Björn Engelbrekt Wahlin

CD8+ T-Cell Content in Diagnostic Lymph Nodes Measured by Flow Cytometry Is a Predictor of Survival in Follicular Lymphoma

Purpose: Follicular lymphoma is a heterogeneous disease with variable prognosis and clinical course. We hypothesized that the presence of nonmalignant T cells in the microenvironment of the tumor may affect the outcome. Experimental Design: Using flow cytometry, we evaluated the T-cell subsets in the lymph node microenvironment of follicular lymphoma. All patients in South Stockholm County with indolent follicular lymphoma and with flow cytometry done on a diagnostic lymph node between 1994 and 2004 were included (N = 139). Diagnosis and grade (1, 2, and 3a) were confirmed by re-review. Flow cytometry results were reanalyzed. Lymphocyte subsets, the Follicular Lymphoma International Prognostic Index, grade, and clinical characteristics were evaluated in univariable and multivariable Cox analysis with respect to overall survival (OS) and disease-specific survival (DSS). Results: Higher CD8+ T-cell levels correlated with longer OS and DSS, independently of the Follicular Lymphoma International Prognostic Index (OS, P = 0.017; DSS, P = 0.020) and independently of all other prognostic factors (OS, P = 0.001; DSS, P = 0.004). Median OS was not reached for patients in the upper quarter of CD8+ T-cell levels (>8.6%), 10.4 years for patients in the middle half (4.2-8.6%), and 6.0 years for patients in the lower quarter (<4.2%). Furthermore, patients who had not required treatment within 6 months from diagnosis had more CD8+ T cells (P = 0.011). Conclusions: Higher levels of CD8+ T cells predict a better prognosis, and these data support an important role for nonmalignant immune cells in the biology of follicular lymphoma. Evaluating the CD8+ T cells by flow cytometry at diagnosis may provide prognostic information.

A Unifying Microenvironment Model in Follicular Lymphoma: Outcome Is Predicted by Programmed Death-1–Positive, Regulatory, Cytotoxic, and Helper T Cells and Macrophages

Purpose: The microenvironment influences outcome in follicular lymphoma. Our hypothesis was that several immune cell subsets are important for disease outcome and their individual prognostic importance should be demonstrable in the same analysis and in competition with clinical factors. Experimental Design: Seventy follicular lymphoma patients with extreme clinical outcome (“poor” and “good” cases) were selected in a population-based cohort of 197. None of the 37 good-outcome patients died from lymphoma, whereas all the 33 poor-outcome patients succumbed in ≤5 years. Furthermore, the good-outcome patients were followed for a long time and needed no or little treatment. A tissue microarray was constructed from diagnostic, pretreatment biopsies. Cellular subsets were quantified after immunostaining, using computerized image analysis, separating cells inside and outside the follicles (follicular and interfollicular compartments). Flow cytometry data from the same samples were also used. Results: Independently of the Follicular Lymphoma International Prognostic Index, CD4+ cells were associated with poor outcome and programmed death-1–positive and CD8+ cells were associated with good outcome. The prognostic values of CD4+ and programmed death-1–positive cells were accentuated when they were follicular and that of CD8+ cells were accentuated when they were interfollicular. Follicular FOXP3+ cells were associated with good outcome and interfollicular CD68+ cells were associated with poor outcome. Additionally, high CD4/CD8 and CD4 follicular/interfollicular ratios correlated with poor outcome. Conclusion: There are many important immune cell subsets in the microenvironment of follicular lymphoma. Each of these is independently associated with outcome. This is the first study showing the effect of the balance of the entire microenvironment, not only of individual subsets. Clin Cancer Res; 16(2); 637–50.

Clinical significance of the WHO grades of follicular lymphoma in a population-based cohort of 505 patients with long follow-up times.

The prognostic value of grading follicular lymphoma has been debated since the 1980s. There is consensus that World Health Organization (WHO) grades 1 and 2 are indolent, but not whether grades 3A or 3B are aggressive. We retrospectively reviewed the follicular lymphoma diagnoses according to the 2008 WHO classification in all diagnostic specimens from a population‐based cohort of 505 patients with a median follow‐up time of 10·0 years (range, 4·6–16·0). After excluding 43 patients with concomitant diffuse large B‐cell lymphoma, 345 remained with grade 1–2, 94 with grade 3A, and 23 with grade 3B follicular lymphoma. Grades 1–2 and 3A seemed equally indolent, with indistinguishable clinical courses, even in patients receiving anthracyclines. Compared with grades 1–3A and independently of clinical factors, grade 3B correlated with higher mortality (P = 0·008), but outcome was improved after upfront anthracycline‐containing therapy (P = 0·015). In contrast to grade 1–3A patients, grade 3B patients experienced no relapses or deaths beyond 5 years of follow‐up. Furthermore, patients with grade 3B were predominantly male and seldom presented with bone‐marrow involvement. We conclude that follicular lymphoma grade 1–3A is indolent and incurable with conventional therapy. Grade 3B appears to be an aggressive but curable disease.

The reliability of immunohistochemical analysis of the tumor microenvironment in follicular lymphoma: a validation study from the Lunenburg Lymphoma Biomarker Consortium

The cellular microenvironment in follicular lymphoma is of biological and clinical importance. Studies on the clinical significance of non-malignant cell populations have generated conflicting results, which may partly be influenced by poor reproducibility in immunohistochemical marker quantification. In this study, the reproducibility of manual scoring and automated microscopy based on a tissue microarray of 25 follicular lymphomas as compared to flow cytometry is evaluated. The agreement between manual scoring and flow cytometry was moderate for CD3, low for CD4, and moderate to high for CD8, with some laboratories scoring closer to the flow cytometry results. Agreement in manual quantification across the 7 laboratories was low to moderate for CD3, CD4, CD8 and FOXP3 frequencies, moderate for CD21, low for MIB1 and CD68, and high for CD10. Manual scoring of the architectural distribution resulted in moderate agreement for CD3, CD4 and CD8, and low agreement for FOXP3 and CD68. Comparing manual scoring to automated microscopy demonstrated that manual scoring increased the variability in the low and high frequency interval with some laboratories showing a better agreement with automated scores. Manual scoring reliably identified rare architectural patterns of T-cell infiltrates. Automated microscopy analyses for T-cell markers by two different instruments were highly reproducible and provided acceptable agreement with flow cytometry. These validation results provide explanations for the heterogeneous findings on the prognostic value of the microenvironment in follicular lymphoma. We recommend a more objective measurement, such as computer-assisted scoring, in future studies of the prognostic impact of microenvironment in follicular lymphoma patients.

T Cells in Tumors and Blood Predict Outcome in Follicular Lymphoma Treated with Rituximab

Purpose: T cells influence outcome in follicular lymphoma, but their contributions seem to be modified by therapy. Their impact in patients receiving rituximab without chemotherapy is unknown. Experimental Design: Using flow cytometry, we evaluated the T cells in tumors and/or blood in a total of 250 follicular lymphoma patients included in two Nordic Lymphoma Group randomized trials that compared single rituximab with IFN-α2a–rituximab combinations. Results: In univariate analysis, higher levels of CD3+, CD4+, and CD8+ T cells in both tumors and blood correlated with superior treatment responses, and in multivariate analysis, tumor-CD3+ (P = 0.011) and blood-CD4+ (P = 0.029) cells were independent. CD4+ cells were favorable regardless of treatment arm, but CD8+ cells were favorable only in patients treated with single rituximab, because IFN-α2a improved responses especially in patients with low CD8+ cell levels. Higher levels of blood-CD3+ (P = 0.003) and blood-CD4+ (P = 0.046) cells predicted longer overall survival, and higher levels of blood-CD8+ cells longer times to next treatment (P = 0.046). Conclusions: We conclude that therapeutic effects of rituximab are augmented by tumor-associated T cells for rapid responses and by systemic T cells for sustained responses. CD4+ and CD8+ cells are both favorable in patients treated with rituximab. IFN-α2a abrogates the negative impact of few CD8+ cells. Clin Cancer Res; 17(12); 4136–44. ©2011 AACR.

Follicular lymphoma in Sweden: nationwide improved survival in the rituximab era, particularly in elderly women - a Swedish Lymphoma Registry study.

Treatment for follicular lymphoma (FL) improved with rituximab. In Sweden, first-line rituximab was gradually introduced between 2003 and 2007, with regional differences. The first national guidelines for FL were published in November 2007, recommending rituximab in first-line therapy. Using the population-based Swedish Lymphoma Registry, 2641 patients diagnosed with FL from 2000 to 2010 were identified and characterized by year and region of diagnosis, age (median, 65 years), gender (50% men), first-line therapy and clinical risk factors. Overall and relative survivals were estimated by calendar periods (2000–2002, 2003–2007 and 2008–2010) and region of diagnosis. With each period, first-line rituximab use and survival increased. Survival was superior in regions where rituximab was quickly adopted and inferior where slowly adopted. These differences were independent in multivariable analyses. Ten-year relative survival for patients diagnosed 2003–2010 was 92%, 83%, 78% and 64% in the age groups 18–49, 50–59, 60–69 and ⩾70, respectively. With increasing rituximab use, male sex emerged as an adverse factor. Survival improved in all patient categories, particularly in elderly women. The introduction and the establishment of rituximab have led to a nationwide improvement in FL survival. However, rituximab might be inadequately dosed in younger women and men of all ages.

The tumour microenvironment influences survival and time to transformation in follicular lymphoma in the rituximab era

The tumour microenvironment influences outcome in patients with follicular lymphoma (FL), but its impact on transformation is less studied. We investigated the prognostic significance of the tumour microenvironment on transformation and survival in FL patients treated in the rituximab era. We examined diagnostic and transformed biopsies from 52 FL patients using antibodies against CD3, CD4, CD8, CD21 (CR2), CD57 (B3GAT1), CD68, FOXP3, TIA1, PD‐1 (PDCD1), PD‐L1 (CD274) and PAX5. Results were compared with a second cohort of 40 FL patients without signs of transformation during a minimum of five years observation time. Cell numbers and localization were semi‐quantitatively assessed. Better developed CD21+ follicular dendritic cell (FDC) meshworks at diagnosis was a negative prognostic factor for overall survival (OS), progression‐free survival (PFS) and time to transformation (TTT) in patients with subsequently transformed FL. Remnants of FDC meshworks at transformation were associated with shorter OS and PFS from transformation. High degrees of intrafollicular CD68+ and PD‐L1+ macrophage infiltration, high total area scores and an extrafollicular/diffuse pattern of FOXP3+ T cells and high intrafollicular scores of CD4+ T cells at diagnosis were associated with shorter TTT. Scores of several T‐cell subset markers from the combined patient cohorts were predictive for transformation, especially CD4 and CD57.

Entourage: the immune microenvironment following follicular lymphoma

In follicular lymphoma, nonmalignant immune cells are important. Follicular lymphoma depends on CD4+ cells, but CD8+ cells counteract it. We hypothesized that the presence of follicular lymphoma is associated with higher CD4+ than CD8+ cell numbers in the tumor microenvironment but not in the immune system. Using flow cytometry, pre-treatment and follow-up CD4/CD8 ratios were estimated in the bone marrow, blood and lymph nodes of untreated follicular lymphoma patients in two independent data sets (N1=121; N2=166). The ratios were analyzed for their relation with bone marrow lymphoma involvement. Bone marrows were also investigated with immunohistochemistry. In either data set, the bone marrow CD4/CD8 ratios were higher in bone marrows involved with lymphoma (P=0.043 and 0.0002, respectively). The mean CD4/CD8 ratio was 1.0 in uninvolved and 1.4 in involved bone marrows. Also higher in involved bone marrows were CD4/CD56 and CD3CD25/CD3 ratios. No blood or lymph node ratios differed between bone marrow-negative and -positive patients. Sequential samples showed increased bone marrow CD4/CD8 ratios in all cases of progression to bone marrow involvement. Immunohistochemistry showed CD4+, CD57+, programmed death-1+, forkhead box protein 3+ and CD21+ cells accumulated inside the lymphoma infiltrates, whereas CD8+, CD56+ and CD68+ cells were outside the infiltrates. This study provides evidence in vivo that the microenvironment changes upon follicular lymphoma involvement.

Hyperferritinemia is associated with low incidence of graft versus host disease, high relapse rate, and impaired survival in patients with blood disorders receiving allogeneic hematopoietic stem cell grafts

High pre-transplantation serum ferritin levels have been reported to be associated with impaired survival post-transplantation in patients with acute myeloid leukemia or myelodysplastic syndrome. We performed a retrospective study of 309 patients who underwent allogeneic hematopoietic stem cell transplantation at two transplantation centers. The aim was to determine the effect of pre-transplantation hyperferritinemia on survival, graft versus host disease, and relapse. In both univariate and multivariate analysis, elevated ferritin levels were significantly associated with shorter overall and relapse-free survival times and increased relapse rate, but lower risk of chronic graft versus host disease. Elevated ferritin levels were not associated with non-relapse mortality. We hypothesize that ferritin may exert an immunosuppressive effect, reducing graft versus host disease and graft versus leukemia effects, resulting in increased risk of relapse and impaired survival.

T-Cell Levels Are Prognostic in Mantle Cell Lymphoma

Purpose: The purpose of this study was to investigate the impact of T-cell subsets on pathologic and clinical features including disease outcome in mantle cell lymphoma (MCL). Experimental Design: Cell populations were investigated using flow cytometry in diagnostic MCL (n = 153) and reactive (n = 26) lymph node biopsies. Levels of tumor cells, T cells, T-cell subsets, and the CD4:CD8 ratio were assessed and related to pathologic and clinical parameters. Results: MCL cases with diffuse and nodular histologic subtypes showed lower levels of T cells, especially CD4+ T cells, than those with mantle zone growth pattern. Both CD3 and CD4 levels were lower in the nodular subtype than in mantle zone (P = 0.007; P = 0.003) and in the diffuse compared with the nodular subtype (P = 0.022; P = 0.015). The CD4:CD8 ratios were inversely correlated to tumor cell proliferation (P = 0.003). Higher levels of CD3+ and CD4+ T cells and higher CD4:CD8 ratios were associated with indolent disease (P = 0.043, 0.021, and 0.003 respectively). In univariate analysis, a high CD4:CD8 ratio, but not the histologic subtype, was correlated to longer overall survival (OS). In multivariate analysis, the CD4:CD8 ratio correlated with OS independently of Mantle Cell Lymphoma International Prognostic Index (MIPI) and high p53 expression (P = 0.023). Conclusion: CD3+, CD8+, and particularly CD4+ T-cell levels are higher in indolent MCL and decrease with more aggressive histology as reflected by a diffuse growth pattern. High CD4:CD8 ratio correlated independently of other high-risk prognostic factors with longer OS, suggesting a prognostic role for T cells in MCL. Clin Cancer Res; 20(23); 6096–104. ©2014 AACR.