Biclonal chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is well characterized clinically and immunophenotypically. Demonstration of a monotypic CD19+, CD5+ B-cell population is central to the diagnosis. We report 2 cases of biclonal CLL. Two elderly men were encountered with an absolute lymphocytosis consisting of the typical CD5+, CD19+, CD23+ B-cell population seen in CLL; however, immunoglobulin light chain restriction by flow cytometry was not apparent as B cells expressed kappa or lambda light chains without a clear monotypic population. Molecular genetic analysis of flow cytometry-sorted cells (kappa and lambda populations) revealed in both cases 2 monoclonal B-cell populations. The characterization of these cases and a review of the issues surrounding biclonal CLL are presented.     

The variable pattern of circulating lymphocyte subpopulations in chronic lymphocytic leukemia.

The percentages of T and B lymphocytes in 13 patients with chronic lymphocytic leukemia were determined at two-week intervals. One patient with "B-cell predominant" disease showed a decrease from 77 to 49 in the percentage of circulating B cells, which bind heat aggregated immunoglobulin and anti-human immunoglobulin. This patient had a comparable increase in T cells, which form rosettes with sheep erythrocytes. The lymphocytes of three other patients, who originally had equal percentages of cells that bound heat-aggregated immunoglobulin and anti-human immunoglobulin, lost lg determinants from 47 to 63 per cent of cells without changing the proportion of cells with receptors for heat-aggregated immunoglobulin or for sheep erythrocytes. The characterization of lymphocytes in chronic lymphocytic leukemia should include sequential determination of cell-surface markers for both T and B cells, since the disease does not represent a proliferation of B cells alone.     

Structural aspects of human IgM antibodies expressed in chronic B lymphocytic leukemia.

BACKGROUND: Malignant B cells from patients with chronic B lymphocytic leukemia (B CLL) generally express both surface IgM and the pan T cell antigen CD5, a characteristic of the B1 population of B lymphocytes. The IgM on the surface of these B CLL cells is frequently polyreactive with respect to its capacity to recognize multiple structurally dissimilar antigens (Ag). OBJECTIVES: To understand the structural characteristics of the polyreactive binding sites of human IgM molecules expressed on B CLL cells by: (1) analyzing the nucleotide and protein sequences of the variable (V) domains of five IgM molecules expressed in cases of B CLL and; (2) utilizing these sequences to generate three-dimensional (3D) models of Fv (VL - VH) molecules. STUDY DESIGN: Peripheral blood leukocytes obtained from five cases of B CLL were tested for polyreactive binding properties by assessing their capacity to bind mouse IgG by indirect immunofluorescence. The V region genes of light and heavy chains were amplified using the polymerase chain reaction, subsequently cloned and their nucleotide sequences obtained. Translated amino acid sequences of the V domains were used to generate homology models of the Fv molecules. RESULTS: Low affinity binding of mouse IgG was demonstrated for all B CLL samples examined, confirming the polyreactive nature of the IgM expressed on these cells. There was an absence or minimal mutation within V region genes when compared to germline Ig genes. Junctional diversity was not observed for VL regions, although truncations and insertions were frequent in D minigenes of VH regions. The binding sites were predicted to form either relatively flat surfaces with occasional protrusions or cavities at the VL - VH domain interface. Aromatic side chains covered a large proportion of the potential binding surfaces in the models of B CLL Fv components. DISCUSSION: Primary DNA sequences can be categorized as germline, suggesting that the B cells involved in B CLL are germline or naive in origin. The medium to large HCDR3s provide the majority of probable contact residues for antigens. While prominent aromatic residues are likely to engage in binding patterns which are conserved (e.g. mouse Ig reactivity), the diverse binding sites predicted for B CLL-derived IgMs also have properties which are conducive to polyreactive antigen binding.     

Human chronic lymphocytic leukemia: Karyotypes in different lymphocyte populations

Karyotypes of different lymphocyte populations from 10 patients with chronic lymphocytic leukemia (CLL) showed two types of chromosome abnormalities. In cultures containing mitogens for T cells (leukoagglutinin (LA)) as well as T and B cells (pokeweed mitogen (PWM); protein A (PA)) random structural aberrations or trisomies were seen. However, one patient had a clone with trisomy 12 in LA- as well as PA-cultured cells. This clone represented about 10–20% of all mitoses, and was observed over a time period of 4 years. Nonrandom rearrangements were found in lipopolysaccharide B (LPS)-stimulated B lymphocytes from one individual in 11 cells analyzed. All these cells had multiple chromosome rearrangements. The cells probably represented a clone, since the karyotype was almost identical in all 11 cells, and the following reciprocal translocations could be identified: t(6;7), t(7;13), and t(11;14).     

Impaired recirculation of B lymphocytes in chronic lymphocytic leukemia

Lymphocytes of the peripheral blood and thoracic duct lymph were studied in 4 patients with chronic lymphocytic leukemia (CLL),1 patient with lymphosarcoma (LSA) and 3 patients with nonhematolo-gical diseases (controls). When stimulated in vitro with phytohemagglu-tinin (PHA) lymph lymphocytes of CLL patients responded markedly as determined by ¹⁴[C]thymidine incorporation, whereas blood lymphocytes showed a delayed and diminished response. The response of blood and lymph lymphocytes of the LSA and control patients was equal. Purified rabbit antisera against κ,λ, μ and γ-chains were labeled with ²⁵I and the labeled cells assessed by autoradiography. In CLL patients, the percentage of lymphocytes bearing κ and μ determinants was higher in the blood than in the lymph. Controls showed a much lower percentage of lymphocytes with immunoglobulins, which was equal in blood and lymph. Furthermore, the membrane dynamics of HL-A-anti-HL-A complexes on the surface of blood and lymph lymphocytes were studied by means of membrane fluorescence. In CLL, the percentage of lymph lymphocytes showing “cap formation” within 2 h was higher in the lymph than in the blood. Using autotransfusion of [³H-]cytidine-labeled blood lymphocytes, it is shown that the recovery of labeled cells in the lymph of CLL patients within 48 h is diminished compared to controls. It is concluded that in CLL, the leukemic cells are B cells whose capacity to recirculate from blood to lymph through the postcapillary venules is impaired. Only a minor population of PHA-responsive T cells appears to recirculate normally. Consequently, the concentration of T cells is higher in the lymph than in the blood and the leukemic B lymphocytes accumulate in the vascular pool. The impaired ability for recirculation and “cap formation” suggests a membrane abnormality of the CLL cell.     

Richter syndrome in B-cell chronic lymphocytic leukemia

Richter syndrome (RS) is well known as a secondary high‐grade lymphoma, mostly diffuse large B‐cell lymphoma (DLBCL) developed in patients with B‐cell chronic lymphocytic leukemia (B‐CLL). In this review, we describe clinicopathological, histological, immunophenotypical and genetic findings of RS. The patients with RS, regardless of transformation of pre‐existing clone or de novo malignant clone, were resistant to conventional combined chemotherapy and died within months of diagnosis. Molecular techniques can provide convincing results for the clonal relationship of RS to pre‐existing B‐CLL. When RS carries a same rearrangement band or a same sequence as B‐CLL by Southern blotting or nucleotide sequence analyses of immunoglobulin heavy and/or light chain genes, it is suggested to that RS transforms from original B‐CLL. These analyses have showed that approximately two‐thirds of RS cases evolved from a B‐CLL clone. How and where does the B‐CLL clone evolve to RS? The genetic alteration of transforming B‐CLL clone into RS has been addressed. Abnormalities of chromosomes 11 and 14 were most frequently involved in RS, but non‐specific. In addition, RS does not include chromosomal translocation between Ig locus and oncogenes or rearrangements of bcl‐6 gene, both of which were found in some de novo DLBCL. Several candidates, such as mutation of p53 gene and abnormalities of cyclin dependent kinase inhibitor, have been proposed to play an important role in the transformation of a part of B‐CLL. However, there is still uncertainly as to how B‐CLL progresses or develops into RS.     

Fluorescent bodies in lymphocytes in chronic lymphocytic leukemia

The relative frequency of lymphocytes with nuclear Y-body-like fluorescent structures (F-bodies) was examined in ten patients with chronic lymphocytic leukemia (CLL) and in ten normal individuals. In each patient, the frequency was significantly higher as compared to that of normal controls. Female CLL patients showed a significantly higher proportion of F-body-positive (F-body +) cells than male CLL patients, whereas in normal males and females similar frequencies of F-body + cells were observed. There was no apparent relationship between the frequency of F-body + lymphocytes and the relative age of an individual. The presence of F-bodies in CLL had no obvious correlation with the presence of karyotypic abnormalities in these patients. Isolated T and B lymphocytes derived from four normal subjects showed no extra F-bodies as found in CLL patients.     

Erythrocyte-rosette receptor expression by chronic lymphocytic leukemia B lymphocytes

Erythrocyte (E) rosette-forming cells have been investigated in three patients with B chronic lymphocytic leukemia whose leukemic lymphocytes were easily identifiable. A small percentage of fresh neoplastic cells formed E rosettes in two patients. In every patient, most unstimulated, cultured leukemic lymphocytes became E+ and this was further enhanced by phytohemagglutinin and pokeweed mitogen stimulation or neuraminidase treatment. These E+ B cells lacked detectable T cell antigens (except for a weak expression of an antigen associated with the helper T cell subpopulation in one case). They were unreactive or weakly reactive by immunofluorescence with a monoclonal antibody to the E receptor. However, this antibody completely inhibited E-rosette formation. The enhanced expression of E-rosette receptors by in vitro cultured cells appeared to be dependent upon the presence of a small number of E-rosetting cells at the beginning of the culture. E-rosette receptor expression by leukemic lymphocytes was most likely in a fourth case (out of 9 patients studied). This finding may account for some of the discrepancies in the study of so-called T cells in B chronic lymphocytic leukemia.     

Chromosome changes in human chronic lymphocytic leukemia

Karyotypes were studied in B- and T-lymphocyte cultures from 66 patients with B-cell CLL and two patients with T-cell CLL. Thirty-one of the B-cell cases had not been treated for their disease; 35 had received radiotherapy, corticosteroids, or cytostatic drugs. Only one of the untreated patients had a clone with an abnormal karyotype. This was present in all her mitotic cells found in cultures containing lipopolysaccharide B (LPS, a B-cell mitogen) and 10% of those in cultures with pokeweed mitogen (PWM, a T- and B-cell mitogen). The karyotype of this clone was 46,XX,t(6;7),t(7;13),t(11;14). Four of the treated patients had clones with specific chromosome changes. These were 47,XY,+12 in 10% of leukoagglutinin (LA, T-cell mitogen) and protein A (PA, T- and B-cell mitogen) cultures in one case; 47,XX,+12,del(14) in 80% of LPS cultures and in all spontaneously dividing cells in another case; 46,XY,t(6;20) in all LPS cultures in another; and 46,XX,t(1;8) in all PA cultures in another. Both structural and numerical nonclonal chromosome aberrations (9%) were found in 24% of the different cultures of cells from untreated patients, and in 15% of the cells in 20% of the different cultures in the patient who had received treatment. Both patients with T-cell CLL had received treatment for their disease, and had a normal karyotype in all cultures.     

CD5 links humoral autoimmunity with B-cell chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) does not result from accumulation of CD5⁺ B cells but, presumably, represents an antigen-driven dynamic process. Autoimmune diseases (AIDs) include hemolytic anemia, thrombocytopenic purpura and immune neutropenia. In turn, CLL and B-cell lymphoma develop in the frame of an AID. Such reciprocal interactions suggest that similar B cells are involved in both disorders. Phenotypic features (i.e., several membrane markers) and functional characteristics of CD5⁺ B cells (i.e., Epstein–Barr virus transformation and expression of apoptotic proteins) distinguish CLL and AID CD5⁺ B cells from their normal counterparts. These cells produce often nonpathogenic polyspecific low-affinity autoantibodies, whereas they present the antigen to antiself B or T cells to feature pathogenic monospecific high-affinity autoantibody. The CD5 molecule itself plays a part by translocating phosphatases to the vicinity of the B-cell antigen receptor, thereby precluding transduction from the B-cell receptor. Such might be the link between CLL and AID, both prevented by the CD5 machinery.     

Mitogen-induced maturation of chronic lymphocytic leukemia B lymphocytes

Peripheral blood lymphocytes from eight patients with untreated B-derived chronic lymphocytic leukemia (CLL) (only one had a serum monoclonal immunoglobulin) were stimulated byNocardia, phytohemagglutinin, and pokeweed mitogen. This stimulation resulted in the occurrence in all but one case of large cytoplasmic immunoglobulin-containing cells with a predominantly lymphoblastic (six cases) or plasmacytic (one case) appearance. These blast cells contained the same immunoglobulin chains as those of the surface immunoglobulins on fresh lymphocytes except for chains that were not found by cytoplasmic immunofluorescence. In three cases, intracytoplasmic immunoglobulin inclusions that were present in both small lymphocytes and stimulated blast cells provided further evidence of the differentiation of the leukemic clone. In another case, both small leukemic B lymphocytes and large B blast cells expressedin vitro a receptor for sheep erythrocytes. A switch from IgM to IgG synthesis was observed in this patient, whose cells showed the strongest response to the activators. The most effective activator wasNocardia, then phytohemagglutinin, whereas an effect of pokeweed mitogen was observed only when the fresh lymphocytes contained a fair percentage of T cells. Leukemic cells, in most patients, were able to differentiate without appreciable B-cell proliferation.This work was presented at the Fourth International Congress of Immunology, Paris, 1980 (Abstract 18.4.22).     

Intractable chronic lymphocytic leukemia and interferon.

The hypothesis that interferon may be useful for intractable chronic lymphocytic leukemia (CLL) is based on two incidental findings: first, the similarity in the in vitro radioresistance of the blood lymphocytes of intractable CLL and of hairy cells; secondly, the similarity of the incidence of intractable CLL patients and the incidence of CLL patients who respond to interferon treatment.     

A clinicopathologic study of familial chronic lymphocytic leukemia.

The familial occurrence of chronic lymphocytic leukemia was studied using morphologic, immunophenotypic, cytogenetic, and immunoglobulin gene rearrangement analyses. Three of six siblings developed chronic lymphocytic leukemia. One (patient 1) died 9 years after the diagnosis of chronic lymphocytic leukemia at age 67 years. The other two patients, ages 64 and 68 years (patients 2 and 3, respectively), are alive after chronic lymphocytic leukemia was diagnosed 11 and 4 years ago, respectively. Using the Rye classification, patient 2 and patient 3 had Stage I and Stage O disease, respectively. In contrast, patient 1 had Stage IV disease. The bone marrow of patient 2 was 90% cellular, with sheets of mature lymphocytes, and that of patient 3 was 70% cellular, with a nodular pattern of similar cells. Both patients 2 and 3 had normal karyotypes. Immunophenotyping studies revealed that patient 3 had an expanded population of B cells with minimal to no detectable expression of surface immunoglobulins and membrane-bound light chains. In contrast, the B-cell population of patient 2 expressed immunoglobulins M, D, and Kappa light chains. Gene rearrangement studies performed on these two patients revealed different but distinct patterns of heavy chain rearrangement. This may represent an evolution of two different clones of chronic lymphocytic leukemia in this family.     

Long-term cultures of chronic lymphocytic leukemia B cells generate B suppressor cells

B suppressor cells (Bs) were generated by stimulation with PHA-P or concanavalin A of B cells from peripheral blood of B-cell chronic lymphocytic leukemia (B-CLL) patients. They suppressed well allogeneic mixed lymphocyte reactions. Long-term colonies of B cells from B-CLL were established for up to 6 weeks in liquid media, with B cells acquiring properties of suppressor cells while being cultured. Two types of cultures were observed: cells either grew in compact multicellular colonies or formed diffuse monolayers. Cells that remained alive in cultures for a number of weeks, but did not multiply, did not develop suppressive characteristics. Bs cells retained their phenotypic markers in cultures. Mitosis and lymphoblasts, but no differentiation into plasma cells, were observed. PHA-P-generated Bs were cultured in long-term colonies, retaining their suppressor properties. The establishment of long-term cultures of B-CLL cells may facilitate a better understanding of the nature and characteristics of normal and leukemic B cells.     

Complex karyotypic evolution in B-cell chronic lymphocytic leukemia.

Cytogenetic analysis at diagnosis in a female patient with chronic B-cell leukemia showed a single abnormal clone with a 4p+ abnormality, 46,XX, -4, +der(4)t(4;?)(p16;?). Six additional clones evolved from this clone during the following 4 1/2 years and showed 3p+, 4p-, and 11q- chromosomes in addition to the 4p+ abnormality. Immunoglobulin heavy chain gene rearrangement studies showed two rearranged bands and a faint germline band. Following splenectomy, a strong germline and faint rearranged bands were seen, suggesting that the majority of cells were normal, whereas cytogenetic studies showed that the karyotypically abnormal cells were still present. The combination of cytogenetic and Ig gene rearrangement studies provides detailed information regarding the number of circulating normal and leukemic cells.     

HLA antigens in chronic lymphocytic leukemia

HLA-A, B, and C phenotypes of 88 white and 14 black patients with chronic lymphocytic leukemia (CLL) were compared with those of 3761 white and 660 black laboratory population controls, and HLA-DR phenotypes were compared with 742 white and 236 black controls from the same population. Several statistically significant associations were found, one of which (a strongly positive association with Cw6 for whites) persisted after correction for the number of antigens tested.     

The pathogenesis of chronic lymphocytic leukemia: analysis of the antibody repertoire

CD5⁺ B cells predominate early in ontogeny and have been associated with autoantibody production. In chronic lymphocytic leukemia (CLL), B lymphocytes express CD5 and frequently produce autoantibodies using developmentally regulated variable (V)-gene segments. Does the self-reactivity observed in CLL reflect transformation of a ‘fetal’ lineage of cells, or could overexpansion of these B cells occur as a consequence of antigen stimulation? Harry Schroeder and Guillermo Dighiero have reviewed the literature describing antibody sequences in CLL and have compared them with the ‘fetal’ repertoire. This analysis indicates that CLL cells use a repertoire characteristics of mature cells, and suggests that antigen may play a role in the pathogenesis of this disease.     

Monocyte and granulocyte defect in chronic lymphocytic leukemia.

Enzymatically homogeneous populations of lymphocytes, monocytes, and neutrophils were isolated by zonal centrifugation from 5 untreated patients with chronic lymphocytic leukemia (CLL) and 2 patients with CLL in full remission. The cells were then quantitatively analyzed for six leukocytic enzymes and compared with cells from normal subjects. CLL monocytes were deficient in beta-glucuronidase (0.06 units; normal, 0.16), myeloperoxidase (0.07 mg; normal, 0.5 mg), and lysozyme (0.7 mg; normal, 3.3 mg). In 2 cases, CLL neutrophils were severely deficient in lysozyme (1 to 2 mg; normal, 7 mg) and myeloperoxidase (2 to 3 mg; normal, 7 mg). Neutrophil alkaline phosphatase and neutral protease were unaffected. CLL lymphocytes shared with the monocytes the deficiency of beta-glucuronidase (0.03 units; normal, 0.09 units). The 2 CLL patients in full remission carried normal enzyme levels in leukocytes of all three cell lines. The CLL lymphocytes of untreated patients were unresponsive to mitogens but became responsive in remission. The CLL monocytes from both untreated and treated patients transformed into macrophages. The pattern of shared enzyme deficiency among lymphocytes, monocytes, and neutrophils of CLL patients and its normalization in all three cell types under remission suggest that the differentiation of the three leukocytic cell lines may be an enzymatically interlinked process and that the deficiency of these enzymes in leukemia may reflect an interrelated aberrant differentiation of the leukemic cells.     

Comparative results with various polyclonal B-cell activators in aneuploid chronic lymphocytic leukemia

The mitotic index and number of abnormal metaphases of cells stimulated with the various B-cell polyclonal mitogens (PBA) in six B cell chronic lymphocytic leukemia (B-CLL) cases were evaluated. The PBA included tetradecanoyl-0-phorbol-13-acetate (TPA), staphylococcus bacterial strain Cowan I (Cowan I), pokeweed mitogen (PWM), Epstein-Barr virus (EBV), and lipopolysaccharide W from E. coli 0.55:B5 (LPS). TPA could stimulate only 1 of 12 samples. Even though Cowan I led to a relatively high mitotic index, abnormal clones were inconsistently obtained with this mitogen. PWM, EBV, and LPS appear to be the most desirable activators of B-CLL cells among the PBA used in this study.