PURPOSE: Patients with indolent non-Hodgkin lymphoma typically respond well to first-line immunochemotherapy. At relapse, single-agent rituximab is commonly administered. Data suggest the immunomodulatory agent lenalidomide could increase the activity of rituximab.METHODS: A phase III, multicenter, randomized trial of lenalidomide plus rituximab versus placebo plus rituximab was conducted in patients with relapsed and/or refractory follicular or marginal zone lymphoma. Patients received lenalidomide or placebo for 12 cycles plus rituximab once per week for 4 weeks in cycle 1 and day 1 of cycles 2 through 5. The primary end point was progression-free survival per independent radiology review.RESULTS: A total of 358 patients were randomly assigned to lenalidomide plus rituximab (n = 178) or placebo plus rituximab (n = 180). Infections (63% v 49%), neutropenia (58% v 23%), and cutaneous reactions (32% v 12%) were more common with lenalidomide plus rituximab. Grade 3 or 4 neutropenia (50% v 13%) and leukopenia (7% v 2%) were higher with lenalidomide plus rituximab; no other grade 3 or 4 adverse event differed by 5% or more between groups. Progression-free survival was significantly improved for lenalidomide plus rituximab versus placebo plus rituximab, with a hazard ratio of 0.46 (95% CI, 0.34 to 0.62; P < .001) and median duration of 39.4 months (95% CI, 22.9 months to not reached) versus 14.1 months (95% CI, 11.4 to 16.7 months), respectively.CONCLUSION: Lenalidomide improved efficacy of rituximab in patients with recurrent indolent lymphoma, with an acceptable safety profile.

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

The diagnosis of malignant lymphoma is a recognized difficult area in histopathology. Therefore, detection of clonality in a suspected lymphoproliferation is a valuable diagnostic criterion. We have developed primer sets for the detection of rearrangements in the B- and T-cell receptor genes as reliable tools for clonality assessment in lymphoproliferations suspected for lymphoma. In this issue of Leukemia, the participants of the BIOMED-2 Concerted Action CT98-3936 report on the validation of the newly developed clonality assays in various disease entities. Clonality was detected in 99% of all B-cell malignancies and in 94% of all T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The combined BIOMED-2 results are summarized in a guideline, which can now be implemented in routine lymphoma diagnostics. The use of this standardized approach in patients with a suspect lymphoproliferation will result in improved diagnosis of malignant lymphoma.

The tumour microenvironment (TME) shapes disease progression and influences therapeutic response. Most aggressive solid tumours have high levels of myeloid cell infiltration, namely tumour associated macrophages (TAM). Recapitulation of the interaction between the different cellular players of the TME, along with the extracellular matrix (ECM), is critical for understanding the mechanisms underlying disease progression. This particularly holds true for prediction of therapeutic response(s) to standard therapies and interrogation of efficacy of TME-targeting agents. In this work, we explored a culture platform based on alginate microencapsulation and stirred culture systems to develop the 3D-3-culture, which entails the co-culture of tumour cell spheroids of non-small cell lung carcinoma (NSCLC), cancer associated fibroblasts (CAF) and monocytes. We demonstrate that the 3D-3-culture recreates an invasive and immunosuppressive TME, with accumulation of cytokines/chemokines (IL4, IL10, IL13, CCL22, CCL24, CXCL1), ECM elements (collagen type I, IV and fibronectin) and matrix metalloproteinases (MMP1/9), supporting cell migration and promoting cell-cell interactions within the alginate microcapsules. Importantly, we show that both the monocytic cell line THP-1 and peripheral blood-derived monocytes infiltrate the tumour tissue and transpolarize into an M2-like macrophage phenotype expressing CD68, CD163 and CD206, resembling the TAM phenotype in NSCLC. The 3D-3-culture was challenged with chemo- and immunotherapeutic agents and the response to therapy was assessed in each cellular component. Specifically, the macrophage phenotype was modulated upon treatment with the CSF1R inhibitor BLZ945, resulting in a decrease of the M2-like macrophages. In conclusion, the crosstalk between the ECM and tumour, stromal and immune cells in microencapsulated 3D-3-culture promotes the activation of monocytes into TAM, mimicking aggressive tumour stages. The 3D-3-culture constitutes a novel tool to study tumour-immune interaction and macrophage plasticity in response to external stimuli, such as chemotherapeutic and immunomodulatory drugs.

Angioimmunoblastic T-cell lymphoma (AITL) is an uncommon, but aggressive nodal peripheral T-cell lymphoma. Little is known of its biology and its natural history has been poorly studied. We report the first comprehensive study on the natural history/histologic progression of AITL by reviewing consecutive biopsies in 31 cases. Immunostaining for CD3, CD20, CD10 and CD21, CD23, CNA-42, CD4, CD8, and Ki 67, in situ hybridization for Epstein-Barr virus (EBV)-encoded RNA and polymerase chain reaction for T-clonality and B-clonality were performed. Histologic progression from AITL with limited nodal involvement and hyperplastic follicles (pattern I) to typical AITL with or without regressed follicles (patterns II and III) was observed in 7 cases, one of which relapsed subsequently as pattern I. Thirteen cases showed typical AITL at presentation and follow-up. Eleven cases where polymerase chain reaction results for T-cell receptor-γ gene rearrangement were directly compared showed an identical band-size in the initial and follow-up biopsies. Seven cases (23%) developed EBV-associated B-cell lymphomas [5 diffuse large B-cell lymphoma (DLBCL) and 2 classic Hodgkin lymphoma]. In 4 cases, a dominant B-cell clone was observed in biopsies lacking evidence of DLBCL. A single case was complicated by EBV-negative DLBCL, whereas another with large cell transformation had a T-cell phenotype. In conclusion, AITL represents a clonal T-cell proliferation with a stable T-cell clone throughout the disease. Partial nodal involvement with hyperplastic follicles is seen in early AITL and at relapse. When “morphologic high-grade transformation” occurs, it is usually due to a secondary (often EBV-associated) B-cell lymphoma, rather than a T-cell neoplasm.

Purpose: The standard treatment of patients with diffuse large B-cell lymphoma (DLBCL) is rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Lenalidomide, an immunomodulatory agent, has shown activity in DLBCL. This randomized phase III trial compared lenalidomide as maintenance therapy with placebo in elderly patients with DLBCL who achieved a complete response (CR) or partial response (PR) to R-CHOP induction. Methods: Patients with previously untreated DLBCL or other aggressive B-cell lymphoma were 60 to 80 years old, had CR or PR after six or eight cycles of R-CHOP, and were randomly assigned to lenalidomide maintenance 25 mg/d or placebo for 21 days of every 28-day cycle for 24 months. The primary end point was progression-free survival (PFS). Results: A total of 650 patients were randomly assigned. At the time of the primary analysis (December 2015), with a median follow-up of 39 months from random assignment, median PFS was not reached for lenalidomide maintenance versus 58.9 months for placebo (hazard ratio, 0.708; 95% CI, 0.537 to 0.933; P = .01). The result was consistent among analyzed subgroups (eg, male v female, age-adjusted International Prognostic Index 0 or 1 v 2 or 3, age younger than 70 v ≥ 70 years), response (PR v CR) after R-CHOP, and positron emission tomography status at assignment (negative v positive). With longer median follow-up of 52 months (October 2016), overall survival was similar between arms (hazard ratio, 1.218; 95% CI, 0.861 to 1.721; P = .26). Most common grade 3 or 4 adverse events associated with lenalidomide versus placebo maintenance were neutropenia (56% v 22%) and cutaneous reactions (5% v 1%), respectively. Conclusion: Lenalidomide maintenance for 24 months after obtaining a CR or PR to R-CHOP significantly prolonged PFS in elderly patients with DLBCL.