The risk of early and late CMV DNAemia associated with Campath use in stem cell transplant recipients.

The risks associated with in vivo and ex vivo use of Campath-1H and -1G in a cohort of 206 stem cell transplant recipients for human CMV (HCMV) DNAemia have been quantified. DNAemia showed a biphasic incidence pattern with an inflexion at day 60. The first phase had a linear risk rate for HCMV DNAemia of 0.3% per day, whereas the second phase had a substantially lower risk rate of 0.058% per day. In multivariable analyses, risk factors for early DNAemia were HCMV serostatus, radiotherapy-based conditioning and CD34 stem cell dose, with the use of in vivo Campath-1H having the most significant risk (hazards ratio=3.68; 95% CI=2.02-6.72; P<0.001). Ex vivo use of Campath was not associated with an increased risk for HCMV DNAemia. Patients receiving either in vivo Campath-1H or -1G experienced HCMV DNAemia earlier (27 and 33 days, respectively) compared with patients receiving no Campath (time to DNAemia, 51 days; P=0.0006). Multivariable analysis of risk factors for HCMV DNAemia occurring beyond 100 days after transplant were older age, acute GVHD>grade II and a lower CD34 stem cell dose, whereas Campath-1H use was not associated with late HCMV DNAemia.

A study to determine the safety profile and maximum tolerated dose of micafungin (FK463) in patients undergoing haematopoietic stem cell transplantation.

This open-label, dose-escalation study assessed the maximum tolerated dose (MTD) of the new antifungal micafungin in patients undergoing haematopoietic stem cell transplantation (HSCT). Participants received 3, 4, 6 or 8 mg/kg/day micafungin intravenously from 7 days to a maximum of 28 days or until neutropaenia resolved. The MTD was defined as the highest dose not causing the same Grade 3 or 4 adverse event in three or more patients. All 36 participants received >/=8 days treatment for a median of 18 days (range: 8-28); 1 patient withdrew consent and a further 11 discontinued to receive another systemic antifungal agent for a suspected infection. No case of confirmed invasive fungal infection occurred. Adverse events were those expected for patients undergoing HSCT and showed no evidence of dose-related toxicity. Criteria for MTD were not met; no patient had a Grade 3 or 4 adverse event considered causally related to micafungin. Thus, the MTD of micafungin can be inferred to be 8 mg/kg/day or higher.

Validation and clinical application of molecular methods for the identification of molds in tissue.

BACKGROUND: Invasive fungal infections due to less-common molds are an increasing problem, and accurate diagnosis is difficult. METHODS: We used our previously established molecular method, which allows species identification of molds in histological tissue sections, to test sequential specimens from 56 patients with invasive fungal infections who were treated at our institution from 1982 to 2000. RESULTS: The validity of the method was demonstrated with the establishment of a molecular diagnosis in 52 cases (93%). Confirmation of the causative organism was made in all cases in which a mold had been cultured from the tissue specimen. Less-common molds were identified in 7% of cases and appear to be an increasing problem. CONCLUSIONS: Our previously established method has proven to be of value in determining the incidence of invasive infection caused by less-common molds. Institutions should continue to pursue diagnosis of invasive fungal infections by means of tissue culture and microbiologic analysis.

Factors affecting reconstitution of the T cell compartment in allogeneic haematopoietic cell transplant recipients.

The factors affecting T cell reconstitution post haematopoietic cell transplantation (HCT) are not well characterised. We carried out a longitudinal analysis of T cell reconstitution in 32 HCT recipients during the first 12 months post transplant. We analysed reconstitution of naïve, memory and effector T cells, their diversity and monitored thymic output using TCR rearrangement excision circles (TRECs). Thymic-independent pathways were responsible for the rapid reconstitution of memory and effector T cells less than 6 months post HCT. Thymic-dependent pathways were activated between 6 and 12 months in the majority of patients with naïve T cell numbers increasing in parallel with TREC levels. Increasing patient age, chronic GVHD and T cell depletion (with or without pretransplant Campath-1H) predicted low TREC levels and slow naïve T cell recovery. Furthermore, increasing patient age also predicted high memory and effector T cell numbers. The effects of post HCT immunosuppression, total body irradiation, donor leucocyte infusions, T cell dose and post HCT infections on T cell recovery were also analysed. However, no effects of these single variables across a variety of different age, GVHD and T cell depletion groups were apparent. This study suggests that future analysis of the factors affecting T cell reconstitution and studies aimed at reactivating the thymus through therapeutic intervention should be analysed in age-, GVHD- and TCD-matched patient groups.

Treatment failure in invasive aspergillosis: susceptibility of deep tissue isolates following treatment with amphotericin B.

OBJECTIVES: To determine whether treatment failure in invasive aspergillosis (IA) is the result of resistance of Aspergillus spp. isolates to amphotericin B. METHODS: Six Aspergillus fumigatus and six Aspergillus flavus isolates cultured from deep tissue biopsies in 11 patients with haematological malignancies during 1991-1998 were tested. A method based on the NCCLS M38-A broth microdilution method, with colorimetric determination of MICs, was used to determine the MICs of amphotericin B and itraconazole. RESULTS: All A. fumigatus isolates were susceptible to amphotericin B (MIC 0.25-0.5 mg/L), as were three A. flavus isolates (MIC 1 mg/L), but three were less susceptible (MIC 2 mg/L). All isolates were susceptible to itraconazole (MIC 0.125-0.25 mg/L). All patients had been treated with amphotericin B, having received a median of 12 days of treatment when the tissue was obtained. CONCLUSION: The difficulty in treating IA may not be because of the susceptibility of the isolates, but because of poor penetration of antifungal agents into infected tissue. Aspergillus spp. invade blood vessels causing thrombosis and tissue infarction, and therefore it may be difficult for antifungal drugs to exceed MICs in infected tissues. This highlights the need for different treatment strategies, such as surgery and the administration of cytokines.

Identification of non-naïve CD4+CD45RA+ T cell subsets in adult allogeneic haematopoietic cell transplant recipients.

The study of thymic-dependent pathways of T cell reconstitution in T cell replete haematopoietic cell transplant (HCT) recipients in previous studies was complicated by the transfer of naïve CD4(+)CD45RA(+) T cells with the stem cell graft. However, direct quantification of thymic output has been enabled by measurement of T cell receptor excision circles (TREC). We analysed T cell reconstitution using T cell phenotyping and TREC quantification in 12 T cell-replete HCT recipients 6-53 years of age during the first 12 months post transplant. We have identified a novel subpopulation of CD4(+)CD45RA(+) T cells in the peripheral blood of these HCT recipients with expansions of this subset being more pronounced in older recipients. The recovery of classical naïve CD4(+)CD45RA(+) T cells was dependent on thymic output whereas this novel CD4(+)CD45RA(+) subpopulation arose independently of thymic output and displayed effector function and phenotype. These results suggest that CD4(+)CD45RA(+) effector populations exist, similar to the CD8(+)CD45RA(+) effector subset, and that the CD45RA antigen should not be used alone to define naïve CD4(+) T cells when monitoring T cell reconstitution in T cell replete HCT recipients. Furthermore, these results raise important questions regarding the role of the thymus in regulating T cell homeostasis in older HCT recipients and normal individuals.

In vitro co-stimulation with anti-CD28 synergizes with IL-12 in the generation of T cell immune responses to leukaemic cells; a strategy for ex-vivo generation of CTL for immunotherapy.

The existence of an immune based graft-versus-leukaemia (GvL) effect highlighted the prospect of managing relapsed leukaemias with T cell-based adoptive immunotherapy. Thus, various strategies have been explored for the in vitro expansion of acute myeloid leukaemia (AML)-specific T cells. In a popular approach, AML blasts have been genetically modified to express co-stimulatory molecules essential for effective T cell priming. One such tactic has been the modification of AML cells to express the B7/CD80 co-stimulatory molecule that binds to CD28 on T cells initiating events that culminate in enhanced cytokine production, proliferation and development of effector functions by T cells. The success of these strategies has been limited by difficulties in attaining sufficient transduction efficiencies and associated high levels of CD80 expression. We demonstrate that these problems can be circumvented by using anti-CD28 monoclonal antibody. Furthermore, we show that the synergistic relationship between CD80/CD28 pathway and interleukin 12 cytokine (IL-12), documented in the generation of cytotoxic T lymphocytes (CTL) for solid tumours, also applies to AML. CD28/IL-12 synergy facilitated the proliferation of allogeneic T cells in response to stimulation with primary AML blasts. The synergy also favoured generation of a Th1-type immune response, evidenced by gamma interferon (IFN-gamma) secretion and facilitated naive and memory T cell proliferation. Unlike some methods of in vitro T cell expansion, use of CD28/IL-12 synergy left T cells in the physiologically appropriate CD45RA-/CCR7- subsets known to be associated with immediate cytotoxic functions.

Granisetron in the control of nausea and vomiting associated with bone marrow transplantation: a review of its efficacy and tolerability.

Cancer patients undergoing bone marrow transplantation (BMT) experience severe nausea and vomiting associated with high-dose chemotherapy agents; these emetic symptoms are compounded by total body irradiation used in many conditioning regimens. This paper reviews clinical experience with the 5-HT(3) receptor antagonist granisetron, both as a single agent and in combination with other anti-emetics, in patients undergoing BMT and peripheral blood stem cell transplantation (PBSCT). Clinical studies demonstrate the efficacy (47-61% with no vomiting and no worse than mild nausea) and tolerability of granisetron. Its long half-life and duration of action may be responsible for its effective 24 h control of nausea and vomiting in BMT patients.

Functional impairment of human T-lymphocytes following PHA-induced expansion and retroviral transduction: implications for gene therapy.

The immune function of retrovirus-mediated gene modified (GM) T cells is critical for a beneficial effect to follow their adoptive transfer into patients. Recent clinical data show that GM T cells expanded with PHA have reduced function in vivo. However, little functional analysis of PHA stimulation is available. Our results show that expansion of T cells with PHA impairs their ability to respond (proliferation, cytotoxicity and IFN gamma and perforin expression) to allogeneic stimulation or viral antigens in vitro. Conversely, CD3/CD28-based protocols can preserve this immune function. Retroviral transduction did not alter the functional profile induced by polyclonal stimulation. We investigated the mechanisms leading to this functional effect, and identified differential effects of PHA and CD3/CD28 on the distribution of CCR7/CD45RA T cell functional subsets, which may explain the functional differences observed. While CD3/CD28 stimulation parallels the lineage differentiation pattern induced by antigens in physiological conditions, PHA induces a skewed distribution of the CCR7/CD45RA functional T cell subsets, with near disappearance of the subpopulations that display the effector phenotype. Overall, this study demonstrates a functional disadvantage for transduction protocols based on PHA, uncovers mechanisms that may explain this functional effect, and provides us with information to design and select transduction protocols with an improved functional outcome.

Toxoplasmosis in bone marrow transplantation: a report of two cases and systematic review of the literature.

Toxoplasma infection represents a rare but often fatal complication in bone marrow transplant (BMT) recipients. We report two cases of toxoplasmosis: one of successfully treated cerebral toxoplasmosis after peripheral blood stem cell transplantation, and a fatal case of pulmonary toxoplasmosis in a BMT recipient. We have systematically reviewed the 110 published cases of toxoplasmosis following BMT. We analyzed the pre-transplant and clinical features of BMT recipients developing toxoplasmosis, together with the diagnostic procedures used and treatment given. By univariate and multivariate statistical analysis we analyzed the risk factors for diagnosis (during life vs post-mortem) and Toxoplasma-related mortality. Ante-mortem diagnosis was made in 47% of cases. Site of infection (P = 0.02; odds ratio 10.8), presence of symptoms at onset (P = 0.01) and conditioning regimen (P = 0.04) were factors influencing whether the diagnosis was made before or after death. Overall mortality rate was 80% and that attributed to toxoplasmosis was 66%. Variables influencing outcome were: site of infection (P = 0.02; odds ratio 5.28), day of onset (P = 0.04) and conditioning regimen (P = 0.04). Underlying disease (P = 0.02; odds ratio 9.45), among patients diagnosed before death, was the most significant factor influencing outcome.

Sequence of two new HLA-Cw alleles: HLA-Cw*0313 and HLA-Cw*1208.

Two new HLA-C alleles have been identified by reverse line blot assay and direct sequencing of polymerase chain reaction (PCR) products. The first polymorphism encodes an amino acid change from lysine in Cw*12022 to asparagine in Cw*1208 at residue 66. The second polymorphism encodes two amino acid changes from isoleucine in Cw*03031 to threonine in Cw*0313 at residue 93 and isoleucine to leucine at residue 94. The functional significance of these polymorphisms on peptide-binding and/or T-cell recognition is unknown.

ETV6/AML1 fusion by FISH in adult acute lymphoblastic leukemia.

Dual-color interphase fluorescence in situ hybridization (FISH) with ETV6 and AML1 probes was used for the first time on a series of 159 adult patients with acute lymphoblastic leukemia (ALL), for detection of the t(12;21)(p13;q22) translocation. Seven patients (4.4%) were found, with 50-100% of positive cells, of whom one of two tested, proved negative for the fusion product by RT-PCR. Two of them, aged 43 and 50 years, are the oldest patients so far confirmed to have the translocation. Three who relapsed at 10, 11 and 24 months, suggest that adults may not enjoy the good short-term prognosis reported for t(12;21)-positive children. Thirty-one-negative cases had signal numbers differing from the two expected for each gene. In 15 cases these results were consistent with the karyotype. In nine cases with uninformative cytogenetics, the numbers were consistent with those for centromeres and indicated a hidden aneuploidy. Loss of ETV6 genes in two cases and AML1 amplification in three others were not suspected from the cytogenetics. In conclusion, FISH proved to be reliable in defining ETV6/AML1 positivity in this group of patients as well as providing valuable insights into negative cases.

Immunotherapy for Hodgkin's disease.

EBV proteins present in the malignant Hodgkin Reed-Sternberg (HR-S) cells of about 40% of patients with Hodgkin's Disease (HD) provide targets for immunotherapy with virus-specific cytotoxic T lymphocytes (CTL). However, Hodgkin tumors use multiple strategies to avoid CTL, including down-regulation of immunodominant EBV antigens, and secretion of cytokines and chemokines such as TGF-beta, that inhibit the activation of CTL and professional antigen-presenting cells (APC). To be effective against this tumor, CTL must resist some or all of these strategies. Thirteen patients with multiply-relapsed HD received EBV-specific CTL, generated ex vivo using the autologous EBV-transformed B cells (LCL) as stimulator cells. After CTL infusion, EBV-specific immunity increased, virus load decreased, CTL homed to sites of malignancy and persisted for up to ten months. Clinically, CTL produced resolution of B symptoms and mixed tumor responses including one complete remission of residual disease remaining after autologous bone marrow transplant. However, no complete remission of bulky disease was achieved. Although LMP2-specific CTL activity could be detected in some of the infused CTL lines, they were present in low frequency. In pre-clinical studies, LMP1 and LMP2-specific CTL could be produced by stimulating PBMC from patients and normal donors with autologous dendritic cells expressing LMP1 or LMP2 from adenoviral vectors. Further, CTL could be rendered resistant to the devastating effects of TGF-beta by transduction with a retrovirus vector expressing a dominant-negative TGF-beta receptor, while transgenic IL-12 could increase the expression of Th1 and decrease that of Th2 cytokines. Future clinical studies will test the efficacy of CTL with improved antigen-specificity and resistance to Hodgkin immune evasion strategies.

Treatment of relapse after allogeneic bone marrow transplantation with reduced intensity conditioning (FLAG +/- Ida) and second allogeneic stem cell transplant.

Acute leukaemias in relapse after allogeneic stem cell transplantation (SCT) respond poorly to donor leucocyte infusions (DLI) compared with chronic myeloid leukaemia (CML), at least in part because of faster disease kinetics. Fludarabine-containing 'non-myeloablative' chemotherapy followed by further allo SCT may offer more rapid and effective disease control. We report 14 patients with relapse after allo SCT for acute leukaemia [seven acute myeloid leukaemia (AML), five acute lymphoblastic leukaemia (ALL)] or refractory anaemia with excess blasts in transformation (RAEB-t, n = 2) treated with fludarabine, high-dose cytosine arabinoside (ara-C) and granulocyte colony-simulating factor (G-CSF) with (n = 10) or without (n = 2) idarubicin (FLAG +/- Ida) or DaunoXome (FLAG-X) (n = 2) and second allo SCT from the original donor. Donors were fully human leucocyte antigen (HLA) -matched in 13 cases with a single class A mismatch in one. Actuarial overall survival was 60% and disease-free survival was 26% at 58 months. Remissions after the second SCT were longer than those after the first bone marrow transplantation (BMT) in eight of the 13 assessable patients to date. Haematopoietic recovery was rapid. Transplants were well tolerated with no treatment-related deaths. The major complication was graft-versus-host disease (GvHD, acute >/= grade II-2 cases, chronic - eight cases, two limited, six extensive) although there have been no deaths attributable to this. FLAG +/- Ida and second allo SCT is a safe and useful approach and may be more effective than DLI in the treatment of acute leukaemias relapsing after conventional allo SCT.

Acute myeloid leukaemia cells secrete a soluble factor that inhibits T and NK cell proliferation but not cytolytic function--implications for the adoptive immunotherapy of leukaemia.

Evidence of an immune mediated graft-versus-leukaemia effect has led to the belief that T and NK cell based adoptive immunotherapy can constitute effective treatment for relapsed leukaemias. However, work on solid tumours has shown this strategy may be hampered, by an immune escape mechanism in which tumour secreted immunosuppressive factors compromise T and NK cell function. Indeed, acute myeloid leukaemia (AML) cells secrete immunosuppressive factors that block the synthesis of Th1 type cytokines in T cells. We demonstrate here that this immunosuppression, mediated by both HL60 AML cell line and primary AML blasts, inhibits T and NK cell proliferation but not cytolytic activity. Supernatants from HL60 cell line and primary AML blasts inhibited T cell proliferation to mitogenic and alloantigen stimulation but had no effect on cytolytic function. Similarly, the proliferation of NK cells to IL-2 and IL-15 stimulation was inhibited whilst their cytolytic function, shown by lysis of AML blasts, K562 and Daudi cells remained unaffected. The failure of T and NK cells to proliferate was not due to effector cell apoptosis. Indeed, removal of lymphocytes from the immunosuppressive environment partially restored their capacity to respond to mitogenic stimulation. T cells exposed to immunosuppressive supernatants did not increase expression of mitotic inhibitory proteins that arrest cell division, thereby ruling this out as a mechanism of operation for this immunosuppression. T cell expansion requires antigen stimulation, usually provided in the form of AML blasts, therefore our data suggest that NK cells may be more practical for the immunotherapy of AML.

Oral ciprofloxacin plus colistin: prophylaxis against bacterial infection in neutropenic patients. A strategy for the prevention of emergence of antimicrobial resistance.

Following a 2-year study, the combination of oral ciprofloxacin and colistin has been used continuously for 10 years without the emergence of resistance. During a 2-year period (1987-1989), we compared ciprofloxacin + colistin (CIP + COL) with neomycin + colistin (NEO + COL) in a randomized trial--combinations chosen because of the potential for prophylaxis of Gram-negative infection by ciprofloxacin, with colistin given to reduce the risk of emergence of resistance. Sixty-four patients with similar demographics in each arm were evaluable for efficacy analysis. Patients on CIP + COL had a significantly lower proportion of neutropenic days with fever (P < 0.001) and neutropenic days on intravenous antibiotics (P < 0.001) than patients on NEO + COL. A total of 54 (15 bacteriologically documented) pyrexial episodes occurred in patients on CIP + COL and 77 (41 bacteriologically documented) in patients on NEO + COL. Only two Gram-negative bacterial infections occurred in the CIP + COL arm compared with 16 in the NEO + COL arm. No Staphylococcus aureus infections occurred in the CIP + COL group compared with 10 in the other patients. Two CIP-resistant Gram-negative bacilli were isolated from patients on CIP + COL compared with 13 NEO-resistant Gram-negative bacilli from patients on NEO + COL. Following a subsequent decade of unchanged use of this prophylactic strategy in neutropenic patients, a 2-year follow-up study between 1 January 1998 and 31 December 1999 showed 66 significant infections during 700 [corrected] neutropenic episodes. Thirty-five of the 111 (31%) isolates were ciprofloxacin-resistant, involving 5% of the neutropenic episodes [corrected].

Caspase 8 activation independent of Fas (CD95/APO-1) signaling may mediate killing of B-chronic lymphocytic leukemia cells by cytotoxic drugs or gamma radiation.

Ligation of the cell-surface Fas molecule by its ligand (Fas-L) or agonistic anti-Fas monoclonal antibodies results in the cleavage and activation of the cysteine protease procaspase 8 followed by the activation of procaspase 3 and by apoptosis. In some leukemia cell lines, cytotoxic drugs induce expression of Fas-L, which may contribute to cell killing through the ligation of Fas. The involvement of Fas, Fas-L, and caspase 8 was studied in the killing of B-cell chronic lymphocytic leukemia (B-CLL) cells by chlorambucil, fludarabine, or gamma radiation. Spontaneous apoptosis was observed at 24-hour incubation, with additional apoptosis induced by each of the cytotoxic treatments. Although Fas mRNA expression was elevated after exposure to chlorambucil, fludarabine, or gamma radiation, Fas protein levels only increased after irradiation. Therefore, Fas expression may be regulated by multiple mechanisms that allow the translation of Fas mRNA only in response to restricted cytotoxic stimuli. None of the cytotoxic stimuli studied here induced Fas-L expression. An agonistic anti-Fas monoclonal antibody (CH-11) did not significantly augment apoptosis induction by any of the death stimuli. A Fas-blocking antibody (ZB4) did not inhibit spontaneous, chlorambucil-, fludarabine-, or radiation-induced apoptosis. However, procaspase 8 processing was induced by all cytotoxic stimuli. These data suggest that the Fas/Fas-L signaling system does not play a major role in the induction of apoptosis in B-CLL cells treated with cytotoxic drugs or radiation. However, Fas-independent activation of caspase 8 may play a crucial role in the regulation of apoptosis in these cells.

Heterogeneity of VH-JH gene rearrangement patterns: an insight into the biology of B cell precursor ALL.

Oligoclonal B cell proliferation, as defined by the presence of more than one leukemic clone, has been detected in approximately 20% to 30% of patients with acute lymphoblastic leukemia (ALL) using PCR or Southern blotting. An accurate assessment of these populations is required to avoid false negative measurements of minimal residual disease (MRD) in follow-up bone marrow (BM) samples of ALL patients. In this study, we analysed 29 ALL patients with two or more immunoglobulin heavy (IGH) chain gene rearrangements in the presentation samples using IGH fingerprinting PCR and sequence analysis. Thirty-nine (51%) of 76 sequences (from 15 patients), shared no VNDNJ homology (ie different CDR3 regions). In the remaining 14 patients, at least two related VH sequences were identified in each patient (identical DNJ sequences). Numerical abnormalities of chromosome 14 was detected in 10 patients. Eight patients were analysed at presentation and relapse. In four of them, expansion of a minor presentation-clone was detected at relapse while the major presentation clone disappeared, confirming 'subclonal evolution'. Finally, in our cohort of patients, the presence of related or unrelated IGH clones did not influence overall survival.