PD-L1 Testing by Immunohistochemistry

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Anja Roden Profile

Anja Roden, M.D.

Professor of Laboratory Medicine and Pathology
Director, Immunohistochemistry Lab
Division of Anatomic Pathology
Mayo Clinic, Rochester, Minnesota



Hello, I am Dr. Anja Roden. I'm a professor of pathology, and I'm one of the thoracic pathologists, and I'm also involved in surgical pathology, autopsy pathology, and thoracic transplant pathology here at Mayo Clinic in Rochester. I am one of the medical directors of the Immunohistochemistry Lab, and I am also the fellowship director of the Pulmonary Pathology Fellowship. Today we will be talking about PD-L1 testing by immunohistochemistry.


I have nothing to disclose. 


We will talk about the role of the PD-L1 interaction in the immune system. And then we will discuss challenges of PD-L1 testing and I will show you the current PD-L1 companion testing in various tumor types.

Immune checkpoint inhibitors

PD-L1 is located on the tumor cell and PD-1 is located on the T-cell. If PD-L1 and PD1 interact, this will lead to hampering of the T-cell and therefore to tumor growth. Because of that, anti-PD1 or anti PD-L1 drugs have been developed to specifically block the PD-1 PD-L1 interaction, and therefore boost the host anti-tumor immune response and inhibit tumor growth. Responses actually have been seen in various tumors. For instance, in non-small cell lung carcinomas, we not only see responses in adenocarcinomas but also in squamous cell carcinomas, which now will give us an additional treatment for squamous cell carcinomas, for which we otherwise don't have a good targeted treatment.

Anti-PD-1 vs. Chemotherapy

Probably one of the seminal studies was by Reck and colleagues that had been published in the New England Journal of Medicine in 2016. This was a phase three randomized trial of over 300 patients with untreated stage IV non-small cell lung carcinoma. All of these tumors had PD-L1 expression using clone 22C3 in at least 50% of tumor cells. These patients then were randomized into either Pembrolizumab treatment, which is anti-PD-1, or chemotherapy. And as you can see in this graph, patients who received Pembrolizumab had a significant better progression-free survival and overall survival, than patients who were treated with chemotherapy.

PD-L1 testing

Many of these studies have been performed in various tumor types and various clones have been used for PD-L1 testing in these studies and therefore, now various PD-L1 clones have been associated with various drugs corresponding to all these clinical trials. Before we go on, we will just talk about what a companion test is and what a complimentary test is. A companion test is PD-L1 testing using an FDA specified clone, and this testing is required before the oncologist can treat the patient with the anti-PD-1 or PD-L1 treatment. In contrast, a complimentary test is PD-L1 testing using an FDA-specified clone, but this testing is only strongly encouraged before treatment, but does not necessarily have to happen. Today, we will be focusing on companion testing.

Definitions for reporting of PD-L1 companion test results

Here are some definitions that we need to know when we report PD-L1 companion test results. We report either tumor proportion score, or TPS, combined positive score, or CPS, percentage of PD-L1 expressing tumor cells, % TC, which is similar to the tumor proportion score, or we report proportion of tumor area occupied by PD-L1 expressing tumor-infiltrating immune cells, which is the % IC. 

Definitions in PD-L1 testing

Any membranous staining counts. So the entire tumor cell does not have to stain, it's enough if only a part of the membrane of the tumor cell stains. We do have to have 100 viable tumor cells to actually interpret the results. If we deal with a metastasis, we need to use the interpretation guidelines of the primary tumor. So for instance, if I see a non-small cell carcinoma of the lung that has metastasized to the liver, and there is a liver biopsy of that tumor, I need to use the interpretation guidelines of the non-small cell lung carcinoma, not of liver carcinomas.

Tumor proportion score

The tumor proportion score, or TPS, is calculated by the number of PD-L1 positive tumor cells divided by the total number of all tumor cells multiplied by 100. 

Here's an example. We appreciate lung adenocarcinoma by these neoplastic glands. We also have another neoplastic gland here. On high power we have the typical tumor cells forming glandular structures, as we would expect in adenocarcinoma. Using clone 22C3 for PD-L1 staining, we can appreciate quite a bit of staining and actually all the tumor cells are positive for PD-L1. As you can see, not the entire tumor cell is surrounded by membranous PD-L1 staining, but the amount of PD-L1 staining is enough on this part of the membrane of the tumor cell. This was a TPS of 100%, as all the tumor cells are positive for PD-L1.

Here's an example of lung adenocarcinoma, metastasizing to the axilla. There are a few areas in which we see carcinoma in these circles here, but you can also see that the carcinoma is associated with lymphocytes as you would expect in an axillary lymph node. And here is another focus of adenocarcinoma. Looking at the PD-L1, again using clone 22C3, we see quite a bit of staining, but if we go on higher power, you can appreciate that the staining is actually in the immune cells, specifically in the lymphocytes, but the tumor cells are actually negative. Also here on the left side, the tumor cells do not show membranous staining. We do see only staining in immune cells. So overall, the tumor proportion score was 0% in this example.

Combined positive score

The combined positive score, or CPS, is calculated by the number of PD-L1 positive cells, including tumor cells, lymphocytes, and macrophages divided by the total number of viable tumor cells multiplied by 100. So for the CPS, you actually can get a result that is greater than 100. 

Immune cells with PD-L1 expression

Just to show you how immune cells will look like with PD-L1 expression, here are essentially lymphocytes, which show PD-L1 expression. 

This is an example of an adenocarcinoma, and we do appreciate all these neoplastic glands, but there are only a few foci of lymphocytes as outlined here by the arrow. On high power, we can see a neoplastic gland, which is associated with a lymphocytic infiltrate.

Now looking at the PD-L1 stain, the red outlines the entire tumor area on the right side. We appreciate the PD-L1 staining, which only stains the immune cells. It also stains a few macrophages here, but it does not stain the tumor cells. Those are negative. So looking again at our tumor area, about 10% of the tumor area is highlighted by the PD-L1, and the PD-L1 in this case only stained the immune cells. So the percent I see is 10 for this example. 

NSCLC companion tests

Here are the current companion tests that are published by the FDA. So on the left side, you see the anti-PD-1 or anti-PD-L1 drug. In the middle is the required clone with which we have to test the tumor so that the oncologist can actually treat with the associated drug on the left side. And for the oncologist, the cutoff of the reporting is really important.

So let's take the example of Pembrolizumab, which is the drug which requires testing of the tumor, using clone 22C3. And we have to report the tumor proportion score. If the oncologist will use it as a second-line therapy, then the tumor has to express at least a score of 1%. So that means at least 1% of the tumor cells have to be showing expression of PD-L1. If the oncologist wants to use the Pembrolizumab in the first line therapy, then at least 50% of the tumor cells have to show expression of PD-L1. You see a few other medications here on the left side that can be used in non-small cell lung carcinoma, such as Cemiplimab, which also requires clone 22C3 reporting TPS. Or Nivolumab combined with an anti-CTLA-4, which requires clone 28-8, and we have to report the TC percentage. Atezolizumab can be used with clones SP142 or SP263. If SP142 is ordered, we have to report a TC and an IC. If clone SP263 is ordered, we have to report a TC and then the clinician will use our reporting to make sure that they really can use that medication. 

Tumor companion tests

These reporting guidelines, the clones, and the cutoffs are different for different tumors. So looking at esophageal squamous cell carcinomas, if the oncologist wants to treat with Pembrolizumab, we have to test using clone 22C3, and we have to report a CPS. If the CPS is 10 or above, then the clinician can use Pembrolizumab for the treatment of esophageal squamous cell carcinoma. Now, the cutoff is different for cervical carcinomas. And then looking at urothelial carcinoma, if the clinician wants to use Atezolizumab, then the clone SP142 has to be used for testing, and we have to report an IC. And if the IC is 5% or over, then the clinician can use Atezolizumab in that patient for the treatment. 

Head & neck SQCC companion tests

And here are the treatment requirements for the head and neck squamous cell carcinomas for Pembrolizumab and Nivolumab. If Pembrolizumab is supposed to be used, we have to use clone 22C3 and report a CPS. If Nivolumab is supposed to be used, we have to test with clone 28-8, and we have to report a TC.

For triple negative breast carcinoma, currently only FDA approved, is Pembrolizumab using clone 22C3 using a cutoff of CPS. Now these requirements change quite frequently. For an up-to-date list, you can go to this FDA webpage and look up the up-to-date testing requirements. You may notice that for instance, for triple-negative breast carcinomas in the past, the clone SP142 was also used, but this was actually withdrawn by the company. And so this has been taken off from the FDA webpage. Very similar in some other tumors, it has been shown that testing is actually not necessary and the oncologist can treat without the testing. And therefore this will not be requiring the PD-L1 testing of the tumor anymore. But again, all of this information can be looked up on the FDA webpage.

Macrophages express PD-L1

There are a few pitfalls in the review of PD-L1 testing. One are the macrophages. So macrophages can actually resemble tumor cells, and therefore we have to correlate our PD-L1 slide with a corresponding H&E slide to differentiate between macrophages and the tumor cells. So for instance, in this case, the macrophages strongly stain with PD-L1, but the tumor cells are negative. 

Good positive controls are tonsils. That's what we use. And of that you can see an example here, and we have a positive control on each slide. Placenta is also possible as a positive control.

PD-L1 clones & expression on tumor cells4

There are some PD-L1 clones which are highly comparable, and these clones include 22C3, 28-8, and SP263. However, these clones should not be interchanged with clone SP142, because it has been shown that SP142 has a much lower sensitivity. These clones also should not be interchanged with clone 73-10, because that clone has been shown to have a higher sensitivity. 

Here's just an example. The same tumor has been stained with SP263 here on the left side and SP142 on the right side. And you can appreciate that the staining intensity and the amount of tumor cells staining is much higher in SP263 here on the lower left-hand side than for SP142 here on the lower right-hand side. So, you would get different results for the PD-L1 scoring.

Heterogeneity of PD-L1 expression5

There's also heterogeneity of PD-L1 expression, specifically within a single tumor. The expression can be focal or patchy. Also it has been shown that PD-L1 expression may be different between independent primary non-small cell lung carcinomas. And indeed, actually in the study, it shows that the agreement in PD-L1 expression between independent primary non-small cell lung carcinoma was only about 50%. So that means if you have multiple independent primary non-small cell lung carcinomas in one specimen, you probably should test all these primary tumors. In contrast, there seems to be a high level of agreement between intrapulmonary metastasis, at least in that one study, the agreement was almost 90%. So overall, sampling may be an issue.

Heterogeneous expression of PD-L1

Here's just an example of heterogeneous expression of PD-L1. This is actually an example of a mesothelioma and we see a lot of tumor here on the H&E. 

If we look on the PD-L1 stain, we see quite patchy staining, and you could imagine if the needle core biopsy would go this way, you might have a different result than if the needle core biopsy would be placed this way. So again, sampling may be an issue. 

PD-L1 companion diagnostics-testing

Also, for PD-L1 companion diagnostics, per FDA, only formalin-fixed paraffin-embedded tissue is actually approved for PD-L1 companion diagnostics. We do test cell blocks and decalcified tissue, even though it's not FDA approved. However, if the results are negative, then another sample, ideally an FFPE tissue block, should be actually tested. Also old FFPE tissue blocks may show reduced or no staining. So again, if the tissue is old, then a newer test or a new specimen should be tested. And actually, as a good rule of thumb, the latest specimen, a recurrence or a metastasis, if tissue is available, should be tested in addition possibly to the original tumor.

PD-L1 evaluation – Our approach

So our approach is, we actually have the luxury that we have all the FDA approved clones available in our lab including 22C3, 28-8, SB263 and SP142. We really need the oncologist involved in the test requisition. The oncologist actually has to request the clone based on what the intended treatment is. We as pathologists then will report the PD-L1 based on the primary tumor site. So that means we have to decide whether we report a TPS, a CPS, a % TC, or a % IC. And we report according to the FDA companion diagnostic guidelines.

Take-home message

So in summary, multiple PD-L1 clones are available for testing. Staining may be heterogeneous and therefore, sampling may be an issue. And the oncologist needs to be involved in the test planning. Specifically, the oncologist needs to communicate which clone should be used for the test and which specimen should be tested. 

Thank you

And with that, please reach out to me by email or a phone call, if you have any questions. And if you are interested, here are some references for you. Thank you very much.


1. Bang YJ, Im SA, Lee KW, et al. Randomized, double-bling phase II trial with prospective classification by ATM protein level to evaluate the efficacy and tolerability of olaparib plus paclitaxel in patients with recurrent or metastatic gastric cancer. J Clin Oncol. 2015;33:2858-65.

2. Kerr K, Nicolson M. Non-small cell lung cancer, PD-L1, and the pathologist. Arch Pathol Lab Med. 2016;140:249-54.

3. Ilie M, Hofman V, Dietel M, et al. Assessment of the PD-L1 status by immunohistochemistry: challenges and perspectives for therapeutic strategies in lung cancer patients. Virch Arch. 2016;468:511-25.

4. Tsao M, Kerr K, Kockx M, et al. PD-L1 immunohistochemistry comparability study in real-life clinical samples: results of blueprint phase 2 project. J Thorac Oncol. 2018;13:1302-11.

5. Mansfield A, Murphy S, Peikert T, et al. Heterogeneity of programmed cell death ligand 1 expression in multifocal lung cancer. Clin Canc Res. 2015;22(9):2177-81.


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