Programmed cell death protein 1 (PD-1, encoded by PDCD1 gene) and one of its two known ligands, the programmed death ligand-1 (PD-L1, encoded by CD274 gene) are among the therapeutically most important checkpoint proteins that mediate tumor-induced immune suppression through T-cell downregulation [1]. Their overexpression has been described in various solid tumors with marked clinical therapeutic effects due to the checkpoint blockade [anti-PD1/PD-L1 antibodies] [2], revolutionizing the treatment of solid malignancies, particularly metastatic melanoma, renal cell carcinoma, and non-small cell lung carcinoma (NSCLC).

Patients with relapsed/refractory malignant lymphomas have limited therapeutic modalities and new therapeutic approaches are immensely important [3]. Recent studies revealed the expression of PD-L1 among various B-cell lymphomas [4–6] with the most remarkable therapeutic benefits of PD-1 blockade in patients with Hodgkin lymphoma [3, 7].

PD-L1 status is usually determined by immunohistochemistry [8–10]. Food and Drug Administration (FDA) has recently approved PD-L1 22C3 antibody (DAKO pharmDx) as a companion diagnostics IHC kit for identifying non-small cell lung cancer (NSCLC) patients that are candidates for treatment with pembrolizumab. Several other antibodies (e.g. 28–8 clone from DAKO; SP142 clone and SP263 clone from Ventana) have been developed and used successfully in clinical trials for detection of PD-L1 protein expression in different tumor types (reviewed in [11]). Although PD-L1 overexpression is associated with greater clinical response (particularly to anti-PD1 antibodies) [11], the available clinical data indicate that only 10–30% tumors with PDL1 over expression respond to the PD-1/PD-L1 checkpoint inhibitors [11–14]. The reasons for this discrepancy might be due to different drugs, different antibody clones (validated for specific platforms, e.g. automated Ventana IHC systems or DAKO IHC autostainer), different thresholds, as well as complex pathophysiological mechanisms behind PD-L1 deregulation due to the interactions between cancer and immune cells [10, 15].

Several recent studies investigated the genetic basis of PD-L1 overexpression in tumors. In Hodgkin lymphoma alterations in chromosome 9p24.1 leads to PD-L1 (CD274) and PD-L2 (PDCDLG2) gene amplification in RS cells [16]. Amplification of PD-L1 gene has also been described in triple-negative breast carcinomas [17, 18] and NSCLC [19, 20]. Green et al. [16] and Roemer et al. [6] also found PD-L1 gene alterations in classical Hodgkin lymphoma (cHL) while Georgiou et al. [21] recently demonstrated various cytogenetic alterations of PD-L1 gene in diffuse large B-cell lymphomas (DLBCL) including gains, amplifications and translocations. Genomic rearrangements of PD-L1 have also been described in primary mediastinal large B-cell lymphomas [22]. A recent comprehensive survey of Budczies et al. [15] revealed frequent PD-L1 copy number variations (gains and amplifications [12%], deletions [31%]) across different cancer subtypes with direct impact on its protein and mRNA expression.

In the present study, we explored the expression of PD-L1 in a diverse group of refractory/relapsed lymphomas. We compared the diagnostic utility of two different anti-PD-L1 clones and also explored the genetic basis of PD-L1 overexpression analyzing PD-L1 (CD274) gene along with PD-L2 and JAK2 genes at 9p24 using in situ hybridization and next-generation sequencing (NGS) assays.

Immune check point blockade with PD-1/PD-L1 has dramatically changed the cancer treatment paradigm with impressive results in several solid tumors as well as in cHL. PD-L1 overexpression has also been described in cancer subtypes beyond those that have been so far considered for immune check point inhibitors [4, 10, 24–26], potentially opening this type of therapy to a larger and more diverse populations of cancer patients. However, predictive value of immunohistochemical biomarkers (thresholds, cellular distribution, methods of analysis) for the PD-1/PD-L1 axis inhibition need still be refined. In some lymphoma subtypes, the identification of PD-L1 positive tumor cells may be challenging due to the abundant reactive/inflammatory cells (e.g. peripheral T-cell lymphomas and cHL). Also, we observed different distribution of PD-L1 expression across the lymphoma subtypes; thus, cHL usually exhibit diffuse and strong PD-L1 positivity on tumor (RS) cells while other lymphoma subtypes rarely exhibit such a pattern of PD-L1 expression.

Patients with relapsed/progressive malignant lymphomas have limited therapeutic modalities and new therapeutic approaches are of crucial importance. Our study revealed over-expression of PD-L1 in cHL, DLBCL (including primary mediastinal B-cell lymphomas), a subset of peripheral T-cell lymphomas and a single case lymphomatoid granulomatosis. These findings are consistent with comprehensive surveys of Menter et al. [clones E1L3N (Cell Signaling, Danvers, MA, USA) and SP142 (Roche/Ventana, Rotkreuz, Switzerland) [5] and Chen et al. (clone 15, #10084-R015, Sino Biological) [4], both of which included a large number of both Hodgkin and non-Hodgkin lymphomas. Another important finding of our study was an excellent concordance between SP142 and SP263 clones using the 5% cut-off as recommended [13, 24]. There are only a few studies that have analyzed the diagnostic utility and comparison of different anti-PD-L1 antibody clones [5, 10, 27–29]. A systematic review of Carbognin et al. [13] confirmed a significant difference in clinical response among solid tumors (melanoma, NSCLC, genitourinary cancers) when a 5% cut-off is used instead of 1% threshold. Hence, development of a standardized threshold for PD-L1 expression in lymphomas requires further investigation.

There is increasing number of studies indicating that alterations at 9p24.1 affect PD-1 ligands (“9p24.1 amplicon”), particularly PD-L1 gene [6]. This amplicon also contains JAK2 (Janus kinase 2) gene involved in JAK2-STAT signaling that further may activate PD-L1 [16] and PD-L2 gene, which is another ligand of PD-1. Similar to previous data, we showed that a subset of PD-L1+ lymphomas (both cHL and non-Hodgkin lymphoma) may harbor genetic alterations at 9p24.1 amplicon, which implies that the remaining non-amplified cases may have “adaptive” PD-L1 overexpression without underlying PD-L1 gene alterations [15]. One case harbored co-amplification of PD-L1 and PD-L2 without JAK2 gene alterations, which is in line with a recent study of Budczies et al. [15]. The authors showed common PD-L1 copy number variations (gains and amplifications [12%], deletions [31%]) across different cancer subtypes with direct impact on its protein and mRNA expression. In particular, cHL are characterized by consistent PD-L1/PD-L2 gene alterations [6]. One of the important exploratory findings in our study is diagnostic utility of FISH assay for detection of PD-L1 gene alterations in cHL. FISH method appears to be a more suitable methodology than NGS for assessing 9p24 status in tumors with a low cancer cell density as it allows for morphologic (visual) identification of single amplified RS cells within heterogeneous (reactive) cell populations in cHL.

We conclude that a substantial proportion of relapsed/refractory B-cell and T-cell non-Hodgkin lymphomas and nearly all classic Hodgkin lymphomas overexpress PD-L1 protein, implying a potential utility of checkpoint blockade therapy in these difficult to treat diseases. Anti-PD-L1 clones SP142 and SP263 exhibit an excellent concordance and both antibodies may be used for IHC detection of PD-L1 in tumors. We also confirm that a subset of refractory, PD-L1 positive lymphomas may harbor genetic alterations of 9p21.4 amplicon affecting PD-L1, PD-L1 and JAK2 genes. FISH assay may be more suitable than NGS assay for determination of PD-L1 alterations in cHL.