Kimberley Jones

Epstein–Barr Virus‐Related Post‐Transplant Lymphoproliferative Disorders: Pathogenetic Insights for Targeted Therapy

Post‐transplant lymphoproliferative disorder (PTLD) is a spectrum of major, life‐threatening lymphoproliferative diseases occurring in the post‐transplant setting. The majority of PTLD is of B‐cell origin and is associated with several risk factors, the most significant being Epstein‐Barr virus (EBV) infection. EBVs in vitro transforming abilities, distinctive latency, clonality within the malignant cells and response to targeted therapies implicate a critical role in the biology of PTLD. This minireview focuses on EBV‐related PTLD pathogenesis, in particular the interplay between aspects of the EBV life cycle and latency with nonviral factors resulting in the wide spectrum of histology and clinical presentations encountered in PTLD. With the increased prevalence of transplantation a rise in the incidence of PTLD may be expected. Therefore the importance of laboratory and animal models in the understanding of PTLD and the development of novel therapeutic approaches is discussed.

Plasma microRNA are disease response biomarkers in classical Hodgkin lymphoma

Purpose: Although microRNAs (miRNA) show potential as diagnostic biomarkers in cancer, their role as circulating cell-free disease response biomarkers remains unknown. Candidate circulating miRNA biomarkers for classical Hodgkin lymphoma (cHL) might arise from Hodgkin–Reed–Sternberg (HRS) cells and/or nonmalignant tumor-infiltrating cells. HRS cells are sparse within the diseased node, embedded within a benign microenvironment, the composition of which is distinct from that seen in healthy lymph nodes. Experimental Design: Microarray profiling of more than 1,000 human miRNAs in 14 cHL primary tissues and eight healthy lymph nodes revealed a number of new disease node–associated miRNAs, including miR-494 and miR-1973. Using quantitative real-time PCR (qRT-PCR), we tested the utility of these, as well as previously identified disease node–associated plasma miRNAs (including miR-21 and miR-155), as disease response biomarkers in a prospective cohort of 42 patients with cHL. Blood samples were taken in conjunction with radiologic imaging at fixed time points before, during, and after therapy. Absolute quantification was used so as to facilitate implementation in diagnostic laboratories. Results: Levels of miR-494, miR-1973, and miR-21 were higher in patients than control (n = 20) plasma (P = 0.004, P = 0.007, and P < 0.0001, respectively). MiR-494 and miR-21 associated with Hasenclever scores ≥3. Strikingly, all three miRNAs returned to normal at remission (P = 0.0006, P = 0.0002, and P < 0.0001 respectively). However, only miR-494 and miR-1973 reflected interim therapy response with reduction being more pronounced in patients achieving complete versus partial responses (P = 0.043 and P = 0.0012, respectively). Conclusion: Our results demonstrate that in patients with cHL, circulating cell-free miRNAs can reflect disease response once therapy has commenced. Clin Cancer Res; 20(1); 253–64. ©2013 AACR.

Serum CD163 and TARC as Disease Response Biomarkers in Classical Hodgkin Lymphoma

Purpose: Candidate circulating disease response biomarkers for classical Hodgkin lymphoma (cHL) might arise from Hodgkin–Reed–Sternberg (HRS) cells or nonmalignant tumor-infiltrating cells. HRS cells are sparse within the diseased node, whereas benign CD163+ M2 tissue-associated macrophages (TAM) are prominent. CD163+ cells within the malignant node may be prognostic, but there is no data on serum CD163 (sCD163). The HRS-specific serum protein sTARC shows promise as a disease response biomarker. Tumor-specific and tumor-infiltrating circulating biomarkers have not been compared previously. Experimental Design: We prospectively measured sCD163 and sTARC in 221 samples from 47 patients with Hodgkin lymphoma and 21 healthy participants. Blood was taken at five fixed time-points prior, during, and after first-line therapy. Results were compared with radiological assessment and plasma Epstein-Barr virus DNA (EBV-DNA). Potential sources of circulating CD163 were investigated, along with immunosuppressive properties of CD163. Results: Pretherapy, both sCD163 and sTARC were markedly elevated compared with healthy and complete remission samples. sCD163 better reflected tumor burden during therapy, whereas sTARC had greater value upon completion of therapy. sCD163 correlated with plasma EBV-DNA, and associated with B symptoms, stage, and lymphopenia. Circulating CD163+ monocytes were elevated in patients, indicating that sCD163 are likely derived from circulating and intratumoral cells. Depletion of cHL CD163+ monocytes markedly enhanced T-cell proliferation, implicating monocytes and/or TAMs as potential novel targets for immunotherapeutic manipulation. Conclusion: The combination of circulating tumor-infiltrate (sCD163) and tumor-specific (sTARC) proteins is more informative than either marker alone as disease response biomarkers in early and advanced disease during first-line therapy for cHL. Clin Cancer Res; 19(3); 731–42. ©2012 AACR.

Expansion of EBNA1-specific effector T cells in posttransplantation lymphoproliferative disorders.

Immunosuppression resulting in impaired Epstein-Barr virus (EBV)-specific T-cell immunity is involved in the pathogenesis of EBV-positive post-transplantation lymphoproliferative disorder (EBV(+) PTLD). Restoration of EBV-specific T-cell immunity by adoptive immunotherapy can induce remission. EBV-nuclear antigen-1 (EBNA1) is unique in being expressed in all cases of EBV(+) PTLD. Recent data demonstrate that EBNA1 is not immunologically silent and can be exploited as a T-cell target. There are no data on EBNA1-specific T cells in PTLD. EBNA1-specific T cells capable of proliferation, interferon-γ release, and CD107a/b degranulation were assayed in 14 EBV(+) PTLD diagnostic blood samples and 19 healthy controls. EBNA1-specific CD4(+) T cells predominated and were expanded in 10 of 14 patients and 19 of 19 controls. Although human leukocyte antigen class I alleles influenced the magnitude of the response, EBNA1-specific CD8(+) effector T cells were successfully generated in 9 of 14 EBV(+) PTLD patients and 16 of 19 controls. The majority of PTLD patients had a polymorphism in an EBNA1 epitope, and T-cell recognition was greatly enhanced when EBNA1 peptides derived from the polymorphic epitope were used. These results indicate that EBNA1-specific T cells should be included in adoptive immunotherapy for PTLD. Furthermore, expansion protocols should use antigenic sequences from relevant EBV strains.

Sodium valproate in combination with ganciclovir induces lysis of EBV‐infected lymphoma cells without impairing EBV‐specific T‐cell immunity

Histone deacytelase inhibitiors (HDACi) represent a new class of anti‐lymphoma therapeutics. Data in the clinical setting regarding on‐ and off‐target effects of these agents are limited. Epstein–Barr virus (EBV)‐positive lymphomas represent a highly defined system in which to make these observations. We present a case of a patient with multiple relapsed EBV‐positive Diffuse Large B‐cell Lymphoma that was chemo‐refractory to anthracylcines, alkylating agents and rituximab. Treatment was commenced with the HDACi sodium valproate (VPA) in combination with the anti‐viral nucleoside analogue ganciclovir (GCV). Therapy resulted in detectable cell‐free unencapsulated circulating EBV‐DNA providing supportive evidence for the first‐time that lysis of virus infected lymphoma cells is induced using this therapeutic combination. EBV‐specific CD8+ effector T‐cell immunity was not impaired by VPA/GCV. Although GCV/VPA was insufficient to induce clinical remission, our data furthers the rationale that more potent HDAC inhibitors such as butyrate or gemcitabine together with GCV, perhaps in combination with chemotherapy, should be further investigated as therapy in relapsed/refractory EBV‐positive lymphomas.

Ratios of T-cell immune effectors and checkpoint molecules as prognostic biomarkers in diffuse large B-cell lymphoma: a population-based study

BACKGROUND Risk-stratification of diffuse large B-cell lymphoma (DLBCL) requires identification of patients with disease that is not cured, despite initial treatment with R-CHOP. The prognostic importance of the revised International Prognostic Index (R-IPI) and cell of origin of the malignant B cell are established in DLBCL. We aimed to develop a novel, easily applicable, tissue-based prognostic biomarker based on quantification of the tumour microenvironment that is independent of and additive to the R-IPI and cell of origin. METHODS We performed digital hybridisation on the NanoString platform to assess the relation between immune effector and inhibitory (checkpoint) genes in 252 formalin-fixed, paraffin-embedded DLBCL tissue specimens obtained from patients treated with R-CHOP. We used a tree-based survival model to quantify net antitumoral immunity (using ratios of immune effector to checkpoint genes) and to generate a cutoff as an outcome predictor in 158 of the 252 patients. We validated this model in tissue (n=233) and blood (n=140) samples from two independent cohorts treated with R-CHOP. FINDINGS T-cell and NK-cell immune effector molecule expression correlated with tumour-associated macrophage and PD-1/PD-L1 axis markers, consistent with malignant B cells triggering a dynamic checkpoint response to adapt to and evade immune surveillance. The ratio of CD4*CD8 to (CD163:CD68[M2])*PD-L1 was better able to stratify overall survival than was any one immune marker or combination, distinguishing groups with disparate 4-year overall survival. 94 (59%) of 158 patients had a score above the cutoff and 4-year overall survival of 92·1% (95% CI 82·9-96·7), and the remaining 64 (41%) patients had a score below the cutoff and 4-year overall survival of 47·0% (32·8-60·5; hazard ratio [HR] 8·3, 95% CI 4·3-17·3; p<0·0001). The CD4*CD8:M2*PD-L1 immune ratio was independent of and added to the R-IPI and cell of origin. Tissue findings in the independent tissue cohort accorded with those in our initial tissue cohort. 139 (60%) of 233 patients had a score above the cutoff and 4-year overall survival of 75·6% (95% CI 64·6-83·6), with the remaining 94 (40%) patients having a score below the cutoff (63·5% [52·5-72·7]; HR 1·9, 95% CI 1·1-3·3; p=0·0067). INTERPRETATION Ratios of immune effectors to checkpoints augment the cell of origin and R-IPI in DLBCL and are applicable to paraffin-embedded biopsy specimens. These findings might have potential implications for selection of patients for checkpoint blockade within clinical trials. FUNDING Leukaemia Foundation of Queensland, Kasey-Anne Oklobdzijato Memorial Fund, the Australasian Leukaemia and Lymphoma Group (Malcolm Broomhead Bequest), the Australian Cancer Research Foundation, and the Cancer Council of Queensland.

Selective accumulation of virus-specific CD8+ T cells within the peripheral blood stem cell compartment.

To the editor: The absence of cellular immunity is central to the pathogenesis of herpesvirus-mediated diseases after allogeneic hemopoietic stem cell transplantation (HSCT).[1][1],[2][2] For both bone marrow (BM)– and granulocyte-colony stimulating factor–mobilized peripheral blood stem cells

Expression profiling of Epstein-Barr virus-encoded microRNAs from paraffin-embedded formalin-fixed primary Epstein-Barr virus-positive B-cell lymphoma samples

Epstein-Barr virus (EBV) is implicated in a range of B-cell malignancies and expresses unique microRNAs (EBV-miRNAs). Due to the requirements for high-quality RNA, studies profiling EBV-miRNA in EBV-positive lymphomas have been restricted to cell-lines or frozen samples. However, the most commonly available archived patient material is paraffin-embedded formalin-fixed (FFPE) tissue. This has impeded the widespread profiling of EBV-miRNA expression in clinical samples. The requirements for accurate EBV-miRNA real-time RT-PCR quantitation in FFPE tissues representing a broad-spectrum of EBV-positive lymphomas were determined systematically, including where the neoplastic cells are sparse relative to the non-malignant infiltrate. The level of cellular EBV-load correlated strongly with the sum of EBV-miRNA expression and the number of EBV-miRNAs detectable. As calibrators for cellular EBV-load, the sum EBV-miRNA was optimal to EBV-genome copy number and EBER2 expression level, with the added advantage of not requiring additional assays. EBV-miRNA was profiled reliably within archival FFPE tissue in 14/23 patients, but not in tissues with low abundance EBV. This method enabled specific and simultaneous detection of numerous EBV-miRNAs in FFPE lymphoma samples that contain EBV at high to medium levels, making it as a useful tool for studies of EBV-miRNA in the majority of diagnostic biopsies.

A Comprehensive Analysis of the Cellular and EBV‐Specific MicroRNAome in Primary CNS PTLD Identifies Different Patterns Among EBV‐Associated Tumors

Primary central nervous system (pCNS) posttransplant lymphoproliferative disorder (PTLD) is a complication of solid organ transplantation characterized by poor outcome. In contrast to systemic PTLD, Epstein–Barr virus (EBV)‐association of pCNS PTLD is almost universal, yet viral and cellular data are limited. To identify differences in the pattern of EBV‐association of pCNS and systemic PTLD, we analyzed the expression of latent and lytic EBV transcripts and the viral and cellular microRNAome in nine pCNS (eight EBV‐associated) and in 16 systemic PTLD samples (eight EBV‐associated). Notably although 15/16 EBV‐associated samples exhibited a viral type III latency pattern, lytic transcripts were also strongly expressed. Members of the ebv‐miR‐BHRF1 and ebv‐miR‐BART clusters were expressed in virtually all EBV‐associated PTLD samples. There were 28 cellular microRNAs differentially expressed between systemic and pCNS PTLD. pCNS PTLD expressed lower hsa‐miR‐199a‐5p/3p and hsa‐miR‐143/145 (implicated in nuclear factor kappa beta and c‐myc signaling) as compared to systemic PTLD. Unsupervised nonhierarchical clustering of the viral and cellular microRNAome distinguished non‐EBV‐associated from EBV‐associated samples and identified a separate group of EBV‐associated pCNS PTLD that displayed reduced levels of B cell lymphoma associated oncomiRs such as hsa‐miR‐155, ‐21, ‐221 and the hsa‐miR‐17‐92 cluster. EBV has a major impact on viral and cellular microRNA expression in EBV‐associated pCNS PTLD.

The impact of HLA class I and EBV latency-II antigen-specific CD8+ T cells on the pathogenesis of EBV+ Hodgkin lymphoma

In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein–Barr virus (EBV) latency‐II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV+cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA‐A*02 is protective in EBV+cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA‐A*02– versus HLA‐A*02+ EBV+cHL patients, suggesting that LMP2A‐specific CD8+ T cell anti‐tumoral immunity may be relatively ineffective in HLA‐A*02– EBV+cHL. To ascertain the impact of HLA class I on EBV latency antigen‐specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV+cHL, the magnitude of ex‐vivo LMP1/2A‐specific CD8+ T cell responses was elevated in HLA‐A*02+ patients. Furthermore, in a controlled in‐vitro assay, LMP2A‐specific CD8+ T cells from healthy HLA‐A*02 heterozygotes expanded to a greater extent with HLA‐A*02‐restricted compared to non‐HLA‐A*02‐restricted cell lines. In an extensive analysis of HLA class I‐restricted immunity, immunodominant EBNA3A/3B/3C‐specific CD8+ T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A‐specific responses were confined largely to HLA‐A*02. Our results demonstrate that HLA‐A*02 mediates a modest, but none the less stronger, EBV‐specific CD8+ T cell response than non‐HLA‐A*02 alleles, an effect confined to EBV latency‐II antigens. Thus, the protective effect of HLA‐A*02 against EBV+cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency‐II antigen‐specific CD8+ T cell hierarchies.