High surface IgM levels associate with shorter response to ibrutinib and BTK bypass in patients with CLL.

Chronic lymphocytic leukemia (CLL) cells have variably low surface IgM (sIgM) levels/signaling capacity, influenced by chronic antigen engagement at tissue sites. Within these low levels, CLL with relatively high sIgM (CLLhigh) progresses more rapidly than CLL with low sIgM (CLLlow). During ibrutinib therapy, surviving CLL cells redistribute into the peripheral blood and can recover sIgM expression. Return of CLL cells to tissue may eventually recur, where cells with high sIgM could promote tumor growth. We analyzed time to new treatment (TTNT) following ibrutinib in 70 patients with CLL (median follow-up of 66 months) and correlated it with pretreatment sIgM levels and signaling characteristics. Pretreatment sIgM levels correlated with signaling capacity, as measured by intracellular Ca2+ mobilization (iCa2+), in vitro (r = 0.70; P < .0001). High sIgM levels/signaling strongly correlated with short TTNT (P < .05), and 36% of patients with CLLhigh vs 8% of patients with CLLlow progressed to require a new treatment. In vitro, capacity of ibrutinib to inhibit sIgM-mediated signaling inversely correlated with pretherapy sIgM levels (r = -0.68; P = .01) or iCa2+ (r = -0.71; P = .009). In patients, sIgM-mediated iCa2+ and ERK phosphorylation levels were reduced by ibrutinib therapy but not abolished. The residual signaling capacity downstream of BTK was associated with high expression of sIgM, whereas it was minimal when sIgM expression was low (P < .05). These results suggested that high sIgM levels facilitated CLL cell resistance to ibrutinib in patients. The CLL cells, surviving in the periphery with high sIgM expression, include a dangerous fraction that is able to migrate to tissue and receive proliferative stimuli, which may require targeting by combined approaches.

Single-nucleotide Fcγ receptor polymorphisms do not impact obinutuzumab/rituximab outcome in patients with lymphoma.

Single-nucleotide polymorphisms (SNPs) have been shown to influence Fcγ receptor (FcγR) affinity and activity, but their effect on treatment response is unclear. We assessed their importance in the efficacy of obinutuzumab or rituximab combined with chemotherapy in untreated advanced follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) in the GALLIUM (www.clinicaltrials.gov #NCT01332968) and GOYA (#NCT01287741) trials, respectively. Genomic DNA was extracted from patients enrolled in GALLIUM (n = 1202) and GOYA (n = 1418). Key germline SNPs, FCGR2A R131H (rs1801274), FCGR3A F158V (rs396991), and FCGR2B I232T (rs1050501), were genotyped and assessed for their impact on investigator-assessed progression-free survival (PFS). In both cohorts there was no prognostic effect of FCGR2A or FCGR3A. In FL, FCGR2B was associated with favorable PFS in univariate and multivariate analyses comparing I232T with I232I, with a more modest association for rituximab-treated (univariate: hazard ratio [HR], 0.78; 95% confidence interval [CI], 0.54-1.14; P = .21) vs obinutuzumab-treated patients (HR, 0.56; 95% CI, 0.34-0.91; P = .02). Comparing T232T with I232I, an association was found for obinutuzumab (univariate: HR, 2.76; 95% CI, 1.02-7.5; P = .0459). Neither observation retained significance after multiple-test adjustment. FCGR2B was associated with poorer PFS in multivariate analyses comparing T232T with I232I in rituximab- but not obinutuzumab-treated patients with DLBCL (HR, 4.40; 95% CI, 1.71-11.32; P = .002; multiple-test-adjusted P = .03); however, this genotype was rare (n = 13). This study shows that FcγR genotype is not associated with response to rituximab/obinutuzumab plus chemotherapy in treatment-naive patients with advanced FL or DLBCL.

Prognostic significance of FCGR2B expression for the response of DLBCL patients to rituximab or obinutuzumab treatment.

Fc γ receptor IIB (FcγRIIB) is an inhibitory molecule capable of reducing antibody immunotherapy efficacy. We hypothesized its expression could confer resistance in patients with diffuse large B-cell lymphoma (DLBCL) treated with anti-CD20 monoclonal antibody (mAb) chemoimmunotherapy, with outcomes varying depending on mAb (rituximab [R]/obinutuzumab [G]) because of different mechanisms of action. We evaluated correlates between FCGR2B messenger RNA and/or FcγRIIB protein expression and outcomes in 3 de novo DLBCL discovery cohorts treated with R plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) reported by Arthur, Schmitz, and Reddy, and R-CHOP/G-CHOP-treated patients in the GOYA trial (NCT01287741). In the discovery cohorts, higher FCGR2B expression was associated with significantly shorter progression-free survival (PFS; Arthur: hazard ratio [HR], 1.09; 95% confidence interval [CI], 1.01-1.19; P = .0360; Schmitz: HR, 1.13; 95% CI, 1.02-1.26; P = .0243). Similar results were observed in GOYA with R-CHOP (HR, 1.26; 95% CI, 1.00-1.58; P = .0455), but not G-CHOP (HR, 0.91; 95% CI, 0.69-1.20; P = .50). A nonsignificant trend that high FCGR2B expression favored G-CHOP over R-CHOP was observed (HR, 0.67; 95% CI, 0.44-1.02; P = .0622); however, low FCGR2B expression favored R-CHOP (HR, 1.58; 95% CI, 1.00-2.50; P = .0503). In Arthur and GOYA, FCGR2B expression was associated with tumor FcγRIIB expression; correlating with shorter PFS for R-CHOP (HR, 2.17; 95% CI, 1.04-4.50; P = .0378), but not G-CHOP (HR, 1.37; 95% CI, 0.66-2.87; P = .3997). This effect was independent of established prognostic biomarkers. High FcγRIIB/FCGR2B expression has prognostic value in R-treated patients with DLBCL and may confer differential responsiveness to R-CHOP/G-CHOP.

Increased circulating resistin levels in early-onset breast cancer patients of normal body mass index correlate with lymph node negative involvement and longer disease free survival: a multi-center POSH cohort serum proteomics study.

BACKGROUND: Early-onset breast cancer (EOBC) affects about one in 300 women aged 40 years or younger and is associated with worse outcomes than later onset breast cancer. This study explored novel serum proteins as surrogate markers of prognosis in patients with EOBC. METHODS: Serum samples from EOBC patients (stages 1-3) were analysed using agnostic high-precision quantitative proteomics. Patients received anthracycline-based chemotherapy. The discovery cohort (n = 399) either had more than 5-year disease-free survival (DFS) (good outcome group, n = 203) or DFS of less than 2 years (poor outcome group, n = 196). Expressed proteins were assessed for differential expression between the two groups. Bioinformatics pathway and network analysis in combination with literature research were used to determine clinically relevant proteins. ELISA analysis against an independent sample set from the Prospective study of Outcomes in Sporadic versus Hereditary breast cancer (POSH) cohort (n = 181) was used to validate expression levels of the selected target. Linear and generalized linear modelling was applied to determine the effect of target markers, body mass index (BMI), lymph node involvement (LN), oestrogen receptor (ER), progesterone receptor and human epidermal growth factor receptor 2 status on patients' outcome. RESULTS: A total of 5346 unique proteins were analysed (peptide FDR p ≤ 0.05). Of these, 812 were differentially expressed in the good vs poor outcome groups and showed significant enrichment for the insulin signalling (p = 0.01) and the glycolysis/gluconeogenesis (p = 0.01) pathways. These proteins further correlated with interaction networks involving glucose and fatty acid metabolism. A consistent nodal protein to these metabolic networks was resistin (upregulated in the good outcome group, p = 0.009). ELISA validation demonstrated resistin to be upregulated in the good outcome group (p = 0.04), irrespective of BMI and ER status. LN involvement was the only covariate with a significant association with resistin measurements (p = 0.004). An ancillary in-silico observation was the induction of the inflammatory response, leucocyte infiltration, lymphocyte migration and recruitment of phagocytes (p < 0.0001, z-score > 2). Survival analysis showed that resistin overexpression was associated with improved DFS. CONCLUSIONS: Higher circulating resistin correlated with node-negative patients and longer DFS independent of BMI and ER status in women with EOBC. Overexpression of serum resistin in EOBC may be a surrogate indicator of improved prognosis.

Evaluation of High-Throughput Genomic Assays for the Fc Gamma Receptor Locus.

Cancer immunotherapy has been revolutionised by the use monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics.

Stimulation of surface IgM of chronic lymphocytic leukemia cells induces an unfolded protein response dependent on BTK and SYK.

B-cell receptor (BCR) signaling plays a key role in the behavior of chronic lymphocytic leukemia (CLL). However, cellular consequences of signaling are incompletely defined. Here we explored possible links between BCR signaling and the unfolded protein response (UPR), a stress response pathway that can promote survival of normal and malignant cells. Compared with normal B cells, circulating CLL cells expressed increased, but variable, levels of UPR components. Higher expression of CHOP and XBP1 RNAs was associated with more aggressive disease. UPR activation appeared due to prior tissue-based antigenic stimulation because elevated expression of UPR components was detected within lymph node proliferation centers. Basal UPR activation also correlated closely with surface immunoglobulin M (sIgM) signaling capacity in vitro in both IGHV unmutated CLL and within mutated CLL. sIgM signaling increased UPR activation in vitro with responders showing increased expression of CHOP and XBP1 RNAs, and PERK and BIP proteins, but not XBP1 splicing. Inhibitors of BCR-associated kinases effectively prevented sIgM-induced UPR activation. Overall, this study demonstrates that sIgM signaling results in activation of some components the UPR in CLL cells. Modulation of the UPR may contribute to variable clinical behavior, and its inhibition may contribute to clinical responses to BCR-associated kinase inhibitors.

The IGHV1-69/IGHJ3 recombinations of unmutated CLL are distinct from those of normal B cells.

IGHV1-69/51p1 is expressed by ∼ 30% of unmutated chronic lymphocytic leukemia (U-CLL) and combines with selected IGHD and IGHJ genes generating stereotypes if HCDR3 amino acid homology is > 60%. We had previously revealed stereotypic IGHV1-69/IGHJ6 rearrangements in normal naive B cells, thereby identifying potential counterparts of U-CLL. A different stereotypic IGHV1-69/IGHD3-16(RF2)/IGHJ3 rearrangement carrying the CAR(GGx)YD motif in the N1-region, recurrent in 6% IGHV1-69+ve CLL, is exceptionally sequence restricted, strongly suggestive of shared antigen recognition. We have now analyzed IGHV1-69/IGHJ3 rearrangements in circulating B cells of healthy individuals using several PCR-based approaches with IGHV1-69/IGHJ3 CLL sequences for reference. Stereotypes were found, but all were distinct from CLL. Remarkably, even a highly sensitive semi-nested PCR, specific for the CLL-expressed IGHV1-69/IGHD3-16(RF2)/IGHJ3 stereotype, failed to identify the CAR(GGx)YD sequence, although similar motifs were found. These highly specific B cells are not apparent in the accessible normal repertoire and may expand in response to rarely expressed antigens important in the pathogenesis of CLL.

Mechanisms and clinical significance of BIM phosphorylation in chronic lymphocytic leukemia.

B-cell receptor and microenvironment-derived signals promote accumulation of chronic lymphocytic leukemia (CLL) cells through increased proliferation and/or decreased apoptosis. In this study, we investigated the regulation of BIM, a proapoptotic BCL2-related protein, which is tightly regulated by phosphorylation. Surface IgM stimulation increased phosphorylation of 2 BIM isoforms, BIM(EL) and BIM(L), in a subset of CLL samples. In contrast, in normal B cells, anti-IgM triggered selective phosphorylation of BIM(EL) only. In CLL, anti-IgM-induced BIM phosphorylation correlated with unmutated IGHV gene status and with progressive disease. Strikingly, it was also associated with progressive disease within the mutated IGHV gene subset. BIM phosphorylation was dependent on MEK1/2 kinase activity, and we identified BIM(EL) serine 69, previously linked to pro-survival responses, as the major site of phosphorylation in CLL and in Ramos cells. BIM(EL)/BIM(L) phosphorylation was associated with release of the pro-survival protein MCL1. Coculture of CLL cells with HK cells, a model of the CLL microenvironment, promoted CLL cell survival and was associated with MEK1/2 activation and BIM(EL) phosphorylation. Hence, BIM phosphorylation appears to play a key role in apoptosis regulation in CLL cells, potentially coordinating antigen and microenvironment-derived survival signals. Antigen-mediated effects on BIM may be an important determinant of clinical behavior.

Surface IgM stimulation induces MEK1/2-dependent MYC expression in chronic lymphocytic leukemia cells.

Although long considered as a disease of failed apoptosis, it is now clear that chronic lymphocytic leukemia (CLL) cells undergo extensive cell division in vivo, especially in progressive disease. Signaling via the B-cell receptor is thought to activate proliferation and survival pathways in CLL cells and also has been linked to poor outcome. Here, we have analyzed the expression of the proto-oncoprotein MYC, an essential positive regulator of the cell cycle, after stimulation of surface IgM (sIgM). MYC expression was rapidly increased after sIgM stimulation in a subset of CLL samples. The ability of sIgM stimulation to increase MYC expression was correlated with sIgM-induced intracellular calcium fluxes. MYC induction was partially dependent on the MEK/ERK signaling pathway, and MYC and phosphorylated ERK1/2 were both expressed within proliferation centers in vivo. Although stimulation of sIgD also resulted in ERK1/2 phosphorylation, responses were relatively short lived compared with sIgM and were associated with significantly reduced MYC induction, suggesting that the kinetics of ERK1/2 activation is a critical determinant of MYC induction. Our results suggest that ERK1/2-dependent induction of MYC is likely to play an important role in antigen-induced CLL cell proliferation.

Fc gamma receptor IIb on target B cells promotes rituximab internalization and reduces clinical efficacy.

The anti-CD20 mAb rituximab is central to the treatment of B-cell malignancies, but resistance remains a significant problem. We recently reported that resistance could be explained, in part, by internalization of rituximab (type I anti-CD20) from the surface of certain B-cell malignancies, thus limiting engagement of natural effectors and increasing mAb consumption. Internalization of rituximab was most evident in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), but the extent of internalization was heterogeneous within each disease. Here, we show that the inhibitory FcγRIIb on target B cells promotes this process and is largely responsible for the observed heterogeneity across a range of B-cell malignancies. Internalization correlated strongly with FcγRIIb expression on normal and malignant B cells, and resulted in reduced macrophage phagocytosis of mAb-coated targets. Furthermore, transfection of FcγRIIb into FcγRIIb negative Ramos cells increased internalization of rituximab in a dose-dependent manner. Target-cell FcγRIIb promoted rituximab internalization in a cis fashion and was independent of FcγRIIb on neighboring cells. It became phosphorylated and internalized along with CD20:anti-CD20 complexes before lysosomal degradation. In MCL patients, high FcγRIIb expression predicted less durable responses after rituximab-containing regimens. Therefore, target-cell FcγRIIb provides a potential biomarker of response to type I anti-CD20 mAb.

Glycosylation of surface Ig creates a functional bridge between human follicular lymphoma and microenvironmental lectins.

Surface Ig (sIg) of follicular lymphoma (FL) is vital for tumor cell survival. We found previously that the Ig in FL is unusual, because the variable region genes carry sequence motifs for N-glycan addition. These are introduced by somatic mutation and are tumor specific. Unexpectedly, added glycans terminate at high mannose, suggesting a potentially important interaction of FL cells with mannose-binding lectins of the innate immune system. We have now identified mannosylated IgM at the surface of primary lymphoma cells. Recombinant lectin domains of the mannose receptor (MR) or DC-SIGN bind mannosylated Igs in vitro and bind to FL cells, signaling sIgM-associated increases in intracellular Ca(2+). Lectins also bind to normal B cells but fail to signal. In contrast, anti-Ig signaled similarly in both FL and normal B cells. Mannosylation patterns were mimicked by FL Ig-derived single-chain Fvs (scFv), providing probes for potential receptors. Mannosylated scFv bound specifically to the lectin domains of the MR and DC-SIGN and blocked signaling. Mannosylated scFv also bound to DC-SIGN on the surface of dendritic cells. This unique lymphoma-specific interaction of sIg with lectins of innate immunity reveals a potential route for microenvironmental support of tumor cells, mediated via the key B-cell receptor.

Surface IgM of CLL cells displays unusual glycans indicative of engagement of antigen in vivo.

Surface IgM (sIgM) has a key influence on the clinical behavior of chronic lymphocytic leukemia (CLL). We now report that it exists in 2 forms with different N-glycosylation patterns in the mu-constant region. One glycoform is similar to normal B cells in bearing mature complex glycans common to most cell-surface glycoproteins. The other is an immature mannosylated form more characteristic of mu chains in the endoplasmic reticulum. Unmutated CLL (U-CLL) expresses a higher proportion of mannosylated surface mu chains than mutated CLL. Normal B cells express only the mature glycoform but can express the immature form after persistent engagement of sIgM, suggesting that glycan modification is a consequence of antigen exposure. CLL cells express variable proportions of the mannosylated form and can revert to the mature form after incubation in vitro. Both glycoforms are able to signal after sIgM engagement in vitro, leading to enhanced tyrosine phosphorylation. These findings support the concept that CLL cells are continuously exposed to antigen in vivo, driving the N-glycosylation pattern of expressed sIgM toward a mannosylated form, especially in U-CLL. Strikingly, this is reminiscent of follicular lymphoma, where mannosylated Ig is expressed constitutively via N-glycosylation sites in the variable region, suggesting a functional asset for this glycoform.

The normal IGHV1-69-derived B-cell repertoire contains stereotypic patterns characteristic of unmutated CLL.

The cell of origin of chronic lymphocytic leukemia (CLL) has long been sought, and immunoglobulin gene analysis provides new clues. In the unmutated subset (U-CLL), there is increased usage of the 51p1-related alleles of the immunoglobulin heavy chain variable 1-69 gene, often combined with selected genes and with immunoglobulin heavy chain diversity IGHJ6. Stereotypic characteristics of the HCDR3 result and suggest antigen selection of the leukemic clones. We have now analyzed 51p1/IGHJ6 combinations in normal blood B cells from 3 healthy persons for parallel sequence patterns. A high proportion (33.3% of sequences) revealed stereotypic patterns, with several (15.0%) being similar to those described in U-CLL. Previously unreported CLL-associated stereotypes were detected in 4.8%. Stereotypes (13.6%) not detected in CLL also were found. The HCDR2-IGHJ6 sequences were essentially unmutated. Junctional amino acids in normal B cells were heterogeneous, as in cases of stereotyped CLL. Phenotypically, normal B cells expressing 51p1-derived immunoglobulin M were naive. This snapshot of the naive B-cell repertoire reveals subsets of B cells closely related to those characteristic of CLL. Conserved patterns in the 51p1-encoded immunoglobulin M of normal B cells suggest a restricted sequence repertoire shaped by evolution to recognize common pathogens. Proliferative pressure on these cells is the likely route to U-CLL.

Monoclonal antibodies directed to CD20 and HLA-DR can elicit homotypic adhesion followed by lysosome-mediated cell death in human lymphoma and leukemia cells.

mAbs are becoming increasingly utilized in the treatment of lymphoid disorders. Although Fc-FcgammaR interactions are thought to account for much of their therapeutic effect, this does not explain why certain mAb specificities are more potent than others. An additional effector mechanism underlying the action of some mAbs is the direct induction of cell death. Previously, we demonstrated that certain CD20-specific mAbs (which we termed type II mAbs) evoke a nonapoptotic mode of cell death that appears to be linked with the induction of homotypic adhesion. Here, we reveal that peripheral relocalization of actin is critical for the adhesion and cell death induced by both the type II CD20-specific mAb tositumomab and an HLA-DR-specific mAb in both human lymphoma cell lines and primary chronic lymphocytic leukemia cells. The cell death elicited was rapid, nonapoptotic, nonautophagic, and dependent on the integrity of plasma membrane cholesterol and activation of the V-type ATPase. This cytoplasmic cell death involved lysosomes, which swelled and then dispersed their contents, including cathepsin B, into the cytoplasm and surrounding environment. The resulting loss of plasma membrane integrity occurred independently of caspases and was not controlled by Bcl-2. These experiments provide what we believe to be new insights into the mechanisms by which 2 clinically relevant mAbs elicit cell death and show that this homotypic adhesion-related cell death occurs through a lysosome-dependent pathway.

Reversible anergy of sIgM-mediated signaling in the two subsets of CLL defined by VH-gene mutational status.

The 2 subsets of chronic lymphocytic leukemia (CLL), of worse or better prognosis, likely derive from pre-GC unmutated B cells, or post-GC mutated B cells, respectively. Different clinical behavior could relate to the ability of tumor cells to respond to surface (sIg)-mediated signals. Unmutated cases (U-CLL) have an increased ability to phosphorylate p72(Syk) in response to sIgM ligation compared to mutated cases (M-CLL). We now confirm and further investigate this differential signaling in a large cohort by [Ca(2+)](i) mobilization. Cases responding to sIgM ligation express higher levels of CD38, ZAP-70, and sIgM. However, CD38 does not influence signaling in vitro or associate with response in bimodal CD38-expressing cases. Similarly, ZAP-70 expression is not required for response in either U-CLL or M-CLL. Strikingly, partially or completely anergized sIgM responses from each subset can recover both sIgM expression and signal capacity spontaneously in vitro or following capping/endocytosis. This provides direct evidence for engagement of putative antigen in vivo. Signaling via sIgD differs markedly being almost universally positive in both U-CLL and M-CLL, with no association with CD38 or ZAP-70 expression. Downstream signaling pathways, therefore, appear intact in CLL, locating anergy to sIgM, mainly in M-CLL. Integration of differential isotype-specific effects mediated by (auto)antigen may determine tumor behavior.

Human follicular lymphoma cells contain oligomannose glycans in the antigen-binding site of the B-cell receptor.

Expression of surface immunoglobulin appears critical for the growth and survival of B-cell lymphomas. In follicular lymphoma, we found previously that the Ig variable (V) regions in the B-cell receptor express a strikingly high incidence of N-glycosylation sequons, NX(S/T). These potential glycosylation sites are introduced by somatic mutation and are lymphoma-specific, pointing to their involvement in tumor pathogenesis. Analysis of the V region sugars from lymphoma-derived IgG/IgM reveals that they are mostly oligomannose and, remarkably, are located in the antigen-binding site, possibly precluding conventional antigen binding. The Fc region contains complex glycans, confirming that the normal glycan processing pathway is intact. Binding studies indicate that the oligomannose glycans occupying the V regions are accessible to mannose-binding lectin. These findings suggest a potential contribution to lymphoma pathogenesis involving antigen-independent interaction of surface immunoglobulin of the B-cell receptor with mannose-binding molecules of innate immunity in the germinal center.

Structural and functional features of the B-cell receptor in IgG-positive chronic lymphocytic leukemia.

PURPOSE: To determine the origin and relationship of the rare IgG+ variant of chronic lymphocytic leukemia (CLL) to the two common IgM+IgD+ subsets that are distinguished by expression of unmutated or mutated V(H) genes, with the former having a worse prognosis. EXPERIMENTAL DESIGN: IgG+ CLL cells were characterized using phenotypic, functional, and immunogenetic analyses. RESULTS: IgG+ CLL was phenotypically similar to mutated IgM+IgD+ CLL (M-CLL) and variably expressed CD38 (4 of 14). ZAP-70, a tyrosine kinase preferentially expressed in unmutated CLL, was found in only 2 of 14 cases. The ability to signal via surface IgM (sIgM) varies between the main subsets of CLL and is associated with expression of ZAP-70. In IgG(+) CLL, 9 of 14 responded to engagement of sIgG with no apparent requirement for expression of CD38 or ZAP-70. However, signal capacity correlated with intensity of sIgG expression. Most switched immunoglobulin variable region genes were somatically mutated without intraclonal variation, and no case expressed activation-induced cytidine deaminase. Derivation from a postgerminal center B cell is, therefore, likely, and a relationship with M-CLL is suggested. This is supported by a shared biased usage of the V4-34 gene. Similar bias in normal B cells developed with age, providing an expanded population for transforming events. However, conserved sequences detected in the CDR3 of V4-34-encoded gamma chains were not found M-CLL, indicating no direct path of isotype switch from M-CLL. CONCLUSION: IgG+ CLL is likely to arise from an age-related expanded pool of B cells, on a path parallel to M-CLL, and perhaps with a similar clinical course.

Antibodies selected from combinatorial libraries block a tumor antigen that plays a key role in immunomodulation.

We searched for cell-surface-associated proteins overexpressed on B cell chronic lymphocytic leukemia (CLL) to use as therapeutic antibody targets. Antibodies binding the immunosuppressive molecule CD200 were identified by cell panning of an antibody phage display library derived from rabbits immunized with primary CLL cells. B cells from 87 CLL patients exhibited 1.6- to 5.4-fold cell-surface up-regulation of CD200 relative to normal B cells. An effect of increased CD200 expression by CLL cells on the immune system was evaluated in mixed lymphocyte reactions. Addition of primary CLL but not normal B cells to macrophages and T cells downregulated the Th1 response, as seen by a 50-95% reduction in secreted IL-2 and IFN-gamma. Antibodies to CD200 prevented downregulation of the Th1 response in most B cell CLL samples evaluated, indicating abrogation of the CD200/CD200R interaction can be sufficient to restore the Th1 response. A disease-progression-associated shift of the immune response from Th1 to Th2 has been observed in numerous cancers. Because this cytokine shift is also believed to promote the induction of regulatory T cells, reverting the immune response to Th1 through direct targeting of the cancer cells may provide therapeutic benefits in CLL by encouraging a cytotoxic T cell response.

The relevance of sequence insertions in the Mcl-1 promoter in chronic lymphocytic leukemia and in normal cells.

We measured the frequency of insertions in the Mcl-1 promoter in chronic lymphocytic leukemia (CLL) and in normal individuals. Insertions were found in 37/54 (69%) of the CLL samples. However, insertions were not associated with prognostic markers and were also detected in 38/66 (58%) of normal controls and in normal cells isolated from CLL patients. Thus, Mcl-1 insertions are not acquired during leukemogenesis and are unlikely to play an important role in this disease.