Systemic diffuse large B-cell lymphoma involving the central nervous system have high rates of defective antigen presentation and immune surveillance.

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Observations from a national sample exchange program for molecular haematology testing.

External quality assessment programs (EQAP) for molecular haematology generally only assess the analytical phase of laboratory testing or provide limited evaluation of post-analytical components. We incorporated comprehensive post-analytical evaluation into an existing national inter-laboratory sample exchange program for molecular haematology due to the increasing complexity of diagnostic molecular testing and interpretation. We report key findings from four years of longitudinal data using this approach. Eighteen participating laboratories enrolled in an annual reciprocal sample exchange program from 2019-2022, which covered conventional and next-generation sequencing (NGS) assays. Participants submitted results on their laboratory information system-generated reports which then underwent central review. Reports were assessed according to consensus values and relevant national and international reporting standards and guidelines. A total of 680 reports were received. Laboratories had high concordance in the analytical phase of testing, with incorrect variant detection observed in a total of six of 680 (0.9%) reports. In contrast, post-analytical concordance was much lower, with at least one discordance observed in 28.9-57.6% of all conventional reports and 33.3-100% NGS reports. The most frequent post-analytical discordances were: (1) not including key technical information on reports (total 41.9% conventional, 47.2% NGS); (2) not using standard gene and variant nomenclature (total 28.2% conventional, 25.6% NGS). NGS reports also demonstrated discrepancies in variant classification (total 20.4%) and interpretation (total 10.2%). The rate of discrepancies generally improved year-on-year. Inter-laboratory concordance for molecular haematology testing is high in the analytical phase, however opportunities exist for improvement in the post-analytical phase. Given that result interpretation is crucial for clinical decision-making and that molecular testing is a complex and evolving field, we suggest that EQAPs should comprehensively evaluate both analytical and post-analytical components of laboratory performance in order to harmonise reporting and to support the accurate interpretation of molecular haematology tests.

Gene Expression Signatures for the Accurate Diagnosis of Peripheral T-Cell Lymphoma Entities in the Routine Clinical Practice.

PURPOSE: Peripheral T-cell lymphoma (PTCL) includes heterogeneous clinicopathologic entities with numerous diagnostic and treatment challenges. We previously defined robust transcriptomic signatures that distinguish common PTCL entities and identified two novel biologic and prognostic PTCL-not otherwise specified subtypes (PTCL-TBX21 and PTCL-GATA3). We aimed to consolidate a gene expression-based subclassification using formalin-fixed, paraffin-embedded (FFPE) tissues to improve the accuracy and precision in PTCL diagnosis. MATERIALS AND METHODS: We assembled a well-characterized PTCL training cohort (n = 105) with gene expression profiling data to derive a diagnostic signature using fresh-frozen tissue on the HG-U133plus2.0 platform (Affymetrix, Inc, Santa Clara, CA) subsequently validated using matched FFPE tissues in a digital gene expression profiling platform (nCounter, NanoString Technologies, Inc, Seattle, WA). Statistical filtering approaches were applied to refine the transcriptomic signatures and then validated in another PTCL cohort (n = 140) with rigorous pathology review and ancillary assays. RESULTS: In the training cohort, the refined transcriptomic classifier in FFPE tissues showed high sensitivity (> 80%), specificity (> 95%), and accuracy (> 94%) for PTCL subclassification compared with the fresh-frozen-derived diagnostic model and showed high reproducibility between three independent laboratories. In the validation cohort, the transcriptional classifier matched the pathology diagnosis rendered by three expert hematopathologists in 85% (n = 119) of the cases, showed borderline association with the molecular signatures in 6% (n = 8), and disagreed in 8% (n = 11). The classifier improved the pathology diagnosis in two cases, validated by clinical findings. Of the 11 cases with disagreements, four had a molecular classification that may provide an improvement over pathology diagnosis on the basis of overall transcriptomic and morphological features. The molecular subclassification provided a comprehensive molecular characterization of PTCL subtypes, including viral etiologic factors and translocation partners. CONCLUSION: We developed a novel transcriptomic approach for PTCL subclassification that facilitates translation into clinical practice with higher precision and uniformity than conventional pathology diagnosis.

Characterisation of immune checkpoints in Richter syndrome identifies LAG3 as a potential therapeutic target.

Richter syndrome (RS), an aggressive lymphoma occurring in the context of chronic lymphocytic leukaemia/small lymphocytic lymphoma, is associated with poor prognosis when treated with conventional immunochemotherapy, therefore, improved treatments are required. Immune checkpoint blockade has shown efficacy in some B-cell malignancies and modest responses in early clinical trials for RS. We investigated the immune checkpoint profile of RS as a basis to inform rational therapeutic investigations in RS. Formalin-fixed, paraffin-embedded biopsies of RS (n = 19), de novo diffuse large B-cell lymphoma (DLBCL; n = 58), transformed indolent lymphomas (follicular [tFL], n = 16; marginal zone [tMZL], n = 24) and non-transformed small lymphocytic lymphoma (SLL; n = 15) underwent gene expression profiling using the NanoString Human Immunology panel. Copy number assessment was performed using next-generation sequencing. Immunohistochemistry (IHC) for LAG3 and PD-1 was performed. LAG3 gene expression was higher in RS compared to DLBCL (P = 0·0002, log2FC 1·96), tFL (P < 0·0001, log2FC 2·61), tMZL (P = 0·0004, log2FC 1·79) and SLL (P = 0·0057, log2FC 1·45). LAG3 gene expression correlated with the gene expression of human leukocyte antigen Class I and II, and related immune genes and immune checkpoints. IHC revealed LAG3 protein expression on both malignant RS cells and tumour-infiltrating lymphocytes. Our findings support the investigation of LAG3 inhibition to enhance anti-tumour responses in RS.

LAG3: a novel immune checkpoint expressed by multiple lymphocyte subsets in diffuse large B-cell lymphoma.

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.

COO and MYC/BCL2 status do not predict outcome among patients with stage I/II DLBCL: a retrospective multicenter study.

In advanced-stage diffuse large B-cell lymphoma (DLBCL), the presence of an activated B-cell phenotype or a non-germinal center (GCB) phenotype, coexpression of MYC and BCL2 by immunohistochemistry, and the cooccurrence of MYC and BCL2 or BCL6 rearrangements are associated with inferior outcomes. It is unclear whether these variables remain prognostic in stage I/II patients. In this retrospective study, we evaluated the prognostic impact of cell of origin (COO), as well as dual-expressor (DE) status and molecular double-hit (DH) status, in stage I/II DLBCL by positron emission tomography with computed tomography (PET-CT). A total of 211 patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone)-like regimens, with or without radiotherapy, was included. The median follow-up in the entire cohort was 4 years (range, 0.4-9.4), with estimated 4-year progression-free survival (PFS) and overall survival (OS) rates of 85% (95% confidence interval [CI], 79-89) and 88% (95% CI, 83-92), respectively. By univariable analysis, DE (PFS: hazard ratio [HR], 1.27; 95% CI, 0.58-2.81, P = .55 and OS: HR, 1.40; 95% CI, 0.60-3.30; P = .44), DH (PFS: HR, 1.21; 95% CI, 0.27-5.31; P = .80 and OS: HR, 0.61; 95% CI, 0.08-4.73; P = .64), and non-GCB status (PFS: HR, 1.59; 95% CI, 0.83-3.03; P = .16 and OS: HR, 1.80; 95% CI, 0.89-3.67; P = .10) were associated with poorer outcomes. In patients with PET-CT-defined stage I/II DLBCL treated with R-CHOP-like therapy, with or without radiation, COO and DE and DH status were not significantly associated with inferior PFS or OS.

The tumour microenvironment is immuno-tolerogenic and a principal determinant of patient outcome in EBV-positive diffuse large B-cell lymphoma.

OBJECTIVE: Epstein-Barr virus-positive diffuse large B-cell lymphoma (EBV-pos DLBCL) is a recently identified entity. Data regarding outcome to frontline immuno-chemotherapy are conflicting. Although the prognostic impact of the tumour microenvironment (TME) in EBV-neg DLBCL is well-established, it remains untested whether the TME influences survival in EBV-pos DLBCL. There are no data with new digital gene expression technologies that simultaneously interrogate the virus, B cells and the tumour microenvironment (TME). METHODS: We used the NanoString™ platform in a population-based cohort of 433 patients to establish if the technology could detect EBV in the tumour biopsies and to investigate the influence that EBV has on the complex tumour microenvironment of DLBCL. RESULTS: Incidence of EBV-pos DLBCL was 6.9% with 5-year survival of 65% vs 82% in EBV-neg DLBCL (P = 0.018). EBV-pos tissues had similar expression of T-cell genes compared to EBV-neg DLBCL but higher levels of the antigen-presenting molecule B2M. This was countered by elevated PD-L1, PD-L2, LAG3 and TIM3 immune checkpoints and a higher CD163/CD68 "M2" macrophage score. CONCLUSION: In EBV-pos DLBCL, the TME is immuno-tolerogenic and may explain the poor outcomes seen in this subtype of DLBCL.

Circulating cell-free miR-494 and miR-21 are disease response biomarkers associated with interim-positron emission tomography response in patients with diffuse large B-cell lymphoma.

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.

Immune evasion via PD-1/PD-L1 on NK cells and monocyte/macrophages is more prominent in Hodgkin lymphoma than DLBCL.

Much focus has been on the interaction of programmed cell death ligand 1 (PD-L1) on malignant B cells with programmed cell death 1 (PD-1) on effector T cells in inhibiting antilymphoma immunity. We sought to establish the contribution of natural killer (NK) cells and inhibitory CD163+ monocytes/macrophages in Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma (DLBCL). Levels of PD-1 on NK cells were elevated in cHL relative to DLBCL. Notably, CD3-CD56hiCD16-ve NK cells had substantially higher PD-1 expression relative to CD3-CD56dimCD16+ cells and were expanded in blood and tissue, more marked in patients with cHL than patients with DLBCL. There was also a raised population of PD-L1-expressing CD163+ monocytes that was more marked in patients with cHL compared with patients with DLBCL. The phenotype of NK cells and monocytes reverted back to normal once therapy (ABVD [doxorubicin 25 mg/m2, bleomycin 10 000 IU/m2, vinblastine 6 mg/m2, dacarbazine 375 mg/m2, all given days 1 and 15, repeated every 28 days] or R-CHOP [rituximab 375 mg/m2, cyclophosphamide 750 mg/m2 IV, doxorubicin 50 mg/m2 IV, vincristine 1.4 mg/m2 (2 mg maximum) IV, prednisone 100 mg/day by mouth days 1-5, pegfilgrastim 6 mg subcutaneously day 4, on a 14-day cycle]) had commenced. Tumor-associated macrophages (TAMs) expressed high levels of PD-L1/PD-L2 within diseased lymph nodes. Consistent with this, CD163/PD-L1/PD-L2 gene expression was also elevated in cHL relative to DLBCL tissues. An in vitro functional model of TAM-like monocytes suppressed activation of PD-1hi NK cells, which was reversed by PD-1 blockade. In line with these findings, depletion of circulating monocytes from the blood of pretherapy patients with cHL and patients with DLBCL enhanced CD3-CD56hiCD16-ve NK-cell activation. We describe a hitherto unrecognized immune evasion strategy mediated via skewing toward an exhausted PD-1-enriched CD3-CD56hiCD16-ve NK-cell phenotype. In addition to direct inhibition of NK cells by the malignant B cell, suppression of NK cells can occur indirectly by PD-L1/PD-L2-expressing TAMs. The mechanism is more prominent in cHL than DLBCL, which may contribute to the clinical sensitivity of cHL to PD-1 blockade.

The T-cell Receptor Repertoire Influences the Tumor Microenvironment and Is Associated with Survival in Aggressive B-cell Lymphoma.

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 TCRß 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.

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

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

Predictors of poor outcome and benefits from antibiotics in children with acute otitis media: pragmatic randomised trial.

OBJECTIVES: To identify which children with acute otitis media are at risk of poor outcome and to assess benefit from antibiotics in these children. DESIGN: Secondary analysis of randomised controlled trial cohort. SETTING: Primary care. PARTICIPANTS: 315 children aged 6 months to 10 years. INTERVENTION: Immediate or delayed (taken after 72 hours if necessary) antibiotics. MAIN OUTCOME MEASURE: Predictors of short term outcome: an episode of distress or night disturbance three days after child saw doctor. RESULTS: Distress by day three was more likely in children with high temperature (adjusted odds ratio 4.5, 95% confidence interval 2.3 to 9.0), vomiting (2.6,1.3 to 5.0), and cough (2.0, 1.1 to 3.8) on day one. Night disturbance by day three was more likely with high temperature 2.4 (1.2 to 4.8), vomiting (2.1,1.1 to 4.0), cough (2.3,1.3 to 4.2), and ear discharge (2.1, 1.2 to 3.9). Among the children with high temperature or vomiting, distress by day three was less likely with immediate antibiotics (32% for immediate v 53% for delayed, chi2=4.0; P=0.045, number needed to treat 5) as was night disturbance (26% v 59%, chi2=9.3; P=0.002; number needed to treat 3). In children without higher temperature or vomiting, immediate antibiotics made little difference to distress by day three (15% v 19%, chi2=0.74; P=0.39) or night disturbance (20% v 27%, chi2=1.6; P=0.20). Addition of cough did not significantly improve prediction of benefit. CONCLUSION: In children with otitis media but without fever and vomiting antibiotic treatment has little benefit and a poor outcome is unlikely.