Intraclonal complexity in chronic lymphocytic leukemia: fractions enriched in recently born/divided and older/quiescent cells.

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.

A novel adoptive transfer model of chronic lymphocytic leukemia suggests a key role for T lymphocytes in the disease.

Chronic lymphocytic leukemia (CLL) is an incurable adult disease of unknown etiology. Understanding the biology of CLL cells, particularly cell maturation and growth in vivo, has been impeded by lack of a reproducible adoptive transfer model. We report a simple, reproducible system in which primary CLL cells proliferate in nonobese diabetes/severe combined immunodeficiency/γc(null) mice under the influence of activated CLL-derived T lymphocytes. By co-transferring autologous T lymphocytes, activated in vivo by alloantigens, the survival and growth of primary CFSE-labeled CLL cells in vivo is achieved and quantified. Using this approach, we have identified key roles for CD4(+) T cells in CLL expansion, a direct link between CD38 expression by leukemic B cells and their activation, and support for CLL cells preferentially proliferating in secondary lymphoid tissues. The model should simplify analyzing kinetics of CLL cells in vivo, deciphering involvement of nonleukemic elements and nongenetic factors promoting CLL cell growth, identifying and characterizing potential leukemic stem cells, and permitting preclinical studies of novel therapeutics. Because autologous activated T lymphocytes are 2-edged swords, generating unwanted graph-versus-host and possibly autologous antitumor reactions, the model may also facilitate analyses of T-cell populations involved in immune surveillance relevant to hematopoietic transplantation and tumor cytoxicity.

Alemtuzumab maintenance may safely prolong chemotherapy-free intervals in chronic lymphocytic leukemia.

This prospective, single-arm study utilized alemtuzumab as a single agent in a novel maintenance schedule in previously treated chronic lymphocytic leukemia patients with the goal of delaying progression of disease and requirement for chemotherapy. In previously treated CLL patients who had achieved stable disease or better, the following schedule of subcutaneous alemtuzumab was administered: a dose escalation in the first week (3, 10 and 30 mg), followed by 7 weeks of 30 mg alemtuzumab once weekly, 16 weeks of 30 mg once every 2 weeks, followed by once every 3 weeks for 24 weeks. Thus, the entire duration of the planned treatment was 48 weeks. A total of 12 patients were enrolled 11 of which had at least one marker of poor prognosis (unmutated, Zap 70+, CD38+, del11q and del17p). The median chemotherapy-free interval was 13 months, and the median time to disease progression was 10 months. Three patients achieved a CR, one achieved nPR, one had a PR, five failed and two had shown a beneficial response but because of recurrent ITP had to stop alemtuzumab. In six of the 10 patients with previously relapsed disease, the chemotherapy-free interval was longer than their prior chemotherapy-free period. One patient had a reactivation of CMV antigenemia, and another had a bacterial pneumonia. There were no grade 3 or 4 toxicities. Alemtuzumab used in a maintenance schedule is a potentially safe and useful tool in delaying disease progression and chemotherapy-free intervals in previously treated CLL patients.

Many chronic lymphocytic leukemia antibodies recognize apoptotic cells with exposed nonmuscle myosin heavy chain IIA: implications for patient outcome and cell of origin.

Many B-cell chronic lymphocytic leukemia (CLL) monoclonal antibodies (mAbs) can be grouped into subsets based on nearly identical stereotyped sequences. Subset 6 CLL mAbs recognize nonmuscle myosin heavy chain IIA (MYHIIA). Herein, we report that during apoptosis, MYHIIA becomes exposed on the cell surface of a subgroup of apoptotic cells, allowing subset 6 CLL mAbs to bind with it. Because other non-subset 6 CLL mAbs interact with apoptotic cells, 26 CLL mAbs, including 24 not belonging to subset 6, were tested for reactivity with MYHIIA-exposed apoptotic cells (MEACs). More than 60% of CLL mAbs bound MEACs well; most of these mAbs expressed unmutated IGHV (15 of 16) and belonged to a stereotyped subset (14 of 16). Binding to MEACs inversely correlated with the degree of IGHV mutation. Interestingly, high binding to MEACs significantly correlated with poor patient survival, suggesting that the basis of IGHV mutation status as a CLL prognostic factor reflects antigen binding. Finally, natural antibodies from human serum also reacted with MEACs. Taken together, our data indicate that a large proportion of CLL clones emerge from natural antibody-producing cells expressing immunoglobulins that recognize MEACs, and that this reactivity is associated with poor clinical outcome.

In vivo intraclonal and interclonal kinetic heterogeneity in B-cell chronic lymphocytic leukemia.

Clonal evolution and outgrowth of cellular variants with additional chromosomal abnormalities are major causes of disease progression in chronic lymphocytic leukemia (CLL). Because new DNA lesions occur during S phase, proliferating cells are at the core of this problem. In this study, we used in vivo deuterium ((2)H) labeling of CLL cells to better understand the phenotype of proliferating cells in 13 leukemic clones. In each case, there was heterogeneity in cellular proliferation, with a higher fraction of newly produced CD38+ cells compared with CD38- counterparts. On average, there were 2-fold higher percentages of newly born cells in the CD38+ fraction than in CD38- cells; when analyzed on an individual patient basis, CD38+ (2)H-labeled cells ranged from 6.6% to 73%. Based on distinct kinetic patterns, interclonal heterogeneity was also observed. Specifically, 4 patients exhibited a delayed appearance of newly produced CD38+ cells in the blood, higher leukemic cell CXC chemokine receptor 4 (CXCR4) levels, and increased risk for lymphoid organ infiltration and poor outcome. Our data refine the proliferative compartment in CLL based on CD38 expression and suggest a relationship between in vivo kinetics, expression of a protein involved in CLL cell retention and trafficking to solid tissues, and clinical outcome.

Characterization of structurally defined epitopes recognized by monoclonal antibodies produced by chronic lymphocytic leukemia B cells.

Despite a wealth of information about the structure of surface membrane immunoglobulin (smIg) on chronic lymphocytic leukemia (CLL) cells, little is known about epitopes reacting with their binding sites. Probing phage-displayed peptide libraries, we identified and characterized mimetopes for Igs of 4 patients with IGHV mutated CLL (M-CLL) and 4 with IGHV unmutated CLL (U-CLL). Six of these mAbs were representatives of stereotyped B-cell receptors characteristic of CLL. We found that mimetic epitopes for U- and M-CLL Igs differed significantly. M-CLL-derived peptides exhibited better amino acid motifs, were more similar to each other, aligned more easily, and formed tighter clusters than U-CLL-derived peptides. Mono-, oligo-, and polyreactivity of peptides correlated with structural changes within antigen-binding sites of selecting M-CLL mAbs. Although M-CLL-isolated peptides and certain U-CLL mAbs bound more effectively to the selecting mAb, others were not as specific, reacting with M-CLL and U-CLL mAbs; these data suggest that in vivo structurally diverse epitopes could bind smIgs of distinct CLL clones, thereby altering survival and growth. Finally, an M-CLL-derived peptide inhibited, in a dose-dependent manner, binding of its homologous mAb to human B lymphocytes; therefore peptides that inhibit or alter the consequences of antigen-smIg interactions may represent therapeutic modalities in CLL.