Multiple productive immunoglobulin heavy chain gene rearrangements in chronic lymphocytic leukemia are mostly derived from independent clones

In chronic lymphocytic leukemia, usually a monoclonal disease, multiple productive immunoglobulin heavy chain gene rearrangements are identified sporadically. Prognostication of such cases based on immunoglobulin heavy variable gene mutational status can be problematic, especially if the different rearrangements have discordant mutational status. To gain insight into the possible biological mechanisms underlying the origin of the multiple rearrangements, we performed a comprehensive immunogenetic and immunophenotypic characterization of 31 cases with the multiple rearrangements identified in a cohort of 1147 patients with chronic lymphocytic leukemia. For the majority of cases (25/31), we provide evidence of the co-existence of at least two B lymphocyte clones with a chronic lymphocytic leukemia phenotype. We also identified clonal drifts in serial samples, likely driven by selection forces. More specifically, higher immunoglobulin variable gene identity to germline and longer complementarity determining region 3 were preferred in persistent or newly appearing clones, a phenomenon more pronounced in patients with stereotyped B-cell receptors. Finally, we report that other factors, such as TP53 gene defects and therapy administration, influence clonal selection. Our findings are relevant to clonal evolution in the context of antigen stimulation and transition of monoclonal B-cell lymphocytosis to chronic lymphocytic leukemia.     

Clonal immunoglobulin gene rearrangements in chronic lymphocytic leukemia: a correlative study

Forty-two patients with chronic lymphocytic leukemia (CLL) were studied for immunoglobulin gene and T-cell receptor gene rearrangements. Immunoglobulin heavy chain gene rearrangements were demonstrable in 41 cases. One rearrangement of the T-cell receptor beta chain gene was detected. Quantification of the relative intensities of germline and rearranged DNA bands suggests that a significant component of the lymphocytosis may be due to cell populations other than the malignant clonal population, particularly in earlier stages of the disease. A direct relationship was found between severity of disease and the relative amount of clonal immunoglobulin heavy chain gene rearrangement. Preliminary data for 12 patients followed sequentially indicated that clinical deteriorations or improvements are reflected in an increase or decrease, respectively, in the proportion of cells with rearranged immunoglobulin genes. Change in the relative proportion of cells with germline versus rearranged genes may provide an additional useful criterion for staging CLL, for more precisely defining the abnormal lymphocyte population, and for monitoring progression of the disease and efficacy of treatment.     

Impaired synthesis of immunoglobulin in patients with chronic lymphocytic leukemia

The cause of hypogammaglobulinemia in patients with chronic lymphocytic leukemia (CLL) is unknown. Experiments were performed to determine if sera, monocytes, or non-T cells from patients with CLL suppress the proliferative response and synthesis of immunoglobulin (Ig) following incubation with pokeweed mitogen (PWM) in cocultures with lymphocytes from normal individuals. The data indicate that sera and monocytes from patients with CLL did not suppress the proliferative response or synthesis of Ig normal non-T cells. When various numbers of normal non-T cells and CLL non-T cells were cocultured with a constant number of normal T cells, the proliferative response and the concentration of supernatant Ig decreased as the proportion of CLL non-T cells increased. Since similar results were obtained when irradiated non-T cells from normal individuals were substituted for non-T cells from patients with CLL, we believe that the decrease in proliferative response and diminished synthesis of Ig is not the result of the suppressor non-T cells but is related to the dilution of normal B cells by inert non-T cells. We conclude that these experiments serve as as in vitro model for patients with CLL and suggest that the hypogammaglobulinemia observed in this disease is related to the diluting out of normal B cells by the accumulation of neoplastic B cells in the peripheral blood, bone marrow, and lymphoid tissue of these patients.     

Restricted immunoglobulin VH region repertoire in chronic lymphocytic leukemia patients with autoimmune hemolytic anemia

Between 10% and 25% of chronic lymphocytic leukemia (CLL) patients have episodes of autoimmune hemolytic anemia (AIHA) during the course of their disease. The anti-erythrocyte autoantibodies in most cases are polyclonal and express a different heavy chain isotype than the malignant clone, indicating that they are secreted by normal autoreactive B lymphocytes. To further investigate the pathogenesis of the AIHA in CLL, we analyzed the lg heavy (H) chain variable region genes expressed by leukemic cells from CLL patients with and without AIHA. Two VH genes were preferentially expressed by the leukemic cells in the CLL cases with AIHA and were present in 9 of the 12 investigated cases. The 51p1/DP-10 gene was expressed in 5 of these cases and was absent in the control group of 12 consecutive CLL cases without AIHA, whereas the DP-50 gene was present in 4 CLL-AIHA cases and only once in the control CLL group. A strikingly similar H-chain CDR3 region that contained a single reading frame of the DXP4 DH gene segment, and N- encoded proline at the DH/JH boundary, and a tyrosine-rich region encoded by the JH6 gene segment was observed in four CLL-AIHA cases. The preferential expression of two VH gene segments and a particular CDR3 region by the leukemic cells of patients with AIHA suggests that the antibodies produced by the CLL cells are directly involved in the pathogenesis of the hemolytic anemia.     

Prognostic significance of serum immunoglobulin pareprotein in chronic lymphocytic leukemia

Objective To investigate the incidence of serum immunoglobulin (Ig) paraprotein in chronic lymphocytic leukemia(CLL) ,and to explore its clinical associated laboratory features and prognostic implication. Methods Serum protein electrophoresis and immunofixation     

Pure red-cell aplasia in patients with chronic lymphocytic leukemia

Four cases of pure red-cell aplasia (PRCA) in association with chronic lymphocytic leukemia (CLL) are summarized. In addition, all reported cases of CLL-PRCA (27, including our 4 cases) are reviewed to consolidate their clinical features and compare them with those of 26 patients with CLL who were anemic (Stage III) and 52 who were not anemic (Stages O-II). The incidence of PRCA in our patients with CLL is about 6%. Eighty-seven percent of patients with PRCA had B-lymphocyte (B-cell) CLL and 13% had T-lymphocyte (T-cell) CLL. In 4 of 17 patients PRCA was the presenting feature, and in 13 of 17 it developed in patients previously diagnosed as having CLL. In the latter group, the average time from the diagnosis of CLL to the development of PRCA was 50.2 months (range, 1 to 96). The average age at presentation with PRCA was 61.6 years (range, 37 to 79), and the male to female ratio was 1.6 to 3:1. Eighty-one percent had splenomegaly and 65% had lymphadenopathy. The average hematocrit was 17 +/- 3.6% with zero reticulocyte count, and the lymphocyte count was 134 X 10(3)/microliter. Normoblasts were absent in the marrow and lymphocytes constituted 50 to 100% of the marrow cells. In patients with B-cell CLL and those with T-cell CLL, the T cells were found to inhibit the growth of the marrow erythroid progenitors in in vitro cultures. These abnormal T cells (Tr cells) possessed IgG Fc receptors on their surface, which may be responsible for induction of PRCA in these patients. Continued administration of cyclophosphamide and prednisone in moderate doses appears to induce remission from PRCA, but not until the leukemic mass and abnormal Tr cells are markedly reduced. Daily or alternate-day cyclophosphamide administration in small to moderate doses seems to be essential to maintain remission.     

Circulating endothelial cells in patients with chronic lymphocytic leukemia

Angiogenesis is increased in B-cell chronic lymphocytic leukemia (B-CLL). We wanted to quantify and characterize the circulating endothelial cells (CECs) in patients with B-CLL and correlate with plasma angiogenesis-related factors. Using a four-color flow cytometry, we prospectively analyzed the CEC in the whole blood of 20 healthy controls and 20 patients with B-CLL. We quantified (CD45−/CD31+/CD146+) and characterized the CECs according to whether they were apoptotic (annexin stain) or activated (CD106+). We also measured plasma levels of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and thrombospondin-1 (TSP-1). Most patients (90%) had Rai stages 0–2 at the time of diagnosis. As a group, B-CLL patients had higher number of CECs (median of 26.5 cells/ml) compared (P = 0.04) to healthy controls (18.5 cells/ml). However, only four (20%) patients had elevated CEC counts, defined as ≥2 SD of the control mean (≥53 cells/ml). The proportions of apoptotic (P = 0.83) and activated (P = 0.12) CECs were similar in both groups. B-CLL patients had higher FGF-2 (P < 0.001), lower TSP-1 (P = 0.004), and similar VEGF (P = 0.27) plasma levels. The number of CECs was not associated with Rai stage, absolute lymphocyte count, or levels of angiogenesis-related factors. CECs are increased in only a small fraction of B-CLL patients in our cohort with low rates of apoptosis and activation. While no correlation was found between CECs and clinical features, more studies in a larger patient sample size and advanced disease are necessary. Supported by the Gundersen Lutheran Center for Cancer and Blood Disorders and the Gundersen Lutheran Medical Foundation. The Greater Richland Area Cancer Elimination (GRACE), Inc. and the Gundersen Lutheran Medical Foundation funded this study.     

Improving treatment for patients with chronic lymphocytic leukemia

The last two decades have been time of tremendous progress in treatment for patients with chronic lymphocytic lymphoma (CLL). Chemoimmunotherapy (CIT) combining anti-CD20 monoclonal antibodies with purine nucleoside analogs has been a substantial advance for patients with CLL and results in increased response rates, progression-free survival, and overall survival. Despite these improved outcomes, only ≈ 45% of patients achieve a complete remission with CIT and nearly all patients eventually relapse and their remains a need to improve efficacy. Although new combinations of traditional agents may lead to incremental progress, more substantive improvements are likely to result through therapeutic targeting of novel pathways critical to CLL B-cell survival including targeting: (1) leukemia cell apoptotic resistance; (2) survival signals mediated through the B-cell receptor; and (3) nurturing interactions with the microenvironment. In this mini-review, we summarize Mayo Clinic's recent efforts to improve CIT for patients with CLL.     

Development of acute myeloid leukemia in patients with untreated chronic lymphocytic leukemia

The development of acute myeloid leukemia (AML) in patients with untreated chronic lymphocytic leukemia (CLL) is rare. We experienced a 65-year-old man who developed AML with aberrant CD7 expression and monoallelic CEBPA mutation during watchful waiting for CLL. He failed to achieve complete response (CR) by standard induction therapy for AML. We retrospectively reviewed 27 patients who developed AML with untreated CLL published between 1973 and 2016. The median age at diagnosis of AML was 68 years, and the median duration between the diagnoses of AML and CLL was 4.2 years. Diagnosis of AML and CLL was made simultaneously in 16 patients. The CR rate of AML was 42.9%, and the median survival was only 1.5 months after the diagnosis of AML. Patients who achieved CR tended to survive longer than those who did not. Our results demonstrated that the development of AML in patients with untreated CLL was associated with a poor response to chemotherapy and an extremely poor prognosis.     

Prognostic significance of immunoglobulin phenotype in B cell chronic lymphocytic leukemia

Seventy-six consecutive untreated patients with B cell chronic lymphocytic leukemia (B-CLL) and classified according to Binet's staging system were studied at the clinical presentation. Several immunologic parameters (number of total and T circulating lymphocytes and their surface membrane immunoglobulin [Smlg] phenotypes and levels of serum Ig) were evaluated with the aim of identifying a biologic marker of prognostic relevance. In this series of persons, Binet staging confirmed its usefulness as a prognostic index (P less than .001). With regard to Smlg, they were mu-type in 41 cases (53.9%), mu-type plus delta-type in 29 cases (38.2%), alpha-type in one case, and not detectable in five cases. No correlations were found between clinical stage and immunoglobulin phenotype, although all but one patient in stage C showed mu-type Smlg alone. On analyzing the survival curves of our patients according to different Smlg phenotypes, we found that patients with only mu-type Smlg had a poorer prognosis (P less than .05) than those with mu-type plus delta-type; this difference was even more significant (P less than .01) in patients in stage A, whereas there were no statistical differences in those in stages B and C. Because the appearance of surface heavy chain of delta-type could be an expression of cell maturation, these results suggest that in B-CLL the presence of phenotypically more mature leukemic cells may correlate with better clinical prognosis, particularly in the early phase of the disease.     

Discordance of ZAP-70 in patients with chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia has a highly variable clinical course with behavior ranging from indolent to aggressive. Identified prognostic markers include IgV(H) mutational status, and CD38 and ZAP-70 expression. In several studies, ZAP-70 expression correlated with IgV(H) mutational status, and predicted disease progression and overall survival. In addition to its prognostic utility, ZAP-70 expression was found to be constant over time, and did not vary between peripheral blood, bone marrow or lymph node specimens in individual patients. In contrast to these reports, we present three CLL patients with discordant ZAP-70 levels. One demonstrated a change in expression with time; the second and third cases had discordant results in blood, lymph node and bone marrow and between blood and bone marrow specimens, respectively, obtained at the same time.     

Rituximab therapy of patients with B-cell chronic lymphocytic leukemia

Rituximab (IDEC-C2B8) is a chimeric antibody that binds to the B-cell surface antigen CD20. Rituximab has significant activity in follicular non-Hodgkin lymphomas. Much less is known about the effects in chronic lymphocytic leukemia (CLL). We have initiated a phase II trial to evaluate the efficacy and safety of rituximab in patients with CD20+ pretreated CLL. To avoid the rituximab-associated toxicity, we restricted the tumor cell load, as measured by the number of circulating lymphocytes and the spleen size, in the first 2 cohorts of patients included in the study. Patients received 4 intravenous infusions of 375 mg/m2 once a week over a period of 1 month. Of the 28 patients evaluable for response, 7 patients showed a partial remission (National Cancer Institute criteria) lasting for a median of 20 weeks, with 1 patient still in remission after 71 weeks. Based on lymphocyte counts only, we found at least a 50% reduction of lymphocyte counts lasting for at least 4 weeks in 13 (45%) of 29 patients. Fifteen patients from 3 institutions were monitored for the immunophenotype profile of lymphocyte subsets. The number of CD5+CD20+ cells decreased significantly and remained low until day 28 after therapy. T-cell counts were not affected. With the exception of one rituximab-related death, adverse events in the remaining patients were mild. The results suggest that rituximab has clinical activity in pretreated patients with B-CLL. Toxicity is tolerable. Response duration after withdrawal of rituximab is rather short. Therefore, other modes of application and the combination with other agents need to be tested.     

Strikingly homologous immunoglobulin gene rearrangements and poor outcome in VH3-21-using chronic lymphocytic leukemia patients independent of geographic origin and mutational status

We recently reported that Swedish V_H3-21-using chronic lymphocytic leukemia (CLL) patients showed restricted immunoglobulin gene features and poor prognosis despite V_H mutation status. To investigate this further, we analyzed the V_H and V_L gene rearrangements in 90 V_H3-21⁺ patients from Sweden, Germany, Italy, United States, Finland, and Australia and correlated these data with survival and other prognostic markers. Sixty-three percent exhibited mutated V_H genes and 37% unmutated V_H genes. Fifty (56%) patients displayed a short and homologous heavy-chain CDR3, many of these with the amino acid motif DANGMDV. Also, a highly biased V2-14 use was evident in 72% of patients with a restricted light-chain CDR3, QVWDS(S/G)SDHPWV. Combined restricted heavy- and light-chain CDR3s were found in patients from all included countries. Although V_H3-21⁺ CLLs have a remarkably predominant expression, analyses of kappa deleting element indicated a conserved light-chain rearrangement order. The overall survival was poor in the V_H3-21⁺ cohort (median survival, 88 months), with no significant difference in relation to mutation status or CDR3 homology. High ZAP-70 and CD38 expression was found in both mutated and unmutated V_H3-21⁺ cases as well as a slight increase of 11q-aberrations. In summary, highly restricted B-cell receptors and worse outcome characterize V_H3-21⁺ CLLs independent of geographic origin and mutation status.      

Genomic complexity identifies patients with aggressive chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) has a variable clinical course. Presence of specific genomic aberrations has been shown to impact survival outcomes and can help categorize CLL into clinically distinct subtypes. We studied 178 CLL patients enrolled in a prospective study at the University of Michigan, of whom 139 and 39 were previously untreated and previously treated, respectively. We obtained unbiased, high-density, genome-wide measurements of subchromosomal copy number changes in highly purified DNA from sorted CD19(+) cells and buccal cells using the Affymetrix 50kXbaI SNP array platform (Santa Clara, CA). Genomic complexity scores were derived and correlated with the surrogate clinical end points time to first therapy (TTFT) and time to subsequent therapy (TTST): measures of disease aggressiveness and/or therapy efficaciousness. In univariate analysis, progressively increasing complexity scores in previously untreated CLL patients identified patients with short TTFT at high significance levels. Similarly, TTST was significantly shorter in pretreated patients with high as opposed to low genomic complexity. In multivariate analysis, genomic complexity emerged as an independent risk factor for short TTFT and TTST. Finally, algorithmic subchromosomal complexity determination was developed, facilitating automation and future routine clinical application of CLL whole-genome analysis.