Introduction: Regulation of the PD-1/PD-L1 immune checkpoint has been under intense investigation in the era of immunotherapy, and PD-1 blocκade has shown promising clinical responses in several neoplasms including classical Hodgkin lymphoma (HL). Frequent amplification of the PD-L1 / PD-L2 gene locus (9p24.1) are found in most HLs, however, in other lymphoma types such as diffuse large B-cell lymphomas (DLBCL), genetic alterations of 9p24.1 locus are detected only in a small subset of cases (< 20%). Therefore, other mechanisms including transcriptional regulation of the PD-L1 gene are currently under investigation. Previous studies have shown that in another CD30+ lymphoma type, namely anaplastic large cell lymphoma of T-cell origin, PD-L1 is highly expressed despite the absence of gene amplification or other chromosomal alterations of 9p24.1 locus, and transcriptional regulation is biologically involved. However, the PD-L1 expression levels, the genetic basis of PD-L1 expression and their clinical significance in CD30+ DLBCLs are not yet studied.
Methods: From a large database of 2110 DLBCLs, 78 patients with CD30+ DLBCL (44 GCB and 34 ABC-type) diagnosed and treated at the University of Texas M.D. Anderson Cancer Center (USA) and Karolinska University Hospital (Sweden) were identified. All patients were treated with R-CHOP regimen and had available clinical data. PD-L1 expression was assessed by immunohistochemistry using a monoclonal antibody and autostainer systems (Dako or Ventana). PD-L1 gene amplification and chromosomal aberrations of its gene locus were assessed by FISH. Mutations of TP53 gene were confirmed by sequencing. Statistical associations of the PD-L1 expression level (% of positive tumor cells) among various patient subgroups were based on the non-parametrical Mann-Whitney and Kruskall-Wallis tests. Survival analyses were performed using the Kaplan-Meier method (log-rank test) and Cox regression models. The DLBCL in vitro system included 3 cell lines (RC-K8, OCI-Ly3, U2932), which were treated with increasing concentrations of Nutlin 3A to stabilize and activate wild-type TP53. Western blot analysis was used to assess the protein levels of p53 and PD-L1 and quantitative RT-PCR was utilized to assess the PD-L1 mRNA level in the cell lines.
Results: Using FISH, PD-L1 gene amplification, chromosomal gains and polyploidy were observed in 6 (7.7%), 3 (3.8%) and 7 (9%) cases, respectively. Expression of PD-L1 protein was found in 66 (85%) CD30+ DLBCLs and the percentage of positive tumor cells ranged from 5 to 100% (median 30%) among the positive cases. In this cohort, TP53 mutations were detected in 14 (18%) CD30+ DLBCL and were not statistically associated with PD-L1 gene amplification or expression levels. However, all PD-L1 negative tumors (n=12) were associated with a wild-type TP53 gene. High expression levels of PD-L1 correlated with ABC phenotype (p=0.04), B symptoms (p=0.03), high serum LDH (p=0.04), ECOG performance status>1 (p=0.015), and the presence of >1 extranodal sites (p=0.04) but not with Ann Arbor stage. Patients who achieved complete remission showed significantly lower PD-L1 expression level (median 20% vs. 40%, p=0.03). Given the significant survival benefit of CD30 expression in DLBCL, PD-L1 expression in this subset abolished advantage of failure-free (FFS) and overall (OS) survival after 48 months of median follow-up. The in vitro data showed that stabilization and activation of p53 in DLBCL cell lines carrying a wild-type p53 gene led to concentration-dependent increase of the PD-L1 mRNA and protein levels. As in silico analysis did not demonstrate a possible TP53 binding site on the PD-L1 gene promoter, an indirect mechanism of p53-dependent regulation of PD-L1 is suggested.
Conclusions: PD-L1 is expressed in most CD30+ DLBCLs, but amplification of the PD-L1 gene locus is not a common mechanism unlike Hodgkin lymphoma. Activation of wild-type TP53 indirectly upregulates PD-L1 in DLBCL and the underlying mechanisms merit further investigation.
Smedby: Janssen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees.
Asterisk with author names denotes non-ASH members.