Clinicopathologic implication of PD-L1 and phosphorylated STAT3 expression in diffuse large B cell lymphoma

Our cohort consisted of 107 patients newly diagnosed with de novo DLBCL, not otherwise specified (NOS) between May 2005 and January 2013 at Seoul National University Bundang Hospital. Clinicopathological information was obtained from clinical records and pathology reports. Large B cell lymphomas with distinct entities including ‘EBV-positive DLBCL, NOS’, ‘DLBCL of the central nervous system’, ‘primary mediastinal large B cell lymphoma’ as well as transformed DLBCL from low grade lymphomas were excluded. The pathologic classification was based on the 2016 Revised 4th edition of World Health Organization classification [29] and further subtyped by Hans algorithm [2] by experienced hematopathologists. The study protocol was approved by the Institutional Review Board of Seoul National University Bundang Hospital.

Hematoxylin and eosin (H & E)-stained slides were reviewed in each case to confirm the original diagnosis and select the most representative sections. A tissue microarray (TMA) was constructed using 2 mm-diameter cores derived from the representative areas of the formalin-fixed paraffin-embedded tissue blocks from each case and from normal tonsils for controls by SuperBioChips Laboratories (Seoul, Korea), as previously described [30].

TMAs were sectioned at a thickness of 4-μm and stained with Benchmark XT and Benchmark ULTRA (Roche Diagnostics) along with normal tonsil specimens as controls for the following antibodies (clones): anti-Bcl-2 (std32, 1:50, Dako), anti-Bcl-6 (std32, ready-to-use, Ventana), anti-CD10 (std32, ready-to-use, Ventana), anti-Mum-1 (mild32, 1:150, Dako), anti-PD-L1 (E1L3N, 1:100, Cell Signaling) and anti-pSTAT3 (#9131, 1:20, Cell Signaling). Using immunochemistry results, the Hans algorithm was applied to each case as described previously [31].

The immunohistochemistry slides were scored by two pathologists (H.J.K and J.H.P). PD-L1 was scored both in tumor B-cells and in non-malignant immune cells. PD-L1 was considered positively expressed in tumor or non-malignant immune cells if membranous staining alone or membranous and cytoplasmic staining together was present. The percentage of stained tumor B-cells and non-malignant immune cells were estimated in each TMA core regardless of intensity. Tumor cells were distinguished from non-malignant immune cells by histologic clues such as nuclear enlargement and atypism, followed by comprehensive interpretation with other immunohistochemical markers including CD20, BCL2, BCL6, CD10 and MUM1. Since an optimal cut-off could not be determined by receiver operating characteristic (ROC) curve analysis, cases were classified by a 30% cutoff for tumor B-cells and 20% cutoff for non-malignant immune cells according to previous studies [6]. pSTAT3 expression was scored in tumor B-cells alone with cutoff value of 40%, which was set based on ROC curve analysis. In addition, the proportion (%) of pSTAT3-positive cells was digitally counted by using digital slide scanner and image analyzer (3DHISTECH, Budapest, Hungary) for correlation analysis.

For fluorescence in situ hybridization (FISH) staining, PD-L1 break-apart probe (9p24.1) (catalog # PDL1BA-20-ORGR) and PD-L1 (orange)/chromosome 9 (green) probe set (9p24.1/9p21.33) (catalog # PDL1-CHR09-20-ORGR) were purchased from empire genomics (Buffalo, NY). The FISH staining process was performed as previously described [32]. Briefly, after deparaffinization and dehydration, slides were immersed in 2 M HCl, boiled using a microwave oven in citrate buffer (pH 6.0), incubated in 1 M NaSCN for 40 min at 80 °C, immersed in a protease buffer and fixed in 10% neutral buffered formalin. After applying the DNA probe set to the slides, they were incubated in a humidified chamber at 83 °C for 3 min to denature the target DNA and probe, and subsequently incubated overnight at 37 °C to achieve hybridization. Following post-hybridization washing, 4,6-diamidino-2-phenylindole (DAPI) and an anti-fade compound (p-phenylenediamine) were applied to the slides as a counter-stain. An Olympus BX51TRF microscope (Olympus Corp., Tokyo, Japan) equipped with the appropriate filter sets was used to analyze the stained cells. For the interpretation of translocation and/or copy number gain/amplification, more than 200 tumor cells in non-overlapping intact nuclei were counted [17, 32]. Separation of orange and green signals in more than 15% of tumor cells were interpreted as translocation [31]. Copy number gain and amplification were defined as PD-L1 gene/chromosome 9 ratio > 2 and > 4, respectively.

The characteristics of 107 patients with DLBCL are summarized on Table 1. Briefly, our cohort consisted predominantly of non-GCB subtype (67%; 72/107) compared to GCB subtype (25%; 27/107) or unclassifiable cases (8%; 8/107) by Hans algorithm. The total cohort mainly included cases with good Eastern Cooperative Oncology Group performance status (ECOG PS) (< 2; 91%; 97/107), absence of B symptoms (79%; 85/107), low international prognostic index (IPI; 66%; 71/107), less than 2 extranodal site involvements (76%; 81/107), absence of bone marrow involvement (79%; 84/107) and non-bulky masses (92%; 98/107). Most of the patients received R-CHOP chemotherapy (87%; 93/107). Compared to the patients with GCB subtype, the patients with non-GCB subtype frequently had elevated serum lactate dehydrogenase levels (p = 0.040). Non-GCB subtype also tended to be associated with a high IPI score (3–5; p = 0.056) and presence of B symptoms (p = 0.088), which did not reach statistical significance.

We investigated the frequencies of PD-L1 expression, PD-L1 gene alteration, i.e., translocation, copy number gain and/or amplification, and pSTAT3 expression in tumor cell nuclei according to clinicopathologic parameters in DLBCL patients (Table 2). The PD-L1 expression was interpreted in two aspects as expression in tumor cells (PD-L1t) and expression in immune cells (PD-L1i). The prevalence of PD-L1 expression was 21% (23/107) in tumor cells and 36% (38/107) in non-malignant immune cells. All cases with PD-L1 gene alteration (PD-L1 GA+) expressed PD-L1 protein in tumor cells (PD-L1t+), and accounted for 10% (10/102) of DLBCLs which includes translocation in 6% (6/102) and gain/amplification in 4% (4/102). Nuclear expression of pSTAT3 (> 40% cutoff) was observed in 41% (44/107) of DLBCLs (Figs. 1 and 2).

In the analysis with clinicopathologic variables (Table 2), PD-L1 GA+ was more frequent in primary extranodal DLBCLs than nodal cases (p = 0.049), and all PD-L1 GA+ cases (n = 10) belonged to non-GCB subtype according to Hans algorithm, while not reaching statistical significance (p = 0.058). As for protein expression, non-GCB subtype also showed more frequent PD-L1 expression (PD-L1t+), as well as pSTAT3 expression, in tumor cells than GCB subtype (p = 0.006 and p = 0.042, respectively). In the analysis with Bcl-2 and each component of Hans algorithm, PD-L1 GA and PD-L1t was significantly or marginally associated with lack of expression of Bcl-6 and/or CD10 (p = 0.019 for PD-L1 GA vs. Bcl-6; p = 0.052 for PD-L1t vs Bcl-6; p = 0.037 for PD-L1t vs CD10), while pSTAT3 expression was mainly related with MUM1 expression (p = 0.006). Of note, PD-L1i was marginally associated with Bcl-2 expression in tumor cells (p = 0.051). Taken together, PD-L1 and/or pSTAT3 signaling pathways are frequently activated in non-GCB subtype or extranodal DLBCLs.

To clarify the associations between PD-L1 gene/protein status and pSTAT3 expression in DLBCLs, we next performed correlation analysis among PD-L1/pSTAT3-related markers including PD-L1t, PD-L1i, PD-L1 GA and pSTAT3 expression with dichotomized variable by 40% cutoff and continuous variable (%) (Table 3). To effectively recognize associations between PD-L1-related markers and pSTAT3, pSTAT3 was analyzed as two different modes of variables: (1) a digitally counted proportional variable (%) of pSTAT3-positive tumor cells and (2) a conventionally interpreted dichotomized variable with 40% cutoff value. In this way, PD-L1t was positively correlated with PD-L1i, PD-L1 translocation and PD-L1 gain/amplification, while PD-L1i was associated only with PD-L1 translocation. Notably, tumor cells of DLBCLs with PD-L1 expression but no PD-L1 gene alteration (PD-L1t+ PD-L1 GA−) had a higher proportion (%) of pSTAT3-positive tumor cells than the rest of the subset (PD-L1t− or PD-L1t+ PD-L1 GA+) (p = 0.033; Fig. 3a), and in comparison to both PD-L1t− subset (p = 0.053) and PD-L1t+ PD-L1 GA+ subset (p = 0.050) with marginal significance as well (Fig. 3b). These results suggest that PD-L1 protein expression in DLBCL tumor cells lacking intrinsic PD-L1 gene activation mechanism, i.e., translocation and gain/amplification, may be induced by STAT3-mediated signaling pathway.

Frequent gene alteration and expression of PD-L1 were known in DLBCLs arising in certain specific sites such as mediastinum and brain [33, 34], both of which were excluded in our cohort. In our cohort, we further analyzed the PD-L1 according to the primary sites, focusing on gastrointestinal tract, testis and adrenal gland. As shown in Table 4, primary testis cases (n = 14) had a higher frequency of PD-L1 gene alteration (29%; 4/14), mainly translocation (21%; 3/14), than non-testis cases (p = 0.029 for PD-L1 GA; p = 0.031 for PD-L1 translocation). Notably, one of the three adrenal gland cases (33%) had a gain/amplification of PD-L1, though with limited significance due to low incidence (p = 0.112). These data suggest that DLBCLs of specific anatomic sites might have preferential alteration patterns of PD-L1 gene.

Survival analysis was conducted in total cohort and separately in the subgroup of patients treated with R-CHOP (n = 93; Table 5). No significance was found in clinical outcome with PD-L1 expression alone in either tumor (PD-L1t) or non-malignant immune cells (PD-L1i) (Fig. 4a–d). However, pSTAT3 expression (> 40%) was significantly associated with inferior progression-free survival in the total cohort (p = 0.021; Fig. 4e) and in the R-CHOP-treated group (p = 0.015; Fig. 4f). Multivariate cox regression that followed revealed pSTAT3 to be an independent prognostic factor in both the total cohort (p = 0.017, HR = 2.724) and R-CHOP-treated patients (p = 0.007, HR = 3.510). Other significant prognostic factors in this multivariate analysis were IPI (p = 0.001, HR = 4.910 in total cohort; p = 0.002, HR = 4.823 in the R-CHOP treated group), Eastern Cooperative Group Performance Status (p = 0.010, HR = 3.717 in the total cohort; p = 0.031, HR = 3.286 in R-CHOP treated group) and Bulky mass (p = 0.010, HR = 4.664 in the R-CHOP treated group). When the R-CHOP cohort (n = 93) was divided into pSTAT3-negative (n = 56) and pSTAT3-positive subgroups (n = 37), PD-L1 expression of non-malignant immune cells (PD-L1i) correlated with poor progression-free survival in pSTAT3-negative patients who received R-CHOP regimen by log-rank test in Kaplan–Meier analysis (p = 0.042; Fig. 5). It did not, however, turn out to be an independent prognostic factor (p = 0.089; Table 6), while ECOG PS was the only significant prognostic factor (p < 0.001, HR = 21.553). Analysis with overall survival revealed no significant results (Additional file 1: Figure S1).