interestingly, the ALK IHC positive but FISH negative cases showed 100% response to crizotinib as compared to 46% response of those wherein IHC was negative and FISH was positive.[23] Similarly, Sun et al. and Peled et al. also showed that patients with IHC positive but FISH negative results had a dramatic response to crizotinib.[30],[31] Moreover, this is in keeping with our experience wherein two patients, one each from the validation and test arm with ALK IHC positive but FISH negative result showed partial response and stable disease status at 12 and 14 months, respectively.

The recent advent of NGS necessitates comparison between highly sensitive ALK IHC (clone D5F3) and NGS. In Marchetti's analysis of 1031 cases, five showed discrepant results of which two were ALK IHC positive FISH negative and three were ALK IHC negative FISH positive. All five discrepant cases were also tested with NGS and the NGS results matched with FISH results in these five cases. However, interestingly, one of their three discrepant cases wherein ALK IHC was negative but which tested postive for both NGS and FISH result, failed to show response to crizotinib; and yet, another case out of those two cases wherein ALK IHC was positive, but both FISH and NGS were negative showed response to crizotinib.[23] Pekar-Zlotin et al. analyzed 51 cases and tested the six discrepant cases with NGS: five of these were IHC positive FISH negative, and in four, the NGS results were concordant with IHC, but the fifth case was NGS negative, yet the patient showed stable disease subsequent to crizotinib.[32] The sixth discrepant case, was IHC negative FISH positive, and NGS negative (outcome not specified). Peled et al. also observed complete response to crizotinib in their ALK IHC/NGS positive patient who was FISH negative.[31] All these aforementioned studies make a strong case for treatment based on stand alone ALK IHC positive result whatever the FISH and NGS results.

As regards a testing algorithm, the existing College of American Pathologists guidelines of 2013 recommends ALK FISH test as the gold standard and a validated ALK IHC test for screening. However, FDA approval of ALK IHC test came later in June 2015 and as yet no new guidelines have been formally announced. The present testing algorithm scenario varies in different institutes in the US and is guided by the belief of the local pathologist and the treating physician (as per informal personal communication obtained by the senior author of the present study NA Jambhekar); whereas some centers rely on a positive ALK IHC result for treatment initiation as is the case in our institute, others rely on parallel testing and insist on a positive ALK FISH result for initiating treatment regardless of the IHC outcome. According to Marchetti et al., all IHC positive cases may be treated with a ALK inhibitor, and as regards, the ALK IHC negative cases, only if the clinicopathological parameters are those of the more frequently reported ALK positive patients such as younger age, nonsmokers, and/or signet ring cell or cribriform histology then alone the ALK FISH test should be done.[23]

 » Conclusion Top

The results of the test arm of the present study, and the overall experience regarding response to crizotinib vis a vis discordant results as published in literature supports the view that, a negative ALK IHC result obviates the need for a subsequent FISH test barring those with a strong clinical profile. Also, a positive ALK IHC result is enough basis for the initiation of treatment with crizotinib or such similar compounds even if ALK FISH is negative. In the Indian scenario, the main requirement for the ALK IHC test would be the availability of the FDA-approved clone and the appropriate automated platform. Thus, finally, to conclude, the FDA approved ALK IHC test results show good concordance with ALK FISH results, and subsequent ALK FISH correlation is optional and need not be mandatory in the testing algorithm.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

 » References Top

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Griffin CA, Hawkins AL, Dvorak C, Henkle C, Ellingham T, Perlman EJ. Recurrent involvement of 2p23 in inflammatory myofibroblastic tumors. Cancer Res 1999;59:2776-80.  Back to cited text no. 3
Debelenko LV, Raimondi SC, Daw N, Shivakumar BR, Huang D, Nelson M, et al. Renal cell carcinoma with novel VCL-ALK fusion: New representative of ALK-associated tumor spectrum. Mod Pathol 2011;24:430-42.  Back to cited text no. 4
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.  Back to cited text no. 5
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.  Back to cited text no. 6
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.  Back to cited text no. 7
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Martelli MP, Sozzi G, Hernandez L, Pettirossi V, Navarro A, Conte D, et al. EML4-ALK rearrangement in non-small cell lung cancer and non-tumor lung tissues. Am J Pathol 2009;174:661-70.  Back to cited text no. 11
Rodig SJ, Mino-Kenudson M, Dacic S, Yeap BY, Shaw A, Barletta JA, et al. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the Western population. Clin Cancer Res 2009;15:5216-23.  Back to cited text no. 12
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Li Y, Pan Y, Wang R, Sun Y, Hu H, Shen X, et al. ALK-rearranged lung cancer in Chinese: A comprehensive assessment of clinicopathology, IHC, FISH and RT-PCR. PLoS One 2013;8:e69016.  Back to cited text no. 15
Desai SS, Shah AS, Prabhash K, Jambhekar NA. A year of anaplastic large cell kinase testing for lung carcinoma: Pathological and technical perspectives. Indian J Cancer 2013;50:80-6.  Back to cited text no. 16
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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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