CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
Sherrie Rice
April 2020—IMpassion130 was the first phase three trial to demonstrate a clinical benefit of cancer immunotherapy in patients with PD-L1-positive metastatic triple-negative breast cancer, and based on the data, atezolizumab plus nab-paclitaxel is approved for this indication.
In the trial, the Ventana SP142 PD-L1 assay with a one percent or greater cutoff was used to evaluate PD-L1 expression in immune cells. But questions remained about how to best identify patients who could benefit from the drug combination, Hope S. Rugo, MD, professor of medicine and director of breast oncology and clinical trials education at the University of California San Francisco Comprehensive Cancer Center, said in a CAP TODAY webinar last November.
To find answers, she and colleagues at institutions in the U.S. and abroad conducted a retrospective post hoc IMpassion130 analysis in which the Dako 22C3 and Ventana SP263 PD-L1 assays were studied for their analytic concordance with SP142. Their results were reported at the ESMO2019 conference last fall.
Dr. Rugo joined Gary Tozbikian, MD, assistant professor and director, Division of Breast Pathology, Department of Pathology, Ohio State University Wexner Medical Center, in the webinar, made possible with support from Genentech.
Dr. Rugo
“The data support that at this point other PD-L1 assays are not interchangeable with SP142,” said Dr. Tozbikian, who provided case examples to demonstrate how the stain is performed and interpreted in clinical practice.
For the post hoc analysis, Dr. Rugo and colleagues looked at PD-L1 by the three IHC assays. “These assays were performed using the respective package inserts,” Dr. Rugo said, “and each slide was read by a single pathologist out of a panel of eight pathologists, all of whom were trained and qualified to read all three assays.”
A positive result for SP142 and SP263 is defined as PD-L1 immune cell positivity in at least one percent of the tumor area. For the Dako 22C3 assay, a combined positive score, or CPS, is used. This includes the number of PD-L1-positive tumor cells, lymphocytes, and macrophages divided by the number of viable tumor cells and multiplied by 100.
“For the purpose of this retrospective post hoc analysis,” Dr. Rugo said, “we used our biomarker-evaluable population, or BEP. This population included 68 percent, or 614 patients, from the intention-to-treat IMpassion130 population who had adequate tissue available to perform this comparison using three different assays.”
In the biomarker-evaluable population, 46 percent of samples were PD-L1 immune-cell–positive versus 41 percent in the intention-to-treat analysis, and the improvement in progression-free survival in the biomarker-evaluable population with the addition of atezolizumab was slightly greater than in the overall population. All other evaluated baseline characteristics were balanced between the BEP and ITT.
The different assays identified different percentages of cases that were positive for PD-L1. More tumors were classified as positive for PD-L1 using the two alternative antibodies and assays (22C3, 81 percent; SP263, 75 percent) than SP142 (46 percent).
“Interestingly, almost all SP142-positive cases are captured by either the 22C3 CPS or the 22C3 assay using the respective cutoffs. Only one percent of tumor samples were positive only using SP142,” Dr. Rugo said.
This combination positivity leads to a suboptimal analytical concordance or overall percentage agreement of only 64 to 69 percent, she said. “An additional 36 percent and 30 percent of cases were positive only for 22C3 CPS or 263, respectively.”
Previous studies demonstrated a correlation between the percent of stromal tumor-infiltrating lymphocytes and clinical outcome in triple-negative breast cancer. In IMpassion130, patients whose tumors tested positive for PD-L1 immune cells using SP142 and one of the other two antibodies, classified as double-positive, had the highest percentage of TILs compared with those whose testing was positive by only one of the other two assays.
“The analysis found an absolute difference in progression-free survival for tumors that were SP142-positive—46 percent of our biomarker population—of 4.2 months, and an absolute difference in overall survival in this subset of our population reported in the overall intention-to-treat analysis of 9.4 months,” Dr. Rugo said.
In contrast, the absolute benefit using 22C3 CPS, which was 81 percent of the BEP, or SP263, which was 75 percent of the BEP, was only 2.1 or 2.2 months for progression-free survival and 2.4 or 3.3 months for overall survival, respectively.
They then looked at both of the combinations of the two different assays and clinical outcomes. Again, more patients had tumors positive for PD-L1 using 22C3 and SP263. “So we’re first looking at the 22C3 CPS of one or greater assay and at tumors that were double-positive for SP142 and 22C3, compared with tumors that were positive only for 22C3 and then the double-negatives.”
In the double-positive population (SP142 and 22C3), the absolute difference in progression-free survival is 4.4 months and overall survival 9.3 months, and this is similar to the finding for tumors positive by SP142 alone.
“In contrast, the differences narrow and become less clinically important in tumors that are single-positive for 22C3 at 1.7 months and essentially no difference for PFS and OS, respectively. And then, as expected based on our overall analysis of this sample and our previously reported data, there was no difference in patients who were double-negative for both assays,” Dr. Rugo said.
The difference in the SP142 and SP263 double-positives that represent a larger population, 45 percent, is 4.2 and 9.4 months for progression-free survival and overall survival, respectively. The difference essentially goes away for the tumors that are SP263-positive alone or, of course, double-negative.
Also studied was the source of tumor and its relationship to outcome in IMpassion130.
Overall, tissue obtained from the breast was more likely to be PD-L1–positive than tissue obtained from different metastatic sites, at 44 percent and 36 percent, respectively.
However, the median time of sample collection to randomization was only 61 days, suggesting that most primary tissue samples were obtained in the metastatic setting.
PD-L1 status also varied by the anatomic site from which tissue was obtained. “Interestingly, the least likely organ to have tissue that was positive for PD-L1 was liver,” Dr. Rugo said, “although this represented only five percent of the total tissues analyzed.” This difference in PD-L1 positivity has been shown in other studies and in other diseases, she said.
Regardless of the source of tissue, however, PD-L1 status predicted benefit from atezolizumab.
Dr. Rugo summed up the findings of the post hoc exploratory biomarker substudy of the IMpassion130 trial:
- Clinical activity was seen in the SP142 PD-L1 immune-cell–positive subgroup regardless of whether the sample came from the primary tissue or metastatic tissue. “Although it’s important to keep in mind that the majority of tumor tissues that were used for PD-L1 testing were obtained within two months of study start,” she said, “we don’t know that there are significant differences between tumor tissue that was obtained a longer time from when you’re intending to analyze and start new treatment.”
- With overall percentage agreements of 64 percent for 22C3 and 69 percent for SP263, the analytic concordance was subpar at less than 90 percent, “and the assays are clearly not equivalent.” “22C3 with a CPS of one or greater and SP263 with an immune cell count of one percent or greater for PD-L1 assays identified a much larger patient population, and the SP142 immune cell one percent or greater group is a subgroup of this larger population.”
- The clinical benefit in 22C3-positive and SP263-positive subgroups was driven by the SP142-positive subgroup. This assay identifies patients with the smallest hazard ratio point estimates and the longest median progression-free and overall survival from atezolizumab and nab-paclitaxel.
- In the United States, the SP142 assay is the approved diagnostic test used to identify patients with metastatic triple-negative breast cancer most likely to benefit from the addition of atezolizumab to nab-paclitaxel.
The staining instrument is the BenchMark Ultra, and it includes the specific staining protocol interpreted according to the interpretation guide. If any component is changed, the lab has to validate the changed system component.
The acceptable specimens for PD-L1 testing are formalin-fixed and paraffin-embedded, archival tissues or tissues obtained recently from resections, excisions, or biopsies, and they can come from primary or metastatic sites. To be considered adequate, they need to contain at least 50 viable tumor cells with associated stroma. “For that reason, cytology specimens are unacceptable. Likewise, any decalcified bony specimens are not acceptable due to lack of validation.”
As for other breast biomarkers, tissues should be fixed in 10 percent neutral buffered formalin with a six- to 72-hour fixation range.
In Dr. Tozbikian’s experience, preanalytics tend to be more of an issue for metastatic cases where a history of breast cancer or a breast cancer met may not be suspected and adherence to fixation requirements may not be 100 percent. “That is something to consider when you are doing PD-L1 testing and considering what specimens to perform the test on,” he said.
When possible, freshly cut unstained slides should be used for the SP142 test. “Try to avoid testing on any archived, precut unstained slides, especially those that have been stored for longer than two months, as these can show staining degradation due to cut sample instability.” In some situations, older archived material may be all that is practically available, but in that case, he advises using freshly cut slides from those stored blocks.
‘You will pick up many more positive cases by looking at the immune
cell component.’
Gary Tozbikian, MD
The prevalence of PD-L1 expression in the immune cell or tumor cell component differs by tumor type. In urothelial carcinoma, non-small cell lung cancer, and triple-negative breast cancer, PD-L1 expression was found to be more prevalent in the immune cell component than the tumor cell component, according to data from the IMvigor210, POPLAR, and IMpassion130 trials, respectively.
For triple-negative breast cancers, 41 percent showed PD-L1 positivity in the immune cell component versus only nine percent in the tumor cell component. “So you will pick up many more positive cases by looking at the immune cell component,” Dr. Tozbikian said.
The 41 percent positive rate is an important number to note, he said. “If you were to initiate testing at your lab and ask the question, ‘When I perform this test using SP142 on triple-negative breast cancers, what positive rate should I expect to observe?,’ the answer is 41 percent. This was the rate that was observed from the data in IMpassion130, and it is the best available current benchmark or positive rate to reference.”
In the triple-negative breast cancers, when the tumor cells were positive, frequently the immune cell component was also positive. Lung carcinomas showed relatively less overlap. “When it comes to PD-L1 testing,” Dr. Tozbikian said, “being aware of which cell component—tumor cell or immune cell or both—is relevant and used for scoring is important, especially since expression rates and scoring systems can differ by site of origin.”