Alexander M. Castellino, PhD

April 21, 2016

NEW ORLEANS — Two immunotherapies that target the cell programmed death (PD) pathway are now available, and both nivolumab (Opdivo, Bristol-Myers Squibb Company) and pembrolizumab (Keytruda, Merck Sharp & Dohme Corp) are approved for treating advanced, refractory, non–small cell lung cancer (NSCLC). Across several studies in patients with NSCLC, response to these agents has been correlated with PD-L1 staining, which determines PD-L1 levels in the tumor tissue. How do the available assays for PD-L1 compare?

The linear correlation between three commercially available assays is good across a range of cutoff points, concluded a presentation at the 2016 American Association for Clinical Research Annual Meeting.

Cutoffs are defined as the percentage of cells expressing PD-L1 when analyzed histochemically. "The dataset builds confidence that the assays may be used according to the cutoff clinically validated for the drug in question," Marianne J. Radcliffe, MD, diagnostic associate director at AstraZeneca, told Medscape Medical News.

"The correlation is good between the assays across the range examined," she added.

However, a recently published study showed a high rate of discordance between another set of PD-L1 assays that were tested.

Dr Marianne Radcliffe

"Different diagnostic tests yield different results, depending on the cutoff for each assay. We need to harmonize the assays so clinicians are talking about the same thing," Brendon Stiles, MD, associate professor of cardiothoracic surgery at Weill Cornell Medicine and New York-Presbyterian Hospital, New York City, told Medscape Medical News.

For Dr Stiles, these studies raise the issue that it is difficult to compare results of diagnostic testing across the different drugs and even with the same drug that are derived from different assays. "More importantly, it raises confusion in clinical practice when a patient's sample stains positive for PD-L1 with one assay and negative with another," he said.

"The commercial strategy for developing companion diagnostics for each drug is not in the best interests of the patients. It generates confusion among both clinicians and patients," Dr Stiles commented. "We need to know if these assays can be used interchangeably," he said.

As new agents come into the clinic, Dr Stiles believes there should be a universal yes-or-no answer, so that clinicians can use the assay to help decide on the use of immunotherapy.

Three Assays Tested

The study presented by Dr Radcliffe and colleagues investigated three commercially available assays, Ventana SP263, Dako 22C3, and Dako 28-8, with regard to how they compare at different cutoffs. Different studies use different cutoffs to express positivity.

Ventana SP263 was developed as a companion diagnostic for durvalumab (under development by AstraZeneca) using a rabbit monoclonal antibody. Positivity is defined as ≥25% staining of tumor cells.

Dako 22C3 was developed, and is approved, as a companion diagnostic for pembrolizumab. It uses a mouse monoclonal antibody. Positivity is defined as ≥1% and ≥50% staining of tumor cells.

Dako 28-8 was developed as a companion diagnostic for nivolumab and uses a rabbit monoclonal antibody (different from the one used in the Ventana SP263). In clinical practice, this assay is used as a complementary diagnostic for nivolumab, but the drug is approved for use regardless of PD-L1 expression. Positivity is defined as ≥1%, ≥5%, or ≥10% staining of tumor cells.

Ventana SP142 was not included in the study because it is not commercially available, Dr Ratcliffe indicated.

Comparing the Assays

The three assays were used on consecutive sections of 500 archival NSCLC tumor samples obtained from commercial vendors. A single pathologist trained by the manufacturer read all samples in batches on an assay-by-assay basis. Samples were assessed per package inserts provided by Ventana and Dako in a Clinical Laboratory Improvement Amendments program-certified laboratory.

Dr Ratcliffe indicated that between reads of samples from the same patient, there was a washout period for the pathologist to remove bias.

The NSCLC samples included patients with stage I (38%), II (39%), III (20%), and IV (<1%) disease. Histologies included nonsquamous (54%) and squamous (43%) cancers.

All three PD-L1 assays showed similar patterns of staining in the range of 0% to 100%, Dr Ratcliffe indicated.

The correlation between any two of the assays was determined from tumor cell membrane staining. The correlation was linear with Spearman correlation of 0.911 for Ventana SP263 vs Dako 22C3; 0.935 for Ventana SP263 vs Dako 28-8; and 0.954 for Dako 28-8 vs Dako 22C3.

"With an overall predictive value of >90%, the assays have closely aligned dynamic ranges, but more work is needed," Dr Ratcliffe said. "In general, scoring of immunohistochemical assays can be more variable between 1% and 10%, and we plan to look at this in more detail," she said. These samples need to be reviewed by an independent pathologist, she added.

Dr Radcliffe said that currently, "Direct clinical efficacy data supporting a specific diagnostic test should still be considered as the highest standard of proof for diagnostic clinical utility."

Why Correlations Are Needed

Pembrolizumab is approved for use only in patients with PD-L1-positive, previously treated NSCLC. A similar patient profile is being considered for nivolumab, for which testing for PD-L1 expression is not required.

For new PD-immunotherapy agents in clinical development, it is not clear whether PD-L1 testing will be mandated.

However, in clinical practice, it is clear that some patients respond to therapy, even if they are PD-L1 negative, as defined from the study. "Is it a failure of the assay, tumor heterogeneity, or is there another time point when PD-L1 expression is turned on?" Dr Stiles asked.

Dr Stiles also pointed out that a recent publication from Yale researchers showed a high a rate of discordance. In this study, PD-L1 expression was determined using two rabbit monoclonal antibodies. Both of these were different from the ones used in the Ventana SP263 and Dako 28-8 assays.

In this study, whole-tissue sections from 49 NSCLC samples were used, and a corresponding tissue microarray was also used with the same 49 samples. Researchers showed that in 49 NSCLC tissue samples, there was intra-assay variability, with results showing fair to poor concordance with the two antibodies. "Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%.

"Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way," the study authors conclude.

The Blueprint Proposal

Coincidentally, a blueprint proposal was announced here at the AACR meeting at a workshop entitled FDA-AACR-ASCO Complexities in Personalized Medicine: Harmonizing Companion Diagnostics across a Class of Targeted Therapies.

The blueprint proposal was developed by four pharmaceutical giants (Bristol-Myers Squibb Company, Merck & Co, Inc, AstraZeneca PLC, and Genentech, Inc) and two diagnostic companies (Agilent Technologies, Inc/Dako Corp and Roche/Ventana Medical Systems, Inc).

In this proposal, the development of an evidence base for PD-1/PD-L1 companion diagnostic characterization for NSCLC would be built into studies conducted in the preapproval stage. Once the tests are approved, the information will lay the foundation for postapproval studies to inform stakeholders (eg, patients, physicians, pathologists) on how the test results can best be used to make treatment decisions.

The blueprint proposal is available online.

Dr Ratcliffe is an employee and shareholder of AstraZeneca. Dr Stiles has disclosed no relevant financial relationships.

American Association for Cancer Research (AACR) 2016 Annual Meeting: Abstract LB-094, presented April 18, 2016.

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