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

Introduction

Background and rationale

The anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that plays a critical role in the development and function of the nervous system. Accordingly, there is protein expression in the nervous system during development and in some nervous tissues in adults. The ALK gene is located on the short arm of chromosome 2 and encodes a transmembrane protein that consists of an extracellular ligand-binding domain, a transmembrane region, and an intracellular kinase catalytic domain. ALKALs have now been reported as ligands for the ALK receptor (1,2). Upon ligand binding, activation of ALK leads to the phosphorylation of downstream signaling molecules, including the phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) pathways, which regulate cell growth, survival, and differentiation. Oncogenic activating fusions were originally described in anaplastic large cell lymphomas and have subsequently been identified in a variety of malignant tumors (3,4).

In 2007, ALK fusions were identified in a small percentage of patients with non-small cell lung carcinoma (NSCLC) (5). The subsequent development and approval of several ALK inhibitors means that the relevance of accurately identifying ALK fusions in patients with NSCLC has never been greater (6,7). Thus, ALK fusions testing in NSCLC has increased over time, but many of the gaps on the implementation of personalized oncology can be traced back to suboptimal biomarker test results (see below) (8).

ALK gene fusions, which are found in approximately 3–5% of the overall NSCLC population, result in aberrant expression of the chimeric ALK proteins (9,10). There is a higher prevalence in never/light smokers, younger age and adenocarcinoma histology (10-13). Interestingly, a very high frequency of signet ring cells has been reported and could be a very good predictive histological feature (14-18). Accordingly, the presence of signet ring cells has been identified in 44.4% to 72% of ALK-positive tumors (median 56%, mean: 57.2%) (14-18). The increasing use of next generation sequencing (NGS) has identified more than 90 different fusion partners, but echinoderm microtubule-associated protein-like 4 (EML4) remains the most frequent partner (approximately 85%) (19,20). The most commonly reported EML4-ALK fusion breakpoints are v1 (30–40%), v3a/b (30–40%) and v2 (10%) (10,20-22). The individual frequencies of non-EML4-ALK fusions are difficult to infer but there are usually below 2%: kinesin family member 5B (KIF5B; range, 0.4–2.2%), striatin (STRN; range, 1–1.4%), huntingtin interacting protein 1 (HIP1; range, 1–3%) or trafficking from ER to golgi regulator (TFG; 1.4%) (21-28). However, some series have found higher frequencies of STRN (6.7%), HIP1 (5%) or TFG (5%), a fact that needs to be taken into consideration when not using NGS as the primary testing modality (see below) (21,29,30).

Knowledge gap and objective

This narrative review is intended to provide pragmatic knowledge of current methods for detecting ALK fusions in patients with NSCLC. Although broad molecular profiling is usually the recommended testing option in most guidelines, NGS is not universally available (4). Moreover, the information presented herein 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 (8). The clinical gaps can be summarized as follows (the three “Ts”): tissue (insufficient tissue or tumor cell overestimated), testing (appropriate assay was not ordered or results were inconclusive or false negative) and time (turnaround time delays) (8). The specific situation of ALK fusions testing in NSCLC is somewhat similar because testing rates have not surpassed 85% among patients’ candidates to ALK inhibition and NGS is combined with single analyte assays in most regions of the world (31-35). Moreover, recent data indicate that 25% of the ALK positive patients begin a non-targeted treatment before receiving the results and surprisingly detection rate is influenced by the type of assay (31,36). Therefore, we hypothesized that in-depth knowledge of the advantages and disadvantages 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, regardless of the histologic subtype (4,37,38). In the era of tumor-agnostic therapies, it is important to highlight that ALK fusions have also been described in many other neoplasms: 50–60% of inflammatory myofibroblastic tumors, 2.2% of papillary thyroid carcinomas, <1% of colorectal adenocarcinomas and a variety of hematolymphoid tumors, including ALK-positive histiocytosis (39-41). This concept is reassuring, as it indicates that we could potentially use the same testing strategies presented herein. We present this article in accordance with the Narrative Review reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-855/rc).

Methods

PubMed was searched for English-language journals from January 1^st, 2012 to February 28^th, 2023 using the following keywords: “ALK” and “lung”, including reviews and our own work (Table 1).

Testing approaches for ALK fusions

The most frequently used tissue assays that are currently available to identify ALK fusions include both traditional single methodologies such as immunohistochemistry (IHC), break-apart fluorescence in situ hybridization (FISH) and real-time PCR, as well as the increasingly popular NGS (42-51). Each of these approaches has advantages and disadvantages that need to be carefully balanced to design sensible testing algorithms. Table 2 summarizes this information (4). Except for “analytical sensitivity” and “diagnostic sensitivity”, all attributes are self-explanatory. The “analytical sensitivity” (often referred to as just “sensitivity”) is the limit of detection. The “diagnostic sensitivity” relates to the comprehensiveness of the assay or the percentage of all ALK fusions described for the gene detectable by the given assay (53).

IHC

There is currently a stand-alone IHC Food and Drug Administration (FDA) approved assay (VENTANA ALK D5F3 CDx Assay, Ventana Medical Systems, Tucson, AZ, USA) (54). Our interpretation of the workflow is presented in Figure 1. Staining requires three sections from the formalin-fixed paraffin embedded (FFPE) material: one hematoxylin and eosin (H&E), a second for the VENTANA anti-ALK (D5F3) CDx assay antibody, and a third for the negative reagent control. System-level controls (previously qualified appendix tissue) are included on each section. If the H&E evaluation indicates that the specimen is not acceptable, a new section should be stained. The assay should be repeated under three circumstances: (I) if the system-level controls fail to show appropriate staining, (II) if the negative reagent control demonstrates a non-specific diffuse staining, and (III) if target cells fail to show appropriate staining. The slide is interpreted using a binary scoring system: positive (strong, granular and cytoplasmic staining in any percentage of tumor cells) or negative (absence of strong granular cytoplasmic staining in tumor cells). Scoring 50 tumor cells may decrease the risk of both false negative and false positive results, but there is no agreement on the number of positive cells required for a positive IHC result (15,55,56). The main advantages of IHC are that it requires only three sections of tissue (a total of 12 µm), and that the turnaround time of its analytical phase is around 4.5 hours, which allows for “real-time” results. If samples have been properly fixed (at least 6 hours in 10% neutral buffered formalin) and both types of controls are included in every patient case (system-level and negative reagent controls), this IHC approach should have very high sensitivity and specificity (15,55). Examples of ALK IHC positive and negative samples are shown in Figure 2A-2H.

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 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.

FISH

FISH using break-apart probes used to be the reference method for the identification of fusions, and as such it is still widely available and recommended by international guidelines (6,7). There is currently an FDA-approved assay (Vysis ALK Break Apart FISH Probe Kit, Abbott Molecular, IL, USA) (57). Our interpretation of the workflow is presented in Figure 3. The assay requires two sections from the FFPE block: one H&E and a second for the FISH assay. If the H&E evaluation shows that the specimen is not acceptable, a new section should be stained. A pathologist should select the best tumor area for hybridization and scoring. If all previous steps are adequate, the stained specimen is scored as depicted in Figure 3. There are two patterns of positivity: (I) break-apart or typical pattern, with one fused signal [orange (i.e., 3') and green (i.e., 5') are either overlapping, adjacent or are less than 2 signal diameters apart] and separated orange and green signals by 2 or more signal diameters; and (II) isolated orange signal or atypical pattern, with one fused signal and one orange signal without the corresponding green signal. The main advantages of FISH are that only two sections of tissue are required, and interpretation is straightforward with very high sensitivity and specificity, particularly if the ALK FDA-approved image analysis algorithm is used (Duet System, BioView, Rehovot, Israel) (15). The use of automated digital scoring workflows has highlighted the prevalence of the atypical pattern, either alone (45%) or in combination with the typical pattern (34%) (15). Finally, we must emphasize two important facts: (I) a precise annotation of the fusion variant is not possible when using IHC or FISH; and (II) detailed IHC and FISH data for most of the rare or uncommon ALK variants is currently lacking (19). Examples of ALK FISH positive and negative samples are shown in Figure 4A-4H.

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 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.

Real-time PCR

RNA-based real-time PCR assays are very popular in regions with a very high prevalence of epidermal growth factor receptor (EGFR) mutations or when tumor content, turnaround time and/or cost preclude the use of NGS (21,43,45,58,59). Taking into consideration the molecular epidemiology of ALK fusions (see above), the design (i.e., breadth) of these kits is probably not as relevant for this discussion as it is for the detection of other actionable fusions (60). Nonetheless, users of these assays should be constantly aware that “pseudo false negatives” (i.e., due to rare or uncommon fusion partners not included in the design of an assay) are unavoidable because the most widely used real-time PCR assays are designed to only identify some of the non-EML4-ALK fusion partners (21,43,45,58,59). Finally, it must be overemphasized that sometimes the fusions are detected by imbalance assessment, a strategy that is a potential source of confusion because its lower specificity (see below).

NGS

NGS should be performed using a clinically validated NGS panel, which ideally should include all guideline recommended biomarkers for patients with advanced NSCLC (6,7). Although there are no recent head-to-head comparisons of all the main available platforms using a large cohort of real-world ALK positive patients, most NGS panels should theoretically have a high performance for the detection of ALK fusions (21,22,29). From a pragmatic stand-point some comments are useful when assessing NGS options or discussing their results. DNA-only hybrid capture methods are fusion-partner agnostic but their sensitivity for the detection of actionable fusions has been challenged (61,62). This situation has prompted many institutions to combine DNA-only NGS with RNA sequencing at least in driver-negative patients. Regarding RNA-based NGS, it is worth considering three facts: (I) is the sequencing method partner-agnostic? (II) is the panel affected by a low quantity and/or quality of the RNA? and (III) what is the turnaround time, including hands-on time and interpretation time? (63). In general, hybrid-capture NGS and anchored multiplex PCR are fusion-partner agnostic but usually require higher quantity and/or quality of RNA, with longer turnaround times (63). In contradistinction, the recent arrival of ultrafast amplicon-based NGS panels is bridging the gap between real-time PCR and targeted NGS (21,52). Finally, a precise annotation of the ALK variant and the molecular context (i.e., the presence of a TP53 mutation) is becoming increasingly important for treatment selection. For example, tumors with EML4-ALK variant 3 are more prone to the development of resistance mutations (24). Accordingly, patients with variant 3 have a worse outcome (64), and frequently TP53 co-mutations are identified within this subgroup (65,66). Nonetheless, the topic is still controversial because other studies have found the same degree of benefit across variants (67), and the predictive value of rare EML4-ALK and non-EML4-ALK fusions is slowly emerging (20,25,68).

Liquid biopsies

Although liquid biopsies encompass a wide range of components, circulating tumor DNA (ctDNA) is the most widely used for detection of actionable findings in patients with NSCLC. Several methodologies for ctDNA analysis have been implemented in clinical practice and research purposes, including PCR-based assays and NGS-based methodologies (69-71). PCR-based approaches have more limited multiplexing capabilities but may deliver faster results with a higher sensitivity for the target genes. NGS-based approaches can provide comprehensive tumor genotyping at a higher turnaround time, cost and complexity (72). Three strategies have been proposed in patients with advanced NSCLC: plasma-first, sequential tissue-first and complementary tissue-first (69). Regardless of the testing strategy, three facts need to be considered to optimize the use of liquid biopsies: (I) ctDNA detection is influenced by the stage of disease (i.e., intrathoracic and intracranial disease have been associated with a higher risk of false-negative results) (73), (II) the sensitivity of ctDNA assays for the detection of ALK fusions ranges from 65% to 78% (74,75), and (III) liquid biopsies are a very effective method for assessing ALK mutations after ALK inhibition (76,77).

Discussion

Tables 3,4 provide real-world considerations to avoid false negative and false positive results when using IHC, FISH, real-time PCR or NGS (4,21-23,55,56,59,62,63,78-81). Although the main potential solutions are shown in the tables and briefly discussed below, 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” (82).

Avoiding false negative results (Table 3)

The use of pre-cut slides is a well-known source of differences in the intensity of IHC staining that can even cause unequivocally false negative results (81). Pathologists should not score ALK IHC slides with less than 50 tumor cells (56). Scoring uncommon FISH patterns can be very challenging, so the use of “molecular redundancy” is advised when encountering such cases (79,80). When using PCR-based assays (real-time PCR or NGS), a precise knowledge of the breadth of the kit/panel and its real-world performance can help rule-out a false negative result (63,78,79). From a practical standpoint, the most important question we need to consider in driver-negative NSCLC patients is: do I need to persevere in the search of actionable findings? In such circumstances, reflex testing with IHC, FISH or a different/larger NGS panel is strongly recommended (4).

Avoiding false positive results (Table 4)

Aberrant ALK expression (i.e., rearrangement negative) has been described in lung neuroendocrine carcinomas (including small cell lung carcinomas) and neural tissue (22,55). Because ALK IHC is a standalone test, reviewing the H&E before scoring the ALK IHC slide is mandatory. FISH slides that only show the atypical pattern of positivity (isolated orange signals) should ideally be scored with an image analysis algorithm to rule out a suboptimal or a very low signal intensity of the green probe, an overlooked reason for ALK FISH false positive results (23,80). Regarding PCR-based methods (real-time PCR or NGS), when an ALK fusion is identified by expression imbalance or with a low number of reads, orthogonal testing is recommended to improve specificity (21,59,63,78,79).

Strengths and limitations of this review

The main strength of this review is that we provide pragmatic and technology-agnostic solutions to avoid false negative and false positive results when detecting ALK fusions (or other actionable fusions for that matter) in patients with NSCLC.

The main limitation is that the available real-world evidence on the performance of the different methods is scarce, due to the constantly changing world of lung cancer biomarker testing.

Conclusions

Through this paper we have described the most frequently used ALK testing modalities: IHC, FISH, real-time PCR and NGS. Although most guidelines recommended broad molecular profiling for patients with advanced NSCLC, single-gene assays are still widely used across the globe. Therefore, the key factor for success (i.e., increase testing rates and avoid false positive and false negative results) is to develop sensible testing algorithms, that level the advantages and disadvantages of the different methodologies. This approach can be reconciled with the fact that RNA sequencing is now becoming the gold standard for fusion identification, because of its superior sensitivity.

Acknowledgments

Fernando Lopez-Rios thanks the Tom Crean Expedition for their constant support.

Funding: This work was partially funded by Instituto de Salud Carlos III, Fondos FEDER and Plan Estatal I+D+I 2008–2011 (No. PI11-02866), 2013–2016 (Nos. PI14-01176, PI17-01001) and 2021–2023 (No. PI22-01700); INGENIO (No. PMP21/00107) and the Next Generation EU funds; Comunidad de Madrid iLUNG Program (Nos. B2017-BMD-3884, S2022-BMD-7437) and Fundacion Mutua Madrileña (No. AP18051-2022).

Footnote

Provenance and Peer Review: This article was commissioned by the Guest Editors (Jessica J. Lin and Justin F. Gainor) for the series “ALK Positive NSCLC” published in Translational Lung Cancer Research. The article has undergone external peer review.

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-855/rc

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-855/prf

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.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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