ALBERT

Description: LAG3 is an immune checkpoint expressed on a variety of immune cells including a sub-population of 'exhausted' effector T cells and TREGs. Early-phase studies of anti-LAG3 mAb show promise in solid and haematological cancers. We have previously demonstrated LAG3 is enriched within the tumor microenvironment in Hodgkin Lymphoma (Gandhi et al. Blood 2006). Data in the aggressive B-cell lymphoma DLBCL is lacking. We used a conventional discovery/ validation approach in two population based Australian cohorts (discovery: Brisbane/Canberra; validation: Sydney) totalling 250 patients treated with R-CHOP with 〉4 year follow-up. Digital gene expression (NanoString) using a consistent LAG3 cut-off showed inferior 5-year overall survival (OS) in both cohorts (discovery: 54% vs. 82%, HR 3.13, p=0.003; validation: 63% vs. 86%, HR 2.95, p=0.025 respectively). In a multivariate model, LAG3HI(p=0.001) was a predictor of OS independent of R-IPI and Cell-of-Origin (by NanoString LST assay). PD-L1 expression was also a predictor of survival though to a lesser degree than LAG3. Notably, LAG3 expression stratified PD-L1HIpatients into two sub-groups with differential survival, with dual LAG3 and PD-L1 positivity conferring particularly poor OS (PD-L1HI/LAG3HI39% vs. 81% PD-L1HI/LAG3LO, HR 3.65, p=0.023). Next, the discovery/validation cohorts were combined with 129 additional DLBCL cases from the ALLG biobank (in whom tissue but no outcome data was available), to test for biological associations and correlations. In these 379 cases, LAG3HIwas enriched in the ABC/UC (66%) subtype vs. LAG3LO(p=0.003). LAG3 was positively correlated with numerous immune checkpoints/effectors including CD4, CD8, PD-1, PD-L1, PD-L2, TIM-3 and CD163 (all p

Description: Background Stage I/II or early-stage follicular lymphoma (ESFL) is considered potentially curable with radiotherapy (XRT). While XRT does achieve local disease control in 〉90% of cases, more than half the patients (pts) relapse by 10 years (yr), generally outside of the radiation field. A recent randomized controlled trial (TROG 99.03) demonstrated that combined modality therapy (CMT), with sequential XRT and systemic therapy, significantly improved PFS but not overall survival (OS) compared to XRT alone in ESFL. However, only half the pts were staged with 18F‐FDG positron emission tomography and computed tomography (PET) and 58% of CMT pts did not receive rituximab.Compared with CT staging, 20-60% of cases are upstaged by PET. Consequentially, there are limitations in applying this trial to modern populations. Despite the support of current guidelines, only one third of pts in clinical practice are treated with XRT. This suggests a need to better understand the role of other treatments, including watchful waiting (WW), in the PETera. Our aim was to compare outcomes with real-world treatment approaches in rigorously staged ESFL patients. Methods We conducted an international, multicenterretrospective study of stage I and II FL pts rigorously staged with bone marrow biopsy and PET. Eligible pts were 〉18yr with newly-diagnosed grade 1-3A FL and ≥3 months follow up. Primary outcome measures were overall response rate (ORR), progression free survival (PFS), OS and risk of transformation. Survival curves were estimated with the Kaplan-Meier method and uni- and multi-variate analysis was performed using Cox regression model. Results A total of 387 pts treated at 13 Australian and 3 Canadian centres between 2005-2017 were studied. Median follow-up was 45 months (range 3.1 - 164.0).5-yrPFS and OS rates were 73.5% (95% CI 66.0-78.5) and 94.4% (95% CI 89.4-93.6) respectively. 22 patients had stage IE duodenal FL with 5-yr PFS and OS rates of 100% and 100% respectively. Considering the unique biology and favorable prognosis of duodenal FL, these cases were excluded from subsequent analyses. Treatment approaches 365 pts included WW (defined as absence of treatment within 6 months from diagnosis) (23.2%), XRT (46.8%), immunochemotherapy (17.2%) and CMT (12.6%). Treatment regimens were: R-CHOP (48.1%), R-CVP (24.4%), BR (9.9%), other (17.6%). First-line therapies for actively treated pts yielded comparable ORRs of 95.6%, 96.7% and 95.9% for XRT, immunochemotherapy and CMT, respectively (P=0.94). Overall, 18.2% of pts relapsed at distant sites, 88.2% of all relapses. Treatment cohorts differed in baseline clinical characteristics. WW pts were significantly older (P=0.007) but otherwise comparable to those treated actively. Compared to chemotherapy or CMT pts, those treated with XRT had more favorable features including fewer B symptoms (4.2% vs 11.2% p=0.029), bulk (≥7cm) (6.8% vs 25.3%, p

Description: Background Primary and secondary central nervous system lymphoma (PCNSL/SCNSL) are rare brain malignancies with an aggressive clinical course and dismal outcomes. The BTK inhibitor ibrutinib has activity in a range of B-cell lymphomas. Phase I and II studies of ibrutinib monotherapy in relapsed/refractory PCNSL have demonstrated promising results, with response rates of up to 81%. Response rates of up to 69% have also been seen in SCNSL. Ibrutinib has been combined with other systemic agents (e.g. rituximab and methotrexate) in phase 1 trials with promising results (Grommes et al Blood 2019); combination with more intensive combination chemotherapy regimens also appears efficacious but has exhibited a potentially limiting toxicity profile, in particular invasive fungal infections. (Lionakis et al Cancer Cell 2017). However, data for ibrutinib in PCNSL and SCNSL outside the clinical trial setting are scarce. Methods We performed a national, multicentre, retrospective study of the clinical outcomes and safety of patients (pts) with PCNSL and SCNSL who received ibrutinib between December 2015 and June 2019. Results The baseline characteristics of the 16 eligible pts are summarised in the table (Figure 1a). 88% (n=14) had relapsed/refractory disease, with two patients receiving ibrutinib as a component of multiagent frontline therapy. The most common target daily dose was 560mg (range 420-840mg); this was reached in all pts. Among all pts, the objective response rate (ORR) was 69%, with a complete remission (CR) rate of 63%. Both patients receiving ibrutinib in combination frontline therapy achieved a CR. ORR in PCNSL pts was 50% (n=4) and SCNSL pts was 88% (n=7), (P=0.28). ORR was 80% (n=4) when ibrutinib was administered as monotherapy, 80% (n=4) when administered with chemotherapy and 75% (n=3) when administered concomitant with whole brain radiotherapy. MYD88L265P mutation at time of starting ibrutinib was only tested in two patients with PCNSL and none with SCNSL. The mutation was detected in both PCNSL cases, and both later attained a CR. With a median follow up of 14 months, calculated using median observation period among patients alive at last follow-up, median progression free survival (PFS) and overall survival (OS) were not reached. 12 month PFS was 56% for the entire cohort (95% confidence interval [CI] 29-76); 50% for PCNSL (95% CI 15-77) and 60% for SCNSL (95% CI 20-85%) (Figure 1b). 12 month OS was 66% for the entire cohort (95% CI 36-85) ; 50% for PCNSL (95% CI 15-77) and 80% for SCNSL (95% CI 20-97%) (Figure 1c). Ten pts had PFS 〉6 months (longest 41.3 months), and 11 pts (69%) remained alive, with 9/11 being free from disease progression. Seven pts remain on ibrutinib at time of data analysis, 3 with PCNSL and 4 with SCNSL. Nine pts (56%) have discontinued therapy; 6 due to progressive disease (PD), 1 due to atrial fibrillation with hypotension requiring inotropic support and 2 in remission, one of whom subsequently underwent autologous stem cell transplant. Dose interruptions or reductions were required in 6 pts (37%), due to bleeding (n=2), infection (n=3) and neutropenia (n=1). Grade 3/4 adverse events were infection (31%, n=5), neutropenia (25%, n=4), febrile neutropenia (12%, n=2) and one each (6%, n=1) of atrial fibrillation, thrombocytopenia, and anaemia. No invasive fungal infections were observed, despite use of 8-16mg daily dexamethasone immediately prior to or during ibrutinib therapy in 10 pts (62%). Conclusions In this small real-world, majority methotrexate-refractory population, ibrutinib demonstrates encouraging efficacy and durable responses despite doses lower than used in clinical trials. No unexpected adverse events were observed. Invasive fungal infections were not seen, despite most patients receiving concurrent dexamethasone and/or chemotherapy. We observed substantial variety in additional therapy during ibrutinib treatment, and the optimal way to use ibrutinib in this heterogenous patient group remains unclear. Disclosures Manos: NovoNordisk Pharmaceuticals: Other: Travel; Janssen: Honoraria. Ho:Celgene: Consultancy, Other: Advisory role. Grigg:Abbvie: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Other: Travel. Gandhi:Amgen: Honoraria; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Research Funding; Roche: Honoraria, Other: Travel Support; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees. Hawkes:Astra Zeneca: Research Funding; Mundi pharma: Research Funding; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck KgA: Research Funding; Merck Sharpe & Dohme: Membership on an entity's Board of Directors or advisory committees; Takeda: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses, Research Funding, Speakers Bureau. Cheah:Roche: Other: Travel expenses; Roche, Janssen, MSD, Gilead, Loxo Oncology, AstraZeneca, TG Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene, Roche, Abbvie: Research Funding. OffLabel Disclosure: Ibrutinib is not currently approved for use in DLBCL/CNS lymphoma.

Description: Key Points DLBCL can be detected in the blood by immunoglobulin high-throughput sequencing (Ig-HTS) with high specificity. Although DLBCL can be detected in leukocytes or plasma by Ig-HTS, plasma has greater sensitivity and more accurately reflects disease.

Description: In classical Hodgkin lymphoma (CHL) the tumor microenvironment (TME) is enriched in T cells that modulate antitumor immunity. PD1 blockade partially restores anti-tumoral T cell function, to induce impressive responses in a proportion of patients with relapsed/refractory CHL (Chen et al JCO 2017). Further characterisation of T cell immune evasion mechanisms in CHL will permit the rational development of enhanced immunotherapeutic strategies. Lymphocyte-activation gene 3 (LAG3) is a cell surface molecule known to be expressed on a subset of immune effector T cells and intratumoral regulatory T cells (Tregs) in solid-organ tumors, with combination PD1/LAG3 mAb blockade showing early promise (Ascierto et al 2017 JCO abst 9520). In contrast, data in haematological malignancies is limited, although it is known that LAG3+ T cells suppress anti-tumoral immunity in CHL and B-CLL (Gandhi et al Blood 2006; Shapiro et al Haematologica 2017). Interestingly, in B-CLL LAG3 is found on both T cells and malignant B cells. Whether Hodgkin Reed-Sternberg (HRS) cells express LAG3 is unknown. To characterise in detail intratumoral and circulating LAG3 in CHL we used a conventional discovery / validation approach. The local institutional discovery cohort was drawn from Princess Alexandra Hospital in Brisbane (Australia), and validated in samples from the prospective randomised phase III international "RATHL" trial (Johnson et al NEJM 2016). Firstly, LAG3 gene expression (GEP) was digitally quantified by NanoString in FFPE tissues in the discovery cohort and compared to normal control nodes and DLBCL tissues. Normalised LAG3 mRNA counts were 5-10-fold higher in CHL than in controls (P 〈 0.001) and 3-5-fold higher than in DLBCL (P 〈 0.001), whereas PD1 and TIM3 mRNA counts did not differ. In CHL samples LAG3 mRNA counts were markedly increased compared to PD-1 axis molecules and TIM3 (P 〈 0.001) (Figure 1a). Higher levels of LAG3 mRNA counts were correlated with infiltration by T cells (CD4 r = 0.55; P 〈 0.00001; CD8 r = 0.51, P 〈 0.0001), and macrophages (CD68 r = 0.45; P = 0.002). Findings were replicated in the RATHL cohort. Next, intratumoral LAG3 cellular distribution was established. Flow cytometry was used to quantify LAG3 in T cell subsets and CD30+CD3- HRS cells in 6 de-aggregated freshly frozen CHL nodes (TILs). LAG3 was evenly distributed between CD8+ T cells, CD127LOCD25HI natural-Tregs (nTregs) and CD127LOCD25LO induced-Tregs (iTregs), but with minimal expression on CD4 non-Tregs, with the latter constituting the majority of intratumoral T cells. LAG3+ T cells typically co-expressed PD1 and/or TIM3. LAG3 was expressed on CD30+CD3+ cells but not on CD30+CD3- cells, consistent with LAG3 expression on activated T cells. Multispectral immunofluorescence (mIF) image analysis confirmed these findings in histological tumor samples (Figure 1b). Also, there was negligible expression of LAG3 on HRS-lines. Finally, the potential role of soluble LAG3 (sLAG3) as a rapid-turnaround circulating biomarker applicable to the routine diagnostic laboratory, was assessed in serum samples using the MSD R-PLEX assay. In the discovery cohort sLAG3 was 3-4-fold increased at pre-therapy compared to controls and 3-6M post-therapy serum (P = 0.001). Results from pre-therapy RATHL serum samples were similar (P 〈 0.05). Notably in RATHL samples at interim restaging after 2 ABVD cycles sLAG3 had reduced by ~5-fold compared to pre-therapy (P 〈 0.0001) in patients with PET/CT responsive disease (Figures 1 c + d). Twelve months post therapy sLAG remained significantly lower than pre-therapy (P 〈 0.05) and was equivalent to control samples. Pre-therapy serum sLAG3 demonstrated a modest correlation with tissue LAG3 mRNA counts (r = 0.45; P = 0.02). In conclusion in CHL, LAG3 mRNA expression was markedly increased relative to control and DLBCL tissues. Within CHL tissues LAG3 mRNA was markedly increased compared to other immune checkpoint molecules. Interrogation of the TME using flow cytometry of TILs and mIF demonstrated LAG3 is evenly distributed between CD8+, nTregs and iTRreg. In tumor samples we did not find evidence of LAG3 expression on HRS cells. To our knowledge this is the first study to demonstrate sLAG3 as a cell free circulating disease response biomarker in CHL. Taken together these findings provide a convincing rationale for further exploration of single and/or combined checkpoint blockade incorporating LAG3 inhibition to treat CHL. Disclosures Abro: Bristol-Myers Squibb: Speakers Bureau; Janssen: Other: education support congress attendance; Celgene: Other: education support congress attendance; Novartis: Consultancy; Amgen: Other: education support congress attendance. Keane:BMS: Research Funding; Roche: Other: Education Support, Speakers Bureau; Celgene: Consultancy, Research Funding; Takeda: Other: Educational Meeting; Merck: Consultancy. Birch:Medadvance: Equity Ownership. Tobin:Amgen: Other: Educational Travel; Celgene: Research Funding. Johnson:Kite: Consultancy; Celgene: Honoraria; Eisai: Research Funding; Incyte: Consultancy; Takeda: Honoraria, Travel, accommodations, expenses; Janssen: Consultancy, Research Funding; Genmab: Consultancy; Novartis: Honoraria; Zenyaku Kogyo: Other: Travel, accommodations, expenses; Bristol-Myers Squibb: Honoraria; Boeringher Ingelheim: Consultancy; Epizyme: Consultancy, Honoraria, Research Funding. Trotman:F. Hoffman-La Roche: Other: Travel to meeting, Unremunerated member of Ad Board, Research Funding; PCYC: Research Funding; Janssen: Other: Unremunerated member of Ad Board, Research Funding; Takeda: Other: Unremunerated member of Ad Board; Celgene: Other: Unremunerated member of Ad Board, Research Funding; Beigene: Research Funding. Bird:Amgen, Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Gill:Janssen: Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau. Gandhi:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Takeda: Honoraria; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: We present the results of a multicenter clinical trial using Epstein-Barr virus (EBV)–specific cytotoxic T lymphocytes (CTLs) generated from EBV-seropositive blood donors to treat patients with EBV-positive posttransplantation lymphoproliferative disease (PTLD) on the basis of the best HLA match and specific in vitro cytotoxicity. Thirty-three PTLD patients who had failed on conventional therapy were enrolled. No adverse effects of CTL infusions were observed and the response rate (complete or partial) in 33 patients was 64% at 5 weeks and 52% at 6 months. Fourteen patients achieved a complete remission, 3 showed a partial response, and 16 had no response at 6 months (5 died before completing treatment). At 5 weeks, there was a significant trend toward better responses with higher numbers of CD4+ cells in infused CTL lines (P = .001) that were maintained at 6 months (P = .001). Patients receiving CTLs with closer HLA matching responded better at 6 months (P = .048). Female patients responded better than male patients, but the differences were not statistically significant. Our results show that allogeneic CTLs are a safe and rapid therapy for PTLD, bypassing the need to grow CTLs for individual patients. The response rate in this poor prognosis patient group is encouraging.

Description: Abstract 5119 Introduction Diffuse Large B cell Lymphoma (DLBCL) is a clinically and biologically heterogeneous lymphoid malignancy, where complete response is achieved in up to 60% of patients, though over half of all patients relapse (Hunt and Reichard, 2007). The prognostic significance of the expression of FOXP1 transcription factor in DLBCL has been highly controversial with some studies describing immunohistochemical FOXP1 expression as a poor prognostic marker (Banham et. all. 2005). Recent transcript analysis has identified over 10 FOXP1 isoforms where some exhibit COOH- or NH3- truncations and also the absence of regulatory domains. These truncated isoforms are highly expressed in ABC-DLBCL (Brown et. al. 2008) and it is suggested that truncated isoforms may aberrantly regulate FOXP1 target genes. Although there have been many studies debating the prognostic significance of FOXP1 overexpression in DLBCL, there has been little examination of the function of FOXP1 in DLBCL, specifically the genes targeted by FOXP1, and how the function of FOXP1 isoforms may contribute to a more aggressive phenotype. Methods Expression constructs for 4 FOXP1 isoforms (isoforms 1, 2, 3 and 8) were kindly provided by Dr. Philip Brown and Dr. Alison Banham. Stable cell lines were established by transfecting the BJAB cell line with either FOXP1 isoform 1 or the empty pBKCMV construct, and selection with G418 antibiotic. Total RNA was extracted from both cell lines and 4 DLBCL tumours (with low to high FOXP1 expression), and gene expression was analysed using Illumina HT12v4 microarray. Genes with 〉2-fold changes in expression between pBKCMV and Isoform 1 stable cell line were investigated. VisANT analysis and Gene set enrichment was performed on differentially expressed genes and candidates were validated by qRT-PCR. Promoter analysis using MatInspector revealed forkhead-binding sites in the promoter region of candidate genes, and dual luciferase reporter gene assays were performed to ascertain promoter regulation by FOXP1 isoforms. Western blot was also performed to validate translational changes in expression. Results In the gene expression microarray analysis of FOXP1 isoform 1 expression compared to baseline levels, 271 genes were identified with 〉2-fold differential expression. High versus low FOXP1 expressing DLBCL patients were also compared, where it was found that 2472 genes were differentially expressed over the 2-fold level. Significantly, comparison of differentially expressed genes in both the cell line and patient samples revealed all 271 genes differentially expressed in the cell line overlapped with the patient genes, indicating these results are translatable to FOXP1 function in DLBCL tumours. Pathway analysis was performed on the differentially expressed genes, and unexpectedly there were no significant enrichment of curated pathways. Alternatively visANT was used to examine interactive networks of the 271 candidate genes, and an integrative analysis using the FOXP1 microarray expression data was used to enrich the interactions. This analysis revealed critical interactions that could not be revealed by pathway analysis, and also identified genes that are likely FOXP1 targets. The promoter regions of 5 genes were investigated using reporter gene assays, and FOXP1 isoforms 3 and 8 were found to be significantly stronger transcriptional repressors compared to isoforms 1 and 2. For example, one of the genes identified, heterogeneous nuclear ribonucleoprotein U (HNRNPU) was highly expressed in the presence of isoform 1, however in the presence of isoform 3 was greatly downregulated. Although this gene has not previously been associated with DLBCL, HNRNPU is a potent regulator of snRNP's thereby having global effects on mRNA production and protein synthesis (Xiao et. al. 2012 Cell Vol. 45(5) 656–668). We are currently investigating the biological significance of other novel FOXP1 target genes, and how the truncated isoforms are involved in the differential regulation of these genes. Conclusion We have identified 271 genes that are differentially expressed in both a cell line model and DLBCL patient tissues with high versus low FOXP1 expression. These are novel genes in DLBCL pathogenesis that also show differential expression in the presence of truncated isoforms, indicating truncated isoforms may have aberrant functions and may contribute to a more pathogenic phenotype. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 93 Lymphomagenesis is a complex process, in part reflecting the nature of the transforming event, as well as the developmental stage of the cell. In the B-cell differentiation represents a continuum that is initiated when a naïve B-cell encounters antigen, undergoes a germinal centre (GC) reaction and ends with terminal differentiation into either a memory or plasma B-cell. Interruption of this process by a transforming event may result in a clonal proliferation where differentiation of the cell is blocked at this stage. The majority of B-cell lymphomas are derived from GC or post-GC B-cells. As physiologically relevant human models that emulate the various stages of B-cell differentiation are lacking we rationalized that in-vitro utilization of the B-cell lymphotrophic Epstein-Barr virus (EBV) would provide insights into this process. In one scenario, EBV infects naïve B-cells and drives a differentiation process paralleling the GC reaction through a well-characterized series of latency gene expression programs. EBV is also implicated in a range of GC and post-GC derived B-cell lymphomas (including Burkitt's, Hodgkin's, PTLD and DLBCL). Using high efficiency EBV infection of isolated naïve B-cells from EBV seronegative subjects, we have demonstrated that EBV infection provides a highly relevant in-vitro model that accurately reflects three distinct phases in the GC differentiation process. Alterations in the expression of a broad range of genes associated with the differentiation of the naïve B-cell were observed within 24 hours of infection and within four days of infection a process exhibiting many similarities to the GC reaction had taken place. These included BCL6, the levels of which were rapidly down-regulated within 24 hours indicating activation of the naïve B-cell. Levels of the memory cell marker CD27 steadily increased over 24 to 96 hours, while BLIMP1 expression increased, peaking at 48 hours. An increase in AID expression over 8 to 48 hours was consistent with somatic hypermutation and isotype switching. Finally a dramatic elevation in expression of the GC associated oncogene LMO2 was observed after two days followed by an equally dramatic downregulation after two weeks. Within two weeks of infection (phase 1), B-cells progressed through a GC-like phase followed by a one week transition state (phase 2) after which continued culture resulted in further differentiation to cells with the phenotypic hallmarks of post-GC cells (phase 3). MicroRNAs (miRNAs) are small non-coding RNAs, which act as negative regulators of gene expression. miRNA expression reflects the developmental lineage and differentiation state of several human cancers and over-expression is implicated in lymphomagenesis. They are also associated with the development of the GC reaction. EBV expresses at least 39 unique miRNAs from the BART and BHRF1 clusters within the viral genome. These EBV miRNAs are differentially expressed in tumour cell lines, suggesting roles during EBV-driven B-cell differentiation and lymphomagenesis. The relationship between EBV miRNAs and the kinetics of EBV driven B-cell differentiation has not been characterized. In our model we find distinct miRNA expression kinetics, coincidental with gene expression changes during B-cell differentiation, suggesting that these regulatory molecules may be involved in the GC process. Although a small number of EBV miRNAs were expressed at low levels early in the GC-like phase 1, the majority were up-regulated during the transition phase 2, exhibiting a subsequent partial down-regulation in the post-GC-like phase 3. The three phases were coincident with differential BART and BHRF1 promoter usage and alternate splicing. Strikingly, application of the infection model to primary patient samples and lymphoma cell-lines revealed that lymphomas clustered within distinct phases, reflecting the full continuum of the B-cell differentiation process. Interestingly, the majority of PTLD samples clustered within the transition phase, whereas Burkitt's and Hodgkin's lymphoma sample segregated with the GC stage. Application of our gene expression and miRNA data to cell-lines and a range of GC and post-GC EBV-positive lymphomas of various histological types indicate that our B-cell differentiation model can be used to accurately classify B-cell lymphomas in a physiologically relevant manner according to the stage of arrested B-cell differentiation. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Early ASCT improves progression-free survival (PFS) but not overall survival (OS) in poor prognosis DLBCL treated with R-CHOP immuno-chemotherapy (Stiff P et al., NEJM 2013). Furthermore, indiscriminate ASCT exposes those poor-risk patients destined to remain in CR with R-CHOP alone to unnecessary toxicity. Here, the role of interim PET/CT to risk-stratify is controversial. One confounding factor is that interim PET/CT is typically performed after 2 (or 3) cycles of chemotherapy when FDG-avidity likely reflects a mix of residual cancer cells and inflammation; in contrast, interim PET/CT after cycle 4 may be more representative of resistant lymphoma. We hypothesized that: 1. assessment of DLBCL patients by interim PET/CT after 4 cycles (iPET4) of R-CHOP-14 allows greater specificity of FDG-avidity; and, 2. for patients with iPET4-positivity, early treatment intensification with R-ICE chemotherapy followed by Zevalin-BEAM conditioning and ASCT results in 2-yr PFS that is equivalent to interim-PET/CT-negative patients treated with R-CHOP alone. Methods: We conducted a prospective multicenter phase 2 study which enrolled DLBCL patients ≤ 70 yrs with either IPI=2-5, or IPI =0-1 with tumor bulk (≥ 7.5 cm), and who were considered fit for ASCT. All patients underwent a diagnostic PET/CT at study entry and were planned to receive 4 cycles of R-CHOP-14 and supported with Pegfilgrastim. Cycle 5 of R-CHOP-14 was delayed 7 days, and an interim PET/CT scan was undertaken at d17-d20 post-4th R-CHOP-14, the delay intended to reduce the impact of rebound inflammation after R-CHOP. All interim PET/CT scans were assessed centrally by a core group of imaging specialists using International Harmonization Project (IHP) criteria with mediastinal blood pool as the reference. Biopsy to confirm residual tumor was not mandated. iPET4-positive patients received 3 cycles of R-ICE followed by ASCT with Zevalin-BEAM conditioning; those who were iPET4-negative received a further 2 cycles of R-CHOP-14 plus 2 doses of Rituximab. Results: A total of 162 patients were enrolled from 20 Australian centers; 11 patients were excluded because of failure to meet inclusion criteria. Baseline characteristics of the 151 evaluable patients included: median age 57 yrs (range, 21 to 69), 40% aged 〉 60 yrs, 62% males, 79% stages 3 or 4, 13% ECOG PS 〉 1, 78% elevated LDH, 48% extranodal sites 〉 1, 54% bulky disease ≥ 7.5 cm, 20% IPI=0-1, 27% IPI=2, 31% IPI=3, and 23% IPI=4-5. No interim PET/CT scan was performed in 8 patients due to progressive disease (PD) (1), bowel perforation (2), toxicity (3), and dose delays ≥ 2 weeks (2). Interim PET/CT scans were undertaken at d17-d20 in 62% and d14-d16 in another 23%. Of the 143 patients with interim PET/CT, 101 (71%) were deemed iPET4-negative and 42 (29%) were iPET4-positive. Interestingly, the baseline characteristics were comparable between iPET4-negative and iPET4-positive patients. Of the 101 iPET4-negative patients, 5 failed to complete therapy due to PD (3), toxicity (1), or infection (1). Of the 42 iPET4-positive patients, 10 failed to complete intensification therapy due to PD (6), 2nd malignancy (1), or consent withdrawal (3). Among iPET4-positive patients undergoing ASCT, there was 1 treatment-related death due to viral infection. At a median follow-up of 35 months, the Kaplan-Meier (KM) estimate of 2-yr PFS and OS for the entire eligible cohort of 151 pts was 72% and 85%, respectively. For the 101 iPET4-negative and 42 iPET4-positive patients, 2-yr PFS was 74% and 67% (P =0.32) (Figure 1), and 2-yr OS was 88% and 78% (P=0.11) (Figure 2), respectively. Among iPET4-negative and iPET4-positive patients with IPI=3-5, 2-yr PFS was 65% and 69% (P=0.74), and 2-yr OS was 81% and 83% (P =0.85), respectively. Conclusions: Patients with poor risk DLBCL who are interim PET/CT-positive after 4 cycles of R-CHOP-14 and switched to intensification with R-ICE followed by ASCT with Zevalin-BEAM have favorable rates of PFS and OS that are equivalent to that seen for patients who are interim PET/CT-negative. This study supports further investigation of treatment intensification in patients with poor risk DLBCL who are interim PET/CT-positive after 4 cycles of immuno-chemotherapy. The study was supported in part by Roche Products Pty. Ltd, Amgen Australia Pty. Ltd, and Bayer Australia Ltd. Figure 1. Progression Free Survival by Interim PET/CT Figure 1. Progression Free Survival by Interim PET/CT Figure 2. Overall survival by Interim PET/CT Figure 2. Overall survival by Interim PET/CT Disclosures Hertzberg: Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Gill:AbbVie: Honoraria; Roche: Research Funding; Sanofi Aventis: Research Funding; Roche: Honoraria. Ho:Celgene: Other: Travel. Cull:Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Travel. Grigg:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lewis:Amgen: Other: Travel; Roche: Honoraria, Other: Travel. Renwick:Amgen: Other: Travel; Bayer: Speakers Bureau. Seymour:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Infinity: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Phebra: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: To investigate the mechanisms of human T-cell reconstitution following allogeneic hemopoietic stem cell transplantation (alloSCT), we analyzed the clonal composition of human cytomegalovirus (HCMV)-specific or Epstein-Barr virus (EBV)-specific CD8+ T cells in 10 alloSC transplant recipients and their donors. All virus-specific CD8+ T-cell clones isolated from recipients after alloSCT contained DNA of donor origin. In all 6 D+/R+ sibling alloSCTs from seropositive donors into seropositive recipients, donor virus-specific clones transferred in the allograft underwent early expansion and were maintained long term in the recipient. In contrast, in 2 of 3 HCMV D+/R- alloSC transplant recipients in whom there was no detectable HCMV infection, donor HCMV-specific clones were undetectable, whereas donor EBV-specific clones were maintained in the same EBV-seropositive recipients, suggesting that transferred clones require antigen for their maintenance. Following D-/R+ transplantation from 3 seronegative donors into seropositive recipients, a delayed primary virus-specific CD8+ T-cell response was observed, in which the T cells contained donor DNA, suggesting that new antigen-specific T cells arose in the recipient from donor-derived progenitors. In 2 of 4 HCMV D+/R+ sibling allograft recipients the clonal composition underwent diversification as compared with their donors, with delayed persistent expansion of HCMV-specific clones that were undetectable in the donor or in the recipient during the early months after transplantation; this diversification may represent expansion of new clones generated from donor-derived progenitors. We conclude that, following alloSCT, late diversification of the HCMV-specific CD8+ T-cell clonal repertoire can occur in response to persistent viral antigen.