Immunotherapy (IO) is rapidly reshaping the treatment landscape for solid tumors, with survival improvements demonstrated in multiple disease types. In the context of metastatic triple-negative breast cancer (mTNBC), the use of IO is based on the results from two phase III clinical trials: Impassion130 and KEYNOTE-355. In both trials, the addition of IO to first-line chemotherapy has shown an improvement in clinical outcomes restricted to patients with PD-L1 expressing tumors. Impassion130 demonstrated a statistically significant improvement in progression-free survival (PFS) with the addition of atezolizumab to nab-paclitaxel in the intention-to-treat (ITT) population, with a greater improvement in the PD-L1 + population, namely PD-L1 stained tumor-infiltrating immune cells (IC) covering ≥1% of the tumor area, as determined by the VENTANA PD-L1 (SP142) as a companion diagnostic test (CDx) [1]. Although the Food and Drug Administration (FDA) granted accelerated approval for the combination of atezolizumab and nab-paclitaxel as frontline treatment of PD-L1 + mTNBC, this study failed to demonstrate a statistically significant improvement in the co-primary endpoint of overall survival (OS) in ITT population due to its hierarchical statistical design, despite a numeric difference in OS [2]. This ultimately led to the withdrawal of the indication in the United States by the agent’s developer. Thus, atezolizumab will no longer be available for the treatment of PD-L1+ mTNBC in the United States, while it is still available in Europe at the time of writing. In contrast, in KEYNOTE-355, the addition of pembrolizumab to chemotherapy improved both progression-free survival (PFS) and overall survival (OS) compared to chemotherapy alone in PD-L1+ (combined positive score, CPS ≥ 10) mTNBC patients. PD-L1 status was defined using the IHC 22C3 pharmDx (Dako North America, Inc.) and a specific scoring system (CPS) [3,4]. Based on these results, the FDA granted full approval to pembrolizumab for locally recurrent unresectable or mTNBC expressing PD-L1 as assessed with 22C3 Dako CDx with a CPS ≥ 10, and the same agent may soon gain approval in Europe as well. Two clinical trials, two drugs, and two CDx to answer the same question: how to identify mTNBC patients who might benefit the most from IO? It is worth noting that such discordance pervades the selection of patients for IO across various tumor histologies [5]. Indeed, there are four anti-PD-(L) 1 agents approved for PD-L1-restricted indications, together with four distinct CDx to assess the expression of PD-L1: nivolumab (28–8, Dako), pembrolizumab (22C3, Dako), atezolizumab (SP142, Ventana), and durvalumab (SP263, Ventana) [5]. Moreover, the complexity of PD-L1 assessment itself presents many key sources of variability (Figure 1) [5]. First of all, PD-L1 expression can be evaluated only on tumor cells (TC), only on IC, or on both (as for CPS). In addition, each CDx uses a different method for scoring calculation (see Table 1) and there is no consensus for the cut-off value, even for the same CDx across different tumor histologies [5]. Another important source of variability is the potential intratumoral heterogeneity in PD-L1 expression within each single lesion, or between primary and metastatic samples (as well as among different metastatic anatomic locations) [6]. Last but not least, different assays use different immunohistochemistry (IHC) staining protocols, primary antibody clones (SP142, SP263, and 22C3), IHC platforms, and detection kits [5]. Hence, the next question easily arises: do different assays ultimately identify different patient populations? Across tumor histologies, SP142 stains fewer TC and IC compared to other clones [7]. In mTNBC, a hint is given by a post hoc analysis from Impassion130, where IHC for PD-L1 status using SP263 and 22C3 was evaluated for analytical concordance with SP142 and patient-associated outcomes in a subset of tumor samples [8]. Prevalence rates using PD-L1 IC ≥ 1% as a threshold for SP142, SP263, and 22C3 were, respectively, 46.4% (95% confidence interval [CI] = 42.5% to 50.4%), 74.9% (95% CI = 71.5% to 78.3%), and 73.1% (95% CI = 69.6% to 76.6%). The prevalence of PD-L1 22C3 CPS ≥ 1 was 80.9%. The analytical concordance, namely overall percentage agreement (OPA) for IC ≥ 1% (+), between SP142 and SP263 or 22C3 was 69.2% and 68.7%, respectively. Even if almost all SP142+ cases were classified as PD-L1+ by other assays (double positive), 29.6% of SP263+ and 29.0% of 22C3+ cases were SP142– (single positive) [8]. Importantly, the IO clinical activity in SP263+ and 22C3+ patients could be mainly ascribed to double-positive cases rather than single-positive cases. Thus,
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