PD1/PD-L1 pathway targeting therapies are nowadays an established treatment option for patients with NSCLC. We assessed whether PD-L1 expression in NSCLC tumor cells was associated with specific ...clinical features or overall survival using four different clones.
A retrospective study included formalin-fixed paraffin embedded (FFPE) surgical tumors from 482 patients. PD-L1 status was assessed with immunohistochemistry in tumor cells on tissue microarrays using clones 28-8, 22C3, SP263 and SP142. Associations with OS were assessed by Kaplan-Meier and multivariate Cox's regression analysis.
Patients' median age: 68 years (39–86); histology: adenocarcinoma (AdCa) 61%, squamous-cell carcinoma (SqCC) 33%, and large cell carcinoma (LCC) 6%; p-stage: IA (46%), IB (30%), IIA (10%), IIB (11,4%), IIIA (1,2%), IIIB – IV (0,4%). PD-L1 positivity (≥1%) in NSCLC for clones 28-8, 22C3, SP263, SP142 was 41.5%, 34.2%, 42.7%, 10.4%, respectively (Pearson Chi-square p < 0.0001). PD-L1 expression was correlated with histology, tumor size and grading. Statistically significant association between PD-L1 expression and OS in NSCLC and Non-AdCa was observed with clone SP142 (log-rank p = 0.045 and p = 0.05, respectively). Statistically significant association between PD-L1 expression and OS in LCC was observed with clones 22C3 (log-rank p = 0.009) and SP263 (log-rank p = 0.050).
Overexpression of the PD-L1 clone SP142 was associated with poor overall survival in NSCLC and Non-AdCa. Clones 22C3 and SP263 were associated with poor prognosis in LCC. PD-L1 status might serve as a prognostic marker in NSCLC.
•PD-L1 status is a validated predictive biomarker for immunotherapy.•PD-L1 staining in tumor cells could be associated with poor prognosis.•PD-L1 (SP142) shows association with poor overall survival in NSCLC.•Validation of the prognostic value of the four commercial PD-L1 clones.
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.
Under normal conditions, the colorectal mucosa exhibits small numbers of scattered lymphocytes and plasma cells in the lamina propria and only few mucosal lymphoid aggregates (MLAs). In Crohn's ...colitis, the number of lymphocytes and plasma cells in the lamina propria and of MLA is substantially increased. In addition, multiple lymphoid aggregates are newly formed in the submucosa (submucosal lymphoid aggregate (SLA)) and deeper. The aim of the present study was to investigate the cellular immune response in MLA, in SLA, and in the lamina propria in Crohn's colitis. Fifty-nine colorectal biopsies/surgical specimens with or without inflammatory diseases were challenged with multiple myeloma 1 (MUM1) that highlights activated T cells, committed B cells, and plasma cells (aT/cB/PC). The number of MUM1-positive aT/cB/PC per high-power field (HPF) in MLA and in SLA was significantly lower in Crohn's colitis than in controls (
p
< 0.05). In contrast, the number of MUM1-positive aT/cB/PC per HPF in the lamina propria was significantly higher in Crohn's colitis and in other forms of chronic colitis than in controls (
p
< 0.05). The paucity of MUM1-positive cells in MLA and in SLA in Crohn's colitis might be caused by an increased number of MUM1-negative precursors. These precursors would eventually migrate into the lamina propria to differentiate into aT/cB/PC, complying thereby with the immunological mucosal demands generated by the on-going chronic inflammation.
In about 60% of Epstein-Barr virus (EBV) carrying nasopharyngeal carcinomas (NPC) LMP1 expressing cells can be detected. The frequency of LMP1 positive cells and the expression level varies from cell ...to cell in the different tumors. Cell lines derived from EBV positive NPCs loose the virus during in vitro culture. The in vitro infected NPC cell line TWO3-EBV used in our study carries the neomycin-resistance gene containing EBV and expresses low level of LMP1. With this cell line it was thus possible to study the regulation of LMP1 expression by modification of chromatin acetylation state.
The TWO-EBV cell line was treated with n -butyrate (NB) or trichostatin A (TSA).
Shown by immunoblotting, the LMP1 level was elevated in the treated samples. Already 2 h after TSA exposure LMP1 expression was higher and it increased up to 24 h. Immunofluorescence staining showed that nearly all cells were LMP1 positive. Neither EBNA2 nor BZLF1 were induced. Tested first 2 h after the treatment, acetylated histone H3 and H4 were already detectable, and their level increased up to 8 h. Chromatin immunoprecipitation (ChIP) verified that the LMP1-promoter (LMP1p) (ED-L1) was acetylated after TSA treatment.
EBV carrying epithelial cells do not express EBNA-2. We showed that LMP1 expression was upregulated by histone deacetylase inhibitors in an in vitro infected, EBV carrier NPC cell line.
In the in vitro infected B-cells six EBV-encoded nuclear antigens (EBNA-1–6) and three latent membrane proteins (LMP-1, -2A, -2B) are expressed (type III latency). In addition, other restricted forms ...of latency occur in the EBV-carrying malignancies. In Burkitt lymphoma (BL) only EBNA-1 is expressed (type I), while in Hodgkin lymphoma (HL), T-, and NK-lymphoma, and nasopharyngeal carcinoma EBNA-1 and LMPs are expressed (type II). B-cells with these three expression patterns have been detected in healthy virus carriers.
While in type III latency two viral transcriptional activators, EBNA-2 and -5, are responsible for LMP-1 expression, the mechanism that controls the expression of LMP-1 in type II latent cells is not known. In order to study the interaction of EBV- and HL-derived cells, we studied the in vitro EBV-converted subline of the KMH2 cells that express only EBNA-1 and LMP-2A. Interestingly, exposure of the KMH2-EBV cells to CD40-ligand and IL-4 induced LMP-1 expression, in the absence of EBNA-2. In BL cell lines lacking EBNA-2 another cytokine, IL-10, could induce LMP-1 expression. IL-10 induced LMP-1 also in tonsillar B-cells infected with the EBNA-2-deleted virus strain P3HR-1.
Our results show that cytokines are responsible for the expression of LMP-1 in type II latent B-cells. These signals are available in the germinal center environment and in the granulation tissue of HLs. Based on these results we propose that LMP-1 expression is induced by extracellular signals and is not a constitutive characteristic of the EBV-carrying type II B-cells. Cytokine mediated induction of LMP-1 may also explain the heterogeneous expression of this viral gene seen in normal and malignant cells.
Proliferation of pluripotent, bone marrow stem cells, which develop to lymphoid and myeloid progenitors, is negatively regulated by estrogen. Although in estrogen deficiency and in estrogen receptor ...knockout mice there is significant alteration in bone marrow hematopoiesis, the effects of aging on estrogen receptor deficiencies in mice have not been investigated yet. In this study we show that by 1.5 years of age, estrogen receptor β knockout (ERβ-/-) mice develop pronounced splenomegaly that is much more severe in females than in males. Further characterization of these mice revealed myelogenous hyperplasia in bone marrow, an increase in the number of granulocytes and B lymphocytes in blood, lymphadenopathy, and infiltration of leukocytes in the liver and lung. Analysis by flow cytometry of the bone marrow cells revealed that the percentage and total number of Gr-1hi/Mac-1hi-positive granulocytes were increased by 15-30% and 100%, respectively. The numbers of B cells in the bone marrow and spleen were significantly higher in ERβ-/-mice than in WT littermates. Some of the ERβ-/-mice also had a severe lymphoproliferative phenotype. Thus the absence of ERβ results in a myeloproliferative disease resembling human chronic myeloid leukemia with lymphoid blast crisis. Our results indicate a previously unknown role for ERβ in regulating the differentiation of pluripotent hematopoietic progenitor cells and suggest that the ERβ-/-mouse is a potential model for myeloid and lymphoid leukemia. Furthermore, we suggest that ERβ agonists might have clinical value in the treatment of leukemia.
B cell type chronic lymphocytic leukaemia (B-CLL) cells carry the Epstein-Barr virus (EBV) receptor CD21 and can be infected in vitro with the virus. The infected cells exhibit an unusual EBV ...program, they express the nuclear proteins but not latent membrane protein 1 (LMP-1). Similar cells were encountered in lymphoid tissues of infectious mononucleosis (IM) patients and in lymphoproliferations of immunosuppressed patients. EBV infected B-CLL cells can be regarded as model for this viral program. In B cells the regulation of LMP-1 is executed mainly by EBV encoded nuclear antigen 2 (EBNA-2), interacting with several cellular proteins and these complexes bind to specific sequences in the LMP-1 promoter. ATF2 and c-Jun were shown to be among the interacting partners of EBNA-2. These molecules can be detected in experimentally infected B lymphocytes. We found c-Jun and/or phosphorylated ATF-2 (p-ATF-2) expression in some B-CLL ex vivo samples. They disappeared or their expression declined promptly in explanted cells, even if they were infected with EBV in vitro. Activation of the infected B-CLL cells by exposure to CD40L was accompanied by p-ATF-2 and c-Jun but not by LMP-1 expression. In one of three clones tested, subsequent treatment with histone deacetylase inhibitors (HDACi), TSA or n-butyrate, could induce LMP-1. Treatment with phorbol-12, 13-dibutyrate (PDB) induced LMP-1 expression in three of four clones. Neither the HDACi nor the PDB treated cells survived.
Depending on stage and risk factors, up to 30% of patients with advanced Hodgkin lymphoma (HL) progress or relapse. Patients with pleural effusions have a particularly poor prognosis and this stage ...of HL is regularly resistant to chemotherapy. All currently available HL cell lines are derived from late stage HL patients. In the present study we measured the sensitivity of these HL lines against the 26 most frequently used cytostatic drugs. We used a novel fluorescent short-term survival assay where the cell was incubated with the drugs for 4 days. The precise number of differentially stained live and dead cells was determined using a custom-built automated laser confocal fluorescent microscope. We found that HL cells, independently of their origin, showed very similar sensitivity patterns for several of the drugs. All HL cell lines were highly sensitive to dactinomycin, paclitaxel and etoposide. Our data suggest that the inclusion of dactinomycin and paclitaxel into chemotherapy protocols against late stage Hodgkin lymphoma with pleural effusion may be justified.
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