Archie G. Prentice

A comparison of low-dose cytarabine and hydroxyurea with or without all-trans retinoic acid for acute myeloid leukemia and high-risk myelodysplastic syndrome in patients not considered fit for intensive treatment

The survival of older patients with acute myeloid leukemia has not improved. Few clinical trials have been available for older patients who are not considered fit for an intensive chemotherapy approach.

Towards a targeted, risk‐based, antifungal strategy in neutropenic patients

The major issues in the management of fungal infection are prevention, diagnosis and treatment. Our main goal must remain prevention, but for a number of reasons prophylaxis against invasive fungal infection (IFI), in the neutropenic patient, remains controversial. We consider this aim to be desirable, given the high mortality rates associated with established infection, due in part to inadequacies and substantial delays in diagnosis. In a meta-analysis Bow et al (1997), found a mortality rate of 47% in patients with invasive fungal infection. In a more recent prospective EORTC survey, Denning et al (1999) found that the 3-month mortality rate in patients with invasive aspergillosis was 64%. However, clinical trials of antifungal prophylaxis have failed to show a reduction in overall mortality in almost every case, other than those conducted in the highest risk patients. Fuel was recently added to the fire of this debate by the publication of a further meta-analysis on the role of prophylaxis and empirical treatment (Gotzsche & Johansen, 1997). We have criticized this report for a number of reasons (Kibber et al, 1997) but do support the contention that more studies are needed using modern diagnostic tools, including PCR, as end-points, given the inadequacy of conventional antemortem methods. In particular, we feel that consideration should be given to the study of targeted prophylaxis and treatment in defined risk groups.

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.

Curability of Patients With Acute Myeloid Leukemia Who Did Not Undergo Transplantation in First Remission

PURPOSE The aims of this study were to quantify the prospects of salvage treatment of patients who did not undergo transplantation in first complete remission (CR1) and to assess the contribution of allograft in second complete remission (CR2) with respect to major risk groups. This evaluation can inform the decision whether to offer a transplant in CR1. PATIENTS AND METHODS Of 8,909 patients who entered the Medical Research Council AML10, AML12, and AML15 trials, 1,271 of 3,919 patients age 16 to 49 years who did not receive a transplant in CR1 relapsed. Of these patients, 19% are alive beyond 5 years compared with 7% of patients who relapsed after an allograft in CR1. Overall survival and the contribution of a transplant in CR2 were assessed overall and by cytogenetic risk group by using Mantel-Byar analysis. RESULTS Fifty-five percent of patients who relapsed entered CR2. This percentage varied by risk group as follows: favorable (82%), intermediate (54%), adverse (27%), and unknown (53%), which resulted in 5-year survivals of 32%, 17%, 7%, and 23%, respectively. Sixty-seven percent of remitters received an allotransplant that delivered superior survival compared with patients who did not receive a stem-cell transplant (42% v 16%). A more-stringent assessment of a transplant by using delayed-entry (Mantel-Byar) analysis confirmed the benefit of transplant overall and within intermediate and adverse risk groups but not the favorable subgroup. CONCLUSION Successful salvage treatment of patients who do not undergo transplantation in CR1 and relapse can be achieved in 19% of patients, which is improved by a transplant except in favorable risk disease. This result suggests that, for intermediate-risk patients in particular, equivalent overall survival can be achieved by delaying transplantation until after relapse, which would require many fewer transplants.

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 αβ (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.

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‐γ 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‐TCRαβ monoclonal antibodies. B‐CLL T cells stimulated with B‐CLL lysate‐pulsed autologous dendritic cells showed a significant (P = 0·0004) increase in IFN‐γ 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‐TCRαβ (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.

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‐α, polyriboinosinic polyribocytidylic acid (Poly(I:C)) and LPS to autologous DCs resulted in the emergence of only a small percentage of CD83+ DCs, IFN‐α having no demonstrable effect. Only the addition of Poly(I:C) to DCs resulted in modestly increased specific cytotoxicity to B‐CLL targets, IFN‐α 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‐γ 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.

p53 intronic point mutation, aberrant splicing and telomeric associations in a case of B-chronic lymphocytic leukaemia

We report a case of chronic lymphocytic leukaemia (CLL) with telomeric associations and a p53 intronic point mutation. Karyotypic analysis revealed clonal and non‐clonal telomeric associations, accompanied by clonal cytogenetic abnormalities and also in isolation. The p53 mutation, which occurred at the invariant base pair −2 of the splice acceptor site in intron 7 resulted in the abolition of correct splicing of exon 7 to exon 8. Multiple aberrant splice products were characterized, all of which differed from wildtype in the DNA binding domain. Fluorescence in situ hybridization demonstrated that the clone retained two copies of the p53 gene and wild‐type p53 transcript was detected on cloning of reverse transcriptase polymerase chain reaction (RT‐PCR) product, indicating that one wild‐type allele remained. However, a plasmid clone with correct splicing at the exon 7/8 boundary, but with a 21 bp deletion in exon 8, was also found at low frequency. This finding indicates clonal evolution, resulting in complete loss of wild‐type p53. The intronic point mutation was not present in DNA extracted from cervical tissue indicating that it was a leukaemic phenomenon. This is the first case of an intronic point mutation to be reported in CLL. This mutation led to chaotic p53 expression and, interestingly, occurred in a case showing telomeric associations, a rare phenomenon in B‐CLL.

Cost Effectiveness of Itraconazole in the Prophylaxis of Invasive Fungal Infections

BackgroundInvasive fungal infections in neutropenic patients treated for haematological malignancies are associated with a high mortality rate and, therefore, require early treatment. As the diagnosis of invasive fungal infections is difficult, effective antifungal prophylaxis is desirable. So far, fluconazole has been the most commonly used.ObjectiveTo assess the cost effectiveness of itraconazole compared with both fluconazole and no prophylaxis for the prevention of invasive fungal infections in haematological patients, mean age 51 years, in Germany and The Netherlands.Study designWe designed a probabilistic decision model to fully incorporate the uncertainty associated with the risk estimates of acquiring an invasive fungal infection. These risk estimates were extracted from two meta-analyses, evaluating the effectiveness of fluconazole and itraconazole and no prophylaxis. The perspective of the analysis was that of the healthcare sector; only medical costs were taken into account. All costs were reported in €, year 2004 values.Cost effectiveness was expressed as net costs per invasive fungal infection averted. No discounting was performed, as the model followed patients during their neutropenic period, which was assumed to be less than 1 year.ResultsAccording to our probabilistic decision model, the monetary benefits of averted healthcare exceed the costs of itraconazole prophylaxis under baseline assumptions (95% CI: from cost-saving to €5000 per invasive fungal infection averted). Compared with fluconazole, itraconazole is estimated to be both more effective and more economically favourable, with a probability of almost 98%.ConclusionsIn specific groups of neutropenic patients treated for haematological malignancies, itraconazole prophylaxis could potentially reduce overall healthcare expenditure, without harming effectiveness, in settings where fluconazole is common practice in the prophylaxis of invasive fungal infections.