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Comprehensive clinical characteristics of Australian patients with classical Hodgkin Lymphoma (cHL) have not previously been systematically collected and described. We report real-world data of 498 eligible patients from the first five years of the Lymphoma and Related Diseases Registry (LaRDR), including baseline characteristics, histologic subtype and treatment patterns in first-line therapy. Patient demographics and distribution of histopathological subtypes of cHL are similar to reported international cohorts. Doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD) was the most common therapy for both early and advanced-stage disease, and 48% of patients with early-stage disease received radiotherapy. Treatment patterns are consistent with international guidelines. In comorbid patients ≥ 60 years of age with advanced-stage disease, there is greater variation in treatment. In patients with a recorded response, the objective response rate (ORR) was 96% in early-stage disease, and 88% in advanced-stage disease. Early progression-free survival data suggest Australian patients with cHL have good outcomes, similar to other international studies.

Here, we present a novel case of a patient with chronic lymphocytic leukemia (CLL) who received CTLA-4 and then PD-1 immune-checkpoint blockade (ICB) as treatment for concomitant metastatic melanoma. Whereas the metastatic melanoma was responsive to ICB, the CLL rapidly progressed (but responded to ICB cessation and ibrutinib). There were no new genetic mutational drivers to explain the altered clinical course. PD-1/PD-L1/PD-L2 and CTLA-4/CD80/CD86 expression was not increased in CLL B cells, CD8+ or CD4+ T-cell subsets, or monocytes. The patient’s CLL B cells demonstrated strikingly prolonged in vitro survival during PD-1 blockade, which was not observed in samples taken before or after ICB, or with other patients. To our knowledge, a discordant clinical course to ICB coupled with these biological features has not been reported in a patient with dual malignancies.

PDF file - 36K, Sensitivity and specificity of sCD163 and sTARC as biomarkers in cHL. (A-D) Receiver Operating Characteristic (ROC) plots demonstrate high sensitivity and specificity of (A) sCD163 pre-therapy cHL versus healthy participant; (B) sCD163 pre-therapy cHL versus CR six months post-therapy; (C) sTARC pre-therapy cHL versus healthy participants; (D) sTARC pre-therapy cHL versus CR six months post-therapy. AUC, area under the curve.

PDF file - 51K, Figure S2. Pre-therapy versus healthy controls absolute quantification of circulating miRNA.

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PDF file - 33K, Figure S3. Circulating miRNA are not significantly influenced by time from collection to processing.

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PDF file - 76K, Figure S1. Comparative quantification of circulating miRNA.

Purpose: To investigate the relationship between the intra-tumoral T-cell receptor (TCR) repertoire and the tumor microenvironment (TME) in de novo diffuse large B-cell lymphoma (DLBCL) and the impact of TCR on survival.Experimental Design: We performed high-throughput unbiased TCRβ sequencing on a population-based cohort of 92 patients with DLBCL treated with conventional (i.e., non-checkpoint blockade) frontline “R-CHOP” therapy. Key immune checkpoint genes within the TME were digitally quantified by nanoString. The primary endpoints were 4-year overall survival (OS) and progression-free survival (PFS).Results: The TCR repertoire within DLBCL nodes was abnormally narrow relative to non-diseased nodal tissues (P < 0.0001). In DLBCL, a highly dominant single T-cell clone was associated with inferior 4-year OS rate of 60.0% [95% confidence interval (CI), 31.7%–79.6%], compared with 79.8% in patients with a low dominant clone (95% CI, 66.7%–88.5%; P = 0.005). A highly dominant clone also predicted inferior 4-year PFS rate of 46.6% (95% CI, 22.5%–76.6%) versus 72.6% (95% CI, 58.8%–82.4%, P = 0.008) for a low dominant clone. In keeping, clonal expansions were most pronounced in the EBV+ DLBCL subtype that is known to express immunogenic viral antigens and is associated with particularly poor outcome. Increased T-cell diversity was associated with significantly elevated PD-1, PD-L1, and PD-L2 immune checkpoint molecules.Conclusions: Put together, these findings suggest that the TCR repertoire is a key determinant of the TME. Highly dominant T-cell clonal expansions within the TME are associated with poor outcome in DLBCL treated with conventional frontline therapy. Clin Cancer Res; 23(7); 1820–8. ©2016 AACR.

Individual results of TCR sequencing for all DLBCL and non-diseased nodal tissues.

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

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 Lymphoma is a malignancy of lymphocytes and lymphoid tissues comprising a heterogeneous group of diseases, with up to 80 entities now described. Lymphoma is the 6th most common cancer in Australia, affecting patients of all ages, with rising incidence rates. With the proliferation of efficacious novel agents, therapeutic strategies are increasingly diverse and survival is improving. There is a clear need for contemporary robust and detailed data on diagnostic, investigational and management strategies for this disease in Australia, New Zealand and worldwide, to inform and benchmark local and international standards of care. Clinical quality registries can provide these data, and support development of strategies to address variations in management, including serving as platforms for clinical trials and research. The Lymphoma and Related Diseases Registry (LaRDR) was developed to capture details of patient demographics, disease characteristics, and management throughout their disease course and therapy and to develop outcome benchmarks nationally and internationally for lymphoma. This report describes the aims, development and implementation of the LaRDR, as well as challenges addressed in the process.Methods The LaRDR was established in 2016 as a multicentre, collaborative project at sites across Australia with a secure online database which collects prospective data on patients with a new diagnosis of lymphoma or chronic lymphocytic leukaemia (CLL). LaRDR development required multidisciplinary participation including specialist haematology, information technology, and biostatistical support, as well as secure funding. Here we describe the database development, data entry, ethics approval process, registry governance and support for participating sites and the coordinating centre.Results To date more than 4,700 patients have been enrolled from 27 sites. Multiple challenges arose during the development, which we describe, along with approaches used to overcome them. Several confirmed international collaborations are now in place, and the registry is providing valuable data for clinicians, researchers, industry and government, including through presentations of results at major national and international conferences.Conclusion Challenges in establishing the LaRDR have been successfully overcome and the registry is now a valuable resource for lymphoma clinicians, researchers, health economists and others in Australia and globally.

Epstein-Barr virus–positive (EBV(+)) diffuse large B-cell lymphomas (DLBCLs) express high levels of programmed death ligand 1 (PD-L1) and PD-L2. MicroRNA (miR) regulation is an important mechanism for the fine-tuning of gene expression via 3′-untranslated region (3′UTR) targeting, and we have previously demonstrated strong EBV miR expression in EBV(+) DLBCL. Whereas the EBV latent membrane protein-1 (LMP1) is known to induce PD-L1/L2, a potential counterregulatory role of EBV miR in the fine-tuning of PD-L1/L2 expression remains to be established. To examine this, a novel in vitro model of EBV(+) DLBCL was developed, using the viral strain EBV WIL, which unlike common laboratory strains retains intact noncoding regions where several EBV miRs reside. This enabled interrogation of the relationship among EBV latency genes, cell of origin (COO), PD-L1, PD-L2, and EBV miRs. The model successfully recapitulated the full spectrum of B-cell differentiation, with 4 discrete COO phases: early and late germinal center B cells (GCBs) and early and late activated B cells (ABCs). Interestingly, PD-L1/L2 levels increased markedly during transition from late GCB to early ABC phase, after LMP1 upregulation. EBV miR–BamHI fragment H rightward open reading frame 1 (BHRF1)–2-5p clustered apart from other EBV miRs, rising during late GCB phase. Bioinformatic prediction, together with functional validation, confirmed EBV miR-BHRF1-2-5p bound to PD-L1 and PD-L2 3′UTRs to reduce PD-L1/L2 surface protein expression. Results indicate a novel mechanism by which EBV miR-BHRF1-2-5p plays a context-dependent counterregulatory role to fine-tune the expression of the LMP1-driven amplification of these inhibitory checkpoint ligands. Further identification of immune checkpoint–targeting miRs may enable potential novel RNA-based therapies to emerge.

Primary central nervous system lymphoma is a clinicopathological disease entity that accounts for 1% of all non-Hodgkin lymphoma (NHL). Advanced patient age, adverse disease biology and complexities of diagnosis and treatment render outcomes markedly inferior to systemic NHL. Despite this, an increasing evidence base, including limited randomised controlled clinical trial data, is informing optimal therapeutic strategies with methotrexate-based induction chemotherapy schedules and intensified consolidation in selected patients. This practice statement represents an evidence-based review of the literature and has been devised to assist healthcare professionals in the diagnosis and management of this disease.

Hodgkin lymphoma (HL) comprises two distinct disease entities based on clinical, morphologic and genotypic characteristics. Relative to classical Hodgkin Lymphoma (cHL), nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is rare, and its tumor microenvironment (TME) is very poorly characterized. With the exception of rituximab, there are no targeted treatments nor advances in the treatment of NLPHL for decades. Unlike cHL, the utility of immune-checkpoint blockade (ICB) has not been evaluated in NLPHL. Diagnostic samples were collected from 49 NLPHL patients from 4 Australian centres and compared with stage-matched cHL patients (and with normal lymph nodes). An integrative transcriptomic, proteomic, T-cell clonal and functional analysis was performed to enable a comparison of the composition of the TME and its contribution to immune-evasion in NLPHL with cHL. 730 cancer-immune related genes were digitally quantified. Relative to cHL, gene set enrichment analysis identified T-cell receptor (TCR) and immune-checkpoint signaling pathway dysregulation in NLPHL. Most striking was prominent differential expression of the immune-transcriptome, particularly enrichment for programmed cell death-1 (PD-1) and T-cell Ig and ITIM domain (TIGIT) in NLPHL versus cHL. These were also over expressed compared to normal lymph nodes. Consistent with this, there was also upregulation of numerous T-cell markers (CD247, CD3D, GZMK, CD28, EOMES) in NLPHL. In contrast, immunosuppressive macrophage (CD163, CD36, CD68, COLEC12, MARCO) and regulatory T-cell genes (FOXP3) were higher in cHL. Importantly, PD-L1 and CD155 (respective ligands for PD-1 and TIGIT) were expressed at the surface of NLPHL and cHL malignant B-cells. Multispectral immunofluorescent microscopy showed intratumoral TIGIT+CD4+ and PD-1+CD4+ T-cells were markedly increased in NLPHL versus cHL and localised within NLPHL follicles. Expanded populations of intratumoral CD4+ T-cell clones were predominantly PD-1+ and frequently also TIGIT+. Multi-parameter flow cytometry and dimensionality reduction was used to establish the distribution of immune checkpoints within circulating T-cell subsets. PD-1+TIGIT+CD4+ T-cells were raised in circulating Treg, TH1 and TH2 subsets in NLPHL versus cHL, and PD-1+TIGIT+ TH2 T-cells displayed raised levels of the exhaustion marker EOMES, collectively indicating systemic T-cell dysfunction. To functionally demonstrate the utility of ICB to stimulate T-cells, an assay using cHL and/or NLPHL cell-lines co-cultured with a genetically engineered effector T-cell-line was developed. This showed that TIGIT/PD-1 dual-blockade was more effective than mono-blockade in inducing NLPHL and cHL tumor-directed CD4+ T-cell activation. Overall, our results indicate that immune-evasion mechanisms in NLPHL are distinct to those operative in cHL, with markedly greater T-cell, PD-1 and TIGIT dysregulation. PD-1 and/or TIGIT blockade warrants evaluation in NLPHL. Citation Format: Jay Gunawardana, Soi C. Law, Muhammed B. Sabdia, Karolina Bednarska, Sandra Brosda, Andreea Zaharia, Hennes Tsang, Lilia M. de Long, Melinda Burgess, Dipti Talaulikar, Justina N. Lee, Emily Jude, Eliza A. Hawkes, Sanjiv Jain, Karthik Nath, Clare Gould, Fiona Swain, Joshua W. D. Tobin, Colm Keane, Simone Birch, Mohamed Shanavas, Cameron Snell, Maher K. Gandhi. The immune checkpoints TIGIT and PD-1 are markedly upregulated in NLPHL compared to classical Hodgkin Lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A17.

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.

Current HLA-typing methods are typically designed to provide exquisitely-detailed identification of multiple HLA-alleles to satisfy the requirements for organ and bone marrow transplantation or genetic studies. Many human immunological studies, on the other hand, focus around only a small number of HLA alleles that are abundant or of relevance to specific diseases. Consequently, for such studies, many HLA typing approaches are not cost-effective and are potentially complicated, slow and not easily performed in-house. Work-flow would be streamlined by a simple, inexpensive and rapid typing method able to be performed in-house. We outline a straightforward approach that provides appropriate data for much immunological research. In a predominantly Caucasian population, flow cytometry using anti-HLA-A2, -B8 and -B7 antibodies consistently and accurately screened for samples carrying the highly-abundant HLA class I alleles HLA-A*02:01, -B*08:01 and -B*07:02 that form the focus of immunological studies. Next, we describe a straightforward and simple strategy for design and use of allele-specific PCR primers to identify, at high-resolution, alleles of interest. When combined with a simple gDNA extraction technique this provides reliable, simple and inexpensive in-house HLA typing demonstrated here for highly-abundant HLA class I alleles.

7516 Background: PD-1 inhibitors are a standard of care in pts with R/R cHL but new approaches are still needed to deepen and lengthen responses. Dual blockade of PD-1 and LAG-3 has demonstrated antitumor activity in preclinical models. The multicohort phase 1/2 MK-4280-003 study (NCT03598608) evaluated the safety and efficacy of favezelimab (MK-4280), a humanized IgG4 LAG-3 inhibitor, plus pembrolizumab (pembro; a PD-1 inhibitor) in pts with R/R hematologic malignancies. This analysis focused on anti–PD-1–naïve pts with R/R cHL (cohort 1). Methods: This study included a safety lead-in phase (part 1) to determine the recommended phase 2 dose (RP2D) followed by a dose-expansion phase (part 2). Eligible pts in cohort 1 must have R/R cHL after autologous stem cell transplantation (ASCT) or be ineligible for ASCT and have had no prior anti–PD-1 therapy. In part 1, patients from all cohorts received pembro IV 200 mg Q3W and favezelimab IV 200 mg or 800 mg Q3W. Dose escalation followed the mTPI design. In part 2, pts received pembro + favezelimab at the established RP2D for up to 35 cycles. Primary end point was safety. Secondary end point was ORR. DOR, PFS, and OS were exploratory end points. Results: Only 1 dose-limiting toxicity (DLT; autoimmune hepatitis [grade 4]) was identified among the first 6 pts from all cohorts in part 1 at the favezelimab 200 mg dose; thus, the dose was escalated to 800 mg. No DLTs were observed in the 15 additional pts treated at the 800 mg dose. The RP2D for the combination was defined as 800 mg Q3W + pembro 200 mg Q3W. In cohort 1, 30 pts were enrolled; median age was 40 years, 53% had ECOG PS 0, and 80% had ≤3 prior lines of therapy. After a median follow-up of 13.5 mo, ORR for cohort 1 was 73% (95% CI, 54-88; CR, 7 pts [23%]; PR, 15 pts [50%]). 28 of 30 pts (93%) had reduction from baseline in target lesions. Median DOR was not reached (NR; 95% CI, 0+ to 23+ mo); 6 pts (51%) had response ≥12 mo. Median PFS was 19 mo (95% CI, 8-NR); 12-mo PFS rate was 57%. Median OS was NR (95% CI, NR-NR); 12-mo OS rate was 94%. As of the database cutoff (Nov 1, 2021), 9 of 30 pts had discontinued (3 AEs; 6 PD). Treatment-related AEs (TRAE) occurred in 26 pts (87%); most common (≥10%) were hypothyroidism (27%), fatigue (20%), infusion-related reactions (20%), headache (17%), and arthralgia, hyperthyroidism, myalgia, and nausea (10% each); grade 3 or 4 TRAEs occurred in 6 pts (20%). 10% of pts discontinued due to TRAEs. No treatment-related deaths occurred. Conclusions: Favezelimab 800 mg + pembrolizumab 200 mg Q3W demonstrated a tolerable safety profile and effective antitumor activity in pts with anti–PD-1–naïve R/R cHL. Further studies will be useful to compare its activity to that of pembrolizumab alone. Clinical trial information: NCT03598608.

7545 Background: PD-1 inhibitors are a standard of care for R/R cHL but optimal therapy after anti–PD-1 therapy failure is yet to be defined. LAG-3/PD-1 coblockade has demonstrated antitumor activity in preclinical models. This multicohort phase 1/2 study (NCT03598608) evaluated the safety and efficacy of favezelimab (MK-4280), a humanized IgG4 LAG-3 inhibitor, plus the PD-1 inhibitor pembrolizumab (pembro) in pts with R/R hematologic malignancies. Cohort 2 focused on pts with R/R cHL refractory to anti–PD-1 therapy. Methods: This study included a safety lead-in phase (part 1) to determine recommended phase 2 dose (RP2D) followed by a dose-expansion phase (part 2). Eligible pts in cohort 2 had R/R cHL, relapsed after or were ineligible for autologous stem cell transplantation (ASCT), and progressed after ≥2 doses of anti–PD-1 therapy (within 12 weeks of last dose). In part 1, pts from all cohorts received pembro IV 200 mg Q3W and favezelimab IV 200 mg or 800 mg Q3W. Dose-finding based on occurrence of dose-limiting toxicities (DLT) was determined using an mTPI design. In part 2, pts received pembro + favezelimab at the established RP2D for up to 35 cycles. Primary end point was safety. Secondary end point was ORR. DOR, PFS, and OS were exploratory. Results: Only 1 DLT (autoimmune hepatitis [grade 4]) was observed among the first 6 pts from all cohorts in part 1 at the favezelimab 200 mg dose; thus, the dose was escalated to 800 mg. No DLTs were observed in 15 additional pts at the 800 mg dose. Favezelimab RP2D was defined as 800 mg Q3W + pembro 200 mg Q3W. In cohort 2, 33 pts were enrolled; median age was 37 yrs, 64% had ECOG PS 0, and 94% had ≥4 prior lines of therapy. After a median follow-up of 16.5 mo, ORR for pts receiving favezelimab 800 mg (n = 29) was 31% (95% CI, 15-51; CR, 2 [7%]; PR, 7 [24%]); 66% of responders had an anti–PD-1–based regimen as most recent line of therapy at study entry. 23 of 29 pts (79%) had reduction from baseline in target lesions. Median DOR for pts who received favezelimab 800 mg was not reached (NR; 95% CI, 0+ to 14+ mo). For all pts in cohort 2, median PFS was 9 mo (95% CI, 5-15); 12-mo PFS rate was 39%. Median OS was 26 mo (95% CI, 26-NR); 12-mo OS rate was 91%. At database cutoff (Nov 1, 2021), 20 pts discontinued treatment (7 AEs, 11 PD/clinical progression, 2, withdrawal/physician decision). TRAEs occurred in 28 pts (85%); most common ( > 10%) were hypothyroidism (18%), nausea and fatigue (15% each), and arthralgia and diarrhea (12% each); grade 3 or 4 TRAEs occurred in 6 pts (18%); 18% discontinued treatment due to TRAEs. No treatment-related deaths occurred. Conclusions: Favezelimab 800 mg + pembro 200 mg Q3W showed a tolerable safety profile and effective antitumor activity in heavily pretreated pts with R/R cHL whose disease had progressed after anti–PD-1 therapy, suggesting that the combination may reinduce a response in these pts. Clinical trial information: NCT03598608.

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?

Blockade of the PD-1 axis has modest efficacy in diffuse large B-cell lymphoma (DLBCL), but data regarding LAG3 are sparse. The impact of LAG3 digital gene expression was tested in 309 patients with DLBCL treated with standard chemoimmunotherapy. Cellular distribution of LAG3 protein was determined by immunohistochemistry and flow cytometry. In tumor-infiltrating lymphocytes (TILs), LAG3 expression was highest on CD4+ regulatory T cells (Tregs) and was also highly expressed on CD8+ T cells compared with CD4+ non-Tregs (both P = .008). LAG3high TILs were enriched in PD-1 and TIM-3. LAG3 was also expressed on a proportion of malignant B cells, and these patients had significantly higher LAG3 messenger RNA in their biopsies (P = .03). LAG3high gene expression was associated with inferior survival in discovery/validation cohorts, independent of cell of origin and the international prognostic index. Patients who were PD-L1high were fivefold more likely to be LAG3high (P < .0001). Patients who were LAG3high/PD-L1high had an inferior progression-free survival (P = .011) and overall survival (P = .005) compared with patients who were LAG3low/PD-L1high. Digital spatial protein analysis confirms LAG3 expression on T cells and, surprisingly, tumor-associated macrophages (TAMs) at higher levels than found on CD20+ B cells in the tumor microenvironment. LAG3 is frequently expressed on CD4+ Tregs and CD8+ TILs, typically with other immune checkpoints, and is also present in a proportion of malignant B cells in DLBCL and in areas enriched for TAMs. LAG3high expression is associated with poor outcome independent of conventional prognosticators.

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.