ALK-Testing in non-small cell lung cancer (NSCLC): Immunohistochemistry (IHC) and/or fluorescence in-situ Hybridisation (FISH)?: Statement of the Germany Society for Pathology (DGP) and the Working Group Thoracic Oncology (AIO) of the German Cancer Society e.V. (Stellungnahme der Deutschen Gesellschaft für Pathologie und der AG Thorakale Onkologie der Arbeitsgemeinschaft Onkologie/Deutsche Krebsgesellschaft e.V.)

doi:10.1016/j.lungcan.2016.11.008

Lung Cancer

Volume 103, January 2017, Pages 1-5

https://doi.org/10.1016/j.lungcan.2016.11.008 Get rights and content

Highlights

•
FISH and IHC are available as validated diagnostic procedures.
•
At individual centers additional NGS-based procedures can be performed.
•
ALK-IHC is at least equivalent to FISH for ALK-activation in NSCLC.
•
Two validated antibodies (5A4 and D5F3) are presently available.
•
Antibodies should be used according to the validation studies.

Abstract

The EML4-ALK pathway plays an important role in a significant subset of non-small cell lung cancer patients. Treatment options such as ALK tyrosine kinase inhibitors lead to improved progression free survival and overall survival. These therapeutic options are chosen on the basis of the identification of the underlying genetic signature of the EML-ALK translocation. Efficient and easily accessible testing tools are required to identify eligible patients in a timely fashion. While FISH techniques are commonly used to detect this translocation, the broad implementation of this type of ALK testing into routine diagnostics is not optimal due to technical, structural and financial reasons. Immunohistochemical techniques to screen for EML4-ALK translocations may therefore play an important role in the near future. This consensus paper provides recommendations for the test algorithm and quality of the respective test approaches, which are discussed in the light of the current literature.

Section snippets

Statement

Alterations within the gene of the anaplastic lymphoma kinase (ALK) occur in about 3–4% of non-small cell lung cancers (NSCLC). Since the approvals (marketing authorizations) of the ALK (MET/ROS1)-inhibitor Crizotinib (Food and Drug Administration (FDA) 2011; European Medicines Agency (EMA) 2012), and the ALK/IGF1 inhibitor Ceritinib (FDA 2014, EMA 2015; for use in patients with progressive tumors under Crizotinib therapy), testing for ALK-activation in advanced-staged, not purely squamous

Summary

Available data from multiple research groups and consortia establish IHC as a valid diagnostic procedure to determine the ALK status in NSCLC. Further quality-assurance measures should accompany the practical application of IHC in this context. Future round robin tests are recommended to test for the diagnostic reliability on a broad base and also to check the potential of NGS in addition to the validated IHC and FISH.

In summary, the following statement can be made based on the available study

References (37)

K.M. Kerr et al.
Second ESMO consensus conference on lung cancer: pathology and molecular biomarkers for non-small-cell lung cancer
Ann. Oncol.
(2014)
A.T. Shaw et al.
Effect of crizotinib on overall survival in patients with advanced non-small-cell lung cancer harbouring ALK gene rearrangement: a retrospective analysis
Lancet Oncol.
(2011)
A. Marchetti et al.
ALK protein analysis by IHC staining after recent regulatory changes: a comparison of two widely used approaches, revision of the literature, and a new testing algorithm
J. Thorac. Oncol.
(2016)
E.S. Yi et al.
Correlation of IHC and FISH for ALK gene rearrangement in non-small cell lung carcinoma: IHC score algorithm for FISH
J. Thorac. Oncol.
(2011)
M. von Laffert et al.
Multicenter immunohistochemical ALK-Testing of non-Small-Cell lung cancer shows high Concordance after harmonization of techniques and interpretation criteria
J. Thorac. Oncol.
(2014)
H. Nitta et al.
New methods for ALK status diagnosis in non-small-cell lung cancer: an improved ALK immunohistochemical assay and a new, Brightfield, dual ALK IHC-in situ hybridization assay
J. Thorac. Oncol.
(2013)
M.W. Wynes et al.
An international interpretation study using the ALK IHC antibody D5F3 and a sensitive detection kit demonstrates high concordance between ALK IHC and ALK FISH and between evaluators
J. Thorac. Oncol.
(2014)
M.I. Ilie et al.
Discrepancies between FISH and immunohistochemistry for assessment of the ALK status are associated with ALK ‘borderline’-positive rearrangements or a high copy number: a potential major issue for anti-ALK therapeutic strategies
Ann. Oncol.
(2015)
M. Ilie et al.
Reply to the letter to the editor ‘ALK FISH rearranged and amplified tumor with negative immunohistochemistry: a rare and challenging case concerning ALK status screening in lung cancer’ by Uguen, et al
Ann. Oncol.
(2015)
S. Savic et al.
Screening for ALK in non-small cell lung carcinomas: 5A4 and D5F3 antibodies perform equally well, but combined use with FISH is recommended
Lung Cancer
(2015)

L.M. Sholl et al.

Combined use of ALK immunohistochemistry and FISH for optimal detection of ALK-Rearranged lung adenocarcinomas

J. Thorac. Oncol.

(2013)

E.C. Minca et al.

ALK status testing in non-small cell lung carcinoma: correlation between ultrasensitive IHC and FISH

J. Mol. Diagn.

(2013)

J.C. Cutz et al.

Canadian anaplastic lymphoma kinase study: a model for multicenter standardization and optimization of ALK testing in lung cancer

J. Thorac. Oncol.

(2014)

K. Gruber et al.

A novel, highly sensitive ALK antibody 1A4 facilitates effective screening for ALK rearrangements in lung adenocarcinomas by standard immunohistochemistry

J. Thorac. Oncol.

(2015)

N. Peled et al.

Next-generation sequencing identifies and immunohistochemistry confirms a novel crizotinib-Sensitive ALK. rearrangemnet in a patient with metastatic non-small-cell lung cancer

J. Thorac. Oncol.

(2012)

F. Cabillic et al.

Parallel FISH and immunohistochemical studies of ALK status in 3244 non-small-cell lung cancers reveal major discordances

J. Thorac. Oncol.

(2014)

X. Gao et al.

Clinical implications of variant ALK FISH rearrangement patterns

J. Thorac. Oncol.

(2015)

C.I. Selinger et al.

Testing for ALK rearrangement in lung adenocarcinoma: a multicenter comparison of immunohistochemistry and fluorescent in situ hybridization

Mod. Pathol.

(2013)

Cited by (23)

Anaplastic lymphoma kinase inhibitor therapy in the treatment of inflammatory myofibroblastic tumors in pediatric patients: Case reports and literature review
2021, Journal of Pediatric Surgery
Citation Excerpt :
However, detection of ALK expression by IHC can be variable and detection of ALK expression related to some ALK fusions can be difficult by IHC. Discrepant results between IHC and FISH have also been reported in lung cancer leading to confusion regarding the effect of such a result on the expectation of therapeutic response to ALK inhibition [33,34]. Positivity for ALK on IHC has also been observed in the case of wild-type ALK indicating the existence of alternative mechanisms for ALK overexpression.
: Inflammatory myofibroblastic tumors (IMTs) are a rare subtype of inflammatory pseudotumor frequently associated with rearrangement of the anaplastic lymphoma kinase (ALK) gene. Their treatment has historically relied on at-times challenging and morbid surgical excision. Recent studies have shown that neo/adjuvant therapy with ALK inhibitors can significantly enhance outcomes in select patients.
: A systematic literature review was performed to characterize comprehensive treatment of ALK-positive IMTs in the pediatric population. This report also includes two patients from our home institutions not previously reported in the literature.
: We identified a total of 27 patients in 12 studies in addition to 2 patients from the senior authors’ institution for a total of 29 patients (median age, 7 years; 52% male). The IMTs comprised a wide range of anatomic locations. Almost half (12, 41.3%) were treated with ALK-inhibitors alone and felt to be in remission. The remainder was treated with ALK-inhibitors either before or after surgery and had a curative response.
: ALK-positive IMTs can be successfully treated with ALK-inhibition alone or in combination with surgical resection. Further genetic characterization may be helpful in determining more precise treatment and defining needed durations thereof.
Continual Improvement of the Reliability of EML4-ALK Rearrangement Detection in Non–Small-Cell Lung Cancer: A Long-Term Comparison of ALK Detection in China
2020, Journal of Molecular Diagnostics
The results of EML4-ALK testing are critical to manage ALK tyrosine kinase receptor inhibitor treatment. Thus, the accurate detection of ALK rearrangement is increasingly becoming a matter of serious concern. To address this issue, a long-term EML4-ALK proficiency testing (PT) scheme was launched in China in 2015, serving as an educational tool for assessing and improving the testing quality of EML4-ALK fusion detection. Responses across 20 different PT samples interrogating three different variants and wild-type samples were collected between 2015 and 2019. Performance was analyzed by evaluating the detection methods, kits, and pre-analytic practices used to further display the landscape of changing conditions of the reliability of EML4-ALK testing. During the 5 years, 3224 results reported from 988 laboratories were evaluated, with an overall error rate of 5.36%. Along with an increasing number of participating laboratories, the error rate within each of the different methods showed a significantly downward trend over the years. No obvious differences in the error rates were found regarding the testing methods or kit manufacturers. Moreover, the individual performance of the laboratories improved when they participated in more PT scheme rounds. The data demonstrated that the performance of individual Chinese laboratories for EML4-ALK testing continuously improved over time by participating PT schemes, regardless of their method. However, care must be taken in standardized operations and validations.
Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology
2018, Journal of Molecular Diagnostics
In 2013, an evidence-based guideline was published by the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology to set standards for the molecular analysis of lung cancers to guide treatment decisions with targeted inhibitors. New evidence has prompted an evaluation of additional laboratory technologies, targetable genes, patient populations, and tumor types for testing.
To systematically review and update the 2013 guideline to affirm its validity; to assess the evidence of new genetic discoveries, technologies, and therapies; and to issue an evidence-based update.
The College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology convened an expert panel to develop an evidence-based guideline to help define the key questions and literature search terms, review abstracts and full articles, and draft recommendations.
Eighteen new recommendations were drafted. The panel also updated 3 recommendations from the 2013 guideline.
The 2013 guideline was largely reaffirmed with updated recommendations to allow testing of cytology samples, require improved assay sensitivity, and recommend against the use of immunohistochemistry for EGFR testing. Key new recommendations include ROS1 testing for all adenocarcinoma patients; the inclusion of additional genes (ERBB2, MET, BRAF, KRAS, and RET) for laboratories that perform next-generation sequencing panels; immunohistochemistry as an alternative to fluorescence in situ hybridization for ALK and/or ROS1 testing; use of 5% sensitivity assays for EGFR T790M mutations in patients with secondary resistance to EGFR inhibitors; and the use of cell-free DNA to “rule in” targetable mutations when tissue is limited or hard to obtain.
Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment With Targeted Tyrosine Kinase Inhibitors: Guideline From the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology
2018, Journal of Thoracic Oncology
In 2013, an evidence-based guideline was published by the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology to set standards for the molecular analysis of lung cancers to guide treatment decisions with targeted inhibitors. New evidence has prompted an evaluation of additional laboratory technologies, targetable genes, patient populations, and tumor types for testing.
To systematically review and update the 2013 guideline to affirm its validity; to assess the evidence of new genetic discoveries, technologies, and therapies; and to issue an evidence-based update.
The College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology convened an expert panel to develop an evidence-based guideline to help define the key questions and literature search terms, review abstracts and full articles, and draft recommendations.
Eighteen new recommendations were drafted. The panel also updated 3 recommendations from the 2013 guideline.
The 2013 guideline was largely reaffirmed with updated recommendations to allow testing of cytology samples, require improved assay sensitivity, and recommend against the use of immunohistochemistry for EGFR testing. Key new recommendations include ROS1 testing for all adenocarcinoma patients; the inclusion of additional genes (ERBB2, MET, BRAF, KRAS, and RET) for laboratories that perform next-generation sequencing panels; immunohistochemistry as an alternative to fluorescence in situ hybridization for ALK and/or ROS1 testing; use of 5% sensitivity assays for EGFR T790M mutations in patients with secondary resistance to EGFR inhibitors; and the use of cell-free DNA to “rule in” targetable mutations when tissue is limited or hard to obtain.
Molecular pathology of non-small cell lung cancer
2017, Diagnostic Histopathology
Citation Excerpt :
This has proved controversial, with some groups claiming to find IHC negative FISH positive cases. Despite this several antibodies are now available [5A4 (Novocastra) and D5F3 (Ventana)] which are generally accepted to show robust performance in combination with FISH or as a stand-alone test in unequivocal cases.18 The significance of IHC-positive, FISH-negative cases remain unclear but there is some evidence to suggest that these cases may respond to ALK targeted TKI therapy and as such these should be reported.
Our increasing understanding of the molecular pathogenesis of non-small cell lung cancer (NSCLC), particularly adenocarcinomas, has opened the door to ‘personalised medicine’ and the advent of new therapeutic strategies. Over the last few years new drugs, or classes of drugs, have been licensed and entered clinical practice for use in advanced NSCLC. The activity of these drugs is dependent on the presence of specific molecular or protein changes in cancer cells which are usually identified using ‘companion diagnostic tests’ specifically designed for this purpose. Pathologists and Pathology Departments have had to forge new links with Clinical Scientists in order to facilitate these additional investigations on the, often limited, tissue obtained for diagnosis. This collaboration plays a critical role in providing the link that allows integration of the traditional morphological diagnosis with the results of these new ‘companion diagnostic’ tests to guide patient management.
CRISPR/Cas9 Technology–Based Xenograft Tumors as Candidate Reference Materials for Multiple EML4-ALK Rearrangements Testing
2017, Journal of Molecular Diagnostics
Citation Excerpt :
In the emerging clinical paradigm of precision medicine, accurate detection is important to make the right decision regarding the treatment of cancer patients, such as in the case of EML4-ALK rearrangements detection for selecting the subgroup of patients for crizotinib therapy.13,14,20,28,29 Among the numerous factors that affect the accuracy of EML4-ALK testing, the lack of proper reference materials stands out.12,20,30,31 Therefore, to ensure the accuracy and reproducibility of detection, the present study seeks to develop a kind of well-characterized candidate reference material for EML4-ALK detection.
The echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (EML4-ALK) rearrangement is an important biomarker that plays a pivotal role in therapeutic decision making for non–small-cell lung cancer (NSCLC) patients. Ensuring accuracy and reproducibility of EML4-ALK testing by fluorescence in situ hybridization, immunohistochemistry, RT-PCR, and next-generation sequencing requires reliable reference materials for monitoring assay sensitivity and specificity. Herein, we developed novel reference materials for various kinds of EML4-ALK testing. CRISPR/Cas9 was used to edit various NSCLC cell lines containing EML4-ALK rearrangement variants 1, 2, and 3a/b. After s.c. inoculation, the formalin-fixed, paraffin-embedded (FFPE) samples from xenografts were prepared and tested for suitability as candidate reference materials by fluorescence in situ hybridization, immunohistochemistry, RT-PCR, and next-generation sequencing. Sample validation and commutability assessments showed that all types of FFPE samples derived from xenograft tumors have typical histological structures, and EML4-ALK testing results were similar to the clinical ALK-positive NSCLC specimens. Among the four methods for EML4-ALK detection, the validation test showed 100% concordance. Furthermore, these novel FFPE reference materials showed good stability and homogeneity. Without limitations on variant types and production, our novel FFPE samples based on CRISPR/Cas9 editing and xenografts are suitable as candidate reference materials for the validation, verification, internal quality control, and proficiency testing of EML4-ALK detection.

View all citing articles on Scopus

^☆: This Statement was already published in german in: -Pathologe. 2016 Mar;37(2):187-91. doi: http://dx.doi.org/10.1007/s00292-016-0152-1; -Pneumologie. 2016 Apr;70(4):277-81. doi: 10.1055/s-0042-102626. Epub 2016 Mar 16.

View full text

Highlights

Abstract

Section snippets

Statement

Summary

Ann. Oncol.

Lancet Oncol.

J. Thorac. Oncol.

J. Thorac. Oncol.

J. Thorac. Oncol.

J. Thorac. Oncol.

J. Thorac. Oncol.

Ann. Oncol.

Ann. Oncol.

Lung Cancer

J. Thorac. Oncol.

J. Mol. Diagn.

J. Thorac. Oncol.

J. Thorac. Oncol.

J. Thorac. Oncol.

J. Thorac. Oncol.

J. Thorac. Oncol.

Mod. Pathol.