Effect of an antimicrobial stewardship programme on antimicrobial utilisation and costs in patients with leukaemia: a retrospective controlled study.

OBJECTIVES: To examine the effectiveness of an antimicrobial stewardship programme on utilization and cost of antimicrobials in leukaemia patients in Canada. METHODS: We conducted a multisite retrospective observational time series study from 2005 to 2013. We implemented academic detailing as the intervention of an antimicrobial stewardship programme in leukaemia units at a hospital, piloted February-July 2010, then fully implemented December 2010-March 2013, with no intervention in August-November 2010. Internal control was the same hospital's allogeneic haematopoietic stem-cell transplantation unit. External control was the combined leukaemia-haematopoietic stem-cell transplantation unit at another hospital. Primary outcome was antimicrobial utilization (antibiotics and antifungals) in defined daily dose per 100 patient-days (PD). Secondary outcomes were antimicrobial cost (Canadian dollars per PD); cost and utilization by drug class; length of stay; 30-day inpatient mortality; and nosocomial Clostridium difficile infection. We used autoregressive integrated moving average models to evaluate the impact of the intervention on outcomes. RESULTS: The intervention group included 1006 patients before implementation and 335 during full implementation. Correspondingly, internal control had 723 and 264 patients, external control 1395 and 864 patients. Antimicrobial utilization decreased significantly in the intervention group (p <0.01, 278 vs. 247 defined daily dose per 100 PD), increased in external control (p = 0.02, 237.4 vs. 268.9 defined daily dose per 100 PD) and remained stable in internal control (p = 0.66). Antimicrobial cost decreased in the intervention group (p = 0.03; $154.59 per PD vs. $128.93 per PD), increased in external control (p = 0.01; $109.4 per PD vs. $135.97 per PD) but was stable in internal control (p = 0.27). Mortality, length of stay and nosocomial C. difficile rate in intervention group remained stable. CONCLUSIONS: The antimicrobial stewardship programme reduced antimicrobial use in leukaemia patients without affecting inpatient mortality and length of stay.

Venous thromboembolism prevention during asparaginase-based therapy for acute lymphoblastic leukemia.

BACKGROUND: Venous thromboembolism (vte) is a recognized complication in patients treated with asparaginase-containing chemotherapy regimens; the optimal preventive strategy is unclear. We assessed the safety and efficacy of prophylaxis using low-dose low molecular weight heparin in adult patients with acute lymphoblastic leukemia in complete remission treated with an asparaginase-based post-remission chemotherapy regimen. METHODS: As part of the intensification phase of the Dana-Farber Cancer Institute 91-01 regimen, asparaginase was administered weekly to 41 consecutive patients for 21-30 weeks; these patients also received prophylaxis with enoxaparin 40 mg daily (60 mg for patients ≥80 kg). Outcomes were assessed against outcomes in a comparable cohort of 99 patients who received the same chemotherapy regimen without anticoagulation prophylaxis. RESULTS: The overall rate of symptomatic venous thrombosis was not significantly different in the prophylaxis and non-prophylaxis cohorts (18.92% and 21.74% respectively). Among patients receiving prophylaxis, vte occurred in higher proportion in those who weighed at least 80 kg (42.86% vs. 4.35%, p = 0.0070). No major bleeding complications occurred in the prophylaxis group (minor bleeding: 8.1%). CONCLUSIONS: Prophylaxis with low-dose enoxaparin during the intensification phase was safe, but was not associated with a lower overall proportion of vte.

A Canadian consensus on the management of newly diagnosed and relapsed acute promyelocytic leukemia in adults.

The use of all-trans-retinoic acid (atra) and anthracyclines (with or without cytarabine) in the treatment of acute promyelocytic leukemia (apl) has dramatically changed the management and outcome of the disease over the past few decades. The addition of arsenic trioxide (ato) in the relapsed setting-and, more recently, in reduced-chemotherapy or chemotherapy-free approaches in the first-line setting-continues to improve treatment outcomes by reducing some of the toxicities associated with anthracycline-based approaches. Despite those successes, a high rate of early death from complications of coagulopathy remains the primary cause of treatment failure before treatment begins. In addition to that pressing issue, clarity is needed about the use of ato in the first-line setting and the role of hematopoietic stem-cell transplantation (hsct) in the relapsed setting. The aim for the present consensus was to provide guidance to health care professionals about strategies to reduce the early death rate, information on the indications for hsct and on the use of ato in induction and consolidation in low-to-intermediate-risk and high-risk apl patients.

The prognostic impact of FLT3-ITD and NPM1 mutations in patients with relapsed acute myeloid leukemia and intermediate-risk cytogenetics.

Internal tandem duplication of the fms-like tyrosine kinase-3 gene (FLT3-ITD) and nucleophosmin-1 (NPM1) mutations have prognostic importance in acute myeloid leukemia (AML) patients with intermediate-risk karyotype at diagnosis, but less is known about their utility to predict outcomes at relapse. We retrospectively analysed outcomes of 70 patients with relapsed, intermediate-risk karyotype AML who received a uniform reinduction regimen, with respect to FLT3-ITD and NPM1 mutation status and first complete remission (CR1) duration. CR1 duration, but not molecular status, was significantly correlated with CR2 rate. On univariate analysis, patients with mutated FLT3-ITD (FLT3+) had significantly worse overall survival (OS) compared with those with neither an NPM1 nor FLT3-ITD mutation (NPM1-/FLT3-). On multivariate analysis, shorter CR1 duration was significantly correlated with inferior OS at relapse (P<0.0001), while FLT3 and NPM1 mutation status and age were not significantly correlated with OS. Patients who subsequently underwent allogeneic stem cell transplant (alloSCT) had a superior OS regardless of CR1 duration, but outcomes were better in patients with CR1 duration>12 months. In intermediate-risk karyotype AML patients receiving reinduction, CR1 duration remains the most important predictor of OS at relapse; FLT3-ITD and NPM1 status are not independent predictors of survival.

A phase I/II study of imatinib plus reinduction therapy for c-kit-positive relapsed/refractory acute myeloid leukemia: inhibition of Akt activation correlates with complete response.

This phase I/II study evaluated imatinib as a c-kit inhibitor combined with mitoxantrone, etoposide and cytarabine therapy for patients with primary refractory or relapsed c-kit+ acute myeloid leukemia (AML). Imatinib was escalated through three dose levels in successive six patient cohorts. The combination was well tolerated up to 400 mg/day imatinib. Of 21 patients treated at this dose, 13 (62%) achieved complete response (CR), 7 (33%) were non-responders and one died during induction. The CR rate was 80% in patients with standard-risk karyotype versus 33% in patients with adverse karyotype. The CR rate for primary non-responders was 6/14 (43%) versus 7/7 (100%) for relapsed patients. AML blasts from peripheral blood were assayed for phosphorylated Akt (pAkt) and phosphorylated ERK (pERK) by flow cytometry before to and after imatinib dosing. Of eight patients achieving CR with reinduction, seven demonstrated marked (≥60%) pAkt inhibition with imatinib therapy. In contrast, all the six non-responders to reinduction demonstrated <60% pAkt inhibition (P=0.005). There was no correlation between pERK inhibition and response to therapy. These results indicate that lack of pAkt inhibition in vivo is associated with resistance to reinduction therapy using this regimen. Further studies using agents that are able to inhibit Akt more effectively are warranted.

A phase I study of tipifarnib combined with conventional induction and consolidation therapy for previously untreated patients with acute myeloid leukemia aged 60 years and over.

Patients aged 60 years and over with previously untreated acute myeloid leukemia were enrolled in a Phase I study combining tipifarnib with standard induction therapy. The regimen consisted of cytarabine 100 mg/m(2)/day continuous intravenous (i.v.) infusion on days 1-7, daunorubicin 60 mg/m(2)/day i.v. push x 3 on days 6-8 and tipifarnib twice daily on days 6-15. Tipifarnib was escalated over four dose levels (200, 300, 400 and 600 mg). Patients achieving complete response (CR) were eligible to receive one consolidation using the same regimen. The following dose-limiting toxicities (DLTs) were identified during induction: dose level I: 2/6 (hyperbilirubinemia, respiratory arrest), level II: 0/3, level III: 0/3 and level IV: 4/10 (one each of diarrhea, neutropenic enterocolitis, arrhythmia and delayed hematologic recovery post-consolidation). There were no DLTs due to delayed hematologic recovery post-induction. Of 22 evaluable patients, there were 10 CR, 2 morphologic leukemia-free state (MLFS), 2 partial remission (PR) and 8 non-responders. Of seven patients with adverse risk cytogenetics, there were four CR/MLFS and one PR. In summary, this regimen was well tolerated and the maximum tolerated dose was not reached, although somewhat more severe gastrointestinal toxicity was seen at dose level IV. Tipifarnib 600 mg b.i.d. is considered the recommended dose for further study using this regimen.

Evidence of functional interaction between NuMA-RARalpha and RXRalpha in an in vivo model of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by reciprocal balanced chromosomal translocations involving retinoic acid receptor-alpha (RARalpha). RARalpha heterodimerizes with the retinoid X receptor-alpha (RXRalpha) and transcriptionally regulates myeloid differentiation in response to ATRA (all-trans retinoic acid). Several lines of evidence suggest that APL fusion proteins interact with RXRalpha. To elucidate the role of RXRalpha in APL, we conditionally knocked out RXRalpha in the hCG-NuMA-RARalpha APL mouse model. Phenotype analysis of NuMA-RARalpha+ mice demonstrated that these mice developed a myeloproliferative disease-like myeloid leukemia within 4 months of birth. While hemizygous and homozygous RXRalpha conditional knockout mice were phenotypically normal as late as 12 months of age, we observed that the leukemic phenotype in NuMA-RARalpha+ mice was dependent on the presence of functional RXRalpha. Bone marrow promyelocyte counts were significantly reduced in NuMA-RARalpha+ mice with RXRalpha knocked down. Significant differences in the accumulations of Gr-1+ and Mac-1+ cells were also seen. We further observed that genes previously identified to be cooperating events in APL were also regulated in an RXRalpha-dependent manner. We therefore propose that the APL fusion protein NuMA-RARalpha cooperates with RXRalpha in the development of leukemia in hCG-NuMA-RARalpha transgenic mice and suggest a novel role for RXRalpha in the pathogenesis of APL.

Assessment of the downstream portion of the mitochondrial pathway of caspase activation in patients with acute myeloid leukemia.

Most chemotherapeutic agents used in the treatment of acute myeloid leukemia (AML) induce apoptosis by triggering the mitochondrial pathway of caspase activation. To investigate the downstream portion of the mitochondrial pathway of caspase activation in patients with AML, cytosolic lysates were stimulated with cytochrome c and dATP and hydrolysis of Ac-DEVD-AFC by effector caspases was measured. Defects in the distal mitochondrial pathway were more common in samples from patients with AML that relapsed rapidly after induction chemotherapy compared to samples from treatment naïve patients. The incidence of blocked pathways did not differ based on response to induction chemotherapy, as even nonresponders generally had an intact pathway. When the distal mitochondrial pathway was blocked, defects were usually at the level of the effector caspases. Thus, functional defects in the distal portion of the mitochondrial pathway of caspase activation may help explain the nature of response and relapse after treatment.

Treatment of elderly patients with acute lymphoblastic leukemia--evidence for a benefit of imatinib in BCR-ABL positive patients.

All patients with acute lymphoblastic leukemia (ALL) over age 60 years seen at Princess Margaret Hospital over an 11-year period were analysed retrospectively. Of 53 patients, 45 received multiagent induction chemotherapy using a variety of regimens. There were 13 BCR-ABL positive patients, 9 of who received imatinib mesylate, either during induction or post-remission therapy. The overall complete remission (CR) rate of all 45-induction patients was 56%, with a 27% induction-related mortality rate. The CR rate was not influenced by induction regimen, age, initial WBC, LDH or BCR-ABL status. The median overall survival of the induction patients was 9 months, while the median progression-free survival (PFS) of the patients achieving CR was 10 months. The estimated overall survival (OS) at 3 years was 18.4% (95% CI: 9.8-34.3%). Age and initial WBC did not significantly predict for OS when evaluating the entire group of induction patients. However, there was a strong trend for BCR-ABL status to favorably predict for PFS, and for OS when only patients treated after July 2000 (when imatinib became available) were evaluated. The results indicate that ALL remains a poor prognosis disease in elderly patients, and that aggressive induction regimens designed for younger patients are very toxic for these patients. These data suggest that BCR-ABL+ ALL is becoming a relatively more favorable prognosis disease in the elderly, likely due to the influence of imatinib therapy. Further regimens should explore the use of less aggressive regimens in elderly patients and should evaluate the optimal way of combining imatinib with conventional agents in BCR-ABL+ patients.

The role of allogeneic bone marrow transplantation in adult patients below the age of 55 years with acute lymphoblastic leukemia in first complete remission: a donor vs no donor comparison.

The role of allogeneic bone marrow transplantation (alloBMT) in adults with acute lymphoblastic leukemia (ALL) in first complete remission (CR1) remains controversial. At our institution, the policy is to offer alloBMT to ALL patients in CR1 up to the age of 55 years if a related donor is available. In addition, unrelated donor transplants are offered to patients with Philadelphia (Ph+) ALL. We report the results on 92 patients with ALL treated according to this policy from September 1992 to October 2001. Of the 87 patients achieving CR1, the comparison of patients with (n=48) or without donors (n=39) was done using an intention-to-treat approach. Of the 48 patients with donors (39 related and nine unrelated), 35 (73%) received alloBMT in CR1. No significant difference in 3-year event-free survival (EFS) (40 vs 39%, P=0.74) or overall survival (OS) (46 vs 58%, P=0.41) was seen in 'donor' vs 'no-donor' groups. For Ph+ patients, 3-year EFS and OS in 'donor' group were 46 and 57%, respectively, none of the patients in 'no-donor' group survived beyond 3 years. With our treatment strategy, 3-year OS of Ph+ patients was equivalent to Ph-negative (Ph-) patients (51 vs 52%, P=0.77). In conclusion, our data show that the policy of performing alloBMT if a sibling donor is available has not resulted in better outcome in Ph- patients.

White cells 2: impact of understanding the molecular basis of haematological malignant disorders on clinical practice.

The molecular basis of many leukaemias is now known, allowing precise diagnosis. Treatment of chronic myeloid leukaemia is now possible by targeting of the BCR-ABL tyrosine kinase. The underlying molecular abnormalities in acute leukaemias allow the outlook for individual patients to be assessed at diagnosis and therapy tailored accordingly. Analysis of V(H) genes in B-cell malignant disorders allows these to be placed in the hierarchy of B-cell development and may provide prognostically valuable information.

Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is believed to play a role in oncogenesis. To elucidate further its physiologic role(s), we have characterized the murine survivin gene and complementary DNA (cDNA). The structural organization of the survivin gene, located on chromosome 11E2, is similar to that of its human counterpart, both containing 4 exons. Surprisingly, 3 full-length murine survivin cDNA clones were isolated, predicting the existence of 3 distinct survivin proteins. The longest open reading frame, derived from all 4 exons, predicts a 140-amino acid residue protein, survivin(140), similar to human survivin, which contains a single IAP repeat and a COOH-terminal coiled-coil domain that links its function to the cell cycle. A second cDNA, which retains intron 3, predicts the existence of a 121-amino acid protein, survivin(121) that lacks the coiled-coil domain. Removal of exon 2-derived sequences by alternative pre-messenger RNA (mRNA) splicing results in a third 40-amino acid residue protein, survivin(40), lacking the IAP repeat and coiled-coil structure. Predictably, only recombinant survivin(140) and survivin(121) inhibited caspase-3 activity. All 3 mRNA species were variably expressed during development from 7.5 days postcoitum. Of the adult tissues surveyed, thymus and testis accumulated high levels of survivin(140) mRNA, whereas survivin(121)-specific transcripts were detected in all tissues, while those representing survivin(40) were absent. Human counterparts to the 3 survivin mRNA transcripts were identified in a study of human cells and tissues. The presence of distinct isoforms of survivin that are expressed differentially suggests that survivin plays a complex role in regulating apoptosis. (Blood. 2000;95:1435-1442)

Early embryonic expression of murine coagulation system components.

The development of the embryonic coagulation system, and its contribution to the maintenance of vascular integrity during the formation of embryonic blood vessels, remain poorly understood. We have characterized the temporal expression patterns of 27 hemostasis-related genes during murine development. We show that, although most coagulation and fibrinolysis-related factors are expressed coordinately by 7.5 dpc, several, including FIX, FXII and PAI2, are not detectable until later developmental timepoints. The expression of hemostasis-specific genes prior to the formation of a functional circulatory system supports the view that some coagulation factors have additional non-hemostatic functions during development. In addition, the discordant expression of some factors suggests that the embryonic hemostatic system may be distinct from that of the adult. These analyses will help to elucidate the regulation of hemostasis during embryonic/vascular development, and will provide a framework to facilitate the interpretation of gene inactivation studies.

CD109 is expressed on a subpopulation of CD34+ cells enriched in hematopoietic stem and progenitor cells.

CD109 is a monomeric cell surface glycoprotein of 170 kD that is expressed on endothelial cells, activated but not resting T-lymphocytes, activated but not resting platelets, leukemic megakaryoblasts, and a subpopulation of bone marrow CD34+ cells. Observing an apparent association between CD109 expression and the megakaryocyte lineage (MK), we sought to determine whether CD109 was expressed on MK progenitors. In fetal bone marrow (FBM), a rich source of MK progenitors, CD109 is expressed on a mean of 11% of CD34- cells. Fluorescence activated cell sorting (FACS) of FBM CD34+ cells into CD109+ and CD109- fractions revealed that the CD34+CD109+ subset contained virtually all assayable MK progenitors, including the colony-forming unit-MK (CFU-MK) and the more primitive burst-forming unit-MK (BFU-MK). The CD34+CD109+ subset also contained all the assayable burst-forming units-erythroid (BFU-E), 90% of the colony-forming units-granulocyte/macrophage (CFU-GM), and all of the more primitive mixed lineage colony-forming units (CFU-mix). In contrast, phenotypic analysis of the CD34+CD109- cells in FBM, adult bone marrow (ABM) and cytokine-mobilized peripheral blood (MPB) demonstrated that this subset comprises lymphoid-committed progenitors, predominantly of the B-cell lineage. CD109 was expressed on the brightest CD34 cells identifiable not only in FBM, but also in ABM and MPB indicating that the most primitive, candidate hematopoietic stem cells (HSC) might also be contained in the CD109+ subset. In long-term marrow cultures of FBM CD34+ cells, all assayable cobblestone area forming cell (CAFC) activity was contained within the CD109+ cell subset. Further phenotypic analysis of the CD34+CD109+ fraction in ABM indicated that this subset included candidate HSCs that stain poorly with CD38, but express Thy-1 (CD90) and AC133 antigens, and efflux the mitochondrial dye Rhodamine 123 (Rho123). When selected CD34+ cells were sorted for CD109 expression and Rho123 staining, virtually all CAFC activity was found in the CD109+ fraction that stained most poorly with Rho123. CD34+ cells were also sorted into Thy-1 CD109+ and Thy-1 CD109+ fractions and virtually all the CAFC activity was found in the Thy-1+CD109+ subset. In contrast, the Thy-1-CD109+ fraction contained most of the short-term colony-forming cell (CFC) activity. CD109, therefore, is an antigen expressed on a subset of CD34+ cells that includes pluripotent HSCs as well as all classes of MK and myelo-erythroid progenitors. In combination with Thy-1, CD109 can be used to identify and separate myelo-erythroid and all classes of MK progenitors from candidate HSCs.

In vitro hematopoietic and endothelial potential of flk-1(-/-) embryonic stem cells and embryos.

Mice deficient in the Flk-1 receptor tyrosine kinase are known to die in utero because of defective vascular and hematopoietic development. Here, we show that flk-1(-/-) embryonic stem cells are nevertheless able to differentiate into hematopoietic and endothelial cells in vitro, although they give rise to a greatly reduced number of blast colonies, a measure of hemangioblast potential. Furthermore, normal numbers of hematopoietic progenitors are found in 7.5-day postcoitum flk-1(-/-) embryos, even though 8. 5-day postcoitum flk-1(-/-) embryos are known to be deficient in such cells. Our results suggest that hematopoietic/endothelial progenitors arise independently of Flk-1, but that their subsequent migration and expansion require a Flk-1-mediated signal.

A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis.

Mouse embryos lacking the receptor tyrosine kinase, Flk1, die without mature endothelial and hematopoietic cells. To investigate the role of Flk1 during vasculogenesis and hematopoiesis, we examined the developmental potential of Flk1-/- embryonic stem cells in chimeras. We show that Flk1 is required cell autonomously for endothelial development. Furthermore, Flk1-/- cells do not contribute to primitive hematopoiesis in chimeric yolk sacs or definitive hematopoiesis in adult chimeras and chimeric fetal livers. We also demonstrate that cells lacking Flk1 are unable to reach the correct location to form blood islands, suggesting that Flk1 is involved in the movement of cells from the posterior primitive streak to the yolk sac and, possibly, to the intraembryonic sites of early hematopoiesis.

Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice.

The receptor tyrosine kinase Flk-1 (ref. 1) is believed to play a pivotal role in endothelial development. Expression of the Flk-1 receptor is restricted to endothelial cells and their embryonic precursors, and is complementary to that of its ligand, vascular endothelial growth factor (VEGF), which is an endothelial-specific mitogen. Highest levels of flk-1 expression are observed during embryonic vasculogenesis and angiogenesis, and during pathological processes associated with neovascularization, such as tumour angiogenesis. Because flk-1 expression can be detected in presumptive mesodermal yolk-sac blood-island progenitors as early as 7.0 days postcoitum, Flk-1 may mark the putative common embryonic endothelial and haematopoietic precursor, the haemangioblast, and thus may also be involved in early haematopoiesis. Here we report the generation of mice deficient in Flk-1 by disruption of the gene using homologous recombination in embryonic stem (ES) cells. Embryos homozygous for this mutation die in utero between 8.5 and 9.5 days post-coitum, as a result of an early defect in the development of haematopoietic and endothelial cells. Yolk-sac blood islands were absent at 7.5 days, organized blood vessels could not be observed in the embryo or yolk sac at any stage, and haematopoietic progenitors were severely reduced. These results indicate that Flk-1 is essential for yolk-sac blood-island formation and vasculogenesis in the mouse embryo.