Leukemia-cell proliferation and disease progression in patients with early stage chronic lymphocytic leukemia.

The clinical course of patients with recently diagnosed early stage chronic lymphocytic leukemia (CLL) is highly variable. We examined the relationship between CLL-cell birth rate and treatment-free survival (TFS) in 97 patients with recently diagnosed, Rai stage 0-II CLL in a blinded, prospective study, using in vivo 2H2O labeling. Birth rates ranged from 0.07 to 1.31% new cells per day. With median follow-up of 4.0 years, 33 subjects (34%) required treatment by NCI criteria. High-birth rate was observed in 44% of subjects and was significantly associated with shorter TFS, unmutated IGHV status and expression of ZAP70 and of CD38. In multivariable modeling considering age, gender, Rai stage, expression of ZAP70 or CD38, IGHV mutation status and FISH cytogenetics, only CLL-cell birth rate and IGHV mutation status met criteria for inclusion. Hazard ratios were 3.51 (P=0.002) for high-birth rate and 4.93 (P<0.001) for unmutated IGHV. The association between elevated birth rate and shorter TFS was observed in subjects with either mutated or unmutated IGHVs, and the use of both markers was a better predictor of TFS than either parameter alone. Thus, an increased CLL birth rate in early stage disease is a strong predictor of disease progression and earlier treatment.

EGR2 mutations define a new clinically aggressive subgroup of chronic lymphocytic leukemia.

Recurrent mutations within EGR2 were recently reported in advanced-stage chronic lymphocytic leukemia (CLL) patients and associated with a worse outcome. To study their prognostic impact, 2403 CLL patients were examined for mutations in the EGR2 hotspot region including a screening (n=1283) and two validation cohorts (UK CLL4 trial patients, n=366; CLL Research Consortium (CRC) patients, n=490). Targeted deep-sequencing of 27 known/postulated CLL driver genes was also performed in 38 EGR2-mutated patients to assess concurrent mutations. EGR2 mutations were detected in 91/2403 (3.8%) investigated cases, and associated with younger age at diagnosis, advanced clinical stage, high CD38 expression and unmutated IGHV genes. EGR2-mutated patients frequently carried ATM lesions (42%), TP53 aberrations (18%) and NOTCH1/FBXW7 mutations (16%). EGR2 mutations independently predicted shorter time-to-first-treatment (TTFT) and overall survival (OS) in the screening cohort; they were confirmed associated with reduced TTFT and OS in the CRC cohort and independently predicted short OS from randomization in the UK CLL4 cohort. A particularly dismal outcome was observed among EGR2-mutated patients who also carried TP53 aberrations. In summary, EGR2 mutations were independently associated with an unfavorable prognosis, comparable to CLL patients carrying TP53 aberrations, suggesting that EGR2-mutated patients represent a new patient subgroup with very poor outcome.

BTK inhibition results in impaired CXCR4 chemokine receptor surface expression, signaling and function in chronic lymphocytic leukemia.

Bruton's tyrosine kinase (BTK) is involved in the regulation of B-cell growth, migration and adhesion. The importance of BTK in cell trafficking is emphasized by the clonal contraction proceeded by lymphocytosis typical for the enzyme inhibitor, ibrutinib, in B-cell malignancies, including chronic lymphocytic leukemia (CLL). Here, we investigated BTK regulation of leukemic B-cell trafficking in a mouse model of aggressive TCL1 CLL-like disease. Inhibiting BTK by ibrutinib reduced surface membrane (sm) levels of CXCR4 but not CXCR5, CD49d and other adhesion/homing receptors. Decreased smCXCR4 levels resulted from blocking receptor signal transduction, which in turn aborted cycling from and to the membrane. This resulted in rapid re-distribution of CLL cells from spleens and lymph nodes into the circulation. CLL cells with impaired smCXCR4 from BTK inhibition failed to home to spleens. These functional changes mainly resulted from inhibition of CXCR4 phosphorylation at Ser339, mediated directly by blocking BTK enzymatic activity and indirectly by affecting the function of downstream targets PLCγ2 and PKCµ, and eventually synthesis of PIM-1 and BTK itself. Our data identify CXCR4 as a key regulator in BTK-mediated CLL-cell retention and have elucidated a complex set of not previously described mechanisms responsible for these effects.

Cytoplasmic myosin-exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability.

The degree of chronic lymphocytic leukemia (CLL) B-cell antigen receptor (BCR) binding to myosin-exposed apoptotic cells (MEACs) correlates with worse patient outcomes, suggesting a link to disease activity. Therefore, we studied MEAC formation and the effects of MEAC binding on CLL cells. In cell line studies, both intrinsic (spontaneous or camptothecin-induced) and extrinsic (FasL- or anti-Fas-induced) apoptosis created a high percent of MEACs over time in a process associated with caspase-3 activation, leading to cytoplasmic myosin cleavage and trafficking to cell membranes. The involvement of common apoptosis pathways suggests that most cells can produce MEACs and indeed CLL cells themselves form MEACs. Consistent with the idea that MEAC formation may be a signal to remove dying cells, we found that natural IgM antibodies bind to MEACs. Functionally, co-culture of MEACs with CLL cells, regardless of immunoglobulin heavy-chain variable region gene mutation status, improved leukemic cell viability. Based on inhibitor studies, this improved viability involved BCR signaling molecules. These results support the hypothesis that stimulation of CLL cells with antigen, such as those on MEACs, promotes CLL cell viability, which in turn could lead to progression to worse disease.

Distinct patterns of novel gene mutations in poor-prognostic stereotyped subsets of chronic lymphocytic leukemia: the case of SF3B1 and subset #2.

Recent studies have revealed recurrent mutations of the NOTCH1, SF3B1 and BIRC3 genes in chronic lymphocytic leukemia (CLL), especially among aggressive, chemorefractory cases. Nevertheless, it is currently unknown whether their presence may differ in subsets of patients carrying stereotyped B-cell receptors and also exhibiting distinct prognoses. Here, we analyzed the mutation status of NOTCH1, SF3B1 and BIRC3 in three subsets with particularly poor prognosis, that is, subset #1, #2 and #8, aiming to explore links between genetic aberrations and immune signaling. A remarkably higher frequency of SF3B1 mutations was revealed in subset #2 (44%) versus subset #1 and #8 (4.6% and 0%, respectively; P<0.001). In contrast, the frequency of NOTCH1 mutations in subset #2 was only 8%, lower than the frequency observed in either subset #1 or #8 (19% and 14%, respectively; P=0.04 for subset #1 versus #2). No associations were found for BIRC3 mutations that overall were rare. The apparent non-random association of certain mutations with stereotyped CLL subsets alludes to subset-biased acquisition of genomic aberrations, perhaps consistent with particular antigen/antibody interactions. These novel findings assist in unraveling specific mechanisms underlying clinical aggressiveness in poor-prognostic stereotyped subsets, with far-reaching implications for understanding their clonal evolution and implementing biologically oriented therapy.

A different ontogenesis for chronic lymphocytic leukemia cases carrying stereotyped antigen receptors: molecular and computational evidence.

Chronic lymphocytic leukemia (CLL) is uniquely characterized by the existence of subsets of cases with quasi-identical, 'stereotyped' B-cell receptors (BCRs). Herein we investigate this stereotypy in 2662 patients with CLL, the largest series yet, using purpose-built bioinformatics methods based on sequence pattern discovery. Besides improving the identification of 'stereotyped' cases, we demonstrate that CLL actually consists of two different categories, based on the BCR repertoire, with important biological and ontogenetic differences. The first ( approximately 30% of cases) shows a very restricted repertoire and is characterized by BCR stereotypy (clustered cases), whereas the second includes cases with heterogeneous BCRs (nonclustered cases). Eleven major CLL clusters were identified with antigen-binding sites defined by just a few critically positioned residues, regardless of the actual immunoglobulin (IG) variable gene used. This situation is closely reminiscent of the receptors expressed by cells participating in innate immune responses. On these grounds, we argue that whereas CLL cases with heterogeneous BCRs likely derive from the conventional B-cell pool, cases with stereotyped BCRs could derive from progenitor cells evolutionarily adapted to particular antigenic challenges, perhaps intermediate between a true innate immune system and the conventional adaptive B-cell immune system, functionally similar to what has been suggested previously for mouse B1 cells.

Microenvironmental influences in chronic lymphocytic leukaemia: the role of antigen stimulation.

Several studies suggest that immune-mediated pathways are important in the pathogenesis of chronic lymphocytic leukaemia (CLL). The in vivo accumulation of leukaemic lymphocytes is facilitated by interactions of CLL cells with other cells and soluble factors that probably occur more often within the microenvironment through classical receptor-ligand interactions. These include CD40L-CD40 and chemokine-chemokine receptor interactions as well as B cell receptor (BCR) engagement by (auto)antigens. Indeed, the categorizations of CLL patients based on immunoglobulin heavy variable (IGHV) gene mutations and structure of the clone's BCR suggest that CLL patient outcome could be a reflection of ongoing BCR signalling in the context of other co-signals.

Clinical and laboratory parameters that define clinically relevant B-CLL subgroups.

B cell-type chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease. This is reflected by the very wide-ranging clinical courses that B-CLL patients experience and by the marked variation in laboratory findings between patients. In this chapter, we will review the various clinical and laboratory parameters that divide B-CLL patients into "subgroups," and correlate the parameters that define them. When feasible, we will also link clinical features to the cellular and genetic characteristics recently defined for these leukemic cells. The discussion is limited to parameters that define phenotypes or subgroups that may relate to disease activity and clinical outcome.

High mobility group box chromosomal protein 1: a novel proinflammatory mediator in synovitis.

OBJECTIVE: High mobility group box chromosomal protein 1 (HMGB-1) is a ubiquitous chromatin component expressed in nucleated mammalian cells. It has recently and unexpectedly been demonstrated that stimulated live mononuclear phagocytes secrete HMGB-1, which then acts as a potent factor that causes inflammation and protease activation. Macrophages play pivotal roles in the pathogenesis of arthritis. The aim of this study was to determine whether synovial macrophage expression of HMGB-1 is altered in human and experimental synovitis. METHODS: Intraarticular tissue specimens were obtained from healthy Lewis rats, Lewis rats with Mycobacterium tuberculosis-induced adjuvant arthritis, and from patients with rheumatoid arthritis (RA). Specimens were immunohistochemically stained for cellular HMGB-1. Extracellular HMGB-1 levels were assessed in synovial fluid samples from RA patients by Western blotting. RESULTS: Immunostaining of specimens from normal rats showed that HMGB-1 was primarily confined to the nucleus of synoviocytes and chondrocytes, with occasional cytoplasmic staining and no extracellular matrix deposition. In contrast, inflammatory synovial tissue from rats with experimental arthritis as well as from humans with RA showed a distinctly different HMGB-1 staining pattern. Nuclear HMGB-1 expression was accompanied by a cytoplasmic staining in many mononuclear cells, with a macrophage-like appearance and an extracellular matrix deposition. Analysis of synovial fluid samples from RA patients further confirmed the extracellular presence of HMGB-1; 14 of 15 samples had HMGB-1 concentrations of 1.8-10.4 microg/ml. CONCLUSION: The proinflammatory mediator HMGB-1 was abundantly expressed as a nuclear, cytoplasmic, and extracellular component in synovial tissues from RA patients and from rats with experimental arthritis. These findings suggest a pathogenetic role for HMGB-1 in synovitis and indicate a new potential therapeutic target molecule.

Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia.

The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression "signature," irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

In vitro stimulation of human tonsillar subepithelial B cells: requirement for interaction with activated T cells.

Human tonsillar subepithelial B cells, which are a marginal zone-equivalent B cell subset, respond readily to T-independent type 2 antigens, but not to polyclonal B cell activators in vitro. In this study, subepithelial (SE) B cells were induced to proliferate and mature into plasma cells when co-cultured with activated T cells. The response of SE B cells was not observed when co-cultures were carried out in transwell chambers or in the presence of blocking anti-LFA-1 antibodies, demonstrating the need for a close T-B cell interaction. The presence of soluble CD40 also prevented the B cell response in vitro suggesting a pivotal role of CD40-CD40 ligand interactions. The data are discussed in terms of the T cell dependence of marginal zone (MZ) B cell response and the possible existence of various MZ B cell subsets.

Analysis of stepwise genetic changes in an AIDS-related Burkitt's lymphoma.

In this study, immunoglobulin variable (Ig V) region genes, c-myc re-arrangement and sequence and p53 status were analyzed in clones derived from a Burkitt's lymphoma cell line (LAM) in which it was previously demonstrated that Epstein-Barr virus (EBV) infection occurred late during lymphomagenesis. Such evidence was based on the finding that 2 groups of cellular clones, characterized by the same c-myc re-arrangement but different EBV-fused termini, were obtained from the LAM cell line. The Ig V gene sequences were identical for the 2 groups of clones with different EBV-fused termini. The Ig variable heavy (V(H)) gene sequence displayed a substantial accumulation of point mutations (but no intra-clonal diversification), whereas the productive Ig V lambda (V(lambda)) gene sequence was virtually unmutated. Studies on the Ig V kappa (V(kappa)) locus suggested a receptor revision event (with a switch from kappa to lambda chain production) prior to EBV infection. Likewise, it was determined that the mutations observed in both p53 alleles and in the re-arranged c-myc gene occurred before EBV infection. Based on these findings, we present a model for the various steps of lymphomagenesis. It is proposed that stimulation by an antigen or a superantigen initially favored the clonal expansion and accumulation of other cytogenetic changes, including those involved in receptor editing. These events occurred prior to or during the germinal center (GC) phase of B-cell maturation. Thereafter, possibly upon exit of the cells from the GC, EBV infection occurred, further promoting lymphomagenesis.

Identification of three subgroups of B cell chronic lymphocytic leukemia based upon mutations of BCL-6 and IgV genes.

Although B cell chronic lymphocytic leukemia (B-CLL) has been traditionally viewed as a tumor of virgin B cells, this notion has been recently questioned by data suggesting that a fraction of B-CLL derives from antigen experienced B cells. In order to further clarify the histogenetic derivation of this lymphoproliferation, we have analyzed the DNA sequences of the 5' non-coding region of BCL-6 proto-oncogene in 28 cases of B-CLL. Mutations of BCL-6 proto-oncogene, a zinc finger transcription factor implicated in lymphoma development, represent a histogenetic marker of B cell transit through the germinal center (GC) and occur frequently in B cell malignancies derived from GC or post-GC B cells. For comparison, the same tumor panel was analyzed for somatic mutations of the rearranged immunoglobulin variable (IgV) genes, which are known to be acquired at the time of B cell transit through the GC. Sequence analyses of BCL-6 and IgV genes allowed the definition of three groups of B-CLL. Group I B-CLL displayed mutations of both BCL-6 and IgV genes (10/28; 36%). Group II B-CLL displayed mutated IgV genes, but a germline BCL-6 gene (5/28; 18%). Finally, group III B-CLL included the remaining cases (13/28; 46%) that were characterized by the absence of somatic mutations of both BCL-6 and IgV genes. Overall, the distribution of BCL-6 and IgV mutations in B-CLL reinforce the notion that this leukemia is histogenetically heterogeneous and that a substantial subgroup of these lymphoproliferations derives from post-germinal center B cells.

Heterogeneity of tonsillar subepithelial B lymphocytes, the splenic marginal zone equivalents.

The VH4 genes expressed by both resting and in vivo-activated subepithelial (SE) B cells from human tonsils were studied. Resting SE B cells were subdivided according to the presence (IgDlow) or absence (IgM-only) of surface IgD. CD27 was abundant on activated SE B cells and low on resting IgM-only B cells. Resting IgDlow SE B cells could be subdivided into CD27low and CD27high cell fractions. Resting IgDlow SE B cells displayed VH4 genes with a substantial number of mutations (13/29 of the molecular clones were mutated), whereas 25/26 of the clones from resting IgM-only SE B cells were unmutated. Moreover, mutated VH4 genes were detected mainly within the CD27high cell fraction of the IgDlow SE B cells. Several identical unmutated VH4DJH sequences (11/32) were found in different molecular clones from resting IgM-only SE B cells, suggesting local cellular expansion. Both unmutated (14/25) and mutated (11/25) sequences were found in mu transcripts of activated SE B cells. Extensive mutation was observed in the gamma transcripts of activated SE B cells. Therefore, SE B cells are heterogeneous, being comprised of B cells with mutated Ig VH4 genes, that are Ag-experienced B cells, and a subset of B cells with unmutated VH4 genes that are either virgin cells or cells driven by Ags that did not induce or select for V gene mutations.

VH gene usage by family members affected with chronic lymphocytic leukaemia.

The excess risk of chronic lymphocytic leukaemia (CLL) in the first-degree relatives of affected patients suggests that familial CLL might constitute a useful model to study the pathogenesis of this disease, as has been demonstrated in numerous other neoplastic disorders. Previous studies have shown non-random utilization of immunoglobulin genes in CLL, some germline in sequence and others containing numerous somatic mutations. To investigate whether familial cases of CLL exhibit similarities in the composition of the B-cell receptor repertoire to the pattern expressed by CLL patients as a whole, we have studied 25 CLL patients belonging to 12 different families (four French and eight Italian), each of which contained at least two affected members. Among familial cases, VH gene segment utilization proved non-random and diverged from the frequencies previously reported among unrelated patients with CLL. Specifically, although the 4-34 and 5-51 gene segments were found repeatedly, the 1-69 and 4-39 gene segments were used sparingly and the 3-23 gene segment presented with increased frequency. Following the pattern detected in studies of unrelated patients, the single 1-69 expressing CLL contained an unmutated H chain sequence and included a long HCDR3 interval. In contrast, 3-23 containing H chains all used JH4, retained at most 93% homology with germline sequence, and included only short HCDR3 intervals. The vast majority of the CLL variable domains contained a high degree of somatic mutation and exhibited an excess of replacement mutations in the CDR intervals. These findings suggest that familial CLL cases may preferentially derive from B-cell progenitors that have responded to antigen.

Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia.

Cellular immunophenotypic studies were performed on a cohort of randomly selected IgM(+) B-chronic lymphocytic leukemia (B-CLL) cases for which Ig V(H) and V(L) gene sequences were available. The cases were categorized based on V gene mutation status and CD38 expression and analyzed for treatment history and survival. The B-CLL cases could be divided into 2 groups. Those patients with unmutated V genes displayed higher percentages of CD38(+) B-CLL cells (>/=30%) than those with mutated V genes that had lower percentages of CD38(+) cells (<30%). Patients in both the unmutated and the >/=30% CD38(+) groups responded poorly to continuous multiregimen chemotherapy (including fludarabine) and had shorter survival. In contrast, the mutated and the <30% CD38(+) groups required minimal or no chemotherapy and had prolonged survival. These observations were true also for those patients who stratified to the Rai intermediate risk category. In the mutated and the <30% CD38(+) groups, males and females were virtually equally distributed, whereas in the unmutated and the >/=30% CD38(+) groups, a marked male predominance was found. Thus, Ig V gene mutation status and the percentages of CD38(+) B-CLL cells appear to be accurate predictors of clinical outcome in B-CLL patients. These parameters, especially CD38 expression that can be analyzed conveniently in most clinical laboratories, should be valuable adjuncts to the present staging systems for predicting the clinical course in individual B-CLL cases. Future evaluations of new therapeutic strategies and drugs should take into account the different natural histories of patients categorized in these manners.

Immunoglobulin V region gene use and structure suggest antigen selection in AIDS-related primary effusion lymphomas.

Primary effusion lymphoma (PEL) is a lymphoproliferation of B cells infected by Kaposi's sarcoma-associated herpes-virus/human herpesvirus-8 and reflecting a late stage of B cell differentiation close to plasma cell. Apart from viral infection, the pathogenesis of PEL is currently unclear. The aim of the present study was to investigate the role of antigen stimulation and selection in the evolution of PEL. In order to assess the specific variable heavy (VH) and light (VL) genes used by PEL and to define the heavy and light chain isotypes expressed by these lymphomas, immunoglobulin (Ig) genes from seven AIDS-related PEL were sequenced (three cell lines and four primary samples). Most of the samples (five out of seven) used lambda light chain genes; the majority of these (n = 4) belonged to the V lambda 3 family. Two cases expressed mu chains, whereas gamma chains were found in two cases. In all cases, significant deviations from the presumed germline counterpart were found in both the expressed VH and VL genes. Statistical evidence for antigen selection was evident in four out of seven samples studied. Evidence for selection was more frequent in the light chain genes than in the heavy chain genes. Collectively, these data indicate that PEL originate from mature, antigen-experienced B cells and bear implications for the pathogenesis and histogenesis of this lymphoma.