A novel PD-L1 assay advances the promise of precision medicine

By Michael B. Lynch, MD

The recent FDA approval of the first companion diagnostic to identify triple-negative breast cancer (TNBC) patients eligible for treatment with atezolizumab immunotherapy brings hope to the 300,000 women around the world, representing 15% of all breast cancer cases, who suffer this aggressive disease with limited treatment options, poor prognosis, and high mortality rates. These patients, who test negative for the three most common markers associated with breast cancer growth—estrogen receptor (ER), human epidermal growth factor receptor-2 (HER2), and progesterone receptor (PR)—are the beneficiaries of recent developments in checkpoint immunotherapy with the goal of boosting immune response against cancer.

This article reviews the use of laboratory testing, in particular immunohistochemistry, in breast cancer diagnosis and treatment. It examines the role of checkpoint inhibitors with a focus on programmed death ligand 1 (PD-L1), the biomarker used to identify patients eligible for treatment with atezolizumab immunotherapy, and discusses highlights from the phase III IMpassion130 study. The concept of companion diagnostics and the value of matching the diagnostic test to the immunotherapy are also discussed.

Cancer in Context

Breast cancer is the leading cause of cancer mortality for women worldwide, killing more than 600,000 in 2018 and afflicting more than 2 million.1 Such outcomes continue to be seen in spite of recent advances in laboratory testing to detect the expression of estrogen and progesterone receptors (ER, PR) and the overexpression of human epidermal growth factor receptor-2 (HER2) protein and, in parallel, the availability of therapeutics to target these subtypes.

Approximately 15% of all breast cancer patients test negative for ER, HER2, and PR, denoted as triple-negative breast cancer, and the outlook for this subset of patients is less favorable. TNBC represents a heterogeneous subtype with poorer prognosis and higher mortality rates.2 In the United States, TNBC is more common among younger women, among those with a BRCA1 gene mutation, and among non-Hispanic black women, than it is among those of other racial or ethnic groups.3 Until now, other than surgery and radiation therapy, chemotherapy has been the primary treatment option for patients diagnosed with TNBC.

Roche-Lynch Figure 1 PD-L1 Immunologic checkpoint_crop1111x651p

Figure 1. The PD-L1 immune checkpoint pathway.

The recent FDA approval of a companion diagnostic, the Ventana PD-L1 (SP142) assay, brings hope to the 300,000 women around the world diagnosed with TNBC. This test assesses expression of the programmed death ligand 1 (PD-L1) in TNBC tissues, and is the first companion diagnostic to identify TNBC patients who can benefit from Tecentriq (atezolizumab) immunotherapy plus chemotherapy. In the IMpassion130 study, a multicenter, randomized, double-blind study, atezolizumab plus nab-paclitaxel prolonged progression-free survival among TNBC patients.4

The Immune Response and Cancer

The latest developments in the diagnosis and treatment of TNBC can best be understood within the framework of the body’s immune response to cancer, and strategies for optimizing immune response to improve survival. In this context, it should be noted that the body responds to cancer cells—or, more specifically, tumor-specific antigens expressed by cancer cells—by activating the immune system. This is the body’s innate defense against the occurrence of primary tumors; the mutation-specific immunity induced by the primary tumor also curbs metastasis.

OptiView diaminobenzidine (DAB) immunohistochemistry (IHC) detection kit.

Figure 2. To perform the Ventana PD-L1 (SP142) assay, formalin-fixed, paraffin-embedded tissue specimens are stained using the OptiView diaminobenzidine (DAB) immunohistochemistry (IHC) detection kit.

Several strategies for enhancing immune response to cancer have been applied with varying degrees of success. Cancer vaccines, for example, expose the body’s immune system to specifically designed antigens and are given to prevent cancer occurrence. Except in the well-known example of vaccination against human papillomavirus, which is preventive of most cervical cancer, this strategy has found limited success.

Oncolytic viruses, engineered to recognize specific antigens and thus selectively infect and kill cancer cells, have also found limited success.

A third strategy, commonly known as chimeric antigen receptor (CAR) T-cell therapy, involves genetic engineering of the patient’s own T cells to recognize cancer cells, and infusing the modified cells back into the patient to fight cancer. The technique has found early success in treating blood cancers (eg, refractory non-Hodgkin lymphoma, pediatric relapsed acute lymphoblastic leukemia), and there are a number of ongoing clinical trials.

The fourth strategy is checkpoint immunotherapy, which targets regulatory pathways in T cells with the goal of circumventing the evasion mechanisms employed by cancer cells to suppress immune response, thereby enhancing antitumor immune response.5 This is an area of active clinical research with thousands of ongoing clinical trials.

The PD-L1 Immunologic Checkpoint

Immunologic checkpoints are the body’s mechanism to attenuate immune response and maintain a fine balance between adequate expression and overexpression of T cells, the lymphocytes that lead the immune response to invaders such as infections and cancers.

BenchMark Ultra

Figure 3. Staining of tissue specimens to be analyzed with the Ventana PD-L1 (SP142) assay is performed on the BenchMark Ultra system, a fully automated instrument for immunohistochemistry and in situ hybridization slide staining.

PD-L1, a transmembrane protein, is a checkpoint inhibitor that downregulates immune response through binding to its two inhibitory receptors, programmed death 1 (PD-1) and B7.1 (Figure 1).

PD-1 is a checkpoint protein, also called an immunoinhibitory receptor, expressed on T cells following T-cell activation (eg, in cancer). PD-1 is activated by binding with PD-L1. It inhibits T-cell proliferation and the production of cytokines—necessary for T-cell proliferation and survival, and cytolytic activity—leading to the functional inactivation or exhaustion of T cells.6

B7.1 is a cell-surface protein ligand on T cells and antigen-presenting cells. PD-L1 binding to B7.1 can mediate downregulation of immune response, including inhibition of T-cell activation and cytokine production.7 Thus, PD-L1 expression can impede antitumor activity, and interruption of the PD-L1/PD-1 pathway is a viable strategy for reinvigorating tumor-specific T-cell immunity suppressed by the expression of PD-L1.

Atezolizumab, an immunotherapeutic agent classified as checkpoint inhibitor, binds to PD-L1 and thus blocks interactions of PD-L1 with both PD-1 and B7.1, restoring T-cell activation. It should be noted that immunotherapeutic agents do not directly kill cancer cells; instead, they prevent the receptors (eg, PD-1 and B7.1) from binding to the ligand (in this case PD-L1) and thus disrupt signaling and immunosuppression. In turn, this process activates the body’s own immune system.


Figure 4. Immunohistochemistry staining of triple-negative breast cancer (TNBC) tissue with the Ventana PD-L1 (SP142) assay. The top image represents TNBC tissue with PD-L1 expression of less than 1% in tumor-infiltrating immune cells; the remaining three slides show TNBC tissue with PD-L1 expression of at least 1% in immune cells. Immune cells often show dark brown punctate or linear staining, which is the immunohistochemistry staining pattern observed in the majority of tissues. Occasionally, circumferential immunohistochemistry membrane staining is also observed, especially in cells that are morphologically consistent with macrophages or dendritic cells.

Determining PD-L1 Status in TNBC Patients

The Ventana PD-L1 (SP142) assay is a qualitative immunohistochemical assay using a rabbit monoclonal primary antibody that binds to PD-L1 in paraffin-embedded tissue sections. The bound antibody at the antigen sites is visualized using immunostaining and amplification reagents included in the kit, which results in a brown diaminobenzidine (DAB) reaction product (Figures 2, 3). The stained specimen is then reviewed by a qualified pathologist under a light microscope.

The scoring algorithm for TNBC, integral to the assay and to assay performance, identifies positive specimens when discernible PD-L1 staining of any intensity is present in tumor-infiltrating immune cells (but not tumor cells) covering at least 1% of the tumor area (Figure 4). It should also be emphasized that the scoring algorithm for the companion diagnostic, as approved, is indication-specific. For example, the scoring algorithm for the Ventana PD-L1 (SP142) assay for TNBC is different from the scoring algorithms for urothelial carcinoma or for non–small cell lung cancer (Figure 5).

The Value of Companion Diagnostics

In the IMpassion130 study, TNBC patients who tested positive for PD-L1 using the Ventana PD-L1 (SP142) assay had significantly longer progression-free survival and higher overall response to atezolizumab–nab-paclitaxel therapy compared with TNBC patients who did not test positive for PD-L1. (Nab-paclitaxel is a chemotherapy drug also known by its brand name, Abraxane.) According to the researchers, “a clinical benefit with atezolizumab–nab-paclitaxel was particularly notable in the PD-L1–positive subgroup, as shown by a median progression-free survival that was significantly longer by 2.5 months . . . by a median overall survival that was 10 months longer at this interim analysis . . . and a numerically higher objective response rate (58.9% versus 42.6%)” when compared to all patients with metastatic TNBC.4

Such results are an example of precision medicine and underscore the value of companion diagnostics for identifying patients most likely to benefit from the treatment. FDA defines a companion diagnostic as one that “provides information that is essential for the safe and effective use of a corresponding drug or biological product.” A positive result is required for the patient to receive the therapeutic.

OptiView amplification reagents

Figure 5. For the Ventana PD-L1 (SP142) assay, bound antibody at the antigen sites is visualized using immunostaining and the OptiView amplification reagents included in the test kit.

By contrast, a complementary diagnostic is defined as one that “aids in the benefit-risk decisionmaking about the use of the therapeutic product.” While a complementary diagnostic also identifies patients likely to benefit from the therapeutic, the clinician is not precluded from prescribing the therapeutic to patients who test negative and may exercise discretion in deciding which patients are likely to benefit.

Precision Medicine and the Laboratory

The promise of precision medicine has spurred research and development of new diagnostics and treatments for many cancers. Already, several checkpoint immunotherapies, all targeting the PD-L1/PD-1 interaction, have been approved by FDA. Each immunotherapy targeting a specific indication is approved with a specific companion or complementary diagnostic. Not surprisingly, several FDA-approved assays for PD-L1 expression are available. Some are approved as complementary diagnostics, some as companion diagnostics. In each case, the diagnostic is approved alongside an immunotherapeutic, and each approval is specific to a type of cancer.

BenchMark Ultra

Figure 6. The BenchMark Ultra system provides continuous-access automated immunohistochemistry and in situ hybridization slide staining to process up to 90 slides in 8 hours, or 120 slides with an overnight run.

The laboratorian has an important role in guiding clinicians through this increasingly complex matrix of diagnostics, immunotherapies, and approved indications for use (Figure 6). Extreme caution must be exercised in ensuring the approved companion diagnostic is used, for several reasons.

The approved diagnostic is the one used to select patients during clinical trials of the corresponding immunotherapy. Thus, the clinical trial results apply specifically to that patient group identified with the specific approved test. Differences in the choice of antibodies against the target antigen can contribute to different test results, as would other factors such as assay procedure or assay design.

Of course, clinicians often have discretion for off-label prescription, as would be the case when a drug is prescribed to patients not tested with the companion diagnostic. With the cost of immunotherapy and, more important, patient outcomes in the balance, clinicians should at least be made aware of the choice. And, as in laboratory testing in general, strict adherence to appropriate procedures for specimen handling, preparation, assay performance, and quality control is paramount.

For the laboratory, precision medicine represents significant opportunities to foster professional advancement through learning and applying the latest technologies, to enhance the lab’s role as consultant to the clinician for navigating the new era of companion diagnostics, to contribute more directly to patient care, and to elevate the visibility of the lab as a key partner in the healthcare system.

Michael B. Lynch, MD, is a pathologist in medical and scientific affairs at Roche Diagnostics. For further information contact CLP chief editor Steve Halasey via [email protected].


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Featured image:  The recent FDA approval of a companion diagnostic brings hope to the 300,000 women around the world diagnosed with triple-negative breast cancer. The Ventana PD-L1 (SP142) assay is a qualitative immunohistochemical assay using a rabbit monoclonal primary antibody that binds to PD-L1 in paraffin-embedded tissue sections.