PD-L1 testing in triple-negative breast cancer: Post hoc IMpassion130 substudy evaluates PD-L1 IHC assay performance

In triple-negative breast cancer, it is only staining in the immune cell component that counts toward scoring. “For the majority of cases, the tumor cell component will be negative. But in a minority of cases, you will observe tumor cell staining that could be present and could potentially increase the interpretive complexity of the case,” Dr. Tozbikian said.

When positive, the immune cells tend to show a more punctate or granular pattern of staining, which appears on the cell membrane. The tumor cells when positive will show a more uniform, complete, honeycomb-like or circumferential staining pattern on the cell surface.

Any immune cell staining counts toward scoring—lymphocytes, macrophages, neutrophils, multinucleated giant cells, and so on—“provided that these immune cells are within the tumor area and are not in necrotic areas,” he said, adding that staining intensity is included in the scoring.

Generally two different patterns of immune cell staining will be seen: aggregate and single cell spread. And intratumoral heterogeneity or regional variation for PD-L1 expression is commonly encountered.

The scoring method for SP142 in triple-negative breast cancer uses a proportion of tumor area scoring system, which is what was used in the clinical trials. “It was the scoring system found to be more reproducible and best correlated with efficacy,” Dr. Tozbikian said.

“You score in the immune cell component only. You calculate the proportion of the tumor area occupied by PD-L1-positive staining immune cells.” The tumor area is defined as the tumor mass itself and includes the associated intratumoral stroma and immediate contiguous peritumoral stroma.

“To interpret the stain, you estimate the proportion of that total tumor area occupied by PD-L1–positive immune cells that are present, either infiltrating the tumor or in the intratumoral stroma or immediate contiguous peritumoral stroma.” If positive immune cell staining occupies greater than or equal to one percent of the total tumor area, the result is considered positive. No staining or less than one percent area staining is negative. “Any tumor cell staining that you observe is ignored.”

The report should specify the PD-L1 immuno­stain and antibody that was performed, he said, and give an overall result interpretation, positive or negative. It should also provide detail about the scoring system used. “For triple-negative breast cancer, it’s a tumor area scoring system with a one percent cutoff scored in immune cells.” Providing a raw score is optional. ”In clinical practice, most positive results you see are going to be in the one to five percent positive range,” he said.

Case No. 1 demonstrates the typical appearance of immune cell staining for PD-L1 SP142. Shown is a low-power view of a core needle biopsy with primary triple-negative breast cancer (Fig. 1a), and a higher power view (Fig. 1b). “This was an invasive ductal carcinoma that was triple negative. You can see a dense, inflammatory infiltrate adjacent to the tumor cell nests and the intratumoral stroma as well as infiltrating the tumor itself,” he said.

Fig. 1c is the corresponding PD-L1 SP142, and PD-L1 expression can be seen in the immune cell component, mainly in the intratumoral stroma. The staining pattern in these immune cells is punctate and granular whereas the tumor cells are negative. “This is the most commonly encountered staining pattern,” Dr. Tozbikian said.

He circled regions of the tumor area that are occupied by PD-L1-positive immune cells (Fig. 1d). To help estimate the proportion of tumor area involvement, he combined the positive regions (Fig. 1e).

The next step is to estimate the combined area as a proportion of the total tumor area (Fig. 1f). “This positive area occupies approximately a little less than one-seventh of the total tumor area, at least in this high-power field. So assuming that the rest of the entire tumor looked like this, the result would be positive with about 13 percent of the total tumor area involved,” he said.

Case No. 2 (Fig. 2a) was taken from a case of a primary breast triple-negative invasive ductal carcinoma and highlights a tumor that will demonstrate both tumor cell and immune cell staining. “You can appreciate an inflammatory infiltrate involving the adjacent intratumoral stroma.” (Fig. 2b).

Fig. 2c is the corresponding PD-L1 immunostain. The majority of the tumor cells are PD-L1-negative, but at the top of the image there is a small cluster of several tumor cells that show PD-L1 expression. In comparison to the immune cells, these tumor cells show a more uniform, complete circumferential staining pattern on the cell surface. The immune cell staining shows a more punctate or granular staining pattern.

Scoring in one high-power field (Fig. 2d) reveals that about five percent of the tumor area is occupied by positive immune cells. “Assuming that the rest of the tumor showed a similar staining pattern, the final result would be positive,” he said.

Case No. 3 (Fig. 3a) came from a lung metastasis by triple-negative breast cancer, and again demonstrates the difference between tumor cell and immune cell staining. At higher power (Fig. 3b), large clusters of epithelioid cells can be seen, as can a dense, inflammatory lymphoplasmacytic infiltrate in the surrounding stroma.

Fig. 3c is the corresponding PD-L1 stain. The majority of the tumor cells are PD-L1-negative, but in the top and right side of the image are several tumor cells showing PD-L1 expression. “Again, in comparison to the immune cells, the tumor cells that are positive show a more uniform or complete circumferential staining pattern.”

“Ignoring the tumor cell staining, still a large proportion of the immune cells in this high-power field are positive.” (Fig. 3d). About 60 percent of the tumor area is involved by PD-L1-positive immune cells. To arrive at a final overall result, he said, the entire tumor area in a case has to be evaluated.

Case No. 4 (Fig. 4) demonstrates an aggregate staining pattern (the more common of the two patterns) in the immune cell. Fig. 4a is a skin recurrence by a triple-negative invasive ductal carcinoma. Fig. 4b is a higher-power view in which a dense, inflammatory cell infiltrate can be seen, mostly in the surrounding stroma but also infiltrating the tumor itself.

Fig. 4c is the corresponding PD-L1 immuno­stain. Positive PD-L1 immune cells can be seen with punctate expression, and the majority of the PD-L1 positivity is seen in aggregates or clusters of positive immune cells located in the intratumoral or peritumoral stroma. This is the aggregate staining pattern.

Case No. 5 (Fig. 5) highlights the single cell spread pattern. Fig. 5a is a low-power view of a corneal biopsy containing a primary triple-negative breast cancer. Fig. 5b is a high-power field. “This is an invasive ductal carcinoma. There is a dense inflammatory infiltrate surrounding the tumor nests. You can appreciate immune cells infiltrating as single cells admixed among the tumor cells and in the stroma.” (Fig. 5c).

Fig. 5d is the corresponding PD-L1 stain, where strong punctate staining can be seen in the immune cells, “but expressed in a more dispersed pattern seen in scattered single cells more diffusely in the TILs that are infiltrating among the tumor cell nests.” This is the single cell spread pattern.

In the same tumor both aggregate and single cell spread patterns of staining can be encountered. “Both of them count,” Dr. Tozbikian said.

Case No. 6 (Fig. 6) demonstrates how to define the total tumor area. Fig. 6a is a low-power view (tumor area circled) of a biopsy of a skin recurrence by triple-negative breast cancer. This is the region where PD-L1 is scored. Fig. 6b is the higher-power view; the entire region in the circle is considered the tumor area. It includes the tumor mass itself, the associated intratumoral stroma, as well as the immediate contiguous peritumoral stroma.

Fig. 6c is the corresponding PD-L1 stain. “You would include any immune cell staining within the circled tumor area to include any PD-L1-positive immune cells that are infiltrating the mass, immune cells that are positive in the intratumoral stroma, and at the periphery of the tumor any PD-L1-positive immune cells in the contiguous peritumoral stroma.”

Case No. 7 (Fig. 7) also demonstrates how to define the tumor area, but this case is a lymph node metastasis by triple-negative breast cancer. It can be seen in Figs. 7a and 7b (low-power view of the lymph node) that the majority of the lymph node has been replaced by metastatic carcinoma. At the bottom left there is a portion of the lymph node that is still uninvolved by metastatic carcinoma. Fig. 7c is a medium-power view of the interface between the metastasis and the uninvolved lymph node. PD-L1 would be scored and assessed inside this circled tumor area.

For lymph node metastases, the scoring method is identical to that of primary and other metastatic sites, he said. “But care should be taken to exclude scoring in the normal or uninvolved lymph node tissue that is not part of the tumor area because native lymph node tissue will show staining for PD-L1 SP142, particularly in lymph node germinal centers. So staining in the uninvolved or native lymph node should be excluded.”

Fig. 7d is the corresponding PD-L1 immuno­stain, where there is accentuated PD-L1–positive expression in the periphery of the tumor and immune cells, and within the metastatic deposit itself. PD-L1 would be scored within the tumor area, and then you would exclude any scoring in the normal, uninvolved lymph node tissue, as the native lymph node tissue will show expression for SP142, particularly in germinal centers.

Case No. 8 (Fig. 8) demonstrates intratumoral heterogeneity for PD-L1 expression. “In clinical practice, it is common to encounter tumors with regional variation for PD-L1 SP142 expression,” he said. For this reason, when assessing PD-L1, “make sure you scan the entire tumor area for PD-L1 expression, evaluating for the presence of intratumoral heterogeneity.”

Case No. 8 is taken from a primary triple-negative breast cancer. Fig. 8a is from an excision specimen, and Fig. 8b is the corresponding PD-L1 immunostain at scanning magnification. This tumor showed significant regional variation for PD-L1 expression. At higher power (Fig. 8c) there is virtually no PD-L1 expression in the immune cell component. If a different region of the tumor is examined (Fig. 8d), “it shows a different story,” Dr. Tozbikian said. On the bottom left side of the tumor at higher power (Fig. 8e), “you can appreciate the presence of PD-L1 immune cells. This is showing a more aggregate staining pattern in the intratumoral stroma. At least in this high-power field, the area involved by PD-L1-positive immune cells is greater than one percent of the tumor area. But you have to consider the entire tumor area when scoring.”

As a practical recommendation, in cases like this, one helpful approach is to do a semiquantitative assessment, he said. “It may be easier to break it up by scoring multiple fields at high power and then take an average.” In this case he used a grid to divide the tumor into separate smaller fields, which could then be assessed separately and from which an average could be taken (Fig. 8f).

“So let’s consider scoring in the bottom left side of the tumor. Going to a more medium-power view, I divided this portion of the tumor into a quadrant-like fashion to help make area quantification easier.” Significant intratumoral heterogeneity is seen (Fig. 8g). The two fields on the right show minimal staining for PD-L1 whereas the two fields on the left show aggregate staining in the immune cells in clusters within the intratumoral and peritumoral stroma, which he circled.

The next step would be to score each separate field. The two fields at right would receive a score of zero percent and on the left 10 and 20 percent. “And you would calculate an average of these: Ten percent plus 20 percent plus two fields with zero percent—the result would be about eight percent overall, at least in this field.”

This is done for the entire tumor and then an overall average is calculated. “I think this approach is useful when you have intratumoral hetero­geneity.”

Sherrie Rice is editor of CAP TODAY. The full webinar is at www.captodayonline.com.