Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotypedBCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of "CLL-biased" features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset. (Blood. 2012;119(19):4467-4475) IntroductionThe analysis of the Ig genes in chronic lymphocytic leukemia (CLL) has contributed significantly toward deciphering the molecular pathogenesis of the disease. Studies from the 1990s provided the first indications for a possible role of Ag(s) in selecting the CLL progenitor cells, through the discovery of a biased Ig heavy variable (IGHV) gene repertoire, different from that of normal B cells, as well as distinctive Ag-binding sites among unrelated cases. [1][2][3][4][5] By the late 1990s, it emerged that the mutational status of the rearranged IGHV genes directly correlated with patient survival. In particular, patients with unmutated IGHV genes were found to follow a more aggressive clinical course and have significantly shorter survival than patients carrying mutated IGHV genes. 6,7 Yet, there were exceptions to this rule: cases using the IGHV3-21 gene, although mostly expressing mutated Ig, had a survival similar to that of unmutated cases. 8 Intriguingly, approximately half of the IGHV3-21 cases were found to display restricted and, in some instances, essentially identical variable heavy complementarity determining region 3 (VH CDR3) sequences and identical light chains, strongly suggesting recognition of a common antigenic determinant. 9 Soon thereafter, the study of Ig sequences in CLL by groups in both Europe and the United States led to the identification of several other subsets of cases carrying highly similar BCR Igs among both mutated and unmutated cases (stereotyped BCR). [10][11][12][13][14] The identification of stereotypy among unrelated and geographically distant cases was widely accepted as evidence for the Submitted November 26, 2011; accepte...

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 g/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.High mobility group box chromosomal protein 1 (HMGB-1; previously called high mobility group 1 [HMG-1] or amphoterin) is an intranuclear factor that facilitates protein interactions with chromatin (1). Hmgb1 knockout mice die shortly after birth because of hypoglycemia secondary to insufficient glucocorticoid receptor expression, which is under HMGB-1-mediated transcriptional control (2). HMGB-1 is ubiquitously present in the nucleus of almost all mammalian cells and is highly conserved between species (3). Beyond this intranuclear role, it has recently been discovered that HMGB-1 is secreted by certain cells, including activated monocytes and macrophages, and plays important roles in inflammation and tumor metastasis (4,5). The molecule is a late mediator of endotoxin lethality in mice and

The failure of chemotherapeutic regimens to eradicate cancers often results from the outgrowth of minor subclones with more dangerous genomic abnormalities or with self-renewing capacity. To explore such intratumor complexities in B-cell chronic lymphocytic leukemia (CLL), we measured B-cell kinetics in vivo by quantifying deuterium ( 2 H)-labeled cells as an indicator of a cell that had divided. Separating CLL clones on the basis of reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) and cluster designation 5 (CD5) revealed that the CXCR4 dim CD5 bright (proliferative) fraction contained more 2 H-labeled DNA and hence divided cells than the CXCR4 bright CD5 dim (resting) fraction. This enrichment was confirmed by the relative expression of two cell cycle-associated molecules in the same fractions, Ki-67 and minichromosome maintenance protein 6 (MCM6). Comparisons of global gene expression between the CXCR4 dim CD5 bright and CXCR4 bright CD5 dim fractions indicated higher levels of pro-proliferation and antiapoptotic genes and genes involved in oxidative injury in the proliferative fraction. An extended immunophenotype was also defined, providing a wider range of surface molecules characteristic of each fraction. These intraclonal analyses suggest a model of CLL cell biology in which the leukemic clone contains a spectrum of cells from the proliferative fraction, enriched in recently divided robust cells that are lymphoid tissue emigrants, to the resting fraction enriched in older, less vital cells that need to immigrate to lymphoid tissue or die. The model also suggests several targets preferentially expressed in the two populations amenable for therapeutic attack. Finally, the study lays the groundwork for future analyses that might provide a more robust understanding of the development and clonal evolution of this currently incurable disease.