Phenotype of chronic lymphocytic leukemia (CLL) B-Cells

Summary In the present report we studied the phenotype of peripheral blood mononuclear cells (PBMC) from 25 patients with B-cell chronic lymphocytic leukemia (CLL). Cells from all the cases expressed monoclonal surface immunoglobulins (SmIg), formed rosettes with mouse erythrocytes (MRFC) and were positive with OKB2 and OKIa monoclonal antibodies. In addition, CCB1 monoclonal antibody was positive in 17 out of 20, Leu-1 in 18 out of 21 and Leu-8 in 23 out of 25 cases. Double labelling experiments confirmed that the Leu-8 antigen was co-expressed on Leu-1+, CCB2+, HLA-DR+ B-CLL cells. Thus, B-CLL cells generally express the SmIg+, MRFC+, Leu-1+, OKB2+, Leu-8+ phenotype. Since it is known that normal peripheral blood B cells may be divided into two subpopulations according to Leu-8 expression, our data indicate that B-CLL cells originate from the more immature Leu-8+ B-cell subset which will respond to anti-IgM, whereas it reacts poorly to pokeweed mitogen.     

More ZAP for chronic lymphocytic leukemia (CLL)

Antigen stimulation of the leukemic clone is increasingly implicated in the pathogenesis of CLL. Chen and colleagues provide evidence that expression of ZAP-70 in CLL B cells renders IgM signaling more effective and thereby could contribute to the more rapidly progressive clinical course of ZAP-70-positive CLL.     

Immunological aspects in chronic lymphocytic leukemia (CLL) development

Chronic lymphocytic leukemia (CLL) is unique among B cell malignancies in that the malignant clones can be featured either somatically mutated or unmutated IGVH genes. CLL cells that express unmutated immunoglobulin variable domains likely underwent final development prior to their entry into the germinal center, whereas those that express mutated variable domains likely transited through the germinal center and then underwent final development. Regardless, the cellular origin of CLL remains unknown. The aim of this review is to summarize immunological aspects involved in this process and to provide insights about the complex biology and pathogenesis of this disease. We propose a mechanistic hypothesis to explain the origin of B-CLL clones into our current picture of normal B cell development. In particular, we suggest that unmutated CLL arises from normal B cells with self-reactivity for apoptotic bodies that have undergone receptor editing, CD5 expression, and anergic processes in the bone marrow. Similarly, mutated CLL would arise from cells that, while acquiring self-reactivity for autoantigens-including apoptotic bodies-in germinal centers, are also still subject to tolerization mechanisms, including receptor editing and anergy. We believe that CLL is a proliferation of B lymphocytes selected during clonal expansion through multiple encounters with (auto)antigens, despite the fact that they differ in their state of activation and maturation. Autoantigens and microbial pathogens activate BCR signaling and promote tolerogenic mechanisms such as receptor editing/revision, anergy, CD5+ expression, and somatic hypermutation in CLL B cells. The result of these tolerogenic mechanisms is the survival of CLL B cell clones with similar surface markers and homogeneous gene expression signatures. We suggest that both immunophenotypic surface markers and homogenous gene expression might represent the evidence of several attempts to re-educate self-reactive B cells.     

Evolution of ibrutinib resistance in chronic lymphocytic leukemia (CLL)

The Bruton tyrosine kinase inhibitor (BTKi) ibrutinib is a new targeted therapy for patients with chronic lymphocytic leukemia (CLL). Ibrutinib is given orally on a continuous schedule and induces durable remissions in the majority of CLL patients. However, a small proportion of patients initially responds to the BTKi and then develops resistance. Estimating the frequency, timing, and individual risk of developing resistance to ibrutinib, therefore, would be valuable for long-term management of patients. Computational evolutionary models, based on measured kinetic parameters of patients, allow us to approach these questions and to develop a roadmap for personalized prognosis and treatment management. Our kinetic models predict that BTKi-resistant mutants exist before initiation of ibrutinib therapy, although they only comprise a minority of the overall tumor burden. Furthermore, we can estimate the time required for resistant cells to grow to detectable levels. We predict that this can be highly variable, depending mostly on growth and death rates of the individual CLL cell clone. For a specific patient, this time can be predicted with a high degree of certainty. Our model can thus be used to predict for how long ibrutinib can suppress the disease in individual patients. Furthermore, the model can suggest whether prior debulking of the tumor with chemo-immunotherapy can prolong progression-free survival under ibrutinib. Finally, by applying the models to data that document progression during ibrutinib therapy, we estimated that resistant mutants might have a small (<2%) mean fitness advantage in the absence of treatment, compared with sensitive cells.Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the Western hemisphere, is characterized by the expansion of CD5⁺CD23⁺ mature monoclonal B cells in the peripheral blood, as well as in lymph nodes and bone marrow. B-cell receptor (BCR) signaling plays a central pathogenic role in CLL, based on structural restrictions of the BCR and BCR-dependent survival and growth of the malignant B cells (1, 2). Bruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase of the Tec kinase family, is essential for BCR signaling. Ibrutinib (previously called PCI-32765) is a potent (IC₅₀, 0.5 nM) BTK inhibitor that inactivates BTK through irreversible covalent bonding to Cys-481 in the ATP-binding domain of BTK (3–5). Early-stage clinical trials found ibrutinib to be particularly active in patients with CLL (6, 7) and mantle cell lymphoma (8), and the drug recently has been Food and Drug Administration-approved for patients with relapsed CLL and mantle cell lymphoma.Preclinical models demonstrated that ibrutinib inhibits CLL cell survival and proliferation (9), as well as leukemia cell migration toward tissue-homing chemokines (CXCL12, CXCL13) and integrin-mediated CLL cell adhesion (10, 11). For patients with CLL, ibrutinib is given orally as a once-daily fixed dose of 420 mg on a continuous schedule until progression or toxicity. In contrast to conventional chemo-immunotherapy, ibrutinib generally is not myelo-suppressive, and responses are not affected by risk factors that predict failure to respond to or short remission durations after chemo-immunotherapy, such as del17p.In CLL, ibrutinib characteristically causes an early redistribution of tissue-resident CLL cells into the peripheral blood, with rapid resolution of enlarged lymph nodes, along with a surge in lymphocytosis (7, 12). This lymphocytosis is asymptomatic, transient, and resolves in most patients during the first few months of therapy. However, the majority of ibrutinib-treated patients do not achieve complete remission (7), and instead the lymphocyte counts stabilize in the long term in many patients at levels that are significantly lower than before treatment, but higher than normal.Although the clinical data so far demonstrate extremely encouraging responses in patients, the question arises as to how long the control of the disease can be maintained during continuous ibrutinib therapy. In particular, drug-resistant mutants can arise that can initiate renewed growth. Indeed, in a minority of patients the growth of drug-resistant mutants has already been documented (13–15). Resistance has been found to be caused by point mutations, and a number of different mutants have been documented. These mutants have been shown to have a mutation in the BTK binding site of ibrutinib or gain-of-function mutations in PLCgγ2, which lead to autonomous BCR activity. The aim of our study was to determine whether, based on previously measured kinetic parameters of the disease in the absence of treatment (16) and during ibrutinib therapy (17), it is possible to use mathematical models to predict the evolutionary dynamics of ibrutinib-resistant mutants, and to predict how long control of CLL with single-agent ibrutinib treatment can be maintained.     

Simultaneous manifestation of chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL)

We report a unique case of 83-year-old Caucasian male with the initial simultaneous manifestation of chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL). The patient presented with absolute lymphocytosis in the blood, asymptomatic generalized lymphadenopathy, and mild splenomegaly. The diagnosis of CLL was suggested from the blood film, but subsequent flow cytometric (FC) analysis on peripheral blood mononuclear cells (PBMNC) revealed two distinct abnormal clones of mature B cells. A small subpopulation (7%) of lymphoid cells expressed CD20, CD11c, FMC-7, CD103, CD25, and kappa surface light chain, consistent with HCL. The larger subpopulation (75%) of lymphoid cells expressed CD19, CD20, CD23, CD5, and lambda light chain, consistent with CLL. The expression of different immunoglobulin light chains on the circulating CLL (lambda) and HCL (kappa) cells suggested two, independent, malignant B-cell clones. Interestingly, FC analysis of bone marrow (BM) cells done 6 months later revealed bright lambda light chain expression on the HCL cells. Despite administration of several different courses of chemotherapy, the HCL subpopulation was not eliminated from the BM but remained stable between 7% and 10% of total BM lymphoid cells. The CLL, responsible for most of clinical symptoms in our patient, responded to combination chemotherapy with fludarabine and cytoxan, and later to monotherapy with rituximab.     

Purine nucleoside phosphorylase in chronic lymphocytic leukemia (CLL).

Purine nucleoside phosphorylase (PNP), the enzyme schematically next to adenosine deaminase in the purine salvage pathway, has been demonstrated cytochemically in peripheral blood lymphocytes of healthy subjects and chronic lymphocytic leukemia (CLL) patients. The enzyme activity is confined to the cytosol. In healthy subjects the majority of lymphocytes are strongly reactive for PNP, whereas the rest are devoid of cytochemically demonstrable activity. The percentage of PNP-positive cells largely corresponds to the number of E rosette-forming cells and is inversely proportional to the number of Ig-bearing cells. In six of seven CLL patients studied only a minor percentage of the lymphocytes showed strong PNP activity, whereas the large majority (88%--98%) possessed trace activity. Such patients have a high number of Ig-bearing cells and a low number of E rosette-forming cells. A different pattern of markers was found in the lymphocytes of the seventh CLL patient: 66% were strongly reactive for PNP, an important number formed E rosettes, and a minor percentage were Ig bearing. These data indicate that PNP can be useful as a "nonmembrane" marker in the differentiation of the B and T cell origin in CLL and deserves to be studied in other lymphoproliferative disorders.     

Abnormal T cell function in early-stage chronic lymphocytic leukemia (CLL) patients

Significant alterations in T cell subpopulations and function occur in chronic lymphocytic leukemia (CLL) patients. We studied whether abnormalities in peripheral blood T cell parameters were present in 15 untreated early stage CLL patients (ie, Rai stage 0, 1, 2). Seven of the nine patients showed decreased T helper support as compared to control T cells for pokeweed mitogen (PWM)-induced control B cell proliferation (ie, patient 6,063 +/- 1,434 cpm vs control 14,894 +/- 121 cpm). All stage 0 and 1 patients showed a marked impairment of T helper activity for control B cell proliferation (patient T = 7,752 +/- 1,137 cpm vs control T = 14,894 +/- 121 cpm). In a separate assay system, six of nine CLL patients showed T suppressor activity for control B cell proliferation greater than control T cell suppressor activity. Four patients were stage 0 and 1. CLL patients demonstrated markedly impaired T cell support for control B cell immunoglobulin synthesis compared to control T cells (188 +/- 28 vs 869 +/- 56 hemolytic plaque-forming cells (HePFC)/culture, respectively). Control T cells showed increasing support for control B cell immunoglobulin synthesis with increasing T:B cell ratios (869 +/- 56 vs 1,265 +/- 48 HePFC/culture, at 1:1 and 2:1 T:B cell ratios, respectively). In contrast, five of eight CLL patients' T cells showed no improvement in control B cell immunoglobulin synthesis with increasing T:B cell ratios (795 +/- 56 vs 569 +/- 48 HePFC/culture, at 1:1 and 2:1 T:B cell ratios, respectively). There was no direct correlation with CLL T cell-mediated suppression of B cell proliferation and suppression of B cell immunoglobulin synthesis. These studies suggest there is a complex array of abnormal immunoregulatory T cell function in early stage CLL. These include a prominent T helper dysfunction and more variable excessive suppressor activity. The relationship of these findings to the basic disease process remains to be elucidated.     

Revision of the guidelines for diagnosis and therapy of chronic lymphocytic leukemia (CLL)

During the past 10 years, significant progress has been achieved in defining new prognostic markers, diagnostic parameters, and treatment options in chronic lymphocytic leukemia (CLL). These developments have led to revision of the National Cancer Institute-sponsored Working Group (NCI-WG) guidelines on CLL established in 1988 and 1996. The update of these guidelines will clarify the role of new prognostic markers in CLL, improve the definitions of response and refractory disease, and add information on the prevention and management of infectious and autoimmune complications.     

Successful labor in the course of chronic lymphocytic leukemia (CLL) and management of CLL during pregnancy with leukapheresis

We describe the successful management of a 30-year-old woman in the second trimester of her pregnancy with chronic lymphocytic leukemia (CLL) in stage IV by using only leukapheresis. We applied three sessions (courses) of leukapheresis throughout the pregnancy. The procedure did not have any significant adverse effect on the patient and the fetus. The patient gave birth vaginally to a healthy boy, weighing 3100 g, at 39 weeks of gestation. Seven months after delivery, Richter's syndrome developed in the patient. We conclude that leukapheresis may provide an alternative for palliative treatment to chemotherapy in pregnant patients with CLL. To our knowledge, this is the fourth reported case of CLL in pregnancy, and the first management of CLL during pregnancy with leukapheresis.     

Prognostic relevance of the FAB morphological criteria in chronic lymphocytic leukemia: correlations with IgVH gene mutational status and other prognostic markers

Morphological examination is the routine first step in the diagnosis of hematological malignancies, including chronic lymphocytic leukemia (CLL). Atypical cell morphology according to the FAB criteria is known to herald disease progression. Several years ago, it was proposed that FAB morphology at diagnosis had a considerable prognostic impact. However, this proposal has not been widely adopted in practice. Thus we questioned the prognostic value of the morphological examination, which was performed retrospectively in 88 patients out of our 110 institutional registry patients (70 males and 40 females, median age 57 yrs) with CLL at diagnosis. We related the results to the more modern prognostic markers. Atypical FAB morphology was shown to correlate with IgVH gene mutation status, trisomy of chromosome 12 and deletion of 17p detected either by conventional G-banding or by fluorescence in situ hybridization (FISH) analysis. The correlation of FAB morphology with CD38 antigen expression or with the histopathological pattern of bone marrow infiltration was not significant. Overall survival (OS) data were available for 84 morphologically examined patients. The patients with atypical morphology (64 patients) had a significantly shorter OS (103 months) than the 20 patients presenting with typical CLL morphology (237 months; p=0.03). Only the mutation status of IgVH genes correlated more closely with OS (p=0.002). Of note, there was no leukemia-related death within "unmutated" cases who had typical FAB morphology (p=0.14), and vice versa, the mutation status had a significant prognostic impact within the morphologically atypical cases (p=0.01). Thus FAB morphology and the mutation status may yield complementary prognostic information. OS was affected both by the presence of cytogenetic aberrations (p=0.03) - most adversely by deletions of 17p and 11q, and by CD38 expression (p=0.003). We conclude that careful examination of peripheral blood smears according to FAB is a simple, cheap and valuable tool in the first-line assessment of prognosis of CLL patients and should not be overlooked even in 3rd millennium when more sophisticated prognostic markers are at hand. This ought to be confirmed in larger prospective studies with multivariate analysis of data.     

Surface antigen expression and correlation with variable heavy-chain gene mutation status in chronic lymphocytic leukemia

Recent studies have demonstrated that B-cell chronic lymphocytic leukemia (CLL) consists of two clinical entities with either somatically hypermutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy-chain (VH) regions. In view of the fact that the cellular biology of these two subsets of disease is currently unexplored, we performed an extensive analysis of the surface antigen expression and correlated this with the VH gene mutation status in a cohort of 32 CLL patients. Using polymerase chain reaction amplification and nucleotide sequencing, the VH genes were shown to be mutated in 10 cases (31%) and unmutated in 22 (69%). The expression of 27 surface membrane antigens in peripheral blood leukemic cells was analyzed by flow cytometry, measuring both the percentage of positive cells as well as the geometric mean fluorescence intensity (GMF). Most of the surface membrane antigens (CD5, CD11c, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD45, VD79b, CD80, CD95, CD122, CD124, CD126, CD130, CD154, IgM, and IgD) showed a similar expression pattern in both UM-CLL and M-CLL patients. The similarity of M-CLL and UM-CLL, as demonstrated here for the first time with many protein markers, indicates a considerably homogeneous phenotype in both subsets. Furthermore, CD27 was strongly expressed in all cases, which may suggest a memory cell phenotype for both M-CLL and UM-CLL. More positive cells in the UM-CLL group were observed regarding CD38, but CD38 was not a good predictor of VH gene mutation status. Seventy percent of the M-CLL cases, but only 36% of UM-CLL cases, were Ig-lambda+. The most striking differential expression, however, was observed in the two slicing variants of the common leukocyte antigen CD45, namely CD45RO and CD45RA. CD45RO expression was significantly associated with M-CLL, whereas the GMF intensity of CD45RA tended to be associated with UM-CLL. The role of these CD45 splicing variants in the pathogenesis of CLL deserves further investigation.     

The use of fluorescence in situ hybridization (FISH) in chronic lymphocytic leukemia (CLL)

As a result of the low proliferative index, only 50% of chronic lymphocytic leukemia cases are adequate for cytogenetic analysis. Of these, about half have clonal abnormalities. The application of fluorescence in situ hybridization (FISH) to CLL has substantially enhanced our ability to detect chromosomal aberrations; the incidence of a number of recurring abnormalities has been established, providing new insights into the pathogenesis of this disease with a direct impact on the prognosis.     

HLA ligandome analysis identifies the underlying specificities of spontaneous antileukemia immune responses in chronic lymphocytic leukemia (CLL)

The breakthrough development of clinically effective immune checkpoint inhibitors illustrates the potential of T-cell–based immunotherapy to effectively treat malignancies. A remaining challenge is to increase and guide the specificities of anticancer immune responses, e.g., by therapeutic vaccination or by adoptive T-cell transfer. By analyzing the landscape of naturally presented HLA class I and II ligands of primary chronic lymphocytic leukemia (CLL), we delineated a novel category of tumor-associated T-cell antigens based on their exclusive and frequent representation in the HLA ligandome of leukemic cells. These antigens were validated across different stages and mutational subtypes of CLL and found to be robustly represented in HLA ligandomes of patients undergoing standard chemo-/immunotherapy. We demonstrate specific immune recognition of these antigens exclusively in CLL patients, with the frequencies of representation in CLL ligandomes correlating with the frequencies of immune recognition by patient T cells. Moreover, retrospective survival analysis revealed survival benefits for patients displaying immune responses to these antigens. These results directly imply these nonmutant self-peptides as pathophysiologically relevant tumor antigens and encourages their implementation for cancer immunotherapy.For a long time peptide-based immunotherapy fell short of its potential to achieve meaningful responses in the clinical setting of cancer therapy (1, 2). A disequilibrium between proposed tumor-associated antigens on the input side (3) and functional vaccines on the output side (4–6) became strikingly apparent. This might in part have been due to the distorted relationship of gene expression and HLA restricted presentation of the corresponding gene product (7, 8). In contrast, a recent study on vaccination with naturally presented tumor-associated peptides identified by direct analysis of primary tumor-derived HLA ligands reported specific, nontoxic vaccine-induced immune responses, which were associated with improved clinical outcome (6). On the other hand, experimental cancer immunotherapy using adoptive transfer of antigen-specific T cells showed high efficiency but also severe toxicity in some patients due to presence of target antigens on normal tissues (7). Both situations underscore the importance and potential of identifying (patho-)physiologically relevant targets by direct differential analysis of the entire landscape of HLA-presented peptides, termed the HLA ligandome.Here, we mapped the nonmutant HLA ligandome of chronic lymphocytic leukemia (CLL) with the aim of developing a CLL-specific multipeptide vaccine. The immunogenicity of CLL, as revealed in GvL-effects and cases of spontaneous remissions (8–10), as well as favorable immune effector-to-target cell ratios present in situations with minimal residual disease (MRD) suggest that CLL might be effectively targeted by T-cell–based immunotherapy (11, 12). Furthermore, the highly variable and in some patients prolonged course of disease indicates an underlying mechanism of tumor control (13). However, so far only very few CLL-associated antigens that are able to elicit specific T-cell responses have been described (14–16).We identified a novel category of ligandome-defined tumor-associated antigens (LiTAAs; source proteins of HLA ligands on tumor cells), which were frequently and exclusively detected in CLL patients. Specific immune recognition of the corresponding HLA ligands (LiTAPs) was observed exclusively in CLL patients, remarkably showing a direct correlation with the frequency of HLA restricted presentation. Furthermore, retrospective survival analysis points to an association of LiTAP-specific immune responses with improved overall survival in CLL patients.     

Lymphocyte surface immunoglobulin density and immunoglobulin secretion in vitro in chronic lymphocytic leukemia (CLL)

Lymphocyte surface immunoglobulin (SIg) and immunoglobulin (Ig) secretion were studied in 14 patients with chronic lymphocytic leukemia (CLL) and 12 healthy subjects. The determination of SIgM-bearing (SIgM+) cells by immunofluorescent staining and the quantification of SIgM by radioimmunoassay (RIA) permitted the calculation of the SIgM density. In 12 normal subjects the percentage of SIgM+ cells averaged 8% (range 4%-12%) and the SIgM density 10.2 ng antigenic equivalent/10(6) SIgM+ cells (SD 4.3). In 12 patients with CLL the respective figures were 68% (range 35%-90%) and 0.68 ng (SD 0.57). Ig secretion from pokeweed mitogen-stimulated CLL cells was markedly diminished as compared with normal lymphocytes. In coculture experiments CLL cells had no suppressive effect on Ig secretion of normal lymphocytes and normal lymphocytes did not enhance Ig secretion leukemic lymphocytes. These results indicate that the impaired secretory activity of CLL cells results from an intrinsic anomaly of these cells.