Christopher P. Fox

Chemoimmunotherapy with methotrexate, cytarabine, thiotepa, and rituximab (MATRix regimen) in patients with primary CNS lymphoma: results of the first randomisation of the International Extranodal Lymphoma Study Group-32 (IELSG32) phase 2 trial

BACKGROUND Standard treatment for patients with primary CNS lymphoma remains to be defined. Active therapies are often associated with increased risk of haematological or neurological toxicity. In this trial, we addressed the tolerability and efficacy of adding rituximab with or without thiotepa to methotrexate-cytarabine combination therapy (the MATRix regimen), followed by a second randomisation comparing consolidation with whole-brain radiotherapy or autologous stem cell transplantation in patients with primary CNS lymphoma. We report the results of the first randomisation in this Article. METHODS For the international randomised phase 2 International Extranodal Lymphoma Study Group-32 (IELSG32) trial, HIV-negative patients (aged 18-70 years) with newly diagnosed primary CNS lymphoma and measurable disease were enrolled from 53 cancer centres in five European countries (Denmark, Germany, Italy, Switzerland, and the UK) and randomly assigned (1:1:1) to receive four courses of methotrexate 3·5 g/m(2) on day 1 plus cytarabine 2 g/m(2) twice daily on days 2 and 3 (group A); or the same combination plus two doses of rituximab 375 mg/m(2) on days -5 and 0 (group B); or the same methotrexate-cytarabine-rituximab combination plus thiotepa 30 mg/m(2) on day 4 (group C), with the three groups repeating treatment every 3 weeks. Patients with responsive or stable disease after the first stage were then randomly allocated between whole-brain radiotherapy and autologous stem cell transplantation. A permuted blocks randomised design (block size four) was used for both randomisations, and a computer-generated randomisation list was used within each stratum to preserve allocation concealment. Randomisation was stratified by IELSG risk score (low vs intermediate vs high). No masking after assignment to intervention was used. The primary endpoint of the first randomisation was the complete remission rate, analysed by modified intention to treat. This study is registered with ClinicalTrials.gov, number NCT01011920. FINDINGS Between Feb 19, 2010, and Aug 27, 2014, 227 eligible patients were recruited. 219 of these 227 enrolled patients were assessable. At median follow-up of 30 months (IQR 22-38), patients treated with rituximab and thiotepa had a complete remission rate of 49% (95% CI 38-60), compared with 23% (14-31) of those treated with methotrexate-cytarabine alone (hazard ratio 0·46, 95% CI 0·28-0·74) and 30% (21-42) of those treated with methotrexate-cytarabine plus rituximab (0·61, 0·40-0·94). Grade 4 haematological toxicity was more frequent in patients treated with methotrexate-cytarabine plus rituximab and thiotepa, but infective complications were similar in the three groups. The most common grade 3-4 adverse events in all three groups were neutropenia, thrombocytopenia, anaemia, and febrile neutropenia or infections. 13 (6%) patients died of toxicity. INTERPRETATION With the limitations of a randomised phase 2 study design, the IELSG32 trial provides a high level of evidence supporting the use of MATRix combination as the new standard chemoimmunotherapy for patients aged up to 70 years with newly diagnosed primary CNS lymphoma and as the control group for future randomised trials. FUNDING Associazione Italiana del Farmaco, Cancer Research UK, Oncosuisse, and Swiss National Foundation.

EBV-associated post-transplant lymphoproliferative disorder following in vivo T-cell-depleted allogeneic transplantation: Clinical features, viral load correlates and prognostic factors in the rituximab era

EBV-associated post-transplant lymphoproliferative disease (PTLD) following Alemtuzumab-based allo-SCT is a relatively uncommon and challenging clinical problem but has not received detailed study in a large cohort. Quantitative-PCR (qPCR) monitoring for EBV reactivation post allo-SCT is now commonplace but its diagnostic and predictive value remains unclear. Sixty-nine patients with PTLD following Alemtuzumab-based allo-SCT were studied. Marked clinicopathological heterogeneity was evident; lymphadenopathy was frequently absent, whereas advanced extranodal disease was common. The median viral load at clinical presentation was 49 300 copies/mL (50–65 200 000 copies/mL) and, notably, 23% and 45% of cases, respectively, had ⩽10 000 and ⩽40 000 copies/mL. The overall response rate to rituximab as first-line therapy was 70%. For rituximab failures, chemotherapy was ineffectual but DLIs were successful. A four-parameter prognostic index predicted response to therapy (OR 0.30 (0.12–0.74); P=0.009] and PTLD mortality (hazard ratio (HR) 1.81 (1.12–2.93) P=0.02) on multivariate analysis. This is the largest detailed series of EBV-associated PTLD after allo-SCT. At clinical presentation, EBV-qPCR values are frequently below customary thresholds for pre-emptive therapy, challenging current paradigms for monitoring and intervention. A four-point score identifies a proportion of patients at risk of rituximab-refractory disease for whom alternative therapy is needed.

Management of Epstein-Barr Virus infections and post-transplant lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines.

Epstein-Barr virus-related post-transplant lymphoproliferative disorders are recognized as a significant cause of morbidity and mortality in patients undergoing hematopoietic stem cell transplantation. To better define current understanding of post-transplant lymphoproliferative disorders in stem cell transplant patients, and to improve its diagnosis and management, a working group of the Sixth European Conference on Infections in Leukemia 2015 reviewed the literature, graded the available quality of evidence, and developed evidence-based recommendations for diagnosis, prevention, prophylaxis and therapy of post-transplant lymphoproliferative disorders exclusively in the stem cell transplant setting. The key elements in diagnosis include non-invasive and invasive methods. The former are based on quantitative viral load measurement and imaging with positron emission tomography; the latter with tissue biopsy for histopathology and detection of Epstein-Barr virus. The diagnosis of post-transplant lymphoproliferative disorder can be established on a proven or probable level. Therapeutic strategies include prophylaxis, preemptive therapy and targeted therapy. Rituximab, reduction of immunosuppression and Epstein-Barr virus-specific cytotoxic T-cell therapy are recommended as first-line therapy, whilst unselected donor lymphocyte infusions or chemotherapy are options as second-line therapy; other methods including antiviral drugs are discouraged.

Fusion of NUP214 to ABL1 on amplified episomes in T-ALL – implications for treatment

and photographed under UV transillumination. To determine the sensitivity of DGGE in detecting RAP1B mutations, random mutagenesis was performed in RAP1B exons 2 and 3. Two mutated PCR products were obtained both in exon 2. Each mutated plasmid was mixed with nonmutated plasmid at different ratios. DGGE analysis detected mutated DNA when representing as low as 10% of the total studied DNA. Using this technique, we did not detect any mutation in RAP1B genomic DNA in the 56 tested samples. We conclude that RAP1A or RAP1B gene mutation is a rare event in MDS bone marrow cells. The main activation mechanisms of p21 Ras proteins in human tumors are single amino acid substitutions at amino acid 12 (glycine), 13 (glycine) and 61 (glutamine) that significantly reduce the intrinsic GTPases activity of Ras proteins and create a transforming protein. Genomic DNA was analysed in the 56 patients by heteroduplex high-pressure liquid chromatography (HPLC) under denaturating conditions, as previously described. Abnormal profiles were sequenced to confirm the mutation. We found a mutation in N-RAS or K-RAS at codons 12/13 or 61 in seven of 56 studied patients (12.5%), which is in accordance with previous studies in the literature. These mutations were identified mainly in high-grade MDS, including RAEB and secondary AML. Deregulation of Rap1 signaling has been identified in malignancies, for example, the Rap1-GEF, CalDAG-GEF, is activated by proviral insertion in a leukemia-prone strain of mice, and the Bcr-Abl fusion tyrosine kinase that plays a central role in chronic myelogenous leukemia, activates both the Ras/ Raf-1 and the Rap1/B-Raf pathways. On the other hand, mutations in RAP1 genes have rarely been investigated in human tumors. In a small series of patients with cold thyroid follicular adenomas, no mutation was identified in either Epac or Rap1. Although RAP1 does not appear as a frequent target for mutagenesis, its activation could participate to malignant phenotype either by activating the B-Raf/MEK/ERK signalling pathway or by stimulating integrin activation and chemokineinduced migration, both favouring hematopoietic cell survival outside of their normal microenvironment in the bone-marrow. Other mechanisms leading to the dysregulated activation of Rap1 could contribute to the pathophysiology of myeloid disorders such as MDS. Acknowledgements

Follicular helper T-cells: expanding roles in T-cell lymphoma and targets for treatment

Follicular helper T‐cells (Tfh cells) are a subset of CD4+ T‐cells that are essential for normal production of high affinity antibodies. Tfh cells characteristically produce IL21 and IL4 and show high expression of surface markers CXCR5, ICOS, PDCD1 (PD‐1) and the chemokine CXCL13. In this review we will focus on the emerging links between Tfh cells and subtypes of T‐cell non‐Hodgkin lymphoma: angioimmunoblastic T‐cell lymphoma (AITL) and ~20% of peripheral T‐cell lymphoma not otherwise specified (PTCL‐NOS) have surface marker features of Tfh cells and share a spectrum of genetic abnormalities. The recurrent genetic abnormalities associated with AITL include mutations in epigenetic modifiers such as TET2 and DNMT3A and the motility and adhesion gene, RHOA, is mutated in up to 70% of cases. ~20% of PTCL‐NOS demonstrate RHOA mutations and have other characteristics suggesting an origin in Tfh cells. The recognition that specific genetic and surface markers are associated with malignant Tfh cells suggests that the next few years will bring major changes in diagnostic and treatment possibilities. For example, antibodies against IL21, PDCD1 and ICOS are already in clinical trials for autoimmune disease or other malignancies and antibodies against CXCL13 are in pre‐clinical development.

IVE (ifosfamide, epirubicin and etoposide) is a more effective stem cell mobilisation regimen than ICE (ifosphamide, carboplatin and etoposide) in the context of salvage therapy for lymphoma

Two commonly used chemotherapy regimens for lymphoma salvage therapy were compared: ICE (ifosphamide, carboplatin and etoposide) ± rituximab and IVE (ifosfamide, epirubicin and etoposide) ± rituximab, for their efficacy in mobilising peripheral blood stem cells for autologous transplantation. Significant differences were observed between the cohorts in terms of number of patients mobilising the stipulated minimum >2 × 106 CD34+/kg (99·2% in IVE group versus 83% in ICE group: P = 0·0002) and also in terms of the number of patients achieving the predetermined target of >5 × 106 CD34+/kg, both in total and during the first apheresis procedure (72% in IVE versus 51% in ICE group and 49% in IVE versus 7% in ICE group: P = 0·02 and P < 0·0001 respectively). This analysis of two similar groups of patients treated within a single‐centre appears to demonstrate that the IVE regimen is a more effective stem cell mobilisation regimen than ICE in the context of salvage therapy for Hodgkin and non‐Hodgkin lymphoma, allowing more patients to achieve the target CD34+ cell collection and proceed to high‐dose therapy and autologous stem cell transplantation.

Memory B-cell reconstitution following allogeneic hematopoietic stem cell transplantation is an EBV-associated transformation event.

Allogeneic stem cell transplantation (allo-HSCT) provides a unique opportunity to track Epstein-Barr virus (EBV) infection in the context of the reconstituting B-cell system. Although many allo-HSCT recipients maintain low or undetectable levels of EBV DNA posttransplant, a significant proportion exhibit elevated and rapidly increasing EBV loads which, if left untreated, may lead to potentially fatal EBV-associated posttransplant lymphoproliferative disease. Intriguingly, this high-level EBV reactivation typically arises in the first 3 months posttransplant, at a time when the peripheral blood contains low numbers of CD27+ memory cells which are the site of EBV persistence in healthy immunocompetent donors. To investigate this apparent paradox, we prospectively monitored EBV levels and B-cell reconstitution in a cohort of allo-HSCT patients for up to 12 months posttransplant. In patients with low or undetectable levels of EBV, the circulating B-cell pool consisted predominantly of transitional and naive cells, with a marked deficiency of CD27+ memory cells which lasted >12 months. However, among patients with high EBV loads, there was a significant increase in both the proportion and number of CD27+ memory B cells. Analysis of sorted CD27+ memory B cells from these patients revealed that this population was preferentially infected with EBV, expressed EBV latent transcripts associated with B-cell growth transformation, had a plasmablastic phenotype, and frequently expressed the proliferation marker Ki-67. These findings suggest that high-level EBV reactivation following allo-HSCT may drive the expansion of latently infected CD27+ B lymphoblasts in the peripheral blood.

Whole-brain radiotherapy or autologous stem-cell transplantation as consolidation strategies after high-dose methotrexate-based chemoimmunotherapy in patients with primary CNS lymphoma: results of the second randomisation of the International Extranodal Lymphoma Study Group-32 phase 2 trial

BACKGROUND The International Extranodal Lymphoma Study Group-32 (IELSG32) trial is an international randomised phase 2 study that addresses two key clinical questions in the treatment of patients with newly diagnosed primary CNS lymphoma. Results of the first randomisation have demonstrated that methotrexate, cytarabine, thiotepa, and rituximab (called the MATRix regimen) is the induction combination associated with significantly better outcome compared with the other induction combinations tested. Here, we report the results of the second randomisation that addresses the efficacy of myeloablative chemotherapy supported by autologous stem-cell transplantation (ASCT), as an alternative to whole-brain radiotherapy (WBRT), as consolidation after high-dose-methotrexate-based chemoimmunotherapy. METHODS HIV-negative patients (aged 18-70 years) with newly diagnosed primary CNS lymphoma and an Eastern Cooperative Oncology Group performance status of 0-3 were randomly assigned to receive four courses of methotrexate 3·5 g/m2 on day 1 plus cytarabine 2 g/m2 twice daily on days 2 and 3 (group A); or the same combination plus two doses of rituximab 375 mg/m2 on days -5 and 0 (group B); or the same methotrexate-cytarabine-rituximab combination plus thiotepa 30 mg/m2 on day 4 (group C), with the three groups repeating treatment every 3 weeks. Patients with responsive or stable disease after induction treatment, with adequate autologous peripheral blood stem-cell collection, and without persistent iatrogenic side-effects, were eligible for the second randomisation between WBRT (photons of 4-10 MeV; five fractions per week; fraction size 180 cGy; started within 4 weeks from the last induction course; group D) and carmustine-thiotepa conditioned ASCT (carmustine 400 mg/m2 on day -6, and thiotepa 5 mg/kg every 12 h on days -5 and -4, followed by reinfusion of autologous peripheral blood stem cells; group E). A permuted block randomised design was adopted for both randomisations, and a computer-generated randomisation list was used within each stratum. No masking after assignment to intervention was adopted. The primary endpoint was 2-year progression-free survival, with induction group and response to induction chemotherapy as stratification parameters. Analyses were done on a modified intention-to-treat basis. This study is registered with ClinicalTrials.gov, number NCT01011920. FINDINGS Between Feb 19, 2010, and Aug 27, 2014, 227 patients were recruited from 53 centres in five countries. 219 of 227 enrolled patients were assessable. Of the 122 patients eligible for the second randomisation, 118 patients were randomly assigned to WBRT or ASCT (59 patients per group) and constitute the study population. WBRT and ASCT were both effective, and achieved the predetermined efficacy threshold of at least 40 progression-free survivors at 2 years among the first 52 patients in both groups D and E. There were no significant differences in 2-year progression-free survival between WBRT and ASCT: 80% (95% CI 70-90) in group D and 69% (59-79) in group E (hazard ratio 1·50, 95% CI 0·83-2·71; p=0·17). Both consolidation therapies were well tolerated. Grade 4 non-haematological toxicity was uncommon; as expected, haematological toxicity was more common in patients treated with ASCT than in those who received WBRT. Two toxic deaths (infections) were recorded, both in patients who received ASCT. INTERPRETATION WBRT and ASCT are both feasible and effective as consolidation therapies after high-dose methotrexate-based chemoimmunotherapy in patients aged 70 years or younger with primary CNS lymphoma. The risks and implications of cognitive impairment after WBRT should be considered at the time of therapeutic decision. FUNDING Agenzia Italiana del Farmaco, Cancer Research UK, Oncosuisse, and Swiss National Science Foundation.

Delineating Outcomes of Patients with Diffuse Large B cell lymphoma using the National Comprehensive Cancer Network‐International Prognostic Index and Positron Emission Tomography‐defined Remission Status; a Population‐based Analysis

The recently devised National Comprehensive Cancer Network International Prognostic Index (NCCN‐IPI) appears superior to the revised IPI (R‐IPI) in delineating outcome in diffuse large B‐cell lymphoma. We examined the outcome of a population‐based cohort of 223 consecutive patients treated with R‐CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone) or R‐CHOP‐like immuno‐chemotherapy between January 2005 and December 2011 by both the NCCN‐IPI and R‐IPI, and further stratified outcome by the achievement of both computerized tomography (CT) and positron emission tomography (PET)‐CT complete remission (CR), with the latter reassessed using blinded central review by an independent nuclear medicine and radiology specialist. The NCCN‐IPI was superior to the R‐IPI in identifying patients at very high risk of systemic and/or central nervous system relapse. Notably, both the NCCN‐IPI and the R‐IPI remained strongly predictive of relapse irrespective of CT or PET‐defined remission status following R‐CHOP. Patients with high‐risk NCCN‐IPI scores (≥6) have a dismal outcome following R‐CHOP therapy regardless of PET‐defined response to R‐CHOP. Moreover, such patients appear refractory to salvage chemotherapy and thus require alternative therapeutic approaches, although age and performance status may, for many patients, preclude the safe delivery of a primary intensified regimen. By contrast, patients with NCCN‐IPI 1–5 who achieve PET‐CR following R‐CHOP have excellent outcomes and may merit reduced follow up frequency.

High-dose therapy and autologous stem cell transplantation for extra-nodal NK/T lymphoma in patients from the Western hemisphere: a study from the European Society for Blood and Marrow Transplantation

Extra-nodal NK/T lymphoma (ENKTL) is rare and more frequently encountered in East Asia. The role of high-dose therapy and autologous stem cell transplantation (HDT-ASCT) for ENKTL is unclear. Twenty-eight evaluable patients who had undergone HDT-ASCT in Europe from 2000–2009 were studied. The median age was 47 years and patients had received a median of two lines of prior therapy. Some 57% of patients were not in complete remission or beyond first complete remission at HDT-ASCT. The 1-year non-relapse mortality (NRM) was 11%; 2-year progression-free survival (PFS) and overall survival (OS) rates were 41% and 52%, respectively. Notably, the 2-year PFS and OS for those with stage III/IV disease were 33% and 40%, respectively, with no relapses beyond 1-year post-HDT-ASCT. This is the largest analysis of HDT-ASCT for patients with ENKTL reported from the Western hemisphere. Survival is comparable to East Asian cohorts and outcomes are encouraging for patients with advanced disease.