p53 and Notch signaling in chronic lymphocytic leukemia: clues to identifying novel therapeutic strategies.

The p53 tumor suppressor protein has a key role in the induction of apoptosis of chronic lymphocytic leukemia (CLL) cells. Abnormalities within the p53 pathway identify a subset of patients with a poor prognosis. This review describes recent advances in understanding the mechanisms that regulate p53 levels and the role of p53 in the control of the cell cycle and of apoptosis. The classical model of p53-mediated apoptosis emphasizes the transcriptional activation of proapoptotic genes. In contrast, a novel model emphasizes p53's non-transcriptional actions as the major route of apoptosis induction, whereas its transcriptional arm predominantly upregulates antiapoptotic genes, thus providing a negative feedback mechanism that limits apoptosis. Further studies have identified the Notch pathway as a candidate p53-induced antiapoptotic mechanism. In contrast to the classical model, the novel model predicts that pharmacological inhibition of p53's transcriptional function or of the Notch signaling pathway will augment apoptosis induction by cytotoxic agents. Therapeutic strategies based on the novel model, which we review here for the first time, may significantly augment the antitumor actions of cytotoxic agents in CLL and in other malignancies.

The sesquiterpene lactone parthenolide induces selective apoptosis of B-chronic lymphocytic leukemia cells in vitro.

We have studied the in vitro actions of the sesquiterpene lactone parthenolide (PTL) on cells isolated from patients with chronic lymphocytic leukemia (CLL). Dye reduction viability assays showed that the median LD(50) for PTL was 6.2 muM (n=78). Fifteen of these isolates were relatively resistant to the conventional agent chlorambucil but retained sensitivity to PTL. Brief exposures to PTL (1-3 h) were sufficient to induce caspase activation and commitment to cell death. Chronic lymphocytic leukemia cells were more sensitive towards PTL than were normal T lymphocytes or CD34(+) haematopoietic progenitor cells. The mechanism of cell killing was via PTL-induced generation of reactive oxygen species, resulting in turn in a proapoptotic Bax conformational change, release of mitochondrial cytochrome c and caspase activation. Parthenolide also decreased nuclear levels of the antiapoptotic transcription factor nuclear factor-kappa B and diminished phosphorylation of its negative regulator IkappaB. Killing of CLL cells by PTL was apparently independent of p53 induction. This is the first report showing the relative selectivity of PTL towards CLL cells. The data here warrant further investigation of this class of natural product as potential therapeutic agents for CLL.

Candida albicans peritonitis in a patient with Felty's syndrome.

A 53 year old man with Felty's syndrome presented with abdominal pain and fever. He underwent a laparotomy after starting broad spectrum antibiotics. An intestinal biopsy showed skip ulcers with fungal hyphae. Peritoneal exudates grew Candida albicans. He was started on intravenous fluconazole and then switched to liposomal amphotericin to which he showed a good clinical response. After one month at home he was readmitted with candidosis and died of a myocardial infarction.

Dendritic cells and their potential therapeutic role in haematological malignancy.

The generation of an effective immune response is dependent on the efficient capture and presentation of antigen by antigen-presenting cells. The most potent antigen-presenting cells are dendritic cells (DC). These cells have the capability of activating naive helper and cytotoxic T cells. In recent years it has been demonstrated that in vivo responses to a number of solid tumours can be generated by DC pulsed with either purified tumour antigen or whole tumour cell lysate. In addition, a number of in vivo studies using DC have also been attempted in solid tumours, with some encouraging results. In haematological malignancies, there is now strong evidence that previous T cell anergy can be reversed and significant anti-tumour immune responses generated, in vitro, against the majority of leukaemias. As far as in vivo studies in haematological malignancies are concerned, although T cell responses have been demonstrated in the majority of cases and some dramatic early clinical responses reported, overall results appear disappointing. However, considering the fact that many of these studies were performed in patients with advanced disease and that such therapeutic strategies are still in their infancy, the overall results are actually quite encouraging. Although there is a real potential for DC immunotherapy in the future, it is important to be realistic about the limitations and obstacles to its development. It is highly unlikely that any form of immunotherapy is going to be effective in advanced disease due to the physical bulk of tumour, the immunosuppressive effects of tumours themselves and to any secondary immunosuppression following standard cancer therapy. The potential for immunotherapy is likely to lie either in adjunctive therapy or for treating minimal residual disease. Even in those situations, one of the major obstacles to be overcome is the state of immunological anergy or tolerance that many tumours seem able to induce. Indeed, there is evidence that, under certain circumstances, DC themselves can present antigen in such a way as to produce this state of anergy. Although, in vitro manipulation of DC and T cells can generate tumour-specific T cells from previously "anergic" cells, once reintroduced in vivo, these cells will be re-exposed to the tumour environment with the risk of being rendered anergic again.

Abnormal T-cell function in B-cell chronic lymphocytic leukaemia.

There is increasing evidence of T cell dysfunction in B cell chronic lymphocytic leukaemia (B-CLL) which may contribute to the aetiology and progress of the disease. An absolute CD8+ lymphocytosis correlates with disease progression and low expression of CD4 and CD8 (as found in autoimmune disease) is seen with abnormal expression of other surface molecules. Although the expression of T cell surface activation markers, CD25 and CD152, may be increased on culture in B-CLL serum, response to the common mitogens, PHA and PWM, is reduced. This and the excess of CD8 cells may explain partly the variable cooperation of T cells with B cell production of immunoglobulin in B-CLL. In the context of T cell cross-talk with antigen presenting cells, B-CLL B cells are poor antigen presenters. But the T cells themselves have significant abnormalities of expression of the many antigens and ligands necessary for this process. In particular, they exhibit variable expression of the low affinity and non-specific adhesion molecules LFA-1 and ICAM-1, variable, clonally restricted and skewed expression of the TCR repertoire (implying repeated antigenic stimulation possibly by CLL antigens), reduced CD28 and CD152 expression (implying impairment of ability to start or stop an immune response) and reduced IL2 and CD25 (IL2 R) expression (critical for positive feed-back in maintenance and expansion of the T cell response to antigen presentation). Although the production of IL2 and other cytokines by the T cell in B-CLL may be impaired, production of the anti-apoptotic cytokine IL4 is not and there may be a unique and expanded subset of CD8/CD30 cells capable of releasing IL4. The relationship of this T cell subset to the malignant B cell in vivo is unknown. However, T cells which are CD4+/CD152+/CCR4+ migrate selectively in vitro in response to the chemokine CCL22 (specific for the receptor CCR4) produced by the malignant B cells and are always seen amongst the malignant cells in bone marrow and lymph nodes from B-CLL patients. Other abnormalities of cytokine secretion are described. These findings suggest that the T cell in B-CLL may be unable to start, maintain and complete an immune response to the malignant B cell and other antigens and may be involved directly in sustaining the tumour. However, autologous tumour specific cytotoxicity has been shown in vitro and T cells which recognise tumour-derived heavy chain fragments circulate in vivo. If adoptive immunotherapy of any nature is to succeed in B-CLL, manipulation to optimise these CTL responses is needed to overcome the profound and variable T cell dysfunction in this disease.

In vitro dendritic cell-induced T cell responses to B cell chronic lymphocytic leukaemia enhanced by IL-15 and dendritic cell-B-CLL electrofusion hybrids.

HLA class II-restricted proliferative and cytotoxic T cell (CTL) responses to B cell chronic lymphocytic leukaemia (B-CLL) can be generated using autologous dendritic cells (DCs) pulsed with tumour cell lysate. In this study a number of different approaches were used to optimize further the in vitro system. First, the effects of a variety of maturation agents were studied. The addition of TNF-alpha, polyriboinosinic polyribocytidylic acid (Poly(I:C)) and LPS to autologous DCs resulted in the emergence of only a small percentage of CD83+ DCs, IFN-alpha having no demonstrable effect. Only the addition of Poly(I:C) to DCs resulted in modestly increased specific cytotoxicity to B-CLL targets, IFN-alpha and LPS having no effect. Secondly, T cells were pretreated with IL-15, prior to culturing with lysate-pulsed autologous DCs. A significant increase in T cell activation (P = 0.038), IFN-gamma secretion (P = 0.030) and specific cytotoxicity to B-CLL targets (P = 0.006) was demonstrated compared to untreated T cells. Thirdly, monocyte derived DCs electrofused with B-CLL B cells were compared with lysate-pulsed DCs. T cells stimulated by fused DCs generated higher levels of specific cytotoxicity to autologous B-CLL B cell targets than those stimulated by lysate pulsed DCs (P = 0.013). Blocking studies demonstrated inhibition of this cytotoxicity by both anti-CD4 (P = 0.062) and anti-CD8 monoclonal antibodies (P = 0.018), suggesting the generation of both HLA class I- and HLA class II-restricted CTL responses. In summary, in vitro B-CLL-specific T cell responses can be enhanced further by preincubating T cells with IL-15 and using autologous fused DC-B-CLL hybrids instead of autologous lysate-pulsed DCs. These preliminary data require confirmation with larger numbers of patients. Such an approach, however, may eventually provide effective immunotherapy for treatment of B-CLL.

Generation in vitro of B-cell chronic lymphocytic leukaemia-proliferative and specific HLA class-II-restricted cytotoxic T-cell responses using autologous dendritic cells pulsed with tumour cell lysate.

Immunotherapy using dendritic cells has shown encouraging results in both haematological and non-haematological malignancies. In this study, monocyte-derived dendritic cells from patients with B-CLL were cultured for 6 days in the presence of IL-4 and GM-CSF. Autologous B-CLL T-cells were cultured alone or with B-CLL lysate-pulsed and unpulsed autologous dendritic cells. IFN-gamma secretion was assessed using ELISA. Cytotoxicity was assessed, after 21 days in culture and re-stimulation, using flow cytometry with and without blockade by anti-HLA class I, anti-HLA class II, anti-CD4, anti-CD8 and anti-TCRalphabeta monoclonal antibodies. B-CLL T cells stimulated with B-CLL lysate-pulsed autologous dendritic cells showed a significant (P = 0.0004) increase in IFN-gamma secretion and a significant (P = 0.0008) increase in specific cytotoxicity to autologous B-cell targets, but none to autologous T cell or B cell targets from healthy individuals. B-CLL T cells cultured with (non-B-CLL) B-cell lysate-pulsed B-CLL dendritic cells showed no significant response. Pulsing dendritic cells from healthy volunteers with an autologous (non-B-CLL) B-cell lysate did not stimulate proliferation, cytokine production or cytotoxicity by autologous T cells. Pulsing B-CLL dendritic cells with allogeneic B-CLL lysates and culturing with autologous T-cells elicited cytotoxicity against autologous B-CLL targets in some cases, but not in others. Cytotoxicity was significantly reduced by blocking with anti-HLA class II (P = 0.001), anti-TCRalphabeta (P = 0.03) and anti-CD4 (P = 0.046) antibodies. Phenotyping of the responding T-cell population demonstrated the majority to be CD4 positive. Our data demonstrate that HLA class II-restricted proliferative and cytotoxic T-cell responses to B-CLL can be generated using autologous dendritic cells pulsed with tumour cell lysate.

Oral antifungals as prophylaxis in haematological malignancy.

In the standard treatment of patients with haematological malignancy, immunosuppressive therapy produces prolonged periods of neutropenia and mucositis, which increase the risk of systemic fungal infection. In allogeneic bone marrow transplantation, this risk extends well beyond the period of neutropenia when graft-versus-host disease, and its treatment, result in prolonged lymphocytopenia. Various agents are used for antifungal prophylaxis and treatment but all have limitations: amphotericin B is restricted by the need for intravenous infusion and the occurrence of adverse events, fluconazole by its narrow spectrum of activity and the emergence of fluconazole-resistant fungi and itraconazole capsules by erratic absorption. Oral administration of antifungals has clear advantages in prophylaxis and an important current strategy is to maximize the extent and reliability of the oral bioavailability of antifungal agents. Mucositis is the main obstacle for success of strategies based on oral delivery. In this review, the ability of these new oral formulations to deliver sufficient antifungal prophylaxis is evaluated.

Analysis of the expression of critical activation/interaction markers on peripheral blood T cells in B-cell chronic lymphocytic leukaemia: evidence of immune dysregulation.

B-cell chronic lymphocytic leukaemia (B-CLL) is characterized by an accumulation of clonal malignant B cells. The intrinsic characteristics that permit this accumulation have been extensively studied and described. However, it is possible that proliferation and survival of this malignant clone is facilitated by a disruption in the interaction between B and T cells that normally regulate the immune system. In this study, using flow cytometry and cell culture techniques, marked abnormalities of the expression of certain key activation and interaction molecules on the peripheral blood T cells of patients with B-CLL were demonstrated. In particular, on comparison with normal controls, there was a marked reduction in the number of circulating T cells expressing CD25 (interleukin 2 receptor) (P = 0.007), CD28 (P = 0.01) and CD152 (CTLA-4) (P = 0.001). There was also a reduction in the number of circulating T cells expressing CD4 (P = 0.03), CD5 (P = 0.05) and CD11a (P = 0.01). There was no difference in the number expressing T-cell receptor alphabeta (P = 0.1), CD8 (P = 0.4), CD54 (P = 0.4) and CD154 (P = 0.5), and the only marker expressed on a greater number of circulating T cells in B-CLL patients was HLA-DR (P = 0.05). These results suggest that there is a profound T-cell dysregulation that may contribute to the survival of the malignant B cells in patients with B-CLL and to the related autoimmune phenomena of the disease.

Are women with polycystic ovary syndrome resistant to activated protein C?

OBJECTIVE: This study was performed to test the hypothesis that an increased prevalence of activated protein C (APC) resistance in women with polycystic ovary syndrome (PCOS) puts them at increased risk of miscarriage and thrombosis. DESIGN: Case control study. SETTING: A district general hospital in the United Kingdom. PATIENT(S): Forty-one women with PCOS and 25 controls. INTERVENTION(S): Clinical histories, ultrasound scans, and venepunctures. MAIN OUTCOME MEASURE(S): Diagnosis of PCOS or control, clinical histories, APC resistance according to an activated partial thromboplastin time-based assay. RESULT(S): There was no significant difference in the proportion of women with APC resistance in both groups (three women in the PCOS group [7%] vs. one woman in the control group [4%]). The prevalence of APC resistance in the entire study population was 6.5%. In the PCOS group, 29% (12/41) gave a positive family history of thrombosis compared with 8% (2/25) in the control group. None of the women with a positive family history of thrombosis had abnormal antithrombin 111, protein C, or protein S levels. CONCLUSION(S): This study suggests that women with PCOS may have the same prevalence of APC resistance as the background population and that APC resistance may not put them at a higher risk of thrombosis or miscarriage compared with the case of the general population.

Immunohistochemical detection of plasminogen activator inhibitor-1 in polycystic ovaries.

Anovulation in women with polycystic ovary syndrome (PCOS) is incompletely understood. The concentration of the glycoprotein plasminogen activator inhibitor-1 (PAI-1) is raised in insulin resistance. This has been described in the granulosa and theca cell layers of the animal but not the human ovary. This study was performed to investigate the location of PAI-1 in the human ovary and investigate whether it may contribute to anovulation in PCOS. PAI-1 was localized immunohistochemically and quantitated using computer image analysis in 17 ovarian follicles from five women with a diagnosis of PCOS and compared with 15 follicles from six normal ovaries. PAI-1 was predominantly found in the granulosa and theca cells in both polycystic and normal ovaries. Image analysis did not reveal a difference in the PAI-1 signal from polycystic compared with normal ovaries. This study shows that PAI-1 plays a role in human ovulation, but its role in PCOS requires further research.

FLUDAP: salvage chemotherapy for relapsed/refractory aggressive non-Hodgkin's lymphoma.

The aim of this study was to investigate the combination of fludarabine phosphate, dexamethasone, cytosine arabinoside and cis-platinum (FLUDAP) in the treatment of patients with relapsed/refractory aggressive non-Hodgkin's lymphoma (NHL). This regimen comprises: dexamethasone 100 mg/d continuous infusion (cont. inf.) d1-3; cytosine arabinoside (ara-C) 1 g/m2/d cont. inf. d 2,3; fludarabine phosphate 30 mg/m2 short inf. 4hr prior to each 24hr ara-C inf.; cis-platinum 50 mg/m2 4hr inf. at the start of each 24hr ara-C inf. G-CSF (lenograstim, Granocyte) is given at 263 microg s.c. daily from day 7 until the neutrophil count reaches 1.0x10(9)/l. The regimen repeats at 21 day intervals. A total of 33 patients were registered (median age 47 years; 24 males, 9 females); the majority (73%) were refractory to their previous treatment and most had advanced disease by Ann Arbor stage. Thirteen (39%) of the 33 enrolled patients (52% of the 25 fully evaluable patients who received at least 2 courses of FLUDAP) responded to treatment. A maximum response of complete remission was achieved in 5 patients, good partial remission in 3, and partial remission in 5. Twelve patients went on to successful stem cell supported intensification therapy. Median survival times were higher in the responding patients, and in those patients transplanted post-FLUDAP. The toxicity associated with the FLUDAP regimen was generally predictable; frequently reported severe events included haematological toxicity and infection. In conclusion, the FLUDAP regimen shows promise as a salvage regimen and increases the available therapeutic options in the treatment of recurrent/refractory aggressive NHL.

Raised plasminogen activator inhibitor-1 (PAI-1) is not an independent risk factor in the polycystic ovary syndrome (PCOS).

BACKGROUND: It has been postulated that an insulin-driven increase in plasminogen activator inhibitor-1 (PAI-1) levels may link insulin resistance to anovulatory infertility in women with PCOS and that it may place them at increased risk of thromboembolic disease. However, previous studies have been conflicting because many have failed to control for body mass index (BMI) which may affect PAI-1. The aim of this study was to investigate PAI-1 activity in women with PCOS and to compare it with unaffected controls of a similar BMI. DESIGN AND PATIENTS: 41 women with PCOS and 25 weight-matched controls participated in this cross-sectional study. Patients were evaluated clinically and by pelvic ultrasound and fasting blood samples were taken for haematological and biochemical tests. MEASUREMENTS: PAI-1 activity, insulin, glucose, triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol, FSH, LH, PRL, testosterone, SHBG, 17-hydroxyprogesterone, plasminogen, fibrinogen (alpha2 antiplasmin, blood pressure and insulin sensitivity with a homeostasis model assessment (HOMA) computer programme. RESULTS: There was no significant difference in BMI or in (log) PAI-1 activity in women with PCOS compared with controls (BMI 29.5 +/- 5.6 vs. 28.4 +/- 6.3 kg/m2, P = 0.25 and PAI-1 2.56 (SD 0.85) vs. 2.14 (SD 0.98) au/ml, P = 0.07). The median fasting insulin level was significantly higher (17 (4.6-134.5) vs. 9.6 (3.7-41.5) mU/l, P < 0.01), and insulin sensitivity significantly lower in the PCOS group, ( 43.17% (5. 48-160) vs. 82.8% (21.8-193), P < 0.01). Women with PCOS also had a significantly higher free androgen index, LH/FSH ratio and a lower HDL/total cholesterol ratio. However blood pressure and all other lipid and haematological measurements were not significantly different between both groups. There were significant positive correlations between (log) PAI-1 activity and BMI (rho = 0.61), triglycerides (rho = 0.49) and fasting insulin (rho = 0.60) and a negative correlation with HDL cholesterol (rho = - 0.46). Triglyceride concentrations showed the most significant relationship with (log) PAI-1 activity on multiple regression. 29% of PCO women (12/41) gave a positive family history of thrombosis compared to 8% (2/25) in the control group. CONCLUSION: Plasminogen activator inhibitor-1 activity is not raised in women with PCOS independent of obesity and these results do not support the hypothesis that it may contribute to their anovulatory infertility, or increase their risk of thrombosis. The only significant metabolic features of the PCOS independent of obesity are insulin resistance, hyperinsulinaemia and lower HDL/total cholesterol ratio. The higher frequency of a positive family history of thrombosis in these women nevertheless requires further explanation.

A randomized controlled trial of itraconazole versus fluconazole for the prevention of fungal infections in patients with haematological malignancies. U.K. Multicentre Antifungal Prophylaxis Study Group.

Fluconazole is widely used as antifungal prophylaxis but it is ineffective against Aspergillus. Itraconazole has a broader spectrum of activity but the capsules give erratic bioavailability in neutropenic patients. We compared itraconazole oral solution (which has an improved pharmacokinetic profile) with fluconazole for antifungal prophylaxis. Adults with haematological malignancies receiving chemotherapy or bone marrow transplants were randomly allocated 5 mg/kg/d itraconazole (itra) solution (288 episodes) or 100 mg fluconazole suspension (flu) (293 episodes) from before the onset of neutropenia until neutrophil recovery or suspected fungal infection. Outcomes were assessed by independent reviewers unaware of the prophylaxis allocation. More proven systemic fungal infections occurred in flu (Aspergillus four, Candida tropicalis one, C. krusei one) than itra (C. albicans one) and more of these were fatal (four versus nil). This difference reached statistical significance when first study episodes were considered separately (six flu versus nil itra, P = 0.03). Significantly more deaths of presumed fungal origin occurred in flu than itra (seven versus nil, P = 0.024). There were significantly more cases of proven aspergillosis in flu than itra (six versus nil, P = 0.038, 5/6 cases were fatal) if those occurring outside the study period are included. Significantly more patients receiving flu required amphotericin B (58 v 39, P = 0.043) but this may have been affected by the fact that the study was not blinded. There were 11 proven mucosal candidal infections in flu and four in itra. Itraconazole solution and fluconazole provide effective prophylaxis against Candida but itraconazole affords greater protection against fatal aspergillosis.

Autoimmune haemolysis in patients with B-CLL treated with chlorodeoxyadenosine (CDA).

We have treated 19 B-chronic lymphocytic leukaemia (B-CLL) patients with CDA (Leustat, Janssen-Cilag). Four patients developed severe autoimmune haemolytic anaemia, and 2 of these had severe reticulocytopenia due to red cell aplasia/hypoplasia. Two patients died as a complication of the haemolysis one during the primary episode, with a clinical course suggestive of transfusion associated graft-versus-host disease (taGVHD), and one following a relapse of haemolysis. The onset of haemolysis occurs within 4 cycles of CDA therapy and is temporally related to the T-lymphocyte nadir induced by CDA. The presence of a positive DAT prior to therapy in 3 of 4 patients developing haemolysis suggests that the CDA induced T-lymphocytopenia may exacerbate the tendency of certain CLL patients to autoimmune haemolysis.