Making the best of PD-L1 IHC testing

The POPLAR trial, recently published, studied the biomarker for atezolizumab, finding an improving positive ratio in favor of atezolizumab response as the degree of either immune cell or tumor cell staining increases. However, Dr. Kerr noted, somewhat different numbers are used to define the cutoffs for immune cell and tumor cell scores, particularly at the high end of the scale. About one-third of the patients were deemed positive because of immune cell staining only.

In the CheckMate trials of nivolu-mab, using an anti-PD-L1 IHC assay, based on a clone known as 28-8, “we have a threshold of definition of positivity of one percent and membrane staining of tumor cells only.” But, Dr. Kerr added, “As definitions of positivity change—above one percent, above five percent, above 10 percent—the chance of patient benefit improves, though in all instances this is better than the docetaxel control arm.”

Although the numbers in the trial are small, “this rather implies that most of the effect, perhaps like pembrolizumab, is being driven by the patients who are highly expressing PD-L1.” (This pattern is for non-squamous NSCLC. Use of PD-L1 IHC for squamous NSCLC is not required, he noted, for nivolumab because the biomarker is not predictive in this setting.)

Some alternative biomarkers are showing promise, Dr. Kerr reported. Immune gene signatures, which measure expression genes by messenger RNA extracted from tumor samples, are one example. Far more data are needed, in part because mRNA is more complicated and may be more fragile to extract in adequate quantity and quality from diagnostic samples. “But it’s certainly an interesting alternative for the future,” he said.

Immune cells themselves, in addition to PD-L1 expression, are also being considered as potential biomarkers. “For example, looking at the presence or absence of particular types of immune cells and where the infiltrate might be in the tumor: Is it amongst the tumor cells or is it only adjacent to the tumor? We know that these factors are prognostic. Are they also predictive of response to therapy? Again, we need more data to draw reasonable conclusions.”

Interferon gamma is also an important regulator of PD-L1 expression, he said. “So more studies are now looking at the presence or absence of other immune checkpoints and interferon gamma expression, possibly as an adjunct to the PD-L1 expression as a biomarker test for these therapies.”

The concept of mutation burden is a fourth possibility, Dr. Kerr said. One study looked at mutation burden as measured by extensive next-generation sequencing or by mutation profiles from the point of view of a molecular smoking signature. “Both of these factors are associated with an improved response or can select a group of patients who seem to respond better to immunotherapy.” Mismatch repair genes and microsatellite instability also have been shown to be associated with improved response in other tumor types. “So at least some of these alternatives may well be usable factors for us in the future.”

Optimally, a biomarker would indicate either a high probability of a patient benefiting from a drug or no probability of benefit, he said. Unfortunately, “PD-L1 IHC represents a biological continuum of protein expression from very low levels through moderate to very high levels. So where do we define our positive and negative groups? We know that different drugs and different trials at different times have used different cutoffs defining positive and negative groups of patients.”

This variability creates a biological scenario that is completely different from the addictive oncogene situation, Dr. Kerr explained. “If we use a cutoff, say, of 50 percent to define a group of patients to whom we would give the drug, the patient who has 52 percent as deemed by the pathologist is going to get the drug. How different is that patient from one who is deemed to have a score of, say, 48 percent who is not going to get the drug? Of course we know the answer to this. Biologically, there is really no significant difference between these two patients, but one will be treated and one will not.”

“This is one of the fundamental reasons why PD-L1 does not look as good to our oncologist colleagues when they compare it to the performance of EGFR mutations or ALK translocation. We have a kind of dose-response relationship to the amount of PD-L1,” which has been consistently shown in all the trials.

Moreover, PD-L1 is heterogeneous and dynamic. In fact, Dr. Kerr said, “Somewhat paradoxically, if you have a large area of tumor to examine with one of these stains, heterogeneity of expression is actually the norm.” Pathologists should be thankful that intensity of expression is not part of this particular biomarker, he added. “It is the proportion of tumor cells that show any staining that is considered the thing we should measure.”

Still, “Heterogeneity and dynamism of expression are not friends as far as the pathologist is concerned when measuring this expression. Sampling error is going to occur, that is for sure.” These factors might have greater impact on lower thresholds, he said, making the biomarker perhaps appear worse than it really is.

The black marks against PD-L1 are easily summarized, Dr. Kerr demonstrated. “Is the drug targeted in this scenario a singular factor in our target system? Absolutely not. Is the biomarker present or absent? No. Is the biomarker stable and functionally unique? No. Is the biomarker easily measured? Well yes, it is, relatively speaking. But it’s not 100 percent predictive.”
PD-L1’s variability, in fact, makes its potential use with other cancers, such as urothelial cancer or squamous cell carcinoma of the head and neck, unclear. “PD-L1 expression shows predictive value in some cancers but not in others. It’s a very curious mixture of signals that we are receiving, and for some oncologists, this is a sign that it’s not a very good biomarker.”

However, “At the moment PD-L1 IHC is all that we really have to go by. We have to make the best of it for the five drugs now available or expected to come along,” he said.

The problem is, “With each drug, we have trials using a different clone, variably different detection systems, and variably different definitions of positivity or scoring mechanisms. This is a huge headache for the pathology world. How are we going to deal with this scenario of four or five drug-assay combinations?”

For one thing, laboratories often have only one staining platform available. It’s unlikely that most laboratories will provide the multiple platforms that would be required, he said. “Other big questions concern how different these assays actually are, and the possibility of laboratory-developed tests, which could be built around trial-validated clones or completely different antibody clones.”

The outcome of any IHC test is a function not only of the primary antibody that is used but also the detection system and all the chemistry that goes behind it, Dr. Kerr said. “So what chance is there that we might have one test to select a variety of different drugs? Is all PD-L1 IHC the same? The answer is definitely no.”

However, he suggested, there is some possibility that one IHC test could be used but scored in multiple different ways to select drugs that are predicated on different definitions of positivity. If that were the case, “pathologists might have to report the PD-L1 IHC according to the number of positive cells indicating a variety of thresholds, and we might actually have to mention individual drugs in our report—something which, in general, we have not done to date.”

For those contemplating developing a test from scratch, “I think we all have to ask ourselves: How safe is it to deviate from trial-validated practice with our current state of knowledge? I don’t have the answer to that question.”

A study known as the BLUEPRINT project, initiated by the International Association for the Study of Lung Cancer and in which several major cancer groups, diagnostic companies, and pharmaceutical firms were involved, found that three of four assays were remarkably similar when the staining of tumor cells was compared, with a close correlation between 28-8, 22C3, and SP263 results. Similar results have been reported in other studies. However, it is difficult to draw conclusions from these data at the moment, Dr. Kerr said.

Immunotherapy drug availability is another factor. “But as we move these drugs into first-line therapy, I think the dynamic around testing will change, and this will change the way pathologists will have to approach this particular marker.”

In the context of biomarker testing, Dr. Kerr said, after the diagnosis of lung cancer is made, “biomarker testing based on IHC may have to incorporate PD-L1 IHC into the overall testing strategy as part of the panoply of tests we may have to deliver. The biological rationale for this therapeutic approach is that the biomarker test is based on our understanding of differences in antigenicity, evidence of immune response, and evidence of an inhibitory mechanism that may be interrupted by the therapy.”

There is skepticism around immunohistochemistry within the oncology community, Dr. Kerr acknowledged. “We’ve had some rough times in the lung cancer world, and I am often reminded of what happened with HER2 testing in breast cancer. That was not a happy situation in the early days, but I think it’s now very much better.”

For PD-L1 testing, he stressed the value of two things: consistent, quality-assured materials for use in testing, and education and training in how to read the slides. Despite the challenges, he noted, a German study found that after training, pathologists who scored cases in 10-percent brackets had very good concordance. “I cannot emphasize enough the importance of training before you begin.”

For the majority of patients, Dr. Kerr said, it’s obvious whether they are above or below the threshold. But even if the test is carried out exactly in the appropriate way, “the human eye and brain are going to have an issue around the allocation of a case that is very close to the threshold.” With a continuous scale, “there is always going to be a gray zone around the threshold and we have to make the call. And we do our best to make that call using all the techniques and skills and experience we’ve developed.”

PD-L1 IHC is a realistic marker, Dr. Kerr concluded. “But in a very complex environment with multiple drugs and assays, there is no doubt that PD-L1 testing presents us with issues. We have to be practical and realistic in our expectations of this particular biomarker.”
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Anne Paxton is a writer and attorney in Seattle.