Birgitta Sander

Molecular subsets of mantle cell lymphoma defined by the IGHV mutational status and SOX11 expression have distinct biologic and clinical features

Mantle cell lymphoma (MCL) is a heterogeneous disease with most patients following an aggressive clinical course, whereas others having an indolent behavior. We conducted an integrative and multidisciplinary analysis of 177 MCL to determine whether the immunogenetic features of the clonotypic B-cell receptors (BcR) may identify different subsets of tumors. Truly unmutated (100% identity) IGHV genes were found in 24% cases, 40% were minimally/borderline mutated (99.9%-97%), 19% significantly mutated (96.9%-95%), and 17% hypermutated (<95%). Tumors with high or low mutational load used different IGHV genes, and their gene expression profiles were also different for several gene pathways. A gene set enrichment analysis showed that MCL with high and low IGHV mutations were enriched in memory and naive B-cell signatures, respectively. Furthermore, the highly mutated tumors had less genomic complexity, were preferentially SOX11-negative, and showed more frequent nonnodal disease. The best cut-off of germline identity of IGHV genes to predict survival was 97%. Patients with high and low mutational load had significant different outcome with 5-year overall survival (OS) of 59% and 40%, respectively (P = 0.004). Nodal presentation and SOX11 expression also predicted for poor OS. In a multivariate analysis, IGHV gene status and SOX11 expression were independent risk factors. In conclusion, these observations suggest the idea that MCL with mutated IGHV, SOX11-negativity, and nonnodal presentation correspond to a subtype of the disease with more indolent behavior.

Is there a role for antigen selection in mantle cell lymphoma? Immunogenetic support from a series of 807 cases

We examined 807 productive IGHV-IGHD-IGHJ gene rearrangements from mantle cell lymphoma (MCL) cases, by far the largest series to date. The IGHV gene repertoire was remarkably biased, with IGHV3-21, IGHV4-34, IGHV1-8, and IGHV3-23 accounting for 46.3% of the cohort. Eighty-four of 807 (10.4%) cases, mainly using the IGHV3-21 and IGHV4-34 genes, were found to bear stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences and were placed in 38 clusters. Notably, the MCL stereotypes were distinct from those reported for chronic lymphocytic leukemia. Based on somatic hypermutation (SHM) status, 238/807 sequences (29.5%) carried IGHV genes with 100% germ line identity; the remainder (569/807; 70.5%) exhibited different SHM impact, ranging from minimal (in most cases) to pronounced. Shared replacement mutations across the IGHV gene were identified for certain subgroups, especially those using IGHV3-21, IGHV1-8, and IGHV3-23. Comparison with other entities, in particular CLL, revealed that several of these mutations were MCL-biased. In conclusion, MCL is characterized by a highly restricted immunoglobulin gene repertoire with stereotyped VH CDR3s and very precise SHM targeting, strongly implying a role for antigen-driven selection of the clonogenic progenitors. Hence, an antigen-driven origin of MCL could be envisaged, at least for subsets of cases.

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 10·0u2003years (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 (Pu2003=u20030·008), but outcome was improved after upfront anthracycline‐containing therapy (Pu2003=u20030·015). In contrast to grade 1–3A patients, grade 3B patients experienced no relapses or deaths beyond 5u2003years 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.

Immunohistochemical prognostic markers in diffuse large B-cell lymphoma: validation of tissue microarray as a prerequisite for broad clinical applications (a study from the Lunenburg Lymphoma Biomarker Consortium).

Background and Aims: The results of class prediction and the determination of prognostic markers in diffuse large B-cell lymphoma (DLBCL) have been variably reported. Apart from biological variations, this may be caused by differences in laboratory techniques, scoring definitions and inter- and intra-observer variation. In this study, an international collaboration of clinical lymphoma research groups has concentrated on validation and standardisation of immunohistochemistry of the currently potentially interesting prognostic markers in DLBCL. Methods: Sections of a tissue microarray with 36 cases of DLBCL were stained in eight laboratories with antibodies to CD20, CD5, bcl-2, bcl-6, CD10, HLA-DR, MUM-1 and Ki-67 according to local methods. The study was performed in two rounds, firstly focused on the evaluation of laboratory staining variation, and secondly on the scoring variation. Results: Different techniques resulted in highly variable results and poor reproducibility for almost all markers. Reproducibility of the nuclear markers was highly sensitive to technical variations, including immunological enhancement techniques (agreements 34%). With elimination of variation due to staining and uniformly agreed on scoring criteria, significant improvement was seen; however less so for bcl-6 and Ki-67 (agreement 53–58%). Absence of internal controls that preclude scoring, significantly influenced the results for CD10 and bcl-6. Conclusion: Semi-quantitative immunohistochemistry for subclassification of DLBCL is feasible, but with varying rates of concordance for different markers and only using optimised techniques and strict scoring criteria. These findings may explain the wide variation in prognostic impact reported in the literature. Harmonisation of techniques and centralised consensus review appears mandatory when using immunohistochemical biomarkers for treatment stratification.

Posttransplant lymphoma—A single-center experience of 500 liver transplantations

Background: Posttransplant lymphoproliferative disease (PTLD) is a serious complication after organ transplantation associated with a high mortality, and is often caused by a primary or reactivated Epstein-Barr virus (EBV) infection. The incidence of PTLD ranges from 1% to 10%, depending on the type of organ transplanted and the immunosuppressive regimens used.Methods: In this retrospective study from a single center, 12 (2.4%) of 500 consecutive recipients of liver grafts developed lymphoma. Patient data were obtained by chart review. All diagnostic biopsies were reviewed by two hematopathologists.Results: The median time between transplantation and the diagnosis of lymphoma was 19.5 (1.5–148) mo. Nine of the patients had been treated with OKT-3 and/or ATG after the transplantation. Two patients had a pretransplant diagnosis of lymphoma. The PTLD was of high grade in all patients, and was associated with EBV in 6 of 9 examined cases. No relation with human herpesvirus-8 could be detected. In all patients, immuno-suppression was reduced at the time of lymphoma diagnosis. Chemotherapy was used in all patients, mostly upfront but in one patient after lymphoma progression after reduction of immunosuppression. Nine patients also got antiviral therapy. Immunotherapy with the monoclonal antibody rituximab was used in one patient. Half of the patients are alive, in complete continuous remission, more than 4 yr after the lymphoma diagnosis. Two patients died of neutropenic sepsis, three of persistent lymphoma, and one of recurrent cirrhosis while in complete remission.Conclusions: PTLD is a significant complication in liver-transplanted patients. Intensive chemotherapy can induce long-term remissions in a substantial number of patients. The role for monoclonal antibodies in this setting should be investigated further.

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.

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.

EXPRESSION OF PTEN AND SHP1, INVESTIGATED FROM TISSUE MICROARRAYS IN PEDIATRIC ACUTE LYMPHOBLASTIC, LEUKEMIA

PTEN and SHP1 are tumor suppressor genes involved in the regulation of cell cycle control and apoptosis. The authors investigated the protein expression of PTEN and SHP1, by immunohistochemistry in tissue microarrays from bone marrow samples in children, diagnosed with acute lymphoblastic leukaemia and nonmalignant controls. PTEN was overexpressed in diagnostic ALL samples, while SHP1 showed a low expression. Both proteins showed a significant difference in expression compared to nonmalignant controls. The roles of PTEN and SHP1 are not well investigated in pediatric leukemia and could in the future play a role as prognostic factors.

Aberrant splicing of genes involved in haemoglobin synthesis and impaired terminal erythroid maturation in SF3B1 mutated refractory anaemia with ring sideroblasts

Refractory anaemia with ring sideroblasts (RARS) is distinguished by hyperplastic inefficient erythropoiesis, aberrant mitochondrial ferritin accumulation and anaemia. Heterozygous mutations in the spliceosome gene SF3B1 are found in a majority of RARS cases. To explore the link between SF3B1 mutations and anaemia, we studied mutated RARS CD34+ marrow cells with regard to transcriptome sequencing, splice patterns and mutational allele burden during erythroid differentiation. Transcriptome profiling during early erythroid differentiation revealed a marked up‐regulation of genes involved in haemoglobin synthesis and in the oxidative phosphorylation process, and down‐regulation of mitochondrial ABC transporters compared to normal bone marrow. Moreover, mis‐splicing of genes involved in transcription regulation, particularly haemoglobin synthesis, was confirmed, indicating a compromised haemoglobinization during RARS erythropoiesis. In order to define the phase during which erythroid maturation of SF3B1 mutated cells is most affected, we assessed allele burden during erythroid differentiation in vitro and in vivo and found that SF3B1 mutated erythroblasts showed stable expansion until late erythroblast stage but that terminal maturation to reticulocytes was significantly reduced. In conclusion, SF3B1 mutated RARS progenitors display impaired splicing with potential downstream consequences for genes of key importance for haemoglobin synthesis and terminal erythroid differentiation.