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Review
. 2023 Jul 31;12(7):1549-1562.
doi: 10.21037/tlcr-22-855. Epub 2023 Jul 11.

A narrative review of methods for the identification of ALK fusions in patients with non-small cell lung carcinoma

Susana Hernandez #  1 , Esther Conde #  2 , Marta Alonso  1 , Adrian Illarramendi  3 , Helena Bote de Cabo  4 , Jon Zugazagoitia  5 , Luis Paz-Ares  6 , Fernando Lopez-Rios  2
Affiliations
Review

A narrative review of methods for the identification of ALK fusions in patients with non-small cell lung carcinoma

Susana Hernandez et al. Transl Lung Cancer Res. .

Abstract

Background and objective: This narrative review is intended to provide pragmatic knowledge of current methods for the search of anaplastic lymphoma kinase (ALK) fusions in patients with non-small cell lung carcinoma (NSCLC). This information is very timely, because a recent survey has identified that almost 50% of patients with advanced NSCLC were not candidates for targeted therapies because of biomarker testing issues.

Methods: PubMed was searched from January 1st, 2012 to February 28th, 2023 using the following keywords: "ALK" and "lung", including reviews and our own work.

Key content and findings: Testing rates have not reached 85% among patients' candidates to ALK inhibition. The advantages and disadvantages of the different analytical options [immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), real-time polymerase chain reaction and next-generation sequencing (NGS)] are discussed. The key factor for success in ALK testing is a deep understanding of the concept of "molecular redundancy". This notion has been recommended and endorsed by all the major professional organizations in the field and can be summarized as follows: "laboratories should ensure that test results that are unexpected, discordant, equivocal, or otherwise of low confidence are confirmed or resolved using an alternative method or sample". In-depth knowledge of the different ALK testing methodologies can help clinical and molecular tumor boards implement and maintain sensible algorithms for a rapid and effective detection of predictive biomarkers in patients with NSCLC.

Conclusions: Multimodality testing has the potential to increase both the testing rate and the accuracy of ALK fusion identification.

Keywords: Anaplastic lymphoma kinase (ALK); fluorescence in situ hybridization (FISH); immunohistochemistry (IHC); next-generation sequencing (NGS); non-small cell lung carcinoma (NSCLC).

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-855/coif). The series “ALK Positive NSCLC” was commissioned by the editorial office without any funding or sponsorship. SH receives honoraria from Roche, Pfizer and Thermo Fisher Scientific and research funding from Roche and Thermo Fisher Scientific. EC receives honoraria from Roche, Thermo Fisher Scientific, Pfizer and Takeda and research funding from Roche and Thermo Fisher Scientific. JZ has served as a consultant for Astra Zeneca, BMS, Roche and Novartis, and received honoraria and travels honoraria from BMS, Roche, Astra Zeneca and Nanostring and research funding from BMS, Astra Zeneca and Roche. LPA has served as a consultant for Lilly, MSD, Roche, Pharmamar, Merck, Astra Zeneca, Novartis, Servier, Amgen, Pfizer, Sanofi, Bayer, BMS, Mirati, GSK, Janssen, Takeda and Daichii, and received honoraria from Astra Zeneca, Janssen, Merck and Mirati and research funding from MSD, AstraZeneca, Pfizer and BMS. FLR receives honoraria from Roche, Thermo Fisher Scientific, Pfizer and Takeda, and research funding from Roche and Thermo Fisher. The authors have no other conflicts of interest to declare.

Figures

Figure 1
Figure 1
ALK immunohistochemistry specimen workflow using the VENTANA ALK (D5F3) CDx Assay. Magnification times: ×200. H&E, hematoxylin and eosin; ALK, anaplastic lymphoma kinase; IHC, immunohistochemistry.
Figure 2
Figure 2
Examples of ALK immunohistochemistry positive (A-D) and negative (E-H) cases using the VENTANA ALK (D5F3) CDx Assay. (A) An ALK-positive tumor showing the typical staining pattern (strong, granular and cytoplasmic staining) (original magnification: ×200); (B,C) positive cases exhibiting atypical staining patterns (B, homogeneous strong but diffuse cytoplasmic staining and C, linear membranous accentuation) (original magnification: ×200); (D) an ALK positive adenocarcinoma with signet ring cells showing a weaker staining (the cytoplasm is replaced by mucin) (original magnification: ×200); (E) an ALK-negative lung adenocarcinoma showing a weak cytoplasmic staining (original magnification: ×200); (F) a small cell lung carcinoma exhibiting an aberrant ALK expression (i.e., fusion negative) (original magnification: ×200); (G) brain metastasis from a lung adenocarcinoma showing positivity in neural tissue but no staining in tumor cells (original magnification: ×200); (H) positivity in alveolar macrophages (original magnification: ×200). ALK, anaplastic lymphoma kinase.
Figure 3
Figure 3
ALK fluorescence in situ hybridization specimen workflow using the Vysis ALK Break Apart FISH Probe Kit. H&E, hematoxylin and eosin; FISH, fluorescence in situ hybridization; ALK, anaplastic lymphoma kinase.
Figure 4
Figure 4
Examples of ALK fluorescence in situ hybridization positive (A-D) and negative (E-H) cases using the Vysis ALK Break Apart FISH Probe Kit. (A) Typical positive or break apart 3'-5' pattern (orange and green signals are separated by 2 or more signal diameters). In some positive cases, tumor nuclei can show more than 1 set of break apart signals; (B) atypical positive or isolated 3' pattern (single orange signals); (C) atypical positive or isolated 3' pattern with ALK copy number gain; (D) atypical positive or isolated 3' pattern. The case also shows a duplication of the ALK 3'end (3'/5'/3') and copy number gain. (E) negative or fused pattern; (F) negative or isolated 5'pattern (single green signal); (G) negative or fused pattern with ALK amplification (≥6 ALK fusion signals per cell in ≥10% of analyzed cells); (H) negative pattern with a duplication of the ALK 3'end (3'/5'/3'). The case also shows a copy number gain (3–5 ALK fusion signals in ≥10% of analyzed cells); ALK FISH assays were scored with an image analysis algorithm (Duet System, BioView, Rehovot, Israel). ALK, anaplastic lymphoma kinase; FISH, fluorescence in situ hybridization.
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