Matthew Kaufman

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.

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.

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.

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 (2H)-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 CXCR4dimCD5bright (proliferative) fraction contained more 2H-labeled DNA and hence divided cells than the CXCR4brightCD5dim (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 CXCR4dimCD5bright and CXCR4brightCD5dim 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 combination of rituximab, cyclophosphamide and dexamethasone effectively treats immune cytopenias of chronic lymphocytic leukemia

Auto-immune hemolytic anemia (AIHA) and idiopathic thrombocytopenic purpura (ITP) are known complications of chronic lymphocytic leukemia (CLL). Rituximab, cyclophosphamide and dexamethasone (RCD) effectively target lymphocytes and inhibit autoimmune processes. We reviewed 21 patients with CLL treated for AIHA alone (n = 18), ITP alone (n = 1) or both (n = 2) with the following RCD regimen: rituximab 375 mg/m2 i.v. infusion given on day 1, cyclophosphamide 750–1000 mg/m2 i.v. on day 2 and dexamethasone 12 mg day 1–7 given every 3 weeks. Response to treatment was seen in all 20 patients with CLL with AIHA. Median hemoglobin pre-treatment was 8 g/dL. The median change in hemoglobin was 5.2 g/dL and the median post-treatment hemoglobin level was noted to be 13.1 g/dL. Median duration of response was 22 months. Nine relapsed patients responded as well. Fifty percent of evaluable patients converted to Coombs negative with median duration of response of 41 months vs. 10 months for those who did not convert. This difference was not statistically significant (p = 0.0674). Steroid-refractory immune thrombocytopenia was present in three patients and all responded to RCD. There were no hospitalisations or infections directly related to RCD. RCD is a safe and effective regimen in the treatment of immune cytopenias associated with CLL.

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.

The challenge of treating complex autoimmune cytopenias in chronic lymphocytic leukemia

The presence of hemolytic anemia (AIHA) and immune-mediated thrombocytopenia (ITP) poses a significant challenge in treating patients with chronic lymphocytic leukemia (CLL). Treatment with purine analogs has been known to induce autoimmune cytopenias, typically hemolytic anemia, but these occurrences are seen de novo as well. The pathophysiology of these autoimmune processes is believed to result from a defect or paucity of normal Tregulatory cells [1‐4]. Not only can the arrest of the autoimmune process be difficult, but its presence can preclude the use of purine analogs—arguably the most effective agents in the treatment of CLL itself. Although autoimmune cytopenias can present at any time in the disease course, they are more frequently seen in the later stages [5], making the requirement of treating both the CLL disease and a concomitant autoimmune process (‘complex autoimmune cytopenias’) a relatively common and difficult scenario. Purine analogs notwithstanding, overlap does exist between the treatments of immune cytopenias and CLL disease. Corticosteroids, and more recently rituximab and alkylating agents, have been shown to be effective in the treatment of AIHA and ITP; these agents are mainstays in the treatment of CLL itself. Recently, we published the results of a retrospective study using the combination of rituximab, cyclophosphamide, and dexamethasone (RCD) in 21 patients with CLL-associated immune cytopenias. The immune cytopenias in all of these patients responded, with a median duration of response of 22 months. Though this combination is effective in the treatment of CLL itself, we did not report on these results [6]. In this issue of Leukemia and Lymphoma, Bowen and colleagues report on 20 patients using the combination of rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP) in the treatment of progressive CLL complicated by autoimmune cytopenias (complex autoimmune cytopenias) [7]. The response rates achieved for both the immune phenomena as well as the progressive CLL are promising. A substantial number of complete responses (CRs) in each condition were achieved as well. The small sample size and the relatively short durations of response are obvious limitations to both the study and the therapy. Despite the limitations, the work of Bowen and colleagues provides an important evidence-based recommendation for treating autoimmune phenomena and CLL simultaneously. To our knowledge, little or no evidencebased guidance in the literature exists for dealing with the practical challenge posed by ‘complex autoimmune cytopenias’ faced by clinicians treating CLL. Additional combinations and newer agents will likely prove useful in these patients. Antibodies affecting the autoimmune phenomena are believed to be polyclonal, and not directly originating from the CLL clone. Nevertheless, newer therapies targeting B-cells in CLL will also likely decrease the activities of the aberrant polyclonal B-lymphocytes. The recent approval of ofatumumab, and the fast-coming addition of other monoclonal agents targeting B-cell antigens, broaden the potential options for new approaches in complex autoimmune cytopenias.