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In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10−5). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10−4) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10−6). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

Detection of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) is becoming increasingly important as treatments improve. An internationally harmonised four-colour (CLR) flow cytometry MRD assay is widely used but has limitations. The aim of this study was to improve MRD analysis by identifying situations where a less time-consuming CD19/CD5/κ/λ analysis would be sufficient for detecting residual CLL, and develop a six-CLR antibody panel that is more efficient for cases requiring full MRD analysis. In 784 samples from CLL patients after treatment, it was possible to determine CD19/CD5/κ/λ thresholds that identified cases with detectable MRD with 100% positive predictive value (PPV). However, CD19/CD5/κ/λ analysis was unsuitable for predicting iwCLL/NCI response status or identifying cases with no detectable MRD. For the latter cases requiring a full MRD assessment, a six-CLR assay was designed comprising CD19/CD5/CD20 with (1) CD3/CD38/CD79b and (2) CD81/CD22/CD43. There was good correlation between four-CLR and six-CLR panels in dilution studies and clinical samples, with 100% concordance for detection of residual disease at the 0.01% (10(-4)) level (n=59) and good linearity even at the 0.001-0.01% (10(-5)-10(-4)) level. A six-CLR panel therefore provides equivalent results to the four-CLR panel but it requires fewer reagents, fewer cells and a much simpler analysis approach.

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Purpose: Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies.Experimental Design: We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted resequencing and explored potential clinical implications in a multinational cohort of 175 patients with SMZL.Results: We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%), and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2 Ã 04 B-cell receptor immunoglobulins, and were associated with a short median time to first treatment (0.12 vs. 1.11 years; P ¼ 0.01).In multivariate analysis, mutations in NOTCH2 [HR, 2.12; 95% confidence interval (CI), 1.02-4.4; P ¼ 0.044] and 100% germline IGHV gene identity (HR, 2.19; 95% CI, 1.05-4.55; P ¼ 0.036) were independent markers of short time to first treatment, whereas TP53 mutations were an independent marker of short overall survival (HR, 2.36; 95 % CI, 1.08-5.2; P ¼ 0.03). Conclusions:We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively.

Hemojuvelin (HJV) positively modulates the iron regulator hepcidin, and its mutations are the major cause of juvenile hemochromatosis (JH), a recessive disease leading to iron overload. Defective HJV reduces hepcidin up-regulation both in humans and in Hjv-deficient mice. To investigate the JH pathogenesis and the functional properties of human HJV we studied the biosynthesis and maturation of 6 HJV pathogenic mutants in HeLa and HepG2 cells. We show that proteolytic processing is defective in mutants F170S, W191C, and G320V, but not in G99V and C119F. Moreover, we show that mutants G99V and C119F are targeted to the cell surface, while F170S, W191C, G320V, and R326X ( IntroductionThe study of hereditary hemochromatosis (HH) has contributed major advances to our understanding of the systemic iron regulation. 1 The key regulatory protein is hepcidin, a small antimicrobial liver peptide 2 that has an inhibitory effect on the release of iron from duodenal cells and macrophages to the circulating transferrin. 3 Mutations of hepcidin cause a recessive form of severe, early-onset hemochromatosis, known as juvenile hemochromatosis (JH; type 2B HH). 4 The same severe phenotype is more commonly caused by mutations of HJV, the gene encoding hemojuvelin (type 2A HH). 3 Studies in humans and mice provide evidence that HJV positively modulates hepcidin expression. Urinary hepcidin levels are remarkably low in patients carrying HJV mutations, 5 and Hjv-deficient mice have dramatically decreased liver hepcidin mRNA. 6,7 The mechanism of hepcidin activation by HJV remains to be clarified. Recently, it has been shown that HJV could act as a bone morphogenetic protein (BMP) coreceptor, enhancing BMPmediated signaling 8 and likely up-regulating hepcidin expression through BMP-activated SMA and MAD proteins (SMAD). 9 HJV is highly homologous to proteins belonging to the family of repulsive guidance molecules (RGMs). In the mouse, the expression of Rgma and Rgmb is limited to developing and adult central nervous system, while Rgmc, the ortholog of human HJV, is detected in the same tissues (skeletal muscle, heart, and liver) where hepcidin is expressed. 3 Similar to RGM members, the HJV gene encodes a protein characterized by multiple domains, including a N-terminal signal peptide, a RGD integrin-binding motif, a partial von Willebrand factor (VWF) type D domain, and a C-terminal glycosilphosphatidylinositol (GPI) anchor domain. All RGM proteins possess a Gly-Asp-Pro-His (GDPH) sequence, 10,11 which undergoes a partial autocatalytic cleavage at the Asp-Pro bond at acidic pH, compatible with that of the late secretory pathway. 12 HJV is retained on the outer layer of the plasma membrane (m-HJV) through the GPI anchor motif, but can also be found as a soluble form (s-HJV) both in vitro and in vivo. 13,14 Recent studies in murine muscle cells and fibroblasts have analyzed the complex biosynthesis of Rgmc during myoblast differentiation. Two classes of GPI anchor Rgmc molecules exist that are differently processed and have distinct fat...

The function of the intracellular protein hematopoietic cell-specific Lyn substrate-1 (HS1) in B lymphocytes is poorly defined. To investigate its role in migration, trafficking, and homing of leukemic B lymphocytes we have used B cells from HS1 ؊/؊ mice, the HS1-silenced human chronic lymphocytic leukemia (

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