Search

Data on the prognostic impact of pretherapy 18F-fluorodeoxyglucose–positron emission tomography (FDG-PET) in follicular lymphoma (FL) is conflicting. The predictive utility of pretherapy total metabolic tumor volume (TMTV) and maximum standardized uptake value (SUVmax) on outcome appears to vary between regimens. Chemoimmunotherapies vary in the extent of T-cell depletion they induce. The role of intratumoral T cells on pretherapy FDG-PET parameters is undefined. We assessed pretherapy FDG-PET parameters and quantified intratumoral T cells by multiple methodologies. Low intratumoral T cells associated with approximately sixfold higher TMTV, and FL nodes from patients with high TMTV showed increased malignant B-cell infiltration and fewer clonally expanded intratumoral CD8+ and CD4+ T-follicular helper cells than those with low TMTV. However, fluorescently labeled glucose uptake was higher in CD4+ and CD8+ T cells than intratumoral B cells. In patients with FDG-PET performed prior to excisional biopsy, SUVmax within the subsequently excised node associated with T cells but not B cells. In summary, TMTV best reflects the malignant B-cell burden in FL, whereas intratumoral T cells influence SUVmax. This may contribute to the contradictory results between the prognostic role of different FDG-PET parameters, particularly between short- and long-term T-cell–depleting chemoimmunotherapeutic regimens. The impact of glucose uptake in intratumoral T cells should be considered when interpreting pretherapy FDG-PET in FL.

PURPOSE Understanding the immunobiology of the 15% to 30% of patients with follicular lymphoma (FL) who experience progression of disease within 24 months (POD24) remains a priority. Solid tumors with low levels of intratumoral immune infiltration have inferior outcomes. It is unknown whether a similar relationship exists between POD24 in FL. PATIENTS AND METHODS Digital gene expression using a custom code set—five immune effector, six immune checkpoint, one macrophage molecules—was applied to a discovery cohort of patients with early- and advanced-stage FL (n = 132). T-cell receptor repertoire analysis, flow cytometry, multispectral immunofluorescence, and next-generation sequencing were performed. The immune infiltration profile was validated in two independent cohorts of patients with advanced-stage FL requiring systemic treatment (n = 138, rituximab plus cyclophosphamide, vincristine, prednisone; n = 45, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone), with the latter selected to permit comparison of patients experiencing a POD24 event with those having no progression at 5 years or more. RESULTS Immune molecules showed distinct clustering, characterized by either high or low expression regardless of categorization as an immune effector, immune checkpoint, or macrophage molecule. Low programmed death-ligand 2 (PD-L2) was the most sensitive/specific marker to segregate patients with adverse outcomes; therefore, PD-L2 expression was chosen to distinguish immune infiltrationHI (ie, high PD-L2) FL biopsies from immune infiltrationLO (ie, low PD-L2) tumors. Immune infiltrationHI tissues were highly infiltrated with macrophages and expanded populations of T-cell clones. Of note, the immune infiltrationLO subset of patients with FL was enriched for POD24 events (odds ratio [OR], 4.32; c-statistic, 0.81; P = .001), validated in the independent cohorts (rituximab plus cyclophosphamide, vincristine, prednisone: OR, 2.95; c-statistic, 0.75; P = .011; and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone: OR, 7.09; c-statistic, 0.88; P = .011). Mutations were equally proportioned across tissues, which indicated that degree of immune infiltration is capturing aspects of FL biology distinct from its mutational profile. CONCLUSION Assessment of immune-infiltration by PD-L2 expression is a promising tool with which to help identify patients who are at risk for POD24.

Objectives A diverse intratumoral T‐cell receptor (TCR) repertoire is associated with improved survival in diffuse large B‐cell lymphoma (DLBCL) treated with rituximab/cyclophosphamide/doxorubicin/prednisolone/vincristine (R‐CHOP) chemoimmunotherapy. We explored the impact of intratumoral TCR repertoire on interim PET (iPET) done after four cycles of R‐CHOP, the relationships between intratumoral and circulating repertoire, and the phenotypes of expanded clonotypes. Methods We sequenced the third complementarity‐determining region of TCRβ in tumor samples, blood at pre‐therapy and after four cycles of R‐CHOP in 35 patients enrolled in ALLGNHL21 trial in high‐risk DLBCL. We correlated the TCR diversity metrics with iPET status, gene expression profiles and HLA‐class I genotypes. We then sequenced the FACS‐sorted peripheral blood T cells in six patients, and pentamer‐sorted EBV‐specific CD8+ T cells in one patient from this cohort. Results Compared with iPET− patients, the intratumoral TCR repertoire in iPET+ patients was characterised by higher cumulative frequency of abundant clonotypes and higher productive clonality. There was a variable overlap between circulating and intratumoral repertoires, with the dominant intratumoral clonotypes more likely to be detected in the blood. The majority of shared clonotypes were CD8+ PD‐1HI T cells, and CD8+ T cells had the largest clonal expansions in tumor and blood. In a patient with EBV+ DLBCL, EBV‐specific intratumoral clonotypes were trackable in the blood. Conclusion This study demonstrates that clonally expanded intratumoral TCR repertoires are associated with iPET+ and that the blood can be used to track tumor‐associated antigen‐specific clonotypes. These findings assist the rationale design and therapeutic monitoring of immunotherapeutic strategies in DLBCL., In a cohort of high‐risk diffuse large B‐cell lymphoma patients enrolled in phase 2 clinical trial, clonally expanded intratumoral TCR repertoires were associated with a positive interim PET scans. Abundant intratumoral clonotypes were identified in peripheral blood at baseline and during chemoimmunotherapy.

Primary central nervous system lymphoma (PCNSL) is confined to the brain, eyes, and cerebrospinal fluid without evidence of systemic spread. Rarely, PCNSL occurs in the context of immunosuppression (eg, posttransplant lymphoproliferative disorders or HIV [AIDS-related PCNSL]). These cases are poorly characterized, have dismal outcome, and are typically Epstein-Barr virus (EBV)-associated (ie, tissue-positive). We used targeted sequencing and digital multiplex gene expression to compare the genetic landscape and tumor microenvironment (TME) of 91 PCNSL tissues all with diffuse large B-cell lymphoma histology. Forty-seven were EBV tissue-negative: 45 EBV− HIV− PCNSL and 2 EBV− HIV+ PCNSL; and 44 were EBV tissue-positive: 23 EBV+ HIV+ PCNSL and 21 EBV+ HIV− PCNSL. As with prior studies, EBV− HIV− PCNSL had frequent MYD88, CD79B, and PIM1 mutations, and enrichment for the activated B-cell (ABC) cell-of-origin subtype. In contrast, these mutations were absent in all EBV tissue-positive cases and ABC frequency was low. Furthermore, copy number loss in HLA class I/II and antigen-presenting/processing genes were rarely observed, indicating retained antigen presentation. To counter this, EBV+ HIV− PCNSL had a tolerogenic TME with elevated macrophage and immune-checkpoint gene expression, whereas AIDS-related PCNSL had low CD4 gene counts. EBV-associated PCNSL in the immunosuppressed is immunobiologically distinct from EBV− HIV− PCNSL, and, despite expressing an immunogenic virus, retains the ability to present EBV antigens. Results provide a framework for targeted treatment.

Introduction Aprotinin is a serine protease inhibitor which is used in cardiac surgery as an anti-fibrinolytic agent to minimise patient bleeding. It was withdrawn from the European market in 2008 due to potential increased mortality when compared to tranexamic acid. Aprotinin was subsequently re-introduced in 2012 with narrow licencing indications, specifically isolated coronary artery bypass graft surgery in high risk patients. Minimisation of intra-operative blood loss plays an important role in patient outcomes, especially in decreasing the need for transfusion which carries its own mortality risks(3,. Tranexamic acid is utilised in the majority of cardiac surgery cases throughout Europe. We looked to quantify and determine indications for aprotinin usage in an Irish tertiary cardiac centre and compare findings to tranexamic acid. Methods Retrospective study of aprotinin usage in cardiac surgery in an Irish tertiary centre over a 3 year period to determine number of cases and indications. Data collection involved operative notes, patient records, pharmacy dispensing accounts. Aprotinin dosage intra-operatively was per the full or half Hammersmith protocol. A cohort which had undergone comparable cardiac surgery procedures with tranexamic acid as the anti-fibrinolytic agent were also studied. Results From 2018-2021, 21 cardiac surgeries were carried out using aprotinin as the sole anti-fibrinolytic agent to minimise patient bleeding in a high risk cohort. For the same time period, to provide scale, 737 cardiac surgeries were carried out using tranexamic acid. Each of the cases which utilised aprotinin represents an emergent, life-threatening operation. The emergency cases comprised: repair of Stanford type A aortic dissection (8, 38%); aortic valve replacement (7, 33.3%); mitral valve replacement (3, 14.3%); tricuspid valve replacement (1, 4.8%); mediastinal revision for cardiac tamponade (1, 4.8%). Valve replacements were complicated by: acute infective endocarditis, aortic root abscess, haematoma, failure of pre-existing prosthetic valves. 1 case utilised aprotinin as per the licenced indication: isolated CABG in patient with high bleeding risk. An ST-elevated myocardial infarction necessitated the emergency CABG procedure in this case. Discussion In summation, 95% of aprotinin usage for cardiac surgery in an Irish tertiary centre falls outside the remit of the licenced indication. Given similar findings in a number of European institutes(5), is it time to re-assess the existing guidelines on aprotinin use, with view to expansion of the licenced indications?

MicroRNA (miRNA)s are dysregulated in Diffuse large B-cell lymphoma (DLBCL), where they reflect the malignant B-cells and the immune infiltrate within the tumor microenvironment. There remains a paucity of data in DLBCL regarding cell-free (c-f) miRNA as disease response biomarkers. Immunosuppressive monocyte/macrophages, which are enriched in DLBCL, are disease response markers in DLBCL, with miRNA key regulators of their immunosuppressive function. Our aim was to determine whether plasma miRNA that reflect the activity of the malignant B-cell and/or immunosuppressive monocytes/macrophages, have value as minimally-invasive disease response biomarkers in DLBCL. Quantification of 99 DLBCL tissues, to select miRNA implicated in immunosuppressive monocytes/macrophage biology, found miR-494 differentially elevated. In a discovery cohort (22 patients), pre-therapy c-f miR-494 and miR-21 but not miR-155 were raised relative to healthy plasma. Both miR-494 and miR-21 levels 3-6 months reduced post immuno-chemotherapy. The validation cohort (56 patients) was from a prospective clinical trial. Interestingly, in sequential samples both miRNAs decreased in patients becoming Positron Emission Tomography/Computerized Tomography (PET/CT)-ve, but not in those remaining interim-PET/CT+. Patient monocytes were phenotypically and functionally immunosuppressive with ex-vivo monocyte depletion enhancing T-cell proliferation in patient but not healthy samples. Pre-therapy monocytes showed an immunosuppressive transcriptome and raised levels of miR-494. MiR-494 was present in all c-f nanoparticle fractions but was most readily detectable in unfractionated plasma. Circulating c-f miR-494 and miR-21 are disease response biomarkers with differential response stratified by interim-PET/CT in patients with DLBCL. Further studies are required to explore their manipulation as potential therapeutic targets.

// Colm Keane 1, 2 , Joshua Tobin 1, 2 , Dipti Talaulikar 3, 4 , Michael Green 5 , Pauline Crooks 6 , Sanjiv Jain 3, 4 and Maher Gandhi 1, 2 1 University of Queensland Diamantina Institute, Translational Research Institute, University of Queensland, Brisbane, Queensland, Australia 2 Princess Alexandra Hospital, Brisbane, Queensland, Australia 3 Canberra Hospital, Canberra, Australian Capital Territory, Australia 4 Australian National University Medical School, Canberra, Australian Capital Territory, Australia 5 MD Anderson Cancer Centre, Houston, Texas, USA 6 Queensland Institute of Medical Research, Brisbane, Queensland, Australia Correspondence to: Colm Keane, email: c.keane@uq.edu.au Keywords: DLBCL; tumor microenvironment Received: January 15, 2018 Accepted: April 06, 2018 Published: May 04, 2018 ABSTRACT Purpose: To test the utility of the circulating Lactate Dehydrogenase (LDH) to absolute lymphocyte count (ALC) ratio (LAR) to predict outcome to conventional first-line chemo-immunotherapy in Diffuse Large B-cell Lymphoma (DLBCL), and investigate its correlation to the tumour immune microenvironment (TME). Experimental Design: A population based cohort of 210 patients (median age: 64, range 18-90 years) with median follow up 3.8 years was analysed. All patients were treated with R-CHOP, and no immunosuppression related cases were included. Tissue for nanoString gene expression was available in 141. Results: High (i.e. adverse) LAR was associated with inferior progression free and overall survival (PFS 45% vs. 78%; OS 56% vs 86%, both p

About half of melanomas respond to immunotherapy, but we currently lack reliable markers to identify likely responders. Therefore, a large number of patients receive this expensive therapy for no benefit. We have previously shown that inhibition of the cell cycle regulator, Checkpoint kinase 1 (CHK1), with subclinical dose of the replication stress inducer hydroxyurea is cytotoxic in a high proportion of melanomas tested. We now report that CHK1 inhibitor + low dose hydroxyurea (LDHU) treatment triggers immunostimulatory cytokine and chemokine expression, and an immunogenic form of cell death. In xenograft models, the drug combination blocks tumor growth for extended periods after the completion of treatment. In syngeneic mouse melanoma models, the drug combination also effectively controls tumor growth and induces regression in a proportion of cases. It also increases immune cell infiltration and activation of a tumor directed cytotoxic T cell response. We also demonstrate that the drug combination does not adversely affect an adaptive immune response or rapidly proliferating human T cells. The mechanism by which drug treatment promotes cytokine expression is unlikely to involve the cGAS-STING signalling pathway which is commonly defective in melanomas. Using two engineered mouse melanoma models we have found that CHK1 inhibitor + LDHU increases cytotoxic CD8+ T cells and NK cell infiltration and activation, but we have also found increased CD4+ Treg tumor infiltration and increased CD8+ T cell PD-1 expression. We have also found increased expression of other immune checkpoint regulators. This indicates a strong anti-tumor immune response is initiated but immune suppression and T cell exhaustion have limited the extent of the response of the tumor infiltrating T cells. However, circulating T cells retained strong anti-tumor activity. The harvested tumors from the drug combination treated mice that do increase in size after treatment show strong evidence of cytotoxic activity. These data provide evidence that CHK1 inhibitors + LDHU is well tolerated and triggers a strong anti-cancer immune response that should combine with immune therapy, although the selection of the target of the immunotherapy will be critical for successfully overcoming the immune suppression triggered. Citation Format: Brian G. Gabrielli, Martina Proctor, Jazmina Gonzalez Cruz, Bijun Zeng, Riccardo Dolcetti, Colm Keane, Nikolas Haass, James Wells. CHKing melanoma: CHK1 inhibitor +low dose hydroxyurea triggers immunogenic cell death and immunostimulatory cytokine expression to drive an anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1443.

Background: Most DLBCL & FL responds well to first line treatment, yet relapsed disease outcomes are poor. Immune checkpoint inhibition (ICI) with PD/PD1 inhibitors (PD1i) yield high response rates in some lymphomas; though single agent PD1i yields a disappointing ORR of 10% in heavily pre-treated DLBCL, some responses are durable. RT stimulates anti-tumour immunity through several mechanisms and may enhance response to ICI. Concurrent ICI & RT is synergistic in preclinical studies & solid tumours, improving local & distant (abscopal) response. RT to multiple disease sites may broaden the spectrum of tumour antigen release and overcome clonal variation between disease sites to further augment the immune response. A dose-response relationship between RT and antigen release has yet to be established. This phase I dose escalation study aims to determine the safety profile of RT in combination with durvalumab, an anti-PD-L1 monoclonal antibody, in relapsed/refractory DLBCL and FL. Study Design and Methods: RaDD (NCT03610061) is a phase I dose escalation study to determine the safety profile of escalating dose and number of sites of RT in combination with durvalumab in relapsed/refractory (RR) DLBCL & FL. Eligible patients (pts) have received ≥ 1 prior line of therapy and are ineligible for or relapsed after autologous stem cell transplant (auto-SCT). Pts with active autoimmune disease, CNS involvement, prior allogeneic-SCT or chronic steroid use are excluded. RT dose and site escalation proceeds according to a 3+3 design with 6 dose levels (cohorts 1-6). Treatment comprises external beam RT to target site(s) daily for 5 days (Cohorts 1-5); Cohort 6 receives a further 5 daily fractions (max 30Gy). Durvalumab 1500mg IV commences day 2 of RT and continues 4-weekly until disease progression. Pts can continue until a second radiological progression if clinical benefit is ongoing. The dose limiting toxicity period is 28 days from start of RT. The primary endpoint is the toxicity, drug pharmacokinetics, maximum tolerated dose (MTD) and recommended phase two dose (RP2D) of simultaneous RT and durvalumab. Secondary endpoints include response rates, progression-free survival and overall survival. Correlative studies will examine the tumour-immune system interaction; an exploratory PET substudy with novel tracers for durvalumab (89Zr-Durvalumab) & CD8+ T cells (89Zr -Df-IAB22M2C) will also be performed. Projected enrolment for determination of the MTD and RP2D is 6-30 pts pending toxicity. Recruitment will continue to 36 pts for secondary endpoint analysis. 22 pts are enrolled to date in the main study, with 2 patients enrolled in the PET-substudy. Acknowledgements: Victorian Cancer Agency (grant funding - TRP16006), Astra Zeneca (durvalumab and funding), Celgene (funding), Imaginab (89Zr -Df-IAB22M2C). Citation Format: Kate Manos, Richard Khor, Geoffrey Chong, Jodie Palmer, Michael MacManus, Colm Keane, Andrew M. Scott, Jake Shortt, David Ritchie, Leonid Churilov, Laura Johnston, Tom Witkowski, Allison Barraclough, Sze Ting Lee, Wendi Lin, Rachel Koldej, Eliza Hawkes. Phase I Dose Escalation Study of Radiotherapy and Durvalumab (MEDI4736) in Relapsed or Refractory Diffuse Large B-cell Lymphoma (DLBCL): The RaDD Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT208.

Background: Novel strategies are needed to improve upon the 60% cure rate of upfront R-CHOP in advanced DLBCL. Single-agent immune checkpoint inhibition (ICI) has limited efficacy in heavily pre-treated DLBCL (response rate Avelumab (Av) is an anti-PDL1 monoclonal antibody with antibody dependent cell cytotoxicity (ADCC) activity which acts synergistically with rituximab (R) in vitro. We report the results of a phase II single arm study assessing safety of 1st line sequential AvR induction, R-CHOP & Av maintenance for DLBCL. Methods: Patients aged ≥18 years, ECOG 0-2 with untreated stage II-IV DLBCL and no active autoimmune disease were treated with AvR induction x2 cycles q2-weekly (Av 10mg/kg IV + R 375mg/m2 IV), followed by R-CHOP21 x 6 cycles then Av 10mg/kg x 6 cycles q2-weekly if in complete metabolic response (CMR) post R-CHOP. The primary endpoint was the rate of grade 3/4 immune-related adverse events (irAE). Secondary endpoints included overall response rate (ORR), failure free survival (FFS), overall survival (OS) and overall toxicity. Response was determined centrally by PET-CT (Lugano 2014 criteria). CMR rates by PET-CT post AvR induction and post C2 R-CHOP were exploratory endpoints. Genomic analysis was performed including next generation sequencing (NGS) based sequence variant detection, copy number analysis and structural variant detection. Results: 28 pts were enrolled from Dec 2017 to Oct 2019. Key baseline characteristics included median age 54 yrs (range 20-79); stage III/IV disease 68%; elevated LDH 61%; IPI ≥2 25%. Histology included 21 DLBCL NOS (75%; 14 GCB, 7 non-GCB by Hans algorithm), 6 primary mediastinal B-cell lymphoma (PMBCL; 21%) and 1 EBV positive DLBCL (4%). The study met its pre-specified primary endpoint of G3/4 irAE ORR post R-CHOP was 89% (all CR) (Figure 1). The ORR to 2 cycles of induction AvR was 60%, including 6 CMR (21%) across all diagnostic/histologic subgroups (n=1 PMBCL, n=2 non-GCB DLBCL, n=3 GCB DLBCL; Figures 1 and 2). Six pts (21%) progressed during AvR induction (with 1 pt completing only 1 x AvR cycle); all subsequently responded to R-CHOP. With a median follow-up of 16 months, 1-year FFS was 76% and OS 89%. Treatment was discontinued early in 5 pts; 2 during R-CHOP due to progressive disease and 3 during Av maintenance (n=1 immune hepatitis; n=1 pulmonary embolism initially reported as pneumonitis; n=1 progressive disease). Alterations in the CD274/PDCDLG2 locus were identified by NGS in 3 of 27 evaluable pts (n=2 PMBCL, n=1 EBV+ DLBCL). Full genomic analysis to identify factors associated with response will be presented. Conclusion: Sequential AvR induction, R-CHOP and Av maintenance in pts with newly diagnosed DLBCL is feasible with a manageable toxicity profile and a high CR rate. Responses to AvR alone were higher than expected based on the relapsed/refractory population and may suggest superior efficacy of ICI in the frontline setting. These results support ongoing sequential studies of immune priming with PD1/PDL1 inhibition prior to R-CHOP in DLBCL. Acknowledgements: Merck KgA for avelumab plus funding. Tour de Cure Scott Canning Early Career Grant (E Hawkes) and Wilson Centre for Lymphoma Genomics for biomarker testing. Disclosures Hawkes: Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding, Speakers Bureau; BMS celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Speakers Bureau; Merck Sharpe &Dohme: Membership on an entity's Board of Directors or advisory committees, Research Funding; takeda: Speakers Bureau; Merck KgA: Research Funding. Chong:Merck Serono: Research Funding; Bristol-Myers Squibb: Research Funding; Hutchison Medipharma: Research Funding; Bayer: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Servier: Research Funding; Isofol: Research Funding. Blombery:Novartis: Consultancy; Janssen: Honoraria; Amgen: Consultancy; Invivoscribe: Honoraria. Barraclough:Roche: Other: Conference sponsorship. Keane:Celgene: Honoraria, Other: Travel; BMS: Research Funding; Roche: Honoraria, Other: Travel, Speakers Bureau; MSD Oncology: Honoraria, Other: Travel; Gilead: Honoraria, Other: Travel, Speakers Bureau. Fong:Pfizer: Honoraria; Astellas: Honoraria; Amgen: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria; AbbVie: Honoraria. Manos:Bristol-Myers Squibb: Other: Conference sponsorship. OffLabel Disclosure: Avelumab is an anti-PDL1 monoclonal antibody. Inhibition of the PD1/PDL1 pathway stimulates anti-tumour immunity.

Neoantigens are generated by mutations acquired in malignant cells. To be immunogenic, neoantigen peptides must be processed and then presented by HLA molecules on the tumor cell surface to evoke a T cell response. Emerging evidence suggests neoantigens are promising new targets of personalised cancer immunotherapies, provided that antigen presentation remains intact. Recently, we (Tobin, JCO 2019) demonstrated the link between the tumor microenvironment (TME) and clinical outcome in patients with advanced-stage Follicular Lymphoma (ASFL). In particular, ASFL patients with favorable outcomes had high levels of immune infiltration (best stratified by PD-L2 expression) with increased intratumoral CD8+ T cell clonotypes suggestive of expanded antigen-specific T cells in response to presentation by HLA class I neoantigens. This is supported by HLA class I pathway genetic aberrations being infrequent (unlike HLA class II) in FL. However, to date there is minimal data on the frequency and nature of putative neoantigens, their relationship with HLA class I antigen presentation and with the TME in any stage of FL. In particular, the immunobiology of early-stage FL (ESFL) has been largely neglected. Here, we present detailed characterization of these parameters in patients with early-stage FL (ESFL) from the TROG99.03 prospective clinical trial (MacManus, JCO 2018). Digital multiplex gene expression (770 gene PanCancer Immune Panel, NanoString) and targeted NGS (365 genes recurrently mutated in hematological malignancies) was performed on 97 FFPE diagnostic tissue samples. Intratumoral CD8A expression cut-off derived by maximally selected rank statistics was associated with >2-fold differential median PFS (CD8Ahi: 11.5 years vs. CD8Alo: 4.9 years, p=0.0042, Figure 1A). In keeping with a relationship between HLA class I immune effector infiltration within the TME and FL tumor antigen presentation/processing, expression of NLRC5 (a transcriptional activator of HLA class I) was also associated with differential median PFS (NLRC5hi: 10.8 years vs. NLRC5lo: 4.9 years, p=0.021, Figure 1B). The impact of expression of CD8A and NLRC5 were independent of mutations in BCL-2,TP53 and all but of one the m7-FLIPI genes. The exception was EZH2, mutations of which were enriched in cases with reduced CD8A (p=0.0066) and NLRC5 (p Next, we investigated the expression of HLA class I and predicted neoantigens. HLA genotyping was performed using a custom sequencing panel on matched germline peripheral blood DNA for 71 TROG99.03 diagnostic samples. For the corresponding somatic coding variants, the strongest HLA class I binding efficiencies were calculated using 8 epitope prediction algorithms. 41% of samples had no detectable neoantigens, whereas 59% of samples had ≥1 neoantigens detected, with 116 neoantigens total. Importantly, unsupervised hierarchical clustering showed 2-fold enrichment of samples with neoantigens among those with elevated HLA A/B expression vs. samples with low HLA A/B (p=0.0023, Figure 1C). Interestingly, roughly one third of neoantigens were due to indels causing frameshift events resulting in the generation of 'non-self' peptides with strong predicted binding affinity. Recently TCGA data has been used to show that frameshift variants occurring at different sites in a gene can converge to generate a handful neo open reading frame peptides ('NOPs') common to multiple cancer patients. Therefore, it has been postulated that these NOPs could have a considerable therapeutic potential as a novel pan-cancer 'semi-personalized' immunotherapy. In keeping with data derived in solid tumors, in ESFL we observed frameshift variants were most frequently observed in KMT2D. Finally, 5 relapsed/transformed FL samples were compared to their paired diagnostic tissue samples. Not only did neoantigens present at diagnosis typically remain detectable, but in several cases additional neoantigens were also found. In summary, we demonstrate for the first time in ESFL that neoantigens are typically associated with intact HLA class I presentation; CD8 infiltration within the TME is related to tumor antigen processing and associated with favorable outcomes; and that a subset of FL tumors have NOPs. These data have important implications for the design of innovative next-generation immunotherapies in FL. Disclosures Keane: BMS: Research Funding; Celgene: Honoraria, Other: Travel; Roche: Honoraria, Other: Travel, Speakers Bureau; MSD Oncology: Honoraria, Other: Travel; Gilead: Honoraria, Other: Travel, Speakers Bureau. Blombery:Amgen: Consultancy; Novartis: Consultancy; Janssen: Honoraria; Invivoscribe: Honoraria. Tobin:Gilead: Research Funding. Seymour:Nurix: Honoraria; Morphosys: Consultancy, Honoraria; Mei Pharma: Consultancy, Honoraria; Gilead: Consultancy; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Gandhi:Celgene: Research Funding; Bristol-Myers Squibb: Research Funding; Merck Sharp & Dohme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Gilead Sciences: Honoraria; Genentech: Honoraria; Mater Research: Current Employment; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Other: Travel, accommodation, expenses .

Despite advances in therapy, still up to one third of patients with diffuse large B-cell lymphoma (DLBCL) will ultimately die of their disease. The revised international prognostic index (R-IPI) is...

De novo diffuse large B-cell lymphoma (DLBCL) presenting with synchronous central nervous system (CNS) and systemic disease (synDLBCL) is not well described and is excluded from clinical trials. We performed a retrospective analysis of 80 synDLBCL patients treated across 10 Australian and UK centres. Of these patients, 96% had extranodal systemic disease. CNS-directed treatment with combination intravenous cytarabine and high-dose methotrexate ("CNS-intensive") (n = 38) was associated with favourable survival outcomes compared with "CNS-conservative" strategies such as intravenous high-dose methotrexate monotherapy, intrathecal therapy and/or radiotherapy (2-year progression-free survival [PFS] 50% vs. 31%, P = 0·006; 2-year overall survival [OS] 54% vs. 44%, P = 0·037). Outcomes were primarily dictated by the ability to control the CNS disease, with 2-year cumulative CNS relapse incidence of 42% and non-CNS relapse 21%. Two-year OS for CNS-relapse patients was 13% vs. 36% for non-CNS relapses (P = 0·02). Autologous stem cell transplantation as consolidation (n = 14) was not observed to improve survival in those patients who received CNS-intensive induction when matched for induction outcomes (2-year PFS 69% vs. 56%, P = 0·99; 2-year OS 66% vs. 56%, P = 0·98). Hyperfractionated or infusional systemic treatment did not improve survival compared to R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone) (2-year OS 49% for both groups). Our study suggests that adequate control of the CNS disease is paramount and is best achieved by intensive CNS-directed induction.

7560 Background: Standard of care immunochemotherapy in front-line (1L) follicular lymphoma (FL) is highly efficacious but not without significant toxicity. High rates of grade 3-5 adverse events (AEs), primarily infection and bone marrow suppression, are experienced in up to 75% of patients. A more tolerable but equally effective approach is required. PD-1 inhibition, in combination with rituximab (R), increases T cell anti-tumour effect & enhances NK cell antibody dependent cell cytotoxicity, with proven efficacy in relapsed FL. The concept of ‘priming’ the immune system with nivolumab (N) prior to tumour-directed therapy has rationale and evidence, but the safety of this approach in 1L FL is not described. Methods: ‘1st FLOR’ (NCT03245021) is an open-label, multi-centre, phase 2, Simon’s 2-stage study of N + R (N = 39). Key eligibility were stage III-IV grade 1-3A FL requiring 1L systemic therapy; ECOG ≤2; adequate organ function. All patients (pts) receive induction N 240mg IV 2-weekly for 4 cycles. Pts with complete response (CR) receive 4 further cycles of 240mg IV N monotherapy then 12 cycles of maintenance N 480mg IV 4-weekly. Pts with < CR had 240mg N plus 375mg/m2 IV R 2-weekly for 4 cycles followed by maintenance N+R (N 480mg 4 weekly for 12 cycles; R 12 weekly for 8 cycles). Primary endpoint (EP) was ≥ G3 toxicity rate during induction. Secondary EPs; response rate by Lugano response criteria, overall toxicity, PFS, OS. Results: Between September 2017 to March 2020, 39 pts were enrolled. Baseline characteristics included median age of 54 (range: 28-79). stage IV disease in 67%, B Symptoms & bulk (≥7cm) in 23% each, intermediate-high risk FLIPI in 74%. The primary EP was met, with only 16 pts (41%) having ≥G3 toxicity at end of induction. Non-immune AEs were predominantly G1-2; most commonly infection (67%) & fatigue (64%). G3-4 Immune-related AEs were infrequent and included pancreatitis plus hepatitis (N = 1), pancreatitis alone (N = 1), rash (N = 1), transaminitis (N = 2), hypocortisolism (N = 1), hyperglycaemia (N = 3) and asymptomatic lipase/amylase increase (N = 3). Median follow-up was 17.5 months (range: 7-39). Overall response rate was 92% (36/39) with CR in 54% (21/39). Median time to CR was 5 months (m) (range: 2-25). Nine pts (23%) discontinued treatment; 7 due to progressive disease (1 pt died of transformed FL), 2 developed constitutional symptoms (1 stable disease, 1 partial response). In 25 evaluable pts, 12m PFS & OS is 72% (CI 51-88) & 96% (CI 80-100). Biomarker analysis is in progress. Conclusions: Immune-priming with single-agent N, then combination N+R in 1L FL is associated with favourable toxicity and high ORR & CR rates potentially providing an alternative to chemotherapy. Acknowledgements: Bristol-myers Squibb provided funding and nivolumab for this study. Clinical trial information: NCT03245021.

The dependence of cancer on an immunotolerant tumor microenvironment (TME) is well established. Immunotherapies that overcome tumor-induced immune suppression have been central to recent advancements in oncology. This is highlighted by the success of agents that interrupt PD-1 mediated immune suppression in a range of cancers. However, while PD-1 blockade has been paradigm-shifting in many malignancies, the majority of cancers show high rates of primary resistance to this approach. This has led to a rapid expansion in therapeutic targeting of other immune checkpoint molecules to provide combination immune checkpoint blockade (ICB), with one such promising approach is blockade of Lymphocyte Activation Gene 3 (LAG-3). Clinically, lymphoproliferative disorders show a wide spectrum of responses to ICB. Specific subtypes including classical Hodgkin lymphoma have demonstrated striking efficacy with anti-PD-1 therapy. Conversely, early trials of ICB have been relatively disappointing in common subtypes of Non-Hodgkin lymphoma. In this review, we describe the TME of common lymphoma subtypes with an emphasis on the role of prominent immune checkpoint molecules PD-1 and LAG3. We will also discuss current clinical evidence for ICB in lymphoma and highlight key areas for further investigation where synergistic dual checkpoint blockade of LAG-3 and PD-1 could be used to overcome ICB resistance.

Mesenchymal stromal cells (MSCs) have a multimodal, immunomodulatory mechanism of action and are now in clinical trials for single organ and systemic sepsis. However, a number of practicalities around source, homogeneity and therapeutic window remain to be determined. Here, we utilised conditioned medium from CD362+-sorted umbilical cord-human MSCs (UC-hMSCs) for a series of in vitro anti-inflammatory assays and the cryopreserved MSCs themselves in a severe (Series 1) or moderate (Series 2+3) caecal ligation and puncture (CLP) rodent model. Surviving animals were assessed at 48 h post injury induction. MSCs improved human lung, colonic and kidney epithelial cell survival following cytokine activation. In severe systemic sepsis, MSCs administered at 30 min enhanced survival (Series 1), and reduced organ bacterial load. In moderate systemic sepsis (Series 2), MSCs were ineffective when delivered immediately or 24 h later. Of importance, MSCs delivered 4 h post induction of moderate sepsis (Series 3) were effective, improving serum lactate, enhancing bacterial clearance from tissues, reducing pro-inflammatory cytokine concentrations and increasing antimicrobial peptides in serum. While demonstrating benefit and immunomodulation in systemic sepsis, therapeutic efficacy may be limited to a specific point of disease onset, and repeat dosing, MSC enhancement or other contingencies may be necessary.

TPS8075 Background: Most DLBCL & FL responds well to first line treatment, yet relapsed disease outcomes are poor. PD1/PDL1 inhibitors yield high response rates in some lymphomas, but single agent therapy in heavily pre-treated pts are disappointing. RT stimulates anti-tumor immunity through several mechanisms and may enhance response to immune checkpoint inhibition (ICI). Concurrent ICI & RT is synergistic in preclinical studies & solid tumors, improving local & distant (abscopal) response. RT to multiple disease sites may broaden the spectrum of tumor antigen release and overcome clonal variation between disease sites to further augment the immune response. Methods: RaDD (NCT03610061) is a phase I, 3+3 dose escalation study to determine the safety profile of escalating dose & number of sites of RT in combination with Durvalumab (anti-PD-L1 antibody) in RR DLBCL & FL. Eligible pts (i.e. ≥1 prior therapy, ineligible for auto-SCT, no contraindication to PDL1i) receive 5 fractions of external beam RT to target site(s). 5 RT dose & site levels are included (dose range 2.5Gy-20Gy to 1-3 sites). Durvalumab 1500mg IV commences day 2 of RT and continues 4-weekly until confirmed disease progression. The DLT period is 28 days from start of RT. Primary endpoint is the recommended phase two dose (RP2D) of RT in combination with durvalumab. Secondary endpoints include response rates, PFS & OS. Correlative studies will examine the tumour-immune system interaction; an exploratory PET substudy with novel tracers for durvalumab (89Zr-Durvalumab) & CD8+ T cells (89Zr -Df-IAB22M2C) will also be performed. Projected enrollment for determination of maximum tolerated dose (MTD) & RP2D is 6-30 pts pending toxicity. Recruitment will continue to 36 pts for secondary endpoint analysis. 9 pts are enrolled across cohorts 1-3 to date. Clinical trial information: NCT03610061 .

Recent studies have shown limited utility of routine surveillance imaging for diffuse large B-cell lymphoma (DLBCL) patients achieving remission. Detection of molecular disease by immunoglobulin high-throughput sequencing (Ig-HTS) from peripheral blood provides an alternate strategy for surveillance. We prospectively evaluated the utility of Ig-HTS within 311 blood and 105 tumor samples from 75 patients with DLBCL, comparing Ig-HTS from the cellular (circulating leukocytes) and acellular (plasma cell-free DNA) compartments of peripheral blood to clinical outcomes and (18)fluoro-deoxyglucose positron emission tomography combined with computed tomography (PET/CT; n = 173). Clonotypic immunoglobulin rearrangements were detected in 83% of patients with adequate tumor samples to enable subsequent monitoring in peripheral blood. Molecular disease measured from plasma, compared with circulating leukocytes, was more abundant and better correlated with radiographic disease burden. Before treatment, molecular disease was detected in the plasma of 82% of patients compared with 71% in circulating cells (P = .68). However, molecular disease was detected significantly more frequently in the plasma at time of relapse (100% vs 30%; P = .001). Detection of molecular disease in the plasma often preceded PET/CT detection of relapse in patients initially achieving remission. During surveillance time points before relapse, plasma Ig-HTS demonstrated improved specificity (100% vs 56%, P < .0001) and similar sensitivity (31% vs 55%, P = .4) compared with PET/CT. Given its high specificity, Ig-HTS from plasma has potential clinical utility for surveillance after complete remission.

Sepsis is a life-threatening syndrome resulting in shock and organ dysfunction stemming from a microbial infection. Sepsis has a mortality of 40% and is implicated in half of all in-hospital deaths. The host immune response to microbial infection is critical, with early-phase sepsis characterized by a hyperinflammatory immune response, whereas the later phase of sepsis is often complicated by suppression. Sepsis has no treatment, and management remains supportive. Stem cells constitute exciting potential therapeutic agents for sepsis. In this review, we examine the rationale for stem cells in sepsis, focusing on mesenchymal stem/stromal cells, which currently demonstrate the greatest therapeutic promise. We examine the preclinical evidence base and evaluate potential mechanisms of action of these cells that are important in the setting of sepsis. We discuss early-phase clinical trials and critically appraise translational barriers to the use of mesenchymal stem/stromal cells in patients with sepsis.

Background: Although ~60% of patients with DLBCL are cured with frontline therapy, outcomes for those with relapsed/refractory disease remain poor. Tumour cells exploit immune checkpoint pathways, including the PD1/PDL1 axis, to evade and inhibit host anti-tumour immune responses. PD1/PDL1 expression and cytogenetic 9p24 alterations in some DLBCL subtypes provide additional rationale for PDL1 inhibition (PD-L1i) in DLBCL. Though single agent PD1i yields a disappointing ORR of 10-30% in heavily pre-treated DLBCL, some responses are durable.1 Radiotherapy (RT) is an established mechanism of stimulating anti-tumour immunity via increased circulating tumour antigen, immunogenic cell death and T-cell recruitment and activation in the tumour microenvironment. Synergy between concomitant immune checkpoint inhibition (ICI) and RT has been demonstrated in preclinical studies2 and solid tumours; a recent study in non-small cell lung cancer demonstrated an ORR of 36% with pembrolizumab + RT compared with 18% with pembrolizumab alone.3 RT hypofractionation appears critical to the abscopal effect when used with ICI,4 and concurrent RT and PD-L1i is more successful than sequential treatment.5 RT to multiple sites may broaden the spectrum of tumour antigen released and overcome clonal variation between disease sites; a dose-response relationship between RT and antigen release has yet to be established. This phase I study aims to determine the safety profile of escalating dose and number of sites of RT in combination with Durvalumab (MEDI4736), an anti-PD-L1 monoclonal antibody, in relapsed/refractory DLBCL, including primary refractory DLBCL and transformed follicular lymphoma. Study Design and Methods: RaDD (NCT03610061) includes eligible pts who have received ≥1 prior line of therapy and are ineligible for or relapsed after autologous stem cell transplantation (SCT). Pts with active autoimmune disease, CNS involvement, prior allogeneic SCT or chronic steroid use are excluded. Treatment comprises external beam RT to target site(s) daily for 5 days (Fig 1). Durvalumab 1500mg IV commences on day 2 of RT and continues 4-weekly until disease progression. Pts can continue until a second radiological progression if clinical benefit is ongoing. The primary endpoint is the toxicity, drug pharmacokinetics, maximum tolerated dose (MTD) and recommended phase two dose (RP2D) of simultaneous RT plus durvalumab. Secondary endpoints are response rates; progression free survival; and overall survival. An exploratory PET substudy will employ novel tracers to characterise the local and systemic immune response via assessment of the biodistribution of durvalumab (with 89Zr-Durvalumab) and CD8+ T cells (with 89Zr -Df-IAB22M2C). Biomarker sample collection is synchronised with PET response assessment. A comprehensive translational substudy will apply high throughput technologies to tissue and sequential blood samples to characterise the tumour-immune system interaction and correlate novel host, tumour and tumour microenvironment factors with treatment responses and toxicity. Findings may inform the RP2D. RT dose and site escalation will proceed according to a 3+3 design with 5 dose levels (cohorts 1-5, Fig 2). The dose limiting toxicity assessment window is the first 28 days. Projected enrolment for determination of MTD and RP2D is 6-30 pts pending toxicity. Recruitment will continue to a total of 36 pts to allow for secondary endpoint analysis. 5 pts have been enrolled to date. Acknowledgements: Victorian Cancer Agency (funding), Astra Zeneca (durvalumab and funding), Celgene (funding), Imaginab (89Zr -Df-IAB22M2C) References: 1. Ansell SM et al. Nivolumab for Relapsed/Refractory Diffuse Large B-Cell Lymphoma in Patients Ineligible for or Having Failed Autologous Transplantation. J Clin Oncol. 2. Deng L et al. Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice. J Clin Invest. 3. Theelen W et al. Effect of Pembrolizumab After Stereotactic Body Radiotherapy vs Pembrolizumab Alone on Tumor Response in Patients With Advanced Non-Small Cell Lung Cancer. JAMA oncology. 4. Golden EB et al. An abscopal response to radiation and ipilimumab in a patient with metastatic non-small cell lung cancer. Cancer Immunol Res. 5. Sharabi AB et al. Radiation and checkpoint blockade immunotherapy: radiosensitisation and potential mechanisms of synergy. Lancet Oncology. Disclosures Hawkes: Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Roche: Research Funding; Takeda: Speakers Bureau; Astra Zeneca: Research Funding; Merck KgA: Research Funding; Mundi pharma: Research Funding; Gilead: 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, Speakers Bureau. Manos:Janssen: Honoraria; Novo Nordisk Pharmaceuticals: Other: Travel. Chong:Merck Serono: Research Funding; Hutchison Medipharma: Research Funding; Pharmacyclics: Research Funding; Novartis: Research Funding; Bayer: Research Funding; BMS: Research Funding. MacManus:National Health and Medical Research Council Australia: Research Funding. Keane:MSD: Consultancy; Celgene: Consultancy; Gilead: Consultancy; BMS: Research Funding; Roche: Consultancy, Other: Travel Grant. Scott:Cancer Council Victoria: Research Funding; Cancer Australia: Research Funding; Avipep: Consultancy; IBA: Consultancy; Paracrine Therapeutics: Equity Ownership, Patents & Royalties; Life Science Pharmaceuticals: Equity Ownership; NHMRC: Research Funding; Abbvie: Consultancy, Patents & Royalties; Cure Brain Cancer: Research Funding; Medimmune: Consultancy; Humanigen: Patents & Royalties. Shortt:Celgene: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Astex: Research Funding; Amgen: Research Funding; Gilead: Speakers Bureau; Takeda: Speakers Bureau. Ritchie:Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; BMS: Research Funding; Takeda: Research Funding; Beigene: Research Funding; Imago: Research Funding; Novartis: Honoraria; Sanofi: Honoraria. Lee:Australian Nuclear Science and Technology Organisation: Membership on an entity's Board of Directors or advisory committees. Koldej:NanoString Technologies: Other: Travel grant. OffLabel Disclosure: Durvalumab is an anti-PD-L1 monoclonal antibody.

Background The time to treatment initiation is determined by tumour burden in patients with follicular lymphoma (FL). The Groupe d'Etude des Lymphomes Folliculaires ('GELF') criteria, defined in the pre-rituximab era, are commonly used to assess tumour burden.2 Patients must meet ≥1 of the following criteria to be considered "high" tumour burden according to GELF: any tumour mass >7 cm; ≥ 3 nodal sites (each >3 cm); B symptoms; splenomegaly; compression syndrome; pleural/peritoneal effusion; leukemic phase or cytopenias. Low tumour burden FL is often excluded from clinical trials, based on data from initial retrospective studies and later randomised trials, demonstrating no survival advantage with chemotherapy compared with observation alone. 1-3 Conversely, it is recommended those with high burden disease receive immediate therapy. The use of GELF in therapeutic decision-making outside of clinical trials is not well described. Methods Cases of newly diagnosed Grade 1-3a FL were retrospectively identified from the Australian Lymphoma and Related Diseases Registry (LaRDR), and 2 additional institutional databases from 2002-2019. Additional data was obtained from electronic hospital records. The primary aim of the study was to determine the utilisation of GELF criteria in guiding therapeutic decisions in FL. The secondary aims were to document frequency of GELF according to stage and treatment and to determine the impact of the number of GELF criteria on PFS. Survival analysis was calculated according to the Kaplan-Meier method. Results 385 cases were identified. Patient characteristics are in table 1. The median follow-up was 2 years (range 0.1-18) with 2-year PFS and OS of 89% (95% CI 85-92%) and 96% (95% CI 92-98%) respectively. 94 (24%) patients underwent a 'watch and wait' approach (W&W), 54 (14%) received radiotherapy alone and 237 (62%) received chemotherapy +/- radiotherapy. 118 (31%) patients had stage I/II disease at diagnosis, of these 36 (30%) underwent a W&W approach, 41 (35%) had radiotherapy alone and 41 (35%) received chemotherapy +/- radiotherapy (with only 3 patients in the latter group being enrolled on clinical trial). 260 (69%) had advanced stage disease, of these 56 (22%) underwent a W&W approach, 13 (5%) had radiotherapy alone and 191(73%) received chemotherapy +/- radiotherapy. In the W&W group, 23% of patients had ≥1 GELF criteria, of these, 38% had limited stage disease. Of the patients who received chemotherapy +/-radiotherapy and radiation therapy, 36% and 57% respectively had no GELF criteria identified with 25% and 80% having limited stage disease respectively (table 2) Where available the reasons for commencing chemotherapy +/- radiotherapy in GELF negative patients were examined and included pain associated with lymph nodes, concerns regarding high-grade transformation (secondary to high SUVmax, size of nodal mass or trajectory of growth), cosmesis, nausea and fatigue. In both the W&W and treated cohorts, the number of GELF criteria did not predict outcome (Figure 1A & B). In a subgroup analysis by treatment modality, patients with no GELF criteria versus those with ≥1 GELF criteria, no statistically significant difference in PFS in the W&W group (PFS: HR 0.69 95% CI 0.14-3.27, P=0.63), the chemotherapy +/- radiotherapy group (PFS: HR 1.66 95% CI 0.79-3.52, P=0.18) and the radiotherapy alone group (PFS: HR 4.53 95% CI 0.51-40.71, P=0.13) was demonstrated. Conclusion One fifth of W&W FL patients had ≥ 1 GELF criteria and 36% of those receiving chemotherapy +/-radiotherapy had no GELF criteria at baseline, suggesting clinicians are using other measures to make therapeutic decisions. By restricting eligibility for clinical trials to those with high tumour burden, using GELF, a significant proportion of patients being treated in the 'real world' are not represented. In our cohort of both treated and untreated patients with FL, the presence of ≥1 GELF criteria did not influence prognosis. References 1. Young RC, et al. The treatment of indolent lymphomas: watchful waiting v aggressive combined modality treatment. Semin Hematol 1988. 2. Brice P, et al. Comparison in low-tumor-burden follicular lymphomas between an initial no-treatment policy, prednimustine, or interferon alfa: a randomized study from the GELF. J Clin Oncol 1997. 3. Ardeshna KM, et al; Long-term effect of a W & W policy versus immediate systemic treatment for asymptomatic advanced-stage NHL. Lancet 2003. Disclosures Cheah: Roche: Other: Travel expenses; Celgene, Roche, Abbvie: Research Funding; Roche, Janssen, MSD, Gilead, Loxo Oncology, Acerta, BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Keane:Roche: Consultancy, Other: Travel Grant; Celgene: Consultancy; MSD: Consultancy; Gilead: Consultancy; BMS: Research Funding. Johnston:Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen, Roche: Membership on an entity's Board of Directors or advisory committees. Dickinson:Merck Sharpe and Dohme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Opat:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Epizyme: Research Funding; CSL: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Beigene: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Amgen: Research Funding; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy. McQuilten:CSL Biotherapies: Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding; Takeda Pharmaceuticals: Research Funding; AbbVie: Research Funding; Janssen-Cilag: Research Funding. Wood:Abbvie, Alexion, Amgen, Bristol-Myers Squibb, Celgene, CSL Behring, Gilead, Janssen, Novartis, Roche,, Sanofi, Takeda: Research Funding. Hawkes:Gilead: 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, Speakers Bureau; Merck KgA: Research Funding; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Research Funding; Takeda: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Mundi pharma: Research Funding; Astra Zeneca: Research Funding; Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees.

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.

Background Recent reports suggest genetic polymorphisms influence susceptibility to rituximab-induced late-onset neutropenia (LON), which in turn may be a predictor of good outcome in B-cell lymphoma. Aims We report the largest study to date assessing FCGR3A-V158F polymorphisms in diffuse large B-cell lymphoma (DLBCL) treated with cyclophosphamide/hydroxydaunorubicin/Oncovin (vincristine)/prednisone/rituximab (CHOP-R). The influence of C1qA-A276G polymorphisms in DLBCL, and the impact of both polymorphisms on susceptibility to LON and outcome were also examined. Methods 115 DLBCL patients treated with CHOP-R were compared with 105 healthy White people controls with regards to FCGR3A-V158F and C1qA-A276G polymorphisms. LON incidence and event-free and overall survival (EFS and OS) were analysed for linkage to either polymorphism. Results The FCGR3A-V158F but not the C1qA-A276G polymorphism influenced the risk of developing LON. 50% of FCGR3A-158V/V patients experienced LON. In contrast, only 7% V/F and 2% F/F experienced LON. The FCGR3A-158V/V genotype was associated with LON compared with V/F (P = 0.028) and F/F genotypes (P = 0.005). Although no patients with either LON or FCGR3A-158V homozygosity relapsed compared with 33% FCGR3A-158F/F and 21% non-LON, this did not translate into improved EFS or OS. Conclusions Polymorphic analysis may be a predictive tool to identify those at high risk of LON. Prospective studies are required to establish definitively if LON or FCGR3A-158V/V genotype influences outcome.

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 LAG3 gene expression was highly correlated with protein expression by tissue microarray based immunohistochemistry (r=0.79, p Finally, levels of soluble LAG3 (sol-LAG3) were quantified within paired plasma of patients enrolled into the ALLG NHL21 PET-adapted prospective DLBCL study. Samples taken pre-therapy and following 4 cycles of R-CHOP (at the time of interim-PET) were compared. Sol-LAG3 levels were higher in patients with DLBCL at diagnosis compared to healthy controls (p In conclusion, high expression of LAG3 in DLBCL is enriched in the non-GCB phenotype, and is associated with poor outcome independent of clinical and biological prognosticators. Dual PD-L1HI/LAG3HIexpression confers particularly poor outcome after conventional front-line immuno-chemotherapy. Intratumoral LAG3 expression is high on PD-1+ CD8+ and TREG subsets. Sol-LAG3 appears to a circulating disease response biomarker. The results combined indicate a key role for LAG3 within the immunobiology of DLBCL and provide a strong rationale for early phase clinical trials utilising anti-LAG3 and anti-PD1 mAb combinations. Disclosures Keane: BMS: Research Funding; Roche: Other: Education Support, Speakers Bureau; Celgene: Consultancy, Research Funding; Takeda: Other: Educational Meeting; Merck: Consultancy. Gould:Novo Nordisk: Other: Educational Travel. Abro:Novartis: Consultancy; Amgen: Other: education support congress attendance; Janssen: Other: education support congress attendance; Bristol-Myers Squibb: Speakers Bureau; Celgene: Other: education support congress attendance. Tobin:Celgene: Research Funding; Amgen: Other: Educational Travel. Birch:Medadvance: Equity Ownership. Talaulikar:Novartis: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Research Funding; Roche: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria. Bird:Amgen, Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hertzberg:Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees. 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.

There is proven pre-clinical and clinical efficacy of mono or combinatorial immune strategies to boost host anti-lymphoma immunity, with classical Hodgkin Lymphoma (cHL) seen as the 'poster child'. Approaches include blockade of immune-checkpoints on exhausted tumor-specific T-cells (via mAb blockade of PD-1, TIM3, LAG3, TIGIT or their ligands), activation of T-cells via mAbs agonistic to CD137, and finally modulation of FOXP3, CTLA-4 and/or LAG3 regulatory T-cells (Tregs) or immunosuppressive tumor-associated macrophages (TAMs). In contrast, studies characterizing the circulating and intra-tumoral microenvironment (TME) of the distinct but rare CD20+ Hodgkin Lymphoma entity (5-8% of HL), Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL), are minimal. Furthermore, to our knowledge no functional profiling studies comparing the host immunity of NLPHL with cHL has been performed. We compared host immunity in 29 NLPHL patients, 30 cHL patients and 10 healthy individuals, with a focus on pertinent and clinically actionable immune parameters. Paraffin-embedded tissue and paired (pre- and post-therapy) peripheral blood mononuclear cells samples were interrogated by digital multiplex hybridization (Nanostring Cancer Immune Profiling Panel) and flow cytometry. Although cytotoxic T-cell gene counts (CD8a, CD8b) were similar, compared to cHL there were higher levels of the immune effector activation marker CD137 (gene counts 439 vs. 287; P Flow cytometry profiling of immune subsets in peripheral blood showed findings consistent with findings in the TME. Specifically, there was elevation of multiple exhaustion markers within CD4, CD8, and NK immune effector cells, with a striking proportion of highly anergic dual-LAG3/PD-1 positive CD8+ T-cells. Also there was elevation of immune-suppressive monocyte/macrophages in cHL relative to NLPHL. Relative to healthy lymph nodes, there was prominent up-regulation of a range of T-cell associated exhaustion markers in both NLPHL and cHL, indicating dysregulated priming of effector immune responses and host immune homeostasis. Comparison between NLPHL and cHL illustrated that NLPHL had a myriad of features that marked its intratumoral TME as a unique immunobiological entity typified by elevated immune checkpoint markers and T-cells with a highly anergic phenotype. Put together, these findings indicate that distinct immune evasion mechanisms are operative within the TME of NLPHL, including markedly higher levels of multiple immune-checkpoints relative to cHL. In contrast, Treg subsets and immune-suppressive monocyte/macrophages were relatively lower than that seen in cHL. T-cells frequently had dual immune-checkpoint expression. The findings from this study provides a compelling pre-clinical rationale for targeting PD-1 or combinatory checkpoint inhibition in NLPHL and sets the basis for future 'chemo-free' rituximab + checkpoint inhibitor clinical trials. Disclosures Tobin: Amgen: Other: Educational Travel; Celgene: Research Funding. Birch:Medadvance: Equity Ownership. Keane:Takeda: Other: Educational Meeting; BMS: Research Funding; Roche: Other: Education Support, Speakers Bureau; Celgene: Consultancy, Research Funding; Merck: Consultancy. Gandhi:BMS: 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; 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.

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.

Follicular Lymphoma (FL) is the most common indolent Non-Hodgkin Lymphoma. Despite generally favorable survival outcomes, 20% of FL patients experience 'Progression of Disease within 24 months' (POD24) and subsequently have poor long-term overall survival (OS) (Casulo, JCO 2015). Unfortunately, POD24 has limited clinical value because it cannot guide up-front clinical decisions. Accurate pre-therapy prognosticators are vital for clinical trial design and are also increasingly being mandated by funding agencies for stratification of patients to emerging front-line treatments. The new 'state-of-the-art' prognosticators 'm7-FLIPI' and POD24-PI' (Pastore, Lancet Oncol 2015; Jurinovic, Blood 2016) supplement clinical parameters with genetic mutational status. However, their applicability to population based cohorts including early-stage and asymptomatic patients remains unknown. Furthermore, there is significant heterogeneity of outcome within these prognostic groupings. The established biological and prognostic importance of the tumor microenvironment (TME) in FL suggests that prognosis would be enhanced by incorporating information on host immunity (Scott, Nat Rev Can 2014). Forty-five pre-treatment FL biopsies were categorized into 'hot' or 'cold' immune nodes by multiplex immunofluorescent imaging and respectively characterized by concordant high or low expression of multiple immune effector and checkpoint-associated proteins. (Fig 1A). Consistent with these findings, gene expression using the Nanostring platform showed that immune effectors (CD4/CD8/TNFa/CD137/CD56) positively correlated with immune checkpoints (PD-1/PD-L1/PD-L2/TIM3/LAG3/CD163/CD68) indicative of an adaptive immune response. Additionally, high-throughput unbiased TCRb sequencing showed the intratumoral TCR repertoire was more clonal in 'hot' compared to 'cold' FL samples (p=0.024), indicative of a skewed T-cell immune response (Fig 1B). We then applied these findings to an independent population-based cohort of 175 cases of FL from the rituximab era with long-term follow-up (median ~7 years), including advanced (n=137) and localized cases (n=38). The aims were to: a) identify new targetable immune parameters of prognostic importance in the rituximab-era; and b) compare and contrast these with published prognostic tools: FLIPI, FLIPI-2, m7-FLIPI, POD24-PI and 'immune survival score' ('ISS', Dave, NEJM 2004). OS was not only inferior in those experiencing POD24 (HR 4.88, p2-fold increase in 5-year patient health costs. Hence, POD24, as well as FFS and TT2T were therefore chosen as the primary outcome measures. M7 mutation frequencies were similar to those previously published (Pastore, Lancet Oncol 2015). However, the prognostic utility of the m7-FLIPI could not be demonstrated, whereas the FLIPI, FLIPI-2, and POD24-PI retained their prognostic value. The POD24-PI was most predictive of FFS (p We have validated the TME in predicting outcome in a population based cohort of FL patients with long-term follow-up treated in the rituximab era. Furthermore, we describe the role of PD-L2 as well as several additional pertinent, clinically-actionable markers of the TME which predict survival to conventional therapies in FL. Low expression of PD-L2 appears to be a surrogate of a broadly co-ordinated downregulation of the intratumoural response. These immune scores are independent of and additive to additive to the FLIPI and POD24-PI. Development of new prognostic models require the incorporation of host immunity along with clinico-genetic features to further improve the specificity, and to accurately risk stratify FL patients. Disclosures No relevant conflicts of interest to declare.

The Hyper-CVAD chemotherapy regimen is being increasingly applied to a number of haematological malignancies. We assessed the impact of Hyper-CVAD on peripheral blood stem cell (PBSC) yields and examined the optimal timing of PBSC collection when using this regimen. Seventy-four consecutive patients were identified in whom an attempt was made to collect PBSC, usually on recovery from cycle A or B. Where PBSC collection was attempted after cycle 3B, only 18% (3/17) of patients successfully mobilized. Fifty-seven patients were mobilized on recovery from cycle 1B (n = 13), 2A (n = 22), 2B (n = 14) or 3A (n = 8). Compared with cycle 2A, 1B was not superior in achieving the minimum ofor =2 x 10(6)/kg CD34+ cells (100% vs. 77%, p = 0.13), but was superior in terms of total CD34+ yield (21.4 vs. 3.2 x 10(6)/kg, p0.001), achieving the target CD34+ cell count ofor =5 x 10(6)/kg (92% vs 36%, p = 0.002), and obtaining both a minimum (92% vs. 18%, p0.001) and target (77% vs. 0%, p0.001) graft with a single apheresis. There were no significant differences in PBSC yields following cycles 2A, 2B and 3A. Hyper-CVAD has substantial stem cell toxicity which can be readily circumvented by using the early chemotherapy cycles for mobilization.

Despite the Revised International Prognostic Index's (R-IPI) undoubted utility in diffuse large B-cell lymphoma (DLBCL), significant clinical heterogeneity within R-IPI categories persists. Emerging evidence indicates that circulating host immunity is a robust and R-IPI independent prognosticator, most likely reflecting the immune status of the intratumoral microenvironment. We hypothesized that direct quantification of immunity within lymphomatous tissue would better permit stratification within R-IPI categories. We analyzed 122 newly diagnosed consecutive DLBCL patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) chemo-immunotherapy. Median follow-up was 4 years. As expected, the R-IPI was a significant predictor of outcome with 5-year overall survival (OS) 87% for very good, 87% for good, and 51% for poor-risk R-IPI scores (P

Elderly patients with acute myeloid leukemia (AML) have a poor prognosis. The authors examined the in vitro and clinical activity of the histone deacetylase inhibitor valproic acid (VA) combined with cytosine arabinoside (AraC) in elderly patients with AML unsuited to intensive therapy. For the in vitro studies, primary AML cells from 11 patients were treated with AraC and VA and analyzed for apoptosis, cytostatic effects, differentiation and acetyl histone H3 induction. VA (alone and with AraC) enhanced apoptosis and induced acetyl histone H3. VA inhibited cell proliferation. For the clinical trial, 15 patients were treated with VA and subcutaneous AraC and assessed for toxicity and response. No complete or partial remissions were achieved. In conclusion, VA has in vitro activity against AML and has additional activity with AraC. However, in this study, this combination demonstrated limited clinical activity in elderly patients with AML.

A 17-year-old girl with ALL developed acute onset left eye swelling and facial pain on day 23 of phase II of the UKALL XII protocol. She had been neutropaenic for 14 days at this time. She had neutropenic fever since day 17 with no obvious cause for this identified up to the time facial swelling appeared. This occurred despite broad-spectrum antibiotic therapy and empiric liposomal amphotericin therapy for her neutropenic fever. The liposomal amphotericin had been commenced 3 days prior to symptoms developing at a dose of 3 mg/kg/day. The eye swelling progressed rapidly with increased pain and closure of left eye within 24 h. Imaging with MRI demonstrated left sided sinusitis (Image 1). The patient was taken to theatre for emergency washout. Findings at surgery demonstrated a soft tissue mass extending onto the skull base, posterior choana and encasing the middle turbinate (Image 2). Microscopy of scrapings from the infiltrate demonstrated an organism with septated hyphae and dichotomous branching (Image 3). Culture of this organism confirmed a diagnosis of Aspergillus flavus sinusitis. Post surgery, the patient continued to improve with high dose liposomal amphotericin (10 mg/kg/day) and caspofungin (50 mg daily). Her pyrexia settled within 36 hr of surgery. She returned to theatre 1 week later for a further washout and at this stage a marked improvement was noted. Neutrophil recovery did not occur until 22-days post initial surgical intervention. During this period she received daily GCSF. Initially, post debridement she received daily granulocyte transfusions for 10 days. Caspofungin was ceased 2 weeks into treatment as her clinical condition had improved. Liposomal amphotericin continued at 5 mg/kg/

8504 Background: In patients with diffuse large B cell lymphoma (DLBCL), tumor specific immunoglobulin genes can be detected in both the cellular and acellular compartments of blood (Green et al AS...

Abstract 3673 Background: Impaired systemic immunity is an established risk factor for the development of B-cell lymphoma. However current understanding of the underlying mechanisms of cell-mediated immune evasion and how this varies during chemo-immunotherapy remains rudimentary. Studies have been hampered by retrospective analysis of heterogenous cohorts containing both new and relapsed patients, with a variety of indolent/aggressive histological subtypes, treated with different treatment regimens. Methods: We performed detailed analysis of circulating immune cell populations in 33 patients (mean age 53 years, range 31 – 69, F:M 11:22) with newly diagnosed high-risk diffuse large B-cell lymphoma (DLBCL) enrolled in a prospective multi-centre Australasian Leukaemia & Lymphoma Group trial. They received 3 cycles of CHOP-R-14, and then after a 4th cycle underwent an interim-PET/CT with centralized review performed at day 17–20 (to enable risk-stratification). Pegylated-GCSF was given on day 2 after each cycle. PET/CT metabolic response categorisation used the harmonisation criteria. 10/33 evaluable patients remained interim-PET/CT avid. Blood samples were taken pre-treatment and on day 21 post-cycle 4, and compared to 23 age/gender-matched healthy control subjects. Results were correlated with interim-PET/CT response status. Laboratory investigators were blinded to clinical results. Results: CD3+, CD8+ and CD4+ T-cells (p=00.21, p=0.005, p=0.0017) among patients with DLBCL were all reduced at pre-treatment compared to controls. After 4 cycles, CD4+ lymphocytes were reduced further (p=0.0003) whereas CD3+ and CD8+ counts remained unchanged. However, CD3+, CD8+ and CD4+ proliferative responses to CD28/CD3 stimulation were equivalent to healthy subjects at both time-points, indicating the absence of an intrinsic functional T-cell defect in response to stimulation. Nor were regulatory T-cells (CD4+CD25hiCD127−) or myeloid-derived suppressor cells (CD33+CD14−HLA-DR−) altered between time-points or as compared to healthy subjects. Circulating monocytes are key regulators of inflammation/ immunity. Absolute monocyte counts were strikingly elevated at pre-treatment compared to healthy subjects (p=0.007), and had a predominantly CD14+HLA-DR−/lo phenotype. Importantly, pre-treatment values for absolute monocytes (p=0.04), monocyte % (p=0.004), CD14+HLA-DR−/lo monocytes (p=0.03), mean fluorescent intensity of HLA-DR on CD14+ monocytes (p=0.025) and CD3+ T-cells (p=0.0374), were all associated with persistent interim-PET/CT avidity. However, all were independent of the international prognostic index (IPI) score, and IPI did not segregate with interim-PET/CT status. Post-cycle 4 monocyte % (p=0.048) and CD3+ T-cells (p= 0.029) were also related to interim-PET/CT avidity. The CD14+HLA-DR−/lo phenotype has been described as immunosuppressive (Lin, Blood 2011). Consistent with this, in-vitro depletion of monocytes from pre-treatment (p=0.03) and post-cycle 4 (p=0.04) samples enhanced CD4+ proliferation. By contrast, monocyte depletion from control samples did not influence CD4+ proliferation. Post-cycle 4 monocytes rose above pre-treatment levels (p=0.0301) as did fluorescent intensity of HLA-DR on CD14+ monocytes (p=0.0027). In line with the post-cycle 4 reduction of the CD14+HLA-DR−/lo ratio (p=0.0135), plasma arginase was reduced relative to pre-treatment (p=0.0035), suggesting that suppression was mediated in part through arginine metabolism. HLA-DR expression on monocytes at the interim time-point was up-regulated from pre-treatment (p=0.01), and accompanied by a relative increase in ex-vivo blood myeloid dendritic cells (BMDCs; p=0.044), suggesting the CD14+HLA-DR−/lo phenotype is associated with an impaired ability to differentiate into BMDCs in-vivo. Conclusion: Monocytes in DLBCL mediate immunosuppression of T-cells and both appear to be associated with interim-PET/CT avidity. Although the role of interim-PET/CT in DLBCL patients treated with chemo-immunotherapy is an area of considerable activity, the study of biological predictors of metabolic response has been relatively neglected. To the best of our knowledge, this is the first prospective study to find an association between cellular markers of inflammation/ immunity and interim-PET/CT avidity in a cohort of uniformly treated high-risk DLBCL patients. Disclosures: No relevant conflicts of interest to declare.

Treatment Of Acute Promyelocytic Leukemia (APML) In The Jehovah’s Witness (JW) Population. Colm Keane, Peter Mollee, Paula Marlton, Devinder Gill. Background: Treatment of acute myeloid leukaemia in JW patients is challenging. The refusal to accept blood products is usually a contraindication to intensive chemotherapy and a potential cure. Methods: We review three cases of APML occurring in JW patients treated at our institution which demonstrate the benefit of newer targeted combination therapies that induce less marrow suppression. Suggested management principles are derived from these anecdotal rare cases. Case 1 was a 39 year old male who was initially treated with ATRA (25 mg/m2) and Darbopoietin 100μg twice weekly with arsenic added on day 10 (0.15mg/kg/day). He initially tolerated therapy well despite severe pancytopenia until (day 20) the hemoglobin fell to 52g/L when he developed chest pain. He was diagnosed to have had a myocardial infarct in the setting of fluid retention. His WCC was 10.4 × 109/L with platelet count of 20 × 109/L. He was managed conservatively with no anticoagulation. His hemoglobin nadir was 44 g/L before rising above 80 g/L by day 38 of therapy at which time he was in cytogenetic and molecular remission. He completed a further 28 weeks of consolidation with ATRA (45mg/m2 for 2 weeks every 4 weeks) and arsenic (0.15mg/kg/day Mon-Fri, d1-28 every 2 months for 4 cycles). He has completed 9 months of planned 2 yrs oral maintenance chemotherapy (ATRA, 6MP, MTX) without further incident. Case 2 was a 62 year old female who was treated with ATRA induction (initially at 25mg/m2 but increased to 45mg/m2 on day 18), plus Erythropoietin (Epo) 4,000U × 3 per week s/c. She was in complete cytogenetic remission on day 37 and complete molecular remission by day 87. She had a hemoglobin nadir of 60 g/L but remained asymptomatic. Consolidation therapy commenced on day 106 consisting 3 cycles of combination therapy with ATRA (45mg/m2/d po for 4 weeks) and arsenic (0.15mg/m2/d iv for 5d/week for 4 weeks). Between consolidation cycles there was a 3-week interval during which all therapy was ceased. This regimen was well tolerated. She remains in complete remission after 8 years. Case 3 was a 28-year-old male who was initially treated with ATRA, low dose cytarabine and Epo 4000U 3-times/week s/c. He developed symptoms of severe anemia when his hemoglobin dropped to 25g/L with syncopal episodes and hypotension. He was subsequently found to have t (11:17) mutation and the leukemia did not respond to ATRA. He died from severe anemia fourteen days after presentation. Case reports published subsequently have shown that ATRA resistance in t(11:17) APML may occasionally be overcome if combined with standard chemotherapy or GCSF. However to date Arsenic has not been useful in this variant form of APML. Recommendations: Therapy. Induction with ATRA 25mg/m2 followed on day 10 by arsenic 0.15mg/kg/day until morphologic remission. Consolidation with alternating cycles of ATRA and arsenic as per Case 1. Maintenance therapy with ATRA, 6MP and MTX for 2 years. Anemia. Our patients tolerated severe anemia reasonably well until hemoglobin dropped to approximately 50g/L (most deaths in the literature due to anemia in the JW population have occurred below this threshold). All JW patients receive maximal erythropoietin stimulation and sparing phlebotomy episodes using paediatric blood collection tubes. Thrombocytopenia and coagulopathy. Antifibrinolytics, DDAVP and newer agents such as FVIIa have been used successfully in JW patients undergoing high risk surgery and may be of benefit in the APML setting for patients with haemorrhagic complications. Differentiation syndrome (DS) Case 1 may have developed DS. The standard management of DS is to commence chemotherapy such as idarubicin, cytarabine or gemtuzumab ozogamicin but because of the potential prolonged myelosuppression such agents are not suitable options for JW patients. We would advocate corticosteroids for DS prophylaxis and if treatment of DS is required, hydroxyurea may be a safer option because of its limited and shorter duration of myelosuppression. Conclusion: Our series demonstrates the benefit of more targeted therapy in APML allowing patients who refuse transfusion of blood products a realistic chance of cure.

PURPOSE: The combination of thalidomide and dexamethasone has been shown to have excellent antimyeloma activity in both relapsed and newly diagnosed patients. This combination offers a potential oral, less toxic alternative to standard regimens. This single-centre retrospective study evaluates the activity of the combination of thalidomide and dexamethasone in patients with newly diagnosed multiple myeloma attending the haematology department of the Mater Misericordiae University Hospital, Dublin. METHODS: Forty-six patients with newly diagnosed multiple myeloma between April 2000 and April 2006 were treated with thalidomide and dexamethasone as first line treatment. The median age of the patients was 70 years (range 46 to 88 years). The median serum paraprotein level was 32g/l (range 2 to 99g/l). The median bone marrow plasma cell percentage was 40% (range 10% to 99%) The first twenty-two patients were commenced on thalidomide at 200mg od with the dose increased by 200mg every two weeks if tolerated to a maximum of 800mg od. The most recent twenty-four patients were commenced on thalidomide at 100mg od with the dose increased to a maximum of 200mg if tolerated. All patients received dexamethasone 40mg per day on days 1–4, 9–12 and 17–20 ot the first cycle and for a four day pulse every four weeks for subsequent cycles. RESULTS: The response rate was 67.4% when assessed by Blade criteria (50% reduction in serum myeloma protein and 90% reduction of urine myeloma protein on at least two occasions for a minimum of 6 weeks). A complete response was observed in 10.9% of patients. There was no significant difference in response rates between the higher thalidomide dose group and the lower thalidomide dose group. The main toxicities included constipation in 57.8%, hyperglycaemia in 33.3%, peripheral neuropathy in 22.2%, drowsiness in 15.6%, peripheral oedema in 15.6%, venous thromboembolism in 11.1%, rash in 11.1% and proximal myopathy in 8.9%. Routine anticoagulation was not used although patients with additional risk factors for venous thromboembolism received prophylactic dose low molecular weight heparin and in total 53.3% of patients were receiving either aspirin, warfarin or low molecular weight heparin at the time of treatment. There was no significant difference between the two thalidomide dose groups in terms of frequency of toxicities. However 27.3% of patients in the higher thalidomide dose group had their treatment stopped within one month due to toxicities compared to 4.2% in the lower dose group. CONCLUSION: The combination of thalidomide and dexamethasone is safe and effective in the treatment of newly diagnosed multiple myeloma. There is less treatment-limiting toxicity and no loss of efficacy with lower doses of thalidomide.

The Hyper-CVAD chemotherapy regimen (JCO2000;18:547) is being increasingly applied to a number of haematological malignancies. We have previously demonstrated Hyper-CVAD to be a highly stem cell toxic regimen which adversely affects peripheral blood stem cell (PBSC) mobilisation and that mobilising efficiency can be significantly improved by collecting PBSC earlier in the treatment course. We now examine the optimal timing of PBSC collection when using this regimen. A retrospective analysis was performed at two centres of consecutive patients (pts) treated with Hyper-CVAD in whom an attempt was made to collect PBSC. The regimen consists of alternating cycles of: (A) cyclophosphamide 300mg/m2 q12hrs for 6 doses d1-3, vincristine 2mg d4&11, doxorubicin 50mg/m2 d4 and dexamethasone 40mg daily d1-4 and d11-14; and (B) methotrexate 1g/m2 over 24 hours d1 and ara-C 1–3g/m2 q12hrs for 4 doses d2&3. PBSCs were collected on recovery from chemotherapy using G-CSF in a daily dose of 10mcg/kg. Target cell number for optimum engraftment was defined as CD34 ≥5 x106/kg, while the minimum threshold for proceeding to ASCT was CD34 ≥2 x106/kg. Pts were analysed according to the timing of mobilisation. 74 pts were identified: median age 44yrs; 69% male; 53% with initial marrow involvement; diagnoses (ALL n=14, lymphoblastic lymphoma n=7, Burkitt’s/Burkitt-like lymphoma n=8, aggressive NHL n=34, mantle cell lymphoma n=11). 28 pts were mobilized off the back of cycle A, 32 pts off the back of cycle B, and 14pts after other mobilising regimens. Overall 66% and 42% of pts obtained a minimum and optimal PBSC collection, respectively. Only 3 of 17 patients (18%) collected beyond cycle 3B successfully mobilised. Two patients mobilised prior to 3B but receiving alternate mobilising chemotherapy were not analysed. The results of stem cell collection on the 55 remaining patients are shown below. Stem cell collection results following hyper-CVAD chemotherapy Timing of stem cell mobilisation Cycle 1B 2A 2B 3A N 13 21 14 7 Total CD34+ (range) 21.36 (4.2–69.1) 3.41 (0–11.18) 5.1 (0–22.3) 4.6 (1.9–8.6) Day of 1st apheresis 12 (11–13) 13.5 (11–16) 13.5 (12–19) 15 (13–19) % with CD34+≥2 x106/kg All patients 100% 81% 64% 86% Obtained in 1 apheresis 92% 19% 36% 14% % with CD34+≥5 x106/kg All patients 92% 38% 50% 39% Obtained in 1 apheresis 77% 0% 21% 0% Cycle 1B compared to 2A was not significantly better in obtaining a minimum graft (100% vs. 81%, p=0.14), but was superior in terms of total CD34+ yield (21.4 vs. 3.4x106/kg, p

8542 Background: Immunity represents a balance between immune-effectors and immune-checkpoints. In DLBCL, circulating lymphocyte:monocyte ratios are prognostic, implicating them as surrogate immune-effectors and immune-checkpoints within the tumor microenvironment (TME). Detailed assessment of blood would enable identification of the optimal TME immune-effector and monocyte/macrophage-checkpoints to assist sub-stratification of conventional prognosticators. Methods: Blood from 140 R-CHOP treated DLBCL patients in the NHL21 Australasian Leukaemia and Lymphoma Group trial were analysed. A circulating immune-effector:monocyte-checkpoint signature segregating interim-PET/CT positivity was identified. Intratumoral applicability was tested in two independent R-CHOP treated DLBCL cohorts, with cell-of-origin (COO) and international prognostic index (IPI) as co-variates. Results: Patient monocytes suppressed T-cell proliferation and NK-cell rituximab-ADCC. Both CD14+HLA-DRlo monocytoid-myeloid derived suppressor ...

Abstract 49 Background: The challenge in Hodgkin Lymphoma (HL) remains to tailor therapies to eradicate malignancy with minimal side-effects, and to simultaneously identify those patients in whom alternate strategies should be instituted early. There are currently no circulating disease response biomarkers for HL. Blood biomarkers may assist interpretation of PET/CT, and can also be performed at each consultation to assess disease response and detection of early relapse. Ideal blood biomarkers must be specific, sensitive, easy to assay and interpret, with rapid turnaround, high reproducibility and comparability between laboratories. Candidate biomarkers might arise either from Hodgkin-Reed Sternberg (HRS) cells or the non-malignant tumor-infiltrate. The relative kinetics of circulating tumor-specific versus tumor-infiltrating biomarkers during therapy has not been compared. HRS cells comprise a minority of the diseased node, in which benign CD163+ M2 macrophages are prominent. Although CD163+ cells within the malignant node may be prognostic, there is no data on serum CD163 (sCD163). The HRS-specific protein TARC (which drives intra-tumoral accumulation of reactive cells) is elevated in serum (sTARC) pre-therapy but its kinetics during therapy are unknown. Methods: The Australasian Leukaemia & Lymphoma Group performed a prospective multi-centre observational study of sCD163 and sTARC in 221 samples from 47 HL patients (mean age: 36 years, range: 18–79; F:M 21:26). Results were compared to 21 age/gender matched healthy participants. Serum protein estimation was performed by ELISA. In EBER-ISH tissue positive patients, results were compared with plasma EBV-DNA (by real-time PCR). Blood was taken at five fixed time-points (prior, during and after therapy), and compared to disease response as adjudged by radiology, and with tissue levels by immunohistochemistry. Therapy was applied as per clinician's preference (85% ABVD, the remainder BEACOPP or ChlVPP). Results: Pre-therapy, both sCD163 and sTARC were markedly elevated compared to healthy and complete remission samples (all p Conclusion: We present the first study demonstrating that the tumor-infiltrate marker CD163 is elevated in the serum of HL patients. The combination of sCD163 with the tumor-specific marker sTARC by ELISA show promise as disease response biomarkers in HL. Furthermore, CD163+ monocytes mediate profound T-cell immunosuppression, indicating a potential therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Abstract 1585 Background: Diffuse Large B-cell Lymphoma (DLBCL) is a heterogeneous disease with variable outcome within international prognostic index (IPI) categories. Previous studies indicate expression of germinal centre B-cell (GCB) markers predict good outcome and that non-GCB sub-types may benefit from alternate therapies. A variety of immunohistochemical combinations have been proposed. However results are inconsistent and there is limited data comparing strategies. The need for a robust, practical method to distinguish GCB-DLBCL remains. We evaluated co-expression of the GCB proteins LMO2 and BCL6 with the Choi, Hans and Tally algorithms in patients treated with R-CHOP chemo-immunotherapy. Patients and Methods: Seventy-five DLBCL patients, chosen solely on availability of material for tissue microarray, were studied using immunohistochemistry for FOXP1, MUM1, CD10, BCL6, LMO2 and GCET1 on original biopsies. Clinical data was obtained from a prospectively maintained database. Results were correlated with event free (EFS) and overall survival (OS). Results: The cohort was typical for DLBCL described in the literature with 63% of patients aged over 60. The International Prognostic Index (IPI) predicted overall survival (p In the subgroup of patients who were positive for BCL6 approximately half were also positive for LMO2. There was a significant difference in overall survival between these BCL6 single positive and BCL6/LMO2 dual positive tumours (p=0.003) with improved outcome seen in the dual positive patients. Similarly those LMO2 positive patients who were BCL6 negative had very poor outcome compared to LMO2/BCL6 dual positive tumours (p=0.0002). Dual positivity for BCL6/LMO2 was strongly associated with germinal centre phenotype with eighty per cent of these patients typing for this cell of origin (p=0.0004). Germinal centre DLBCL as typed by the Choi algorithm could be separated into two significantly different groups with different overall survival based on LMO2/BCL6 dual positivity (p=0.01) Conclusion: LMO2/BCL6 dual positivity is a prognostic marker in DLBCL treated with R-CHOP that is independent of IPI, and predicts outcome in GCB typed tumours. Should these findings be prospectively validated, the strategy would be easily transferable to the diagnostic laboratory. Disclosures: No relevant conflicts of interest to declare.

8539 Background: Patients with DLBCL reveal striking heterogeneity in clinical outcomes. Plasma cell-free DNA (cfDNA) levels have been shown to be adversely prognostic. Both circulating tumor DNA (...