A high LDH to absolute lymphocyte count ratio in patients with DLBCL predicts for a poor intratumoral immune response and inferior survival

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 < 0.001) at 5-years. Patients with a high LAR had a strikingly different TME compared to patients with a low ratio. Low LAR was associated with a good-risk TME immune gene signature (p < 0.0001), including high CD8 and lower M2 macrophage infiltration. COO classification was not significantly different between high and low LAR patients. LAR was predictive of outcome independent of cell of origin and the international prognostic index (IPI). In particular, LAR discriminated patients with high IPI (3- 5), showing 5-year PFS and OS of 32% vs. 74% (p=0.0006), and 43% vs. 81% (p=0.0003). A combined nanoString based immune score and the LAR allowed better prediction of outcome than either prognosticator alone (p < 0.0001). Conclusions: The LAR reflects the TME within DLBCL, and is a strong predictor of outcome in DLBCL treated with conventional first-line therapy that is independent of and additive to the IPI. Further studies are required to determine if this easily applicable blood assay can determine patients that might benefit from immune checkpoint blockade

A high LDH to absolute lymphocyte count ratio in patients with DLBCL predicts for a poor intratumoral immune response and inferior survival

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 < 0.001) at 5-years. Patients with a high LAR had a strikingly different TME compared to patients with a low ratio. Low LAR was associated with a good-risk TME immune gene signature (p < 0.0001), including high CD8 and lower M2 macrophage infiltration. COO classification was not significantly different between high and low LAR patients. LAR was predictive of outcome independent of cell of origin and the international prognostic index (IPI). In particular, LAR discriminated patients with high IPI (3- 5), showing 5-year PFS and OS of 32% vs. 74% (p=0.0006), and 43% vs. 81% (p=0.0003). A combined nanoString based immune score and the LAR allowed better prediction of outcome than either prognosticator alone (p < 0.0001). Conclusions: The LAR reflects the TME within DLBCL, and is a strong predictor of outcome in DLBCL treated with conventional first-line therapy that is independent of and additive to the IPI. Further studies are required to determine if this easily applicable blood assay can determine patients that might benefit from immune checkpoint blockade

Non-parametric Heat Map Representation of Flow Cytometry Data: Identifying Cellular Changes Associated With Genetic Immunodeficiency Disorders

Genetic primary immunodeficiency diseases are increasingly recognized, with pathogenic mutations changing the composition of circulating leukocyte subsets measured by flow cytometry (FCM). Discerning changes in multiple subpopulations is challenging, and subtle trends might be missed if traditional reference ranges derived from a control population are applied. We developed an algorithm where centiles were allocated using non-parametric comparison to controls, generating multiparameter heat maps to simultaneously represent all leukocyte subpopulations for inspection of trends within a cohort or segregation with a putative genetic mutation. To illustrate this method, we analyzed patients with Primary Antibody Deficiency (PAD) and kindreds harboring mutations in TNFRSF13B (encoding TACI), CTLA4, and CARD11. In PAD, loss of switched memory B cells (B-SM) was readily demonstrated, but as a continuous, not dichotomous, variable. Expansion of CXCR5+/CD45RA- CD4+ T cells (X5-Th cells) was a prominent feature in PAD, particularly in TACI mutants, and patients with expansion in CD21-lo B cells or transitional B cells were readily apparent. We observed differences between unaffected and affected TACI mutants (increased B cells and CD8+ T-effector memory cells, loss of B-SM cells and non-classical monocytes), cellular signatures that distinguished CTLA4 haploinsufficiency itself (expansion of plasmablasts, activated CD4+ T cells, regulatory T cells, and X5-Th cells) from its clinical expression (B-cell depletion), and those that were associated with CARD11 gain-of-function mutation (decreased CD8+ T effector memory cells, B cells, CD21-lo B cells, B-SM cells, and NK cells). Co-efficients of variation exceeded 30% for 36/54 FCM parameters, but by comparing inter-assay variation with disease-related variation, we ranked each parameter in terms of laboratory precision vs. disease variability, identifying X5-Th cells (and derivatives), naïve, activated, and central memory CD8+ T cells, transitional B cells, memory and SM-B cells, plasmablasts, activated CD4 cells, and total T cells as the 10 most useful cellular parameters. Applying these to cluster analysis of our PAD cohort, we could detect subgroups with the potential to reflect underlying genotypes. Heat mapping of normalized FCM data reveals cellular trends missed by standard reference ranges, identifies changes associating with a phenotype or genotype, and could inform hypotheses regarding pathogenesis of genetic immunodeficiency.This study was supported by a National Health and Medical Research Council (NHMRC) of Australia Centre of Research Excellence (APP1079648)

Routine use of ancillary investigations in staging diffuse large B-cell lymphoma improves the International Prognostic Index (IPI)

<p>Abstract</p> <p>Background</p> <p>The International Prognostic Index (IPI) is used to determine prognosis in diffuse large B-cell lymphoma (DLBCL). One of the determinants of IPI is the stage of disease with bone marrow involvement being classified as stage IV. For the IPI, involvement on bone marrow is traditionally defined on the basis of histology with ancillary investigations used only in difficult cases to aid histological diagnosis. This study aimed to determine the effect of the routine use of flow cytometry, immunohistochemistry and molecular studies in bone marrow staging upon the IPI.</p> <p>Results</p> <p>Bone marrow trephines of 156 histologically proven DLBCL cases at initial diagnosis were assessed on routine histology, and immunohistochemistry using two T-cell markers (CD45RO and CD3), two B-cell markers (CD20 and CD79a) and kappa and lambda light chains. Raw flow cytometry data on all samples were reanalysed and reinterpreted blindly. DNA extracted from archived paraffin-embedded trephine biopsy samples was used for immunoglobulin heavy chain and light chain gene rearrangement analysis. Using immunophenotyping (flow cytometry and immunohistochemistry), 30 (19.2%) cases were upstaged to stage IV. A further 8 (5.1%) cases were upstaged using molecular studies. A change in IPI was noted in 18 cases (11.5%) on immunophenotyping alone, and 22 (14.1%) cases on immunophenotyping and molecular testing. Comparison of two revised IPI models, 1) using immunophenotyping alone, and 2) using immunophenotyping with molecular studies, was performed with baseline IPI using a Cox regression model. It showed that the revised IPI model using immunophenotyping provides the best differentiation between the IPI categories.</p> <p>Conclusion</p> <p>Improved bone marrow staging using flow cytometry and immunohistochemistry improves the predictive value of the IPI in patients with DLBCL and should be performed routinely in all cases.</p

Death from mantle cell lymphoma limits sequential therapy, particularly after first relapse: Patterns of care and outcomes in a series from Australia and the United Kingdom

Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma characterised by a heterogeneous clinical course. Patients can often receive sequential treatments, yet these typically yield diminishing periods of disease control, raising questions about optimal therapy sequencing. Novel agents, such as chimeric antigen receptor T-cell therapies and bispecific antibodies, show promise in relapsed MCL, but are often reserved for later treatment lines, which may underserve patients with aggressive disease phenotypes who die early in the treatment journey. To assess the problem of patient attrition from lymphoma-related death limiting sequential treatment, we performed a multicentre retrospective cohort analysis of 389 patients treated at Australian and UK centres over a 10-year period. Deaths from MCL increased after each treatment line, with 7%, 23% and 26% of patients dying from uncontrolled MCL after first, second and third lines respectively. Patients with older age at diagnosis and early relapse after induction therapy were at particular risk of death after second-line treatment. This limitation of sequential treatment by lymphoma-related death provides support for the trial of novel therapies in earlier treatment lines, particularly in high-risk patient populations