Skip to main content

11-04-2022 | ALK-mutated NSCLC | Interview | Article

Pathologic testing for ALK and ROS1 aberrations in NSCLC


Speaker: Frédérique Penault-Llorca

Frédérique Penault-Llorca outlines the importance of the ALK and ROS1 driver alterations in NSCLC and the available methods for their detection.

This content is intended only for healthcare providers and was made possible by educational funding provided by F. Hoffmann-La Roche Ltd.


Hello. My name is Frédérique Penault-Llorca. I am a pathologist and a molecular pathologist. I work in a Comprehensive Cancer Center, Centre Jean Perrin in Clermont-Ferrand, France, and I'm in charge of the molecular diagnostic platforms in my institution. Identifying which mutation contributes to cancer development is a key step in understanding tumor biology and developing targeted therapies.

Gene alterations, such as mutations, amplification, fusions that provide a selective growth advantage and thus promote cancer development, are termed "driver alteration." Those alterations that do not promote cancer development are termed passenger alterations. They are just the reflection of genomic instability.

ALK gene is on chromosome 2. ALK gene is encoding receptor tyrosine kinase, which belongs to the insulin receptor superfamily. The protein is a transmembrane receptor, so it has an extracellular domain and hydrophobic stretch corresponding to a single plasma membrane region and an intracellular kinase domain.

This is the target for the targeted therapies. It plays an important role in the development of brain, and it exerts its effect on specific neurons in the nervous system. Sometimes this gene is rearranged, so there is a fusion with another gene, and in nonsquamous cell lung cancer ALK gene is rearranged in 5% of the cases.

The host gene is on chromosome 6. It encodes a receptor tyrosine kinase, and we don't know exactly the physiological role in humans. We have chromosomal rearrangements resulting in ROS1 fusion in 1% to 2% of nonsquamous cell lung cancer, and this is the main mechanism underlying ROS1-driven oncogenesis.

Most ROS1 mutation and amplification of an unknown significance and dysregulated host expression is probably, at most, a secondary oncogenic mediator. So for ROS1, it's really the fusion that is important in oncogenesis.

Those chromosomal rearrangements, they are the most common genetic alteration in those two genes, and they result in activation of oncogenic pathways that are important for proliferation, cell survival, so they are highly oncogenic.

When we have a fusion gene, this is giving a receptor, but it's permanently activated, and it is leading to a continuous cell proliferation signaling, and it's also interfering with normal apoptotic cell signaling. So the receptors that have been coded by those fusion genes are highly oncogenic.

ALK gene rearrangement occurs in 3% to 5% of adenocarcinoma, and ROS1 fusions in 1% to 2%, also of adenocarcinoma, and they are very important because they predict response to ALK and ROS1-targeted inhibitors.

The clinical presentation of patients with ALK and ROS1 fusions in nonsquamous cell lung cancer is quite superimposable so they are more frequently found in patients who are never smokers or light former smokers.

There is a higher incidence in young patients, higher incidence in female patients, but maybe it's due to the smoking habits, and then on morphology, they are more frequently found in solid, acinar, cribriform, papillary carcinoma, so this is more found in the Asian population.

And in the Western population, they also have another rare histology that is a signet-ring pattern, and they express by immunohistochemistry TTF-1 like many adenocarcinoma. So this is the classical clinical presentation. However, those clinical-pathological features are insufficiently sensitive to screen for testing an individual patient.

A significant proportion of ALK and ROS1 rare tumors may go undetected, and very frequently, we find those alterations in patients that are with another histology or are like 60 or 70 years old, so that's why we don't select, we don't use those clinical and pathological criteria alone for the selection of patients.

What is for sure is that ALK and ROS1 rearrangement are very infrequent in squamous cell carcinoma. They have been reported in adenosquamous carcinoma. So those adenosquamous carcinoma is a mix of squamous cell carcinoma and adenocarcinoma, and those ALK and ROS1 rearrangement are largely mutually exclusive with EGFR or KRAS mutations.

So for ALK, we have many drugs. Some of them we use for first-line therapy like alectinib, crizotinib, other that we use for a second option of first-line like ceritinib. And then, if we have a resistance, we need to rebiopsy or to perform a liquid biopsy to elucidate the mechanism of resistance.

And in particular, we have a type of mutation in ALK that is found. That is called the G1202R resistance mutation, and those ALK resistance mutation, they have different percentage of occurrence depending on the type of drug.

We can also use other drugs like brigatinib, for instance, or alectinib, and the effect of those drugs can lead to other mutations.

So in the event of a first progression under a tyrosine kinase inhibitor, it's possible to adapt the treatment to the molecular profile of resistance if this is available.

Or we can switch to another tyrosine kinase inhibitor that was not previously used but can be chosen from alectinib, brigatinib, ceritinib, or lorlatinib. For ROS1, the first-line treatment is usually based on crizotinib and the second line on lorlatinib or on entrectinib.

Again, we have, frequently, resistance. And in one third of the cases, there are resistance mutations, and the most frequent one for ROS1 is the G2032R. And also, we have other out-of-target resistance mechanism, also for ALK because it has MET amplification, so they will guide potential treatment options.

So we have several tools that we can use for testing for ALK and ROS1 alteration. We have immunohistochemistry, looking at the protein, so protein overexpression, in situ hybridization, looking at the rearrangement, or NGS. So the situation is different for ALK and for ROS1.

For ALK, immunohistochemistry may be sufficient to establish the diagnosis, subject, of course, to compliance to the quality criteria of immunohistochemistry. So if we have a strong expression of ALK, in many guidelines, we say that we can consider that this patient has an ALK rearrangement. So it's what we called a 3+ by immunohistochemistry.

It is, nevertheless, prudent because there is a non-zero risk of false positive to confirm the presence of the rearrangement by another technique, such as FISH or NGS.

But a strong immunohistochemistry for ALK could be enough to select the patient for a tyrosine kinase treatment. Of course, if it's a 2+ or a lower expression, or if it's negative by immunohistochemistry, but if it's a nonsmoker, a young patient, we will use another technique to be sure that we don't miss a patient with a rearrangement of ALK.

For ROS1, the situation is different. In fact, ROS immunohistochemistry does not currently have the same performance, and it is not sufficient. So usually, we use, in parallel, ROS1 immunohistochemistry and FISH or ROS1 immunohistochemistry and NGS.

And in the guidelines, it's very clear that platforms that cannot provide FISH or NGS must send the sample to other platforms because we really need to have ALK rearrangement and the ROS1 rearrangement status for the first-line treatment of metastatic non-small-cell lung cancer.

Right now, we consider that NGS is the gold standard because we have many targets to look at in lung cancer, several mutations such as the different mutation in EGFR, some specific mutations in RAS, alteration in ERBB2, alterations that could be also a potential resistance like MET and also fusions in ALK and ROS1, but also fusions in RET in and NTRK, in NRG1.

So today, NGS is considered as probably the best option because it's all in one test, so it's more or less the gold standard option. For NGS, we have two possibilities, broad DNA NGS or RNA sequencing.

So in fact, if a large, broad DNA NGS panel fails to identify drivers, we have to conduct RNA sequencing. Usually, it's targeted RNA sequencing to maximize the detection of fusions because we know that with DNA, we don't have the maximum sensitivity to detect fusions, so it's better to run also an RNA-Seq for more specificity.

We will use liquid biopsy in case of tissue insufficiency if it's impossible to biopsy the patient. If the patient has very low tumor content in the biopsy, so we will use liquid biopsy.

And today, also, the use of liquid biopsy is desirable when we want to monitor resistance so at progression.

And this will be, of course, very important as a first-line diagnostic tool, particularly in the nonsmoking patient, to be sure that we cover all of the potential targeted alteration in the nonsquamous cell lung cancer.

In fact, RNA today, with the huge number of targets that we have to screen for nonsquamous cell lung cancer, now is considered, when it's accessible, of course, as the gold standard for the screening of those alterations in squamous cell lung cancer, and it's, and it's better to offer both DNA and RNA NGS.

So for the first-line treatment in metastatic non-small-cell lung cancer, we must have at least EGFR status, ALK, and ROS1 status. And we need for the second line to have other biomarkers, so it's really something that we need to have at diagnosis.

And then, after progression, it's very important to be able to look at the resistance mechanism to be able to target the treatment because we have options for the resistance cases.

Unfortunately, for tyrosine kinase inhibitors, we know that, in most of the cases, we will have resistance, and those resistance can occur by different mechanisms.

So one mechanism will be on-target, so it will be through mutations in ALK genes or ROS1 gene, or It can be off-target, meaning that it will be an activation of other pathway like MET, for instance, MET amplification.

Or it can be also sometimes a change in the phenotype of the tumor. By that, I mean that an adenocarcinoma will switch to a small-cell carcinoma, and this will be the default mechanism of resistance to tyrosine kinase inhibitors.

It's important to monitor and to diagnose that because we know that the second-generation, third-generation of tyrosine kinase inhibitors can be sensitive and can work for the patient with those resistance mutations to the first-line treatment.

Copy number alterations - at the beginning for ALK, we were thinking that copy number variation, alteration, were thought, to identify patients that may respond to ALK inhibitors, but in fact, today, we have more and more data showing that could be a mechanism of resistance.

And for ROS1, copy number variation in ROS1 doesn't seem to have any impact on resistance or response to treatment. For the future, I think that ALK and ROS1 testing should be included in a targeted RNA-Seq panel at diagnosis and also for the screening for mutation and other markers at progression.

And I think that this will be through NGS so NGS on the tumor. I think it's important, at least, to have at the beginning of a disease an evaluation of the tissue because we need, also, to have other parameters. So we need to have at least one biopsy of the tissue also because we need to have PD-L1.

So this is immunohistochemistry. We need to look at that on the tissue. But after, I think that liquid biopsy should be extremely-- a noninvasive tool that should be used more.

And of course, when we don't have access to the tissue or if it will take too long to get a tissue biopsy for a patient, liquid biopsy with a large NGS panel, a broad NGS panel is also a very interesting option for our patients.

I have started to test ALK and ROS1 now for many years, and we were doing sequential testing, so immunohistochemistry then FISH. And we were doing PCR to look at KRAS, to look at EGFR, and then we had new biomarkers coming.

And then, we observed that NGS was becoming less expensive. Also, it was available, at least in my country, in France, in many places, because we had an incentive from the government to install NGS platforms.

So now I think that in a view of maximizing small biopsies, it's better now to start directly with NGS with the exception of PD-L1, because PD-L1 we need to do immunohistochemistry. But for all the other biomarkers, we can do molecular biology.

And the combination of DNA and RNA NGS is really, for me now, what should be performed because it's really cost effective, and it's cost effective in many ways. It's cost effective because it helps us to have all the biomarkers at the same time.

So it's also possible for the oncologists to foresee the second line of the third line of treatment because some drugs are not available for the first line. Not all the drugs are available as first-line options. And it's also tools that now, as molecular biologists, we know well how to use them, how to interpret data.

So we are able to give the result within 1 or 2 weeks, depending on the complexity or if we have DNA and RNA-Seq at the same time, or first DNA sequencing and then RNA sequencing. But now, with very good timelines for the management of the first-line treatment for the patient with advanced or metastatic non-small-cell lung cancer.

About the contributor

Frédérique Penault-Llorca

Professor Frédérique Penault-Llorca is Head of the Department of Pathology and Tumor Biology at the Centre Jean Perrin in Clermont-Ferrand, France.

Full biography


New additions to the Adis Journal Club

A selection of topical peer-reviewed articles from the Adis journals, curated by the editors.

Attention: This content is intended for healthcare professionals only

In order to proceed please confirm whether you are a healthcare professional.

No, I am not a
healthcare professional
Yes, I am a
healthcare professional