Splenic marginal-zone B cells, marginal-zone B cells of Peyer’s patches in the gut, and nodal marginal-zone B cells (also identified as monocytoid B cells) share a similar morphology and immunophenotype. These cells likely represent a distinct subset of B cells in humans and rodents, but their precise ontogenetic relationship as well as their origin from B cells of the germinal center is still debated. To study this, we performed a mutation analysis of the rearranged immunoglobulin variable genes (VH) of microdissected single nodal and splenic marginal-zone cells. In addition, we investigated the presence of proliferating cells and B-cell clones in the human splenic and nodal marginal zone as well as adjacent germinal centers. This was performed by immunohistochemical staining for the Ki-67 antigen and denaturing gradient gel analysis of amplified immunoglobulin heavy chain genes’ complementarity determining region 3 of microdissected cell clusters. A variable subset of nodal and splenic marginal-zone B cells showed somatic mutations in their rearranged VH genes, indicating that both virgin and memory B cells are present in the nodal and splenic marginal zone. Nodal and splenic marginal-zone B cells preferentially rearranged VH3 family genes such as DP47, DP49, DP54, and DP58. A preferential rearrangement of the same VH genes has been shown by others in the peripheral CD5− IgM+ B cells. These data suggest that the splenic and nodal marginal-zone B cells are closely related B-cell subsets. We also showed that marginal-zone B cells may cycle and that clones of B cells are frequently detected in the nodal as well as the splenic marginal zone. These clones are not related to those present in adjacent germinal centers. These data favor the hypothesis that clonal expansion occurs in the marginal zone. Whether the somatic hypermutation mechanism is activated during the clonal expansion in the marginal zone and which type of immune response triggers the clonal expansion need to be elucidated.

Survival of patients with high-risk diffuse large B-cell lymphoma (DLBCL) is suboptimal, and the risk of central nervous system (CNS) progression is relatively high. We conducted a phase 2 trial in 139 patients aged 18 to 64 years who had primary DLBCL with an age-adjusted International Prognostic Index (aaIPI) score of 2 to 3 or site-specific risk factors for CNS recurrence. The goal was to assess whether a dose-dense immunochemotherapy with early systemic CNS prophylaxis improves the outcome and reduces the incidence of CNS events. Treatment consisted of 2 courses of high-dose methotrexate in combination with biweekly rituximab (R), cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP-14), followed by 4 courses of R-CHOP-14 with etoposide (R-CHOEP) and 1 course of high-dose cytarabine with R. In addition, liposomal cytarabine was administered intrathecally at courses 1, 3, and 5. Coprimary endpoints were failure-free survival and CNS progression rates. Thirty-six (26%) patients experienced treatment failure. Progression occurred in 23 (16%) patients, including three (2.2%) CNS events. At 5 years of median follow-up, failure-free survival, overall survival, and CNS progression rates were 74%, 83%, and 2.3%, respectively. Treatment reduced the risk of progression compared with our previous trial, in which systemic CNS prophylaxis was given after 6 courses of biweekly R-CHOEP (hazard ratio, 0.49; 95% CI, 0.31-0.77; P = .002) and overcame the adverse impact of an aaIPI score of 3 on survival. In addition, outcome of the patients with BCL2/MYC double-hit lymphomas was comparable to the patients without the rearrangements. The results are encouraging, with a low toxic death rate, low number of CNS events, and favorable survival rates. This trial was registered at www.clinicaltrials.gov as #NCT01325194.

Primary cold agglutinin disease (CAD) is a hemolytic anemia mediated by monoclonal anti-I autoantibodies. CAD is caused by an underlying low grade B-cell lymphoproliferative disease of the bone marrow with a typical histology that is different from lymphoplasmacytic lymphoma and, accordingly, does not display the MYD88 L265P mutation (Randen et al., Haematologica, 2014). Since CAD is a clonal lymphoproliferative disorder, we studied the mutational landscape to further characterize the disease and identify potential novel treatment approaches. We prospectively collected bone marrow samples of CAD patients, enrolled in a clinical trial (CAD5; www.clinicaltrials.gov, NCT02689986). Exome sequencing of six cases was performed and findings were confirmed in ten additional cases using targeted sequencing. For these analyses, clonal B cells and normal T cells, used as control, were purified from bone marrow samples using fluorescent activated cell sorting. All mutations were verified by Sanger sequencing. Whole-exome sequencing was performed at BGI Tech Solutions (Hongkong) using the Agilent SureSelect Human All Exon V4 Reagent Kit and Illumina HiSeq technology. The bioinformatics pipeline consistent of BWA alignment tool (aligned to hg19); Picard tools FixMateInformation and MarkDuplicates; the Genome Analysis Toolkit (GATK) IndelRealigner and BaseRecalibrator; somatic variant detection tools Strelka, MuTect and Pindel; the annotation tool SnpEff. Recurrent somatic mutations were found in KMT2D (11/16 cases, 69%) and CARD11 (5/16, 31%) (Table 1, Figure 1-2). 7/16 (44%) of KMT2D mutations were deemed high impact mutations by SnpEff and to result in inactive protein. 2/16 (12,5%) of KMT2D mutations are missense mutations and are predicted to impair SET domain function. 2/16 (12,5%) of KMT2D mutations are classified by SnpEff as low impact mutations of which functional tests are necessary to demonstrate potential consequences for protein function. Of interest, two additional patients showed rare germline KMT2D variants that have also been seen in Kabuki syndrome patients, although these patients do not have Kabuki syndrome. CARD11 was somatically mutated in 5/16 (31%) cases. Four of those patients had a concurrent KMT2D mutation. All CARD 11 mutations were classified as moderate impact mutations by SnpEff. Mutations were tightly clustered in a 20bp sequence of the coiled-coil domain sequence (Table 1, Figure 2). KMT2D is a histone lysine methyl transferase that represses B cell lymphoma development. Mono-allelic mutations of KMT2D seem to act in a dominant fashion and cause partial loss of protein expression with cell growth advantage. KMT2D is frequently mutated in follicular lymphoma, diffuse large B cell lymphoma and nodal marginal zone lymphoma. Mono-allelic constitutional mutations cause Kabuki syndrome, characterized by distinct facial characteristics and multiple organ malformations. Patients frequently develop immune-mediated thrombocytopenia as well as auto-immune hemolytic anemia. CARD11 coiled-coil domain mutations result in constitutive NF-kB activation and enhanced NF-kB activity upon antigen receptor stimulation. Mutations were previously detected in diffuse large B cell lymphomas of activated B cell origin. Mono-allelic CARD11 coiled coil mutations are not oncogenic per se in mice and humans, but result in B-cell proliferation and auto-antibody production. Since four CAD patients showed concurrent KMT2D and CARD11 mutations, it seems likely that the mutations act in concert with anti-I B-cell receptor stimulation to contribute to CAD-associated lymphoproliferative disease. In conclusion, we demonstrated a high frequency of KMT2D and CARD11 mutations in the bone marrow B cell lymphoproliferative disease of patients with CAD. These results confirm that CAD-associated B cell lymphoproliferative disease is a distinct disease different from other known B cell lymphoproliferative diseases of the bone marrow, most notably lymphoplasmacytic lymphoma. The identification of these recurrent mutations in CAD may allow the design of novel treatment modalities. Table 1 Mutations in KMT2D and CARD11 gene in CAD. Table 1. Mutations in KMT2D and CARD11 gene in CAD. Figure 1 Mutations within KMT2D gene and protein detected in CAD patients. Figure 1. Mutations within KMT2D gene and protein detected in CAD patients. Figure 2 Mutations within CARD11 protein detected in CAD patients. Figure 2. Mutations within CARD11 protein detected in CAD patients. Disclosures No relevant conflicts of interest to declare.

PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR5y) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p < 0.0001), 29 (HzR 2.7, p < 0.0001), and 79 (HzR 3.5, p < 0.0001) associated with hazard of relapse adjusted for age, cytogenetics, and WBC. The early (day 15) response associated with CIR5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10−2 versus 5.2 × 10−3, p < 0.0001). Undetectable FCM- and/or PCR-MRD on day 29 identified patients with a very good outcome (CIR5y = 3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD > 10−4 associated with a CIR5y = 22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.