Extended follow-up and impact of high-risk prognostic factors from the phase 3 RESONATE study in patients with previously treated CLL/SLL.

In the phase 3 RESONATE study, ibrutinib demonstrated superior progression-free survival (PFS), overall survival (OS) and overall response rate (ORR) compared with ofatumumab in relapsed/refractory CLL patients with high-risk prognostic factors. We report updated results from RESONATE in these traditionally chemotherapy resistant high-risk genomic subgroups at a median follow-up of 19 months. Mutations were detected by Foundation One Heme Panel. Baseline mutations in the ibrutinib arm included TP53 (51%), SF3B1 (31%), NOTCH1 (28%), ATM (19%) and BIRC3 (14%). Median PFS was not reached, with 74% of patients randomized to ibrutinib alive and progression-free at 24 months. The improved efficacy of ibrutinib vs ofatumumab continues in all prognostic subgroups including del17p and del11q. No significant difference within the ibrutinib arm was observed for PFS across most genomic subtypes, although a subset carrying both TP53 mutation and del17p had reduced PFS compared with patients with neither abnormality. Reduced PFS or OS was not evident in patients with only del17p. PFS was significantly better for ibrutinib-treated patients in second-line vs later lines of therapy. The robust clinical activity of ibrutinib continues to show ongoing efficacy and acceptable safety consistent with prior reports, independent of various known high-risk mutations.

Chronic lymphocytic leukaemia: current first-line therapy.

There is a major evolution in progress in the first-line therapy of chronic lymphocytic leukaemia. Several recent, large, clinical trials have documented superior outcomes with fludarabine-based therapy compared with treatment with alkylating agents. Monoclonal antibodies, especially rituximab, are establishing an important role for targeted treatment. It is expected that chemoimmunotherapy will become the preferred treatment for many patients in the near future. Specific challenges remain to be answered, however, especially the optimal treatment for the elderly, patients with autoimmune haemolysis and those with P53 deletions and mutations.

Fludarabine induces differential expression of proteins in human leukemia and lymphoma cells.

The purine analog fludarabine (FdAMP) is widely used for chemotherapy of B-lymphoid malignancies, and multiple mechanisms of action leading to apoptosis have been proposed. We examined changes at the protein level induced in the Raji cell line (Burkitt's lymphoma) by fludarabine nucleoside (FdA). Raji cells are sensitive to FdA. Raji cells treated with FdA (3 micro M, 24 hours), accumulate multiple phosphorylated forms of p53 in the nucleus that in turn degrade to phosphorylated forms of p40. Using CD antibody microarrays to determine surface expression profiles for Raji cells treated with FdA, we found up-regulation of the following CD antigens: CD20, CD54, CD80, CD86, and CD95. FdA thus induces changes in the genetic program of the cells that might be exploited to obtain synergy with therapeutic antibodies.

Immunophenotyping of leukemias using a cluster of differentiation antibody microarray.

Different leukemias express on their plasma membranes particular subsets of the 247 defined cluster of differentiation (CD) antigens, which may resemble those of precursor cells along the lineages of differentiation to mature myeloid and lymphoid leukocytes. The extent of use of CD antigen expression (immunophenotyping) for identification of leukemias has been constrained by the technique used, flow cytometry, which commonly specifies only three CD antigens in any one assay. Currently, leukemias and lymphomas are diagnosed using a combination of morphology, immunophenotype, cytochemistry, and karyotype. We have developed a rapid, simple procedure, which enables concurrent determination of 50 or more CD antigens on leukocytes or leukemia cells in a single analysis using a microarray of antibodies. A suspension of cells is applied to the array, and cells only bind to antibody dots for which they express the corresponding CD antigen. For patients with significantly raised leukocyte counts, the resulting dot pattern then represents the immunophenotype of those cells. For patients at earlier stages of disease, the diagnosis depends on recognition of dot patterns distinct from the background of normal leukocytes. Distinctive and reproducible dot patterns have been obtained for normal peripheral blood leukocytes, chronic lymphocytic leukemia (CLL), hairy cell leukemia, mantle cell lymphoma, acute myeloid leukemia, and T-cell acute lymphoblastic leukemia. The consensus pattern for CD antigen expression found on CLL cells taken from 20 patients in descending order of cells bound was CD44, HLA-DR, CD37, CD19, CD20, CD5, CD52, CD45RA, CD22, CD24, CD45, CD23, CD21, CD71, CD11c, and CD9. The antigens that provided the best discrimination between CLL and normal peripheral blood leukocytes were CD19, CD20, CD21, CD22, CD23, CD24, CD25, and CD37. Results obtained for the expression of 48 CD antigens from the microarray compared well with flow cytometry. The microarray enables extensive immunophenotyping, and the intact cells captured on antibody dots can be further characterized using soluble, fluorescently labeled antibodies.

Transendothelial migration of lymphocytes in chronic lymphocytic leukaemia is impaired and involved down-regulation of both L-selectin and CD23.

Chronic lymphocytic leukaemia (B-CLL) is characterized by a progressive accumulation of B lymphocytes in blood and bone marrow and high concentrations of soluble CD23 and L-selectin are found in the serum of these patients. In this study lymphocytes from normal subjects and patients with B-CLL were allowed to undergo transendothelial migration across confluent layers of human umbilical vein endothelial cells. Lymphocytes in B-CLL samples showed an impaired capacity to migrate while the minor proportion of normal T cells was enriched by a mean of 2.5-fold in the transmigrated lymphocytes. In contrast, the ratio of B to T lymphocytes in normal preparations was unchanged in the transmigrated population. The expression of adhesion molecules on B-CLL lymphocytes before and after transendothelial migration was studied by flow cytometry which showed that 71 +/- 5% of L-selectin was lost from the surface of transmigrated lymphocytes. T and B cells from normal subjects also showed a major loss of L-selectin after transmigration. B-CLL lymphocytes and normal B cells expressed CD23 but this molecule was down-regulated following transendothelial migration, whereas the expression of VLA-4, ICAM-1, LFA-1 and CD44 was unchanged. Lymphocytes incubated with oxidized ATP, an irreversible inhibitor of P2Z/P2X7 purinoceptors, retained their capacity for transendothelial migration and showed the same loss of L-selectin as control leukaemic lymphocytes. Our results show that B-CLL lymphocytes have impaired ability for transendothelial migration compared to normal peripheral blood lymphocytes. Moreover, transendothelial migration involves a universal loss of L-selectin and CD23 from lymphocytes which suggests that the high serum levels of soluble L-selectin and CD23 observed in B-CLL may be generated by shedding during the process of transendothelial migration.

B-cell chronic lymphocytic leukaemia with CD8 expression: report of 10 cases and immunochemical analysis of the CD8 antigen.

We report 10 cases of B-cell chronic lymphocytic leukaemia (B-CLL) with expression of the T-cell antigen CD8. The majority of patients had typical B-cell CLL with stable and non-progressive stage A(O) disease except for more common expression of lambda light chain and CD25. Two patients had progressive disease and required therapy, one with atypical morphological and phenotypic features. The incidence of CD8 expression was approximately 0.5% of B-CLL patients from our institutions. Immunoprecipitation of the CD8 antigen from four of these B-CLLs showed identity to the CD8 antigen expressed on T cells with precipitation of CD8alpha bands of molecular weight approximately 34 kD. In view of the known intracellular signalling mechanism of CD8 using the tyrosine kinase p56-lck, we studied p56-lck expression by Western blot and found lack of consistent expression of the CD8 surface antigen, with most lacking p56-lck. Our report indicates that CD8 expression in B-CLL is probably underrecognized but is not a marker of disease progression. The CD8 on the B-CLL surface is immunochemically identical to the antigen on T cells, but is not accompanied by its usual signalling mechanism of p56-lck tyrosine kinase and therefore is unlikely to be a functionally active receptor.

Comparative effects of cladribine, fludarabine and pentostatin on nucleotide metabolism in T- and B-cell lines.

BACKGROUND AND AIMS: the purine nucleoside analogues cladribine (CdA), fludarabine (F-Ara-AMP) and pentostatin (dCf), are effective therapy for a range of T- and B-cell lymphoid malignancies. The effects upon nucleotide metabolism in human CCRF-CEM T-cell leukaemia and Raji B-cell lymphoma cell lines of these drugs have been compared to assess possible mechanisms of cytotoxicity. METHODS: Leukaemia cells were exposed to a purine nucleoside analogue and perchloric acid extracts were analysed by HPLC for 2'-deoxynucleoside-5'-triphosphates (dNTPs), nucleoside-5'-triphosphates (NTPs) and drug metabolites. RESULTS: After addition of a purine nucleoside analogue, CdA-TP and F-Ara-ATP accumulate in cells while the levels of dCf-TP formed were not detectable by ultra-violet absorbance. In response to accumulating concentrations of drug triphosphate, the cellular levels of dNTPs initially decrease (0-4 h), then accumulate above their initial levels (4-10 h) before slowly declining beyond 10 h. NTPs also accumulate during the period 4-10 h before declining at later times. CONCLUSION: The temporal effects on the levels of dNTPs and NTPs of the 3 purine nucleoside analogues are similar against CCRF-CEM and Raji cells. However, CdA induces major depletions of dTTP, dGTP and dATP in CCRF-CEM cells and F-Ara-A induces a major accumulation of dATP in Raji cells.

In vivo binding of mouse IgG via polyreactive surface IgM abrogates progressive lymphocytosis in prolymphocytic leukemia.

Surface IgM expressed by malignant CD5+ B-cells from patients with B-chronic lymphocytic leukemia (B-CLL) has previously been shown to bind mouse Ig in what appears to be an example of polyreactive antigen-binding activity. This report demonstrates the in vitro and in vivo binding of mouse Ig to the surface of malignant B-cells from a patient with B-cell prolymphocytic leukemia (B-PLL). In vitro studies showed that K121, a mouse monoclonal antibody, bound to the B-PLL cells via the same low-affinity binding interaction demonstrated to occur between mouse Ig and surface IgM expressed by B-CLL cells rather than in the conventional sense against a specific antigen via its antigen-binding site. With the view to using this phenomenon to target malignant B-cells, it was important to determine whether the low-affinity interaction also occurred in vivo. Infusions of K121 totalling 286 mg were administered to a B-PLL patient over 7 days. Binding of K121 to circulating B-PLL cells was demonstrable after the administration of 36 mg of antibody and was preceded by the appearance of free antibody in the serum. Throughout the period of the infusion, the rapid rise in the peripheral blood white cell count normally observed after leukopheresis was abrogated. However, the count rose markedly after cessation of the antibody infusion in parallel with a decrease in both free and cell-bound K121. There were no observable side effects and no host immune response to either species specific or idiotypic determinants on the mouse Ig was detected. The in vivo binding of mouse Ig together with the previous in vitro data suggest the potential for a novel targeting mechanism using a region of the mouse Ig molecule to target polyreactive Ig expressed by malignant cells in B-CLL and B-PLL.

Chronic lymphocytic leukaemia with upper airway obstruction.

We report a case of B-cell chronic lymphocytic leukemia in a 58 year old female in whom the clinical course was dominated by upper airway obstruction due to massive enlargement of the palatine and later the lingual tonsils. The peripheral blood morphology and immunophenotype were typical of chronic lymphocytic leukaemia with expression of CDl9+, CD20+, CD5+, CD23+ and HLA-DR+ together with weak, surface immunoglobulin with monoclonal lambda light chain. Therapy included surgical removal of the palatine tonsils and later chemotherapy, both of which provided temporary relief of obstruction before recurrence of obstruction at the site of the lingual tonsils. Lasting relief from mass effect and obstruction only occurred following localised radiotherapy to Waldeyer's ring.

The myelodysplastic syndromes: an analysis of prognostic factors in 226 cases from a single institution.

Two hundred and twenty-six patients were diagnosed with myelodysplastic syndrome (MDS), according to the French-American-British (FAB) criteria, over a 13-year period, and studied retrospectively in a single institution in order to study indicators which were prognostically significant. Analysis of clinical and laboratory data indicated that the FAB classification, the Bournemouth, Dusseldorf, Goasguen, Sanz and FAB Scoring Systems were all good predictors of survival. We found advancing age, haemoglobin (Hb) < or = 9 g/dl, platelet count < or = 50 x 10(9)/l, increased peripheral total white cell count (WCC) and monocytosis, increased bone marrow blasts, dysgranulopoiesis, and bone marrow fibrosis were significant adverse prognostic variables. The commonest complication and cause of death was infection; however, infective episodes were not significantly associated with low neutrophil counts (either < or = 1.5 x 10(9)/l or < or = 0.8 x 10(9)/l) and there was also no significant association between neutropenia and survival. These findings indicate that neutrophil dysfunction plays an important role in the clinical progression of patients with MDS. The effect of new therapeutic modalities, such as the haemopoietic growth factors, on reducing infective episodes may be as significant as their effect on increasing neutrophil counts.

Splenic lymphoma with villous lymphocytes: natural history and response to therapy in 50 cases.

We studied the natural history and response to treatment in 50 patients with splenic lymphoma with villous lymphocytes followed for a minimum of 6 months and up to 15 years (median 3.7 years). The disease occurs in the elderly (median 68 years) and affects males more than females (M/F ratio 1.77). The median survival for the group was not reached but 82% were surviving at 3 years and 78% at 5 years. Twelve patients (24%) died, one-third of deaths were disease related (four patients) and one-half were due to cardiovascular disease or another malignancy (six patients). The outcome was worse for males (31% died) than females (11% died). Fourteen patients were not treated and 10 remain alive between 1 and 6 years from diagnosis. The remaining patients were treated by chemotherapy, splenic irradiation or splenectomy. The response to chemotherapy was poor and only eight of 22 (36%) patients treated achieved a good response. Splenic irradiation was employed in seven patients and three benefited from it. Splenectomy seems to be the treatment of choice, with significant improvement seen in 19 of 20 patients with one post-operative death. This response, lasting from 6 months to 7 years (median 4 years), was seen irrespective of whether splenectomy was the initial treatment or used later in management.

B-ly-7, a monoclonal antibody reactive with hairy cell leukemia, also defines an activation antigen on normal CD8+ T cells.

We undertook a study to determine the specificity of the monoclonal antibody, B-ly-7, for hairy cell leukemia (HCL) by examining the expression in 150 samples from B-cell lymphoproliferative diseases as well as screening for reactivity in a number of other hematologic malignancies. Within the B-cell lineage we found that the expression of B-ly-7 was highly specific for HCL and reacted with all 28 cases examined, as well as with 3 of 9 cases of a variant form of HCL. Cells of other closely related B-cell disorders, prolymphocytic leukemia, and splenic lymphoma with villous lymphocytes were negative. Investigation of the peripheral blood and bone marrow of patients with HCL before and after treatment with alpha-interferon or deoxycoformycin suggests that B-ly-7 may be useful in the assessment of minimal disease after therapy. In addition to HCL, we found that B-ly-7 was positive with cells of three mature, CD4+ T-cell malignancies. In view of the reactivity with malignancies of activated B and T cells, we searched for the expression of B-ly-7 on activated, normal B and T cells and found that B-ly-7 reacted specifically with activated normal peripheral blood CD8+ T cells. B-ly-7 has a number of applications, including the precise classification of mature B-cell neoplasia and the diagnosis HCL and its assessment after treatment. In addition, B-ly-7 recognizes a small subset of T-cell disorders. Its expression on these malignancies and on in vitro activated peripheral blood CD8+ T cells suggests that B-ly-7 detects a lymphocyte activation antigen.

Exercise-induced CD8 lymphocytosis: a phenomenon associated with large granular lymphocyte leukaemia.

This report describes a patient with a large granular lymphocyte leukaemia (CD8 + lymphoproliferative disease) and severe neutropenia (less than 0.5 x 10(9)/l) in whom exercise resulted in a marked lymphocytosis, a phenomenon which has not previously been recorded. The lymphocyte count at rest was within normal limits (2.2 x 10(9)/l), then fell to the resting level within 15 min of cessation of exercise. The peripheral blood mononuclear cells showed the morphology of large granular lymphocytes (LGL) by light and electron microscopy both at rest (30%) and to a much greater extent during exercise (70%). Immunophenotyping of these lymphocytes during exercise demonstrated that the predominant cell was CD3+, CD8+, CD57+ (Leu7)/CD4-, CD16-, CD25-. In the resting state, despite a total lymphocyte count within the normal range, surface marker studies indicated an excess of cells with the CD8+/CD57 + T cell phenotype (26%; cf. normal range less than or equal to 10%). Functional assays revealed a minimal increase in natural killer (NK) activity during exercise. T cell receptor beta chain gene rearrangement was demonstrable in the peripheral blood at rest and during exercise. Although severe neutropenia was present, the growth of normal colony forming units, granulocyte-macrophage (CFU-GM) was not inhibited by patient lymphocytes and no anti-neutrophil antibodies were demonstrated. Finally, hyposplenism has developed and the relationship of this to the LGL leukaemia is discussed. In summary, the findings demonstrated large granular lymphocyte leukaemia as the primary disorder for which the primary manifestation, apart from the neutropenia, was a marked exercise-induced lymphocytosis.

Human B cells: differentiation and neoplasia.

Human B cells differentiate from stem cells to immunoglobulin secreting plasma cells and during this course a series of discrete phases can be recognised. These stages are mirrored in the various malignancies that arise from the B cell lineage and therefore a knowledge of the cellular and molecular events in B cell differentiation are important for a full appreciation of B cell neoplasia. This review intends to provide an overview of human B cell differentiation and the related clinical spectrum of B cell leukaemia and lymphoma. The review will discuss the phenotype of B cells utilizing the expression of surface immunoglobulin and the expression of molecules recognised by monoclonal antibodies including recent cluster designations that are B cell restricted. Immunoglobulin genes and the process of rearrangement which they undergo will be considered as well as molecular defects recognised by the Southern blotting technique. Finally, the most commonly encountered phenotype patterns from each of the recognized entities within the spectrum of B cell neoplasia will be described.