Recovery Of Peripheral Blood Counts Research Articles

Phase IIB Randomized Study of CPX-351 Versus Conventional Cytarabine + Daunorubicin in Newly Diagnosed AML Patients Aged 60-75: An Interim Analysis

Abstract Introduction CPX-351 is a liposomal formulation that encapsulates cytarabine and daunorubicin at a 5:1 molar ratio. This molar ratio was found, in vitro, to be consistently synergistic and nonantagonistic across multiple leukemic and solid tumor cell lines. A randomized phase II study comparing CPX-351 with conventional cytarabine + daunorubicin (7 + regimen) in patients with acute myeloid leukemia (AML) aged 60-75 years is currently in follow-up. This report presents interim safety and efficacy data for 85 of the 126 subjects treated on this study. Patients and Methods Eligible patients included those with de novo or secondary AML, Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0-2, SCr 50% by echo or multigated acquisition (MUGA). Exclusions were made for acute promyelocytic leukemia (t[15;17]), known favorable cytogenetics (eg, t[8;21] and inv[16]), > 368 mg/m 2 of prior anthracycline exposure, New York Heart Association (NYHA) class III/IV myocardial impairment, uncontrolled infections, prior treatment for AML (except for hydroxyurea), active central nervous system (CNS) leukemia, and conditions preventing ability to give informed consent. Results Data collected by 15 August 2009 for the first 85 patients enrolled were evaluated for interim safety and efficacy. Fifty-seven patients were randomized to CPX-351 and 28 patients to 7 + 3 control. The median age was 68 years; most patients were male (70.6%), had ECOG PS of 0 or 1 (83.5%), and had de novo AML (66%). The CPX-351 arm had more patients with ECOG PS of 2 (21.1% vs. 7.1%) and a higher proportion of patients with baseline white blood cell count (WBC) > 20,000/μL (22.9% vs. 14.3%). All 85 patients experienced at least 1 adverse event. Mortality at 30 days was low in both study arms (5.3% and 3.6%). Serious adverse events (SAEs) were more frequent in the CPX-351 arm and consisted predominantly of cytopenia-related events associated with treatment or leukemia, including fever and febrile neutropenia, sepsis, pneumonia, major bleeding episodes, and a number of minor infections. Although SAEs appeared to be more frequent following CPX-351, there was no evidence for this difference leading to an increase in deaths on study. Among other adverse events, CPX-351 appeared to have comparable rates of skin rash (57.9% vs. 57.1%) and gastrointestinal adverse events (nausea: 56.1% vs. 57.1%; vomiting: 17.5% vs. 28.6%; stomatitis: 21.1% vs. 21.4%; diarrhea: 61.4% vs. 64.3%). As noted in the phase I study, grade 3 gastrointestinal adverse events were uncommon. Peripheral blood count recovery was slower for the CPX-351 arm for both absolute neutrophil count (ANC) recovery to > 500/μL (37.5 days vs. 28 days) and platelet count recovery to > 20,000/μL (28 days vs. 21 days). CPX-351 treatment resulted in higher rates of CR + CRi (61.4% vs. 50%). This interim analysis has produced encouraging signs of antileukemic activity for CPX-351 consistent with earlier clinical findings. The study has completed enrollment and follow-up for CR duration and percent survival at 1 year will be completed by the end of 2010.

European Development of Clofarabine as Treatment for Older Patients With Acute Myeloid Leukemia Considered Unsuitable for Intensive Chemotherapy

Treatment options for older patients with acute myeloid leukemia (AML) who are not considered suitable for intensive chemotherapy are limited. We assessed the second-generation purine nucleoside analog, clofarabine, in two similar phase II studies in this group of patients. Two consecutive studies, UWCM-001 and BIOV-121, recruited untreated older patients with AML to receive up to four or six 5-day courses of clofarabine. Patients in UWCM-001 were either older than 70 years or 60 to 69 years of age with poor performance status (WHO > 2) or with cardiac comorbidity. Patients in BIOV-121 were >or= 65 years of age and deemed unsuitable for intensive chemotherapy. A total of 106 patients were treated in the two monotherapy studies. Median age was 71 years (range, 60 to 84 years), 30% had adverse-risk cytogenetics, and 36% had a WHO performance score >or= 2. Forty-eight percent had a complete response (32% complete remission, 16% complete remission with incomplete peripheral blood count recovery), and 18% died within 30 days. Interestingly, response and overall survival were not inferior in the adverse cytogenetic risk group. The safety profile of clofarabine in these elderly patients with AML who were unsuitable for intensive chemotherapy was manageable and typical of a cytotoxic agent in patients with acute leukemia. Patients had similar prognostic characteristics to matched patients treated with low-dose cytarabine in the United Kingdom AML14 trial, but had significantly superior response and overall survival. Clofarabine is active and generally well tolerated in this patient group. It is worthy of further evaluation in comparative trials and might be of particular use in patients with adverse cytogenetics.

Cost Burden Analysis of Ineffective Induction Chemotherapy in Elderly Patients with AML.

Abstract 4531 IntroductionAccording to the Surveillance, Epidemiology, and End Results (SEER) database 12,810 patients will be diagnosed with acute myeloid leukemia (AML) in 2009 with an incidence rate of 3.5 per 100,000 persons in the United States. The SEER data estimates that 55% of those diagnosed are 65 years or older. Standard first line therapy for the treatment of AML consists of at least one round of chemotherapy commonly referred to as induction. A significant portion of those patients who receive standard induction chemotherapy will have leukemia that fails to show a complete response as defined by less than 5% leukemia blasts in the bone marrow with normal tri-lineage hematopoiesis and peripheral blood count recovery (Mueller S, et al. BMC Cancer 2006, 6:143). Age, patient performance status, the presence of secondary AML, cytogenetics, and molecular markers are prognostic for particular cohorts of patients, however, there is currently no means to predict whether an individual's leukemia will or will not undergo a complete response (CR) after the administration of standard AML induction chemotherapy. MethodsA budget impact model was designed to assess the incremental societal cost and incremental cost to a typical national health plan of treating patients who will fail induction therapy. The analysis only evaluated elderly patients over the age of sixty five. Data were gathered from the peer reviewed literature. All costs have been updated to 2009 dollars using the medical care component of the consumer price index. The patient population was determined to be 6,981 patients in 2009 with 30% receiving induction therapy (Menzin J, et al. Arch Intern Med. 2002; 162:1597-1603) and only 38% demonstrating a CR (Appelbaum FR, et al. Blood 2006 107:3481-5) resulting in 1,303 patients failing during induction therapy. The costs were taken from Menzin et al., which used Medicare claims data to determine the associated costs of AML patients during the first two years post diagnosis. In the model Medicare payments are used as a proxy for direct costs to society and a national health plan. These reimbursements are able to capture the costs of the initial hospital visit, lab/diagnostic/radiology, supportive care, drugs, and adverse events through the observation of payments across all appropriate settings of care (inpatient hospitalization, skilled nursing facility, outpatient hospital/clinical, physician's office, home health, and hospice). A patient undergoing chemotherapy incurred costs of $120,468 over two years, while a patient avoiding chemotherapy only incurred costs of $40,720. ResultsThe incremental cost of a patient receiving induction therapy was calculated to be $79,748. For a national health care plan with an assumed average of one million members results in a cost of $283,856 to treat patients whose leukemia would not respond to induction chemotherapy. The overall societal impact of treating this patient population with ineffective therapy is $104 million. ConclusionsPatients who are subjected to ineffective chemotherapy face the cytotoxic effects of the treatment, none of the benefits of treatment response, and impose a significant cost burden to themselves and the healthcare payment system. Diagnostics currently in development that can identify patients at the time of diagnosis with disease unresponsive to therapy may have the ability to alleviate unnecessary costs, while steering patients to better tailored and more effective therapies. Disclosures:Cleary Cohen:Nodality, Inc: Employment. Middlebrook:Nodality Inc: Employment.

Epigenetic Priming with Decitabine Prior to Standard Induction Chemotherapy in Less-Than-Favorable Risk Acute Myelogenous Leukemia (AML).

Abstract 3079Poster Board III-16Epigenetic silencing of tumor suppressor genes (TSGs) is among the most common acquired abnormalities observed in AML and is prototypically linked to DNA methylation. Aberrant DNA methylation is most prevalent in adverse risk AML patients (Melki, Vincent et al. 1999; Toyota, Kopecky et al. 2001). Reactivated expression of TSGs is commonly implicated in the clinical activity of DNA hypomethylating agents since these genes often have roles in cellular differentiation, apoptosis, DNA repair, and checkpoint control (Jones and Taylor 1980; Pinto, Zagonel et al. 1989; Petti, Mandelli et al. 1993; Schimmer, Pedersen et al. 2003; Schmelz, Sattler et al. 2005; Schmelz, Wagner et al. 2005; Tamm, Wagner et al. 2005). We, and others, have demonstrated that DNA hypomethylating agents can sensitize resistant cancer cells to cytotoxic agents in vitro (Avramis, Mecum et al. 1989; Anzai, Frost et al. 1992; Plumb, Strathdee et al. 2000; Fulda, Kufer et al. 2001; Niitsu, Hayashi et al. 2001; Arnold, Goel et al. 2003; Eramo, Pallini et al. 2005; Kanda, Tada et al. 2005; Qiu, Mirkin et al. 2005) yet this approach has been sparsely studied in clinical trials. We hypothesized that pre-treatment with a hypomethylating agent would increase the efficacy of standard induction chemotherapy for AML. DesignWe conducted a phase I dose-escalation study of decitabine administered just prior to a single, standard induction with infusional cytarabine (100 mg/m2 for 7 days) and daunorubicin (60 mg/m2 × 3 doses) as initial treatment for patients with less-than favorable risk AML. Three dose levels of decitabine (20 mg/m2 for 3, 5 or 7 days), delivered either as a bolus (Arm A) or by continuous infusion (Arm B), were used as priming for standard induction chemotherapy. PatientsAt current data cut-off, 25 (of 30 patients planned) were fully evaluable: median age was 56 (range 23–60); 21 (84%) patients had adverse risk AML because of karyotype (No. 15, 60%), or other adverse features (FLT3-ITD or antecedent hematologic disorder, No. 11, 44%), and the remaining 4 (16%) patients had intermediate risk AML. Toxicity was similar to that of standard induction chemotherapy alone. Two patients treated at the highest dose level had grade 3 mucositis that resolved prior to count recovery. We observed no excess hematologic toxicity and all patients had peripheral blood count recovery within 30 days or were found to have persistent AML. There was no induction mortality and no patients required ICU transfer. A maximum tolerated dose was not achieved. ResponseTwenty-one patients (84%) responded to therapy: 14 CR (56%), 7 PR (28%). Of the patients with PR to protocol treatment, 5 achieved remission with a second, standard induction bringing the overall CR rate to 76%. The final cohort of patients is currently accruing. All patients went on to receive additional AML therapy and 14 have received an allogeneic HSCT. Pharmacodynamic analyses of DNA hypomethylation prior to and immediately following priming demonstrate genome-wide DNA hypomethylation at all dose levels. Detailed analyses of DNA methylation are ongoing. ConclusionComplete remission is requisite for the cure of AML and this study demonstrates that it is safe to combine the epigenetic modifier, decitabine, with full-dose, standard, cytotoxic chemotherapy as an approach to improve remission rates. Our results suggest that epigenetic priming is potentially superior to standard induction therapy alone as initial therapy for patients with less than favorable risk AML and provide the foundation for a subsequent phase II study. DisclosuresOff Label Use: Phase I study of decitibine use in AML.

Connexin-43 Regulates the Cell Cycle Entry of Hematopoietic Stem Cells within the Stem Cell Niche.

Abstract 1500Poster Board I-523Bone marrow (BM) osteoblasts and stromal (O/S) cells are crucial in the establishment of the hematopoietic niches in the BM. Connexin 43 (Cx43) is expressed by BM stromal cells and by hematopoietic stem cells and progenitors (HSC/P) and is overexpressed in the BM endosteal space upon administration of chemotherapy or radiotherapy. We have previously reported that Cx43 is critical in fetal liver and in BM hematopoiesis. Since Cx43 is expressed by both HSC and the hematopoietic microenvironment, we dissected out the cellular mechanisms responsible for Cx43 function in the BM. We analyzed the hematopoiesis of mice deficient in Cx43 in the O/S cells (Collagen 1α-Creflox/flox; O/S-Cx43-deficient) or in the hematopoietic cells (Vav1-Creflox/flox; H-Cx43-deficient). Upon basal conditions, analysis of the HSC compartment of H-Cx43-deficient mice showed a ∼30% decreased content of immunophenotypically defined long-term HSC (LT-HSC) in BM of H-Cx43KO mice compared with their WT littermates, whereas there was not significant variation in the ST-HSC population content. The reduced LT-HSC population in H-Cx43KO mice was associated with a modest increased quiescence (∼12% increase of LT-HSC in G0). Interestingly, the expression of cyclin D1 and p21cip1 in the H-Cx43KO LT-HSC were 50% reduced and 4-fold increased, respectively, suggesting a decreased ability to enter cell cycle. While we found no significant engraftment difference in primary recipients of competitive repopulation assays, we found a marked reduction (>50%) in the engraftment ability of LT-HSC Cx43-deficient cells when transplanted into secondary recipients. When submitted to stress by 5-fluorouracil (5-FU) administration, H-Cx43KO mice showed a severely decreased hematopoietic recovery of peripheral blood (PB) counts for neutrophils and platelets accompanied with a marked reduction in the BM cellularity and hematopoietic progenitor content on day +14 after treatment. This defect was associated with a dramatic decreased (∼75 %) in the proliferation of the Cx43-deficient, LT-HSC population by 48 hours post-5-FU administration and a relative decrease of the expansion of the ST-HSC/MPP pool as early as 6 days post-5-FU administration. Interestingly, O/S-Cx43-deficient mice also showed severely delayed hematological recovery after 5-FU administration, with reduction in cellularity and hematopoietic progenitor content, suggesting that the increased hematopoietic toxicity induced by 5-FU in the context of Cx43 deficiency may depend on HSC-to-O/S Cx43 homotypic communication. This communication would be responsible of control of the G1 restriction checkpoint in LT-HSC. In summary, our findings suggest that Cx43 expression plays a crucial role controlling the LT-HSC pool size and fitness in response to stress. Disclosures:Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

Synergistic protecting effect of cord blood CD34+ cells over-expressing both interleukin-3 and Flt3 ligand on lethally irradiated mice

The objective of the present work was to study the effect of interleukin-3 (IL-3) and Flt3 ligand (FL) over-expression in human cord blood CD34(+) cells on rescuing lethally irradiated mice by transplantation. CD34(+) cells were isolated from human umbilical cord blood (UCB) and infected with recombinant retrovirus expressing FL, IL-3 or FL/IL-3 genes, respectively. Stably transduced CD34(+) cells were transplanted i.v. into NOD/SCID/IL2rgamma(null) mice that had been conditioned with 8.0 Gy of irradiation. At 6 weeks post-irradiation, chimerism in the animal bone marrow (BM) and the spleen were investigated. Recovery of peripheral blood cell and animal survival were recorded 2, 4, and 6 weeks post-irradiation. The chimerism was further investigated by serial transplantation assay. At 6 weeks post-transplantation, each of the three transduced human UCB CD34(+) cell types could be observed in all examined BM of chimeric mice; however, more human cells could be found in BM, spleen and peripheral blood of those mice transplanted with CD34(+) cells over-expressing IL-3/FL cells. Over-expression of IL-3 and FL at mRNA and protein levels could be detected in the BM cells of chimeric mice. Accordingly, hIL-3/FL co-overexpressing mice showed greater recovery of peripheral blood counts as early as 2 weeks after irradiation, and a much higher survival rate (11/12) than other groups at 6 weeks post-irradiation. Serial transplantation assay indicated that human cord blood CD34(+) cells could recover potentials of proliferation and self-renewal after being transplanted into mice. Our results suggested that transplant of CD34(+) cord blood cells over-expressing IL-3/FL genes improved rescue of radiation-injured mice, which probably results from the synergistic effect between hIL-3 and hFL causing the rapid reconstitution of hematopoiesis.

High-Dose Etoposide: From Phase I to a Component of Curative Therapy

High-Dose Etoposide: From Phase I to a Component of Curative Therapy

Hemophagocytic Syndrome in an Adolescent With Crohn Disease Receiving Azathioprine and Infliximab

Hemophagocytic Syndrome in an Adolescent With Crohn Disease Receiving Azathioprine and Infliximab

Non-Myeloablative Conditioning Regimen (NMCR) for Allogeneic Bone Marrow Transplantation (BMT) in Patients with Severe Aplastic Anemia (SAA).

Allogeneic bone marrow transplantation is a curative form of therapy for patients (pts) with acquired severe aplastic anemia. Current preparative therapies are associated with early and late sequelae such as organ injury, and secondary tumors. Recent studies showed that BMT following reduced-intensity or NMCR may result in long-term survival for a fraction of pts with hematologic malignancies (Giralt, Biol. Blood Marrow Transplant , 13:884, 2007). However, with the exception of BMT for pts With Fanconi's anemia, little is known about using NMCR for patients with non-malignant disorders. We report the use of NMCR in patients with SAA.Patients and Methods: Four female pts ages 6–12 years, diagnosed with SAA, had allogeneic BMT from an HLA-identical sibling (SIB) (Pts #1 and #2) or a matched unrelated donor (MUD) (pts #3 and #4). The reasons to offer NMCR were: delay in results of chromosome fragility studies (Pt #1), abnormal pulmonary function (Pt #2), history of recent life threatening infection (Pt #3), and failure to respond to immunosuppressive therapy (Pt #4). The NMCR consisted of fludarabine (FLU) (30 mg/m2 x 4), low dose cyclophosphamide (LDC) (5 mg/kg x 4) and rabbit antithymocyte globulin (rATG) (1.5 mg/kg x 4) in patients with SIB donor and FLU, LDC, at a higher dose of 15 mg/kg x 4), rATG and a single fraction of total body irradiation at 200 cGy in patients with a MUD donor. Supportive care, prophylactic anti-microbial therapy, and treatment for prevention of aGvHD were given according to the institution standard guidelines.Results: The NMCR was well tolerated in all 4 patients. Pts #1 and #2 who had a SIB BMT had no transplant-related toxicities, including mucositis or alopecia. Toxicities in the MUD BMT patients included mild mucositis and partial alpecia in both pts. Pt#3 had reactivation od Enterobacter cloacae sepsis with typhlitis and later CMV viremia. Myeloid and platelet engraftment were uneventful in pts #1, #2, and #4. The recovery of peripheral blood counts was slow in Pt #3 following typhlitis and CMV viremia. Myeloid engraftment occurred on day +19 (range 15–33 days). The median time to a platelet count >20,000 unsupported by transfusion was day +33, (range 12–76 days). Periodic engraftmen anlyses using short tandem repeat (STR) by PCRT continue to show full donor chimerism in all 4 pts. There were no signs for acute or chronic graft-vs-host disease (aGvHD or chGvHD, respectively) in pts with SIB BMT. Both patients continue to do well with a fully recovered hematopoietic system 17 months and 42 months post transplant. There were no aGVHD.or chGVHD in Pt#3. Pt #4 had aGVHD of the skin, clinical grade II, which responded well to immunosuppressive therapy. Both MUD BMT pts are well 5 and 3 months post-transplant, respectively, with partial hematopoietic recovery in Pt #3 and normal counts in Pt #4.Conclusion: This data suggests that a non-myeloablative, immunosuppressive regimen is sufficient to provide a stable engraftment in the patients with SAA. This approach may be associated with decreased transplant-related, short- and long-term, toxicities. A larger study is needed to fully evaluate the outcome and the toxicity associated with this conditioning.

A Phase I Trial of the Small Molecule Pan-Bcl-2 Family Inhibitor Obatoclax Mesylate (GX15-070) Administered by Continuous Infusion for up to Four Days to Patients with Hematological Malignancies.

Obatoclax antagonizes the BH3-binding groove of the bcl-2 family of anti apoptotic proteins. It is active in chronic lymphocytic leukemia(CLL; O’Brien et al, ASH 2005) with a recommended phase II dose of 28 mg/m2 given over 3 h every 3 weeks with DLT of grade 3 infusional CNS toxicities. We evaluated prolonged infusions to minimize these toxicities while maintaining clinical activity. An initial trial established a Phase II dose of 28 mg/m2 over 24 h every 2 weeks with dose limiting toxicities (DLTs) at 40 mg/m2 and responses in 4/8 MDS patients (Borthakur et al., ASH 2006). We report on 21 patients who received obatoclax 20–28 mg/m2/24 h weekly (n=9) or 20 mg/m2/24 h for 2, 3 or 4 consecutive days every 2–3 weeks (n=12). The final dose level of 28 mg/m2/24 h for 4 days is now open. Median age was 61 (range 26–75) and 11 patients were male. Diagnoses included: acute myelogenous leukemia (AML; 13), myelodysplastic syndromes (MDS; 6), acute lymphocytic leukemia (ALL; 1) and CLL (1). A total of 180 infusions of 24 h were administered. The most common adverse events (AE) were euphoric mood (57%), fatigue (57%), febrile neutropenia (43%), gait disturbance (43%), chills (33%), diarrhea (33%), nausea (33%), somnolence (33%), dizziness (29%). All were of Grades 1–2 with the exception of 7 grade 3 AEs of febrile neutropenia (unrelated to obatoclax administration) and 1 each of diarrhea, fatigue and gait disturbance. There were no DLTs. Multiple day infusions were well tolerated; for the 20 mg/m2/24 h x 4days dose level, 4/6 patients received ≥ 4 cycles, with two ongoing at this time. Plasma concentrations of obatoclax reached a steady state before end of infusion. Mean Cmax values at 20 mg/m2/24 h during1x24, 2x24, 3x24,4x24 h, and 28 mg/m2/24 h during 1x24 h infusions was 15.3, 8.4, 10.1, 15.7 and 10.8 ng/mL, respectively. The mean AUC(0–tlast) values at 20 mg/m2/24 h associated with 1x24, 2x24, 3x24, 4x24 h, and 28 mg/m2/24 h associated with 1x24 h infusions were 264.3, 396.9, 624.3, 1109.3 and 211.1 ng · hr/mL, respectively, increasing as expected with the duration of the infusion. One patient with treatment-related AML with a t(9;11)(p22;q23) translocation achieved a cytogenetic complete response (CR) with complete hematological recovery and transfusion independence on Day 9 following the start of weekly 24 h infusions of obatoclax. This CR was sustained > 8 months while the patient received a total of 35 weekly infusions of 20 mg/m2 without cumulative toxicities. Conclusions: obatoclax can be administered by prolonged infusions without eliciting novel or cumulative toxicities. The dramatic response observed in a patient with AML with immediate recovery of peripheral blood counts supports that the cytotoxic effects of obatoclax are specific to malignant cells while sparing normal bone marrow, as did our earlier reports of improvement in cytopenias in patients with MDS and CLL. The infusional schedules described here will be attractive to combine with standard chemotherapy regimens for AML and other indications to improve treatment outcomes where they may be limited by overexpression of Bcl-2 family members.

Dasatinib (BMS-354825) in Patients with Chronic Myeloid Leukemia (CML) and Philadelphia-Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL) Who Are Resistant or Intolerant to Imatinib: Update of a Phase I Study.

Imatinib mesylate resistance in CML and Ph+ ALL is frequently associated with BCR-ABL mutations that interfere with the ability of imatinib to inhibit BCR-ABL. Dasatinib (BMS-354825) is a novel, oral, multi-targeted kinase inhibitor, which targets BCR-ABL and SRC kinases. Dasatinib is 325-fold more potent versus imatinib in cells transduced with wild-type BCR-ABL, and has demonstrated preclinical activity against 18 of 19 imatinib-resistant BCR-ABL mutants (O'Hare, et al. Cancer Res 65(11):4500–5, 2005; Shah et al, Science , 305:399–401, 2004). Here we present an update of a Phase I dose-escalating study, initiated in November 2003, of dasatinib in imatinib-resistant/intolerant patients with CML in late chronic phase (CP) or advanced disease (accelerated phase [AP], myeloid blast crisis [MBC] or lymphoid blast crisis [LBC]) or with Ph+ ALL. Data are available for 84 patients (40 CP, 10 AP, 23 MBC, 11 LBC/Ph+ ALL). As of this writing, 40 CP patients, with 8 years' median duration of CML (range: 1–17 yrs), have been treated with dasatinib (15–180 mg/day, once-daily [QD] or twice daily [BID]) for a median of 13 months. The rate of complete hematologic response (CHR) in CP is 88% (35/40). Major cytogenetic responses (MCyR) were observed in 40% (16/40), with complete CyR (CCyR) in 33% (13/40). In advanced disease, 44 patients have been treated with dasatinib (70–240 mg/day, BID) for a median of 3–7 months, depending on the cohort (see Table); 2 patients (1 MBC and 1 LBC) are not evaluable for response, but are included in the analysis of time to progression. The rate of major hematologic response (MHR) (bone marrow blasts <5%) is 80% (8/10) in AP, 77% (17/22) in MBC and 60% (6/10) in LBC/Ph+ ALL. The CHR rate (<5% bone marrow blasts, with recovery of peripheral blood counts) is 50% (5/10) in AP, 18% (4/22) in MBC and 50% (5/10) in LBC/Ph+ ALL. The overall rates of MCyR and CCyR in advanced disease were 36% (15/42) and 21% (9/42), respectively. Cytogenetic responses were seen in patients with a wide spectrum of BCR-ABL mutations, as well as in patients with minimal or no prior CyR with imatinib. Treatment was well tolerated. Reversible grade 3–4 myelosuppression was observed in all cohorts [CP (38%); AP (70%); MBC and LBC/Ph+ ALL (91%)]. 8 patients developed unexplained pleural effusions (2 CP; 6 BC), which were easily managed without treatment discontinuation. No patients have gone off study for toxicity. Responses are durable in CP and AP patients, but relapses have occurred in the MBC and LBC/Ph+ ALL cohorts, often due to dasatinib-resistant BCR-ABL mutations. These data support the therapeutic potential of dasatinib in patients with imatinib-resistant or -intolerant CML/Ph+ ALL. Phase II studies (the ‘START' [SRC/ABL Tyrosine kinase inhibition Activity: Research Trials of dasatinib] program) are currently ongoing in imatinib-resistant/intolerant patients with all phases of CML and Ph+ ALL.Phase of CML/ALLPatients remaining in response at July 31, 2005Follow-up, median (range) daysCP (N=40)36 (90%)392 (95–588)AP (N=10)8 (80%)200 (39–365)MBC (N=23)9 (39%)144 (2–364)LBC/Ph+ ALL (N=11)1 (9%)77 (8–233)

Phase II Study of Decitabine in Combination with Imatinib Mesylate in Patients with Accelerated (AP) or Blastic Phase (BP) of Chronic Myeloid Leukemia (CML).

Decitabine, a hypomethylating agent, has clinical activity in imatinib refractory chronic myeloid leukemia (Issa et al. JCO 2005). The objective of this phase II study is to investigate the activity of decitabine in combination with imatinib in patients with Ph-positive CML-AP or non-lymphoid CML-BP. To be eligible for this study, patients who had previously been treated with imatinib must have clinical evidence of imatinib failure. Treatment schedule was imatinib orally at 600mg daily and decitabine intravenously at 15mg /m2 x 5 days/week for two consecutive weeks every 6 weeks. At least two courses were planned for all patients. Global DNA methylation of peripheral blood mononuclear cells was measured using a LINE1 bisulfite/pyrosequencing assay. From January 2003 to July 2005, 27 patients (8 BP, 19 AP) were enrolled into this study. Clonal evolution was observed in 21 patients (77%). Twenty-four patients (88%) had failed prior therapy with imatinib, and 3 (12%) were newly diagnosed patients with CML (2 AP and 1 BP). All patients received at least 1 cycle of the treatment (median 3, range 1–12). Toxicity was evaluated in all patients; grade 3/4 toxicity included infection (n=9), CNS bleed (n=2), GI bleed (n=2), dyspnea (n=1), diarrhea (n=1), and edema (n=1). Seven patients (26%) received only 1 cycle of treatment, owing to early death from acute disease progression (n=5), recurrent subdural hematoma (n=1), and a patient's decision due to grade 3 diarrhea (n=1). Twenty patients (74%) received 2 or more treatment cycles and were evaluated for response. Nine patients (33%) achieved complete hematologic response (CHR: BM blast <5%, normalization of peripheral blood and disappearance of signs and symptoms of the disease), and 3 patients (11%) achieved partial hematologic response (PHR: same criteria except for persistence of immature cells, splenomegaly or thrombocytosis but <50% of pretreatment). These included 7 patients (26%) that achieved cytogenetic response (3 complete cytogenetic response (Ph 0%), 1 partial cytogenetic response (Ph<35%), and 3 minor cytogenetic response (Ph<90%)). Median response duration was 13 weeks (range 4–107+). Four patients (15%) who achieved CHR underwent allogeneic stem cell transplantation. Median overall survival of patients with AP and BP were similar in this study (49 and 53 weeks, respectively). Median overall survival of patients with cytogenetic response (n=7), hematologic response only (n=5), and non-responders including those who received only 1 cycle (n=15) were 86, 63, and 18 weeks, respectively (p<0.01). LINE1 methylation decreased from 71.6%±0.9% (mean±standard error of mean) to 60.4%±2.0% on day 5, 60.5%±1.8% on day 12, and returned to 68.8%±1.4% at recovery of peripheral blood counts. Absolute decrease in LINE1 methylation trended greater in non-responders than responders on day 5 (15.4%±3.9% vs 7.6%±2.1%, p=0.14) or day 12 (14.4%±4.5% vs 9.3%±1.8%, p=0.34), though the difference was not statistically significant. In summary, decitabine in combination with imatinib is an active regimen for CML-AP and BP, including patients previously treated with imatinib. Global hypomethylation was observed after decitabine therapy, both in responders and non-responders.

Molecular Analysis of Dasatinib Resistance Mechanisms in CML Patients Identifies Novel BCR-ABL Mutations Predicted To Retain Sensitivity to Imatinib: Rationale for Combination Tyrosine Kinase Inhibitor Therapy.

Point mutations within the BCR-ABL kinase domain represent the most common mechanism of resistance to imatinib in patients with CML. Preclinical studies have shown that dasatinib (BMS-354825) is effective at inhibiting the kinase activity of imatinib-resistant BCR-ABL mutants with the notable exception of the T315I mutation, which remains highly resistant to imatinib, dasatinib, and AMN107 (Gorre et al, Science 2001; Shah et al, Science 2004; Weisberg et al, Cancer Cell, 2005). Clinical data from Phase I and II studies of dasatinib in CML confirms the in vitro findings. Each of three imatinib-resistant patients bearing the T315I mutation (CP=1; AP=2) did not achieve objective hematologic or cytogenetic responses during treatment with dasatanib on a Phase I study. Additionally, each of two phase II patients with the T315I mutation (CP=1; LBC=1) treated at UCLA showed no evidence of objective response. We have also detected the T315I mutation in each of two cases of acquired resistance in a phase II (LBC =2) study, and in seven of nine patients with acquired resistance to dasatinib in phase I and II studies (CP=1; MBC=3; LBC=2; Ph+ ALL=1).Notably, we detected a novel BCR-ABL mutation, T315A, in one of the two patients who relapsed without a detectable T315I mutation. The patient is a 53 year-old female whose chronic phase CML had progressed to myeloid blast phase while being treated with imatinib. The imatinib-resistant mutation M244V was identified prior to dasatinib treatment. The patient achieved a major hematologic response (<5% blasts with partial recovery of peripheral blood counts) on dasatinib 90 mg orally given twice daily, but relapsed with MBC after six months. Sequence analysis of the BCR-ABL kinase domain at the time of relapse revealed the presence of the imatinib-resistant mutation M244V as well as the novel mutation T315A. This finding is of particular interest because T315A and several other novel BCR-ABL mutations were recently recovered in a saturation mutagenesis study designed to define potential mechanisms of dasatinib resistance. Remarkably, many of these mutations retain sensitivity to imatinib in vitro (Burgess et al, PNAS, 2005). Through periodic molecular monitoring of other dasatinib-treated patients, we have identified a second novel BCR-ABL mutant, F317I, that developed in an imatinib-resistant CP patient after 9 months of treatment. Similar to T315A, F317I was isolated in the saturation mutagenesis screen for dasatinib resistance and is predicted to retain sensitivity to imatinib. Taken together, our findings implicate the T315I mutation as the principle mechanism of resistance to dasatinib, but more importantly, strongly support the use for combination kinase inhibitor therapy in CML to prevent emergence of drug resistant clones. A phase I trial to assess the safety of combining imatinib with dasatinib is planned.

Bone Marrow Connexin-43 Expression Is Critical for Hematopoietic Regeneration after Chemotherapy.

Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be crucial in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. Gap junctions (GJs) represent the best described intercellular communication (IC) system, and they are characterized by the existence of plaques of narrow channels between contacting cells. Each cell contributes with one hemichannel, which is composed of six proteins, called connexins. Connexin 43 (Cx43) is expressed by BM OS cells. Cx43 has been associated with the cadherin/β-catenin signaling pathway, recently reported as relevant in the OS/HSC interaction at the stem cell niche. Multiple osteogenic defects have been reported in human Cx43 mutations and Cx43 has been shown to be essential in controlling osteoblast functions. BM Cx43 expression is up-regulated up to 100-fold by 5-fluorouracil (5-FU) treatment. Due to the perinatal death of Cx43 germline null mice, a conditional genetic approach was employed to study the role of Cx43 in stem cell proliferation and differentiation. The interferon-inducible Mx1 gene is expressed by both hematopoietic and stromal BM cells. Therefore, we crossed Cx43flox/+ mice with Mx1-Cre transgenic (Mx1-CreTg/−) mice. Cx43+/+:Mx1-CreTg/−and Cx43flox/flox:Mx1-CreTg/− littermates have been analyzed. Gene deletion was induced in vivo by injecting the interferon-inducer polyI:C (8 injections of 300 μg every other day), generating control (Cx43+) and Cx43-deficient (Cx43KO) mice. After one week, Cx43+ and Cx43KO mice were injected with 5-FU (150 mg/Kg i.v.). Cx43 expression in Cx43KO BM was markedly reduced (>80%) as analyzed on day +14 post-5-FU treatment. Cx43 deficiency did not induce a significant change in peripheral blood counts before 5-FU treatment, but the hematopoiesis recovery after 5-FU treatment was severely impaired as demonstrated by absence of recovery of peripheral blood counts, including profound neutropenia, anemia with reticulocytopenia, thrombocytopenia and a 5 to 8-fold decrease of cellularity and hematopoietic progenitor content (granulomacrophagic colony-forming-units -CFU-GM-, erythroid burst forming units (BFU-E) and mixed colony forming units -CFU-mix-) in BM and spleen on day +14 post-5-FU treatment. However, the femoral content of Lin−/c-kit+/Sca1+ cells in Cx43KO BM was maintained when compared to Cx43+ BM (139±19 vs 117±32 x 103 Lin−/c-kit+/Sca1+ cells per femur, respectively). Short-term competitive repopulation ability of Cx43KO BM cells was diminished as compared to Cx43+ mice (5.9±0.35% vs 22.2±4.6%, respectively, p<0.01), specifically for myeloid (9.2±1.4% vs 35.8±4.3%, respectively, p<0.001) and B lymphoid (0.4±0.2% vs 3.0±1.0%, respectively, p<0.01) cells, but showed spared long-term (6-month) competitive repopulation ability with roughly normal hematopoietic differentiation. Altogether, these data suggest that hematopoietic regeneration after cycle-specific chemotherapy is blocked in Cx43-deficient mice at the long-term HSC repopulating level. Cx43 expression within the BM appears to be crucial in the development of an efficient response to hematopoietic stress.

Bone Marrow Connexin-43 Expression Is Critical for Hematopoietic Regeneration After Chemotherapy

Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be crucial in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. Gap junction channels (connexons) are formed by polypeptides (connexins) arranged in hexamers and represent the best described intercellular communication system. Connexin-43 (Cx43) is expressed by BM OS cells and has been associated with the cadherin/beta -catenin signaling pathway, recently reported as relevant in the OS/HSC interaction at the stem cell niche. Here, we employed an inducible gene-targeted murine approach to study the role of Cx43 in HSC proliferation and differentiation in vivo. Mx-Cre/Cx43+/+ and Mx-Cre/Cx43flox/flox littermates have been analyzed after gene deletion induced in vivo by the interferon-inducer poly (I)-poly (C), generating control (Cx43+) and Cx43-deficient (Cx43-/-) mice. After one week, Cx43+ and Cx43-/- mice were treated with 5-fluorouracil (5-FU). Cx43 expression in Cx43-/- BM was markedly reduced (> 90%) as analyzed on day +14 post-5-FU treatment. Cx43 deficiency did not induce a significant change in peripheral blood counts before 5-FU treatment, but the hematopoiesis recovery after 5-FU treatment was severely impaired as demonstrated by absence of recovery of peripheral blood counts, including profound neutropenia, anemia with reticulocytopenia, thrombocytopenia and a 5- to 8-fold decrease of cellularity and hematopoietic progenitor content (granulomacrophagic colony-forming-units (CFU-GM-), erythroid burst forming units (BFU-E) and mixed colony forming units (CFU-mix-) in BM and spleen on day +14 post-5-FU treatment. However, the femoral content of Lin-/c-kit+/Sca1+ cells in Cx43-/- BM was maintained when compared to Cx43+ BM. Short-term competitive repopulation ability of Cx43-/- BM cells was diminished as compared to Cx43+ mice, specifically for myeloid and B lymphoid cells, but showed spared long-term competitive repopulation ability with roughly normal hematopoietic differentiation. These data suggest that hematopoietic regeneration after cycle-specific chemotherapy is blocked in Cx43-deficient mice at the long-term HSC repopulating level. Cx43 expression within the BM appears to be crucial in the development of an efficient response to hematopoietic stress.

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TLK199 is a novel glutathione analog that is a selective inhibitor of the enzyme glutathione S-transferase (GST) P1-1. TLK199 treatment induces hematopoietic cell proliferation and differentiation through activation of the MAP kinase signaling pathway leading to activation of JNK and ERK2. In rodent models of chemotherapy induced neutropenia, treatment with TLK199 accelerated recovery of white cell counts at rates comparable to treatment with G-CSF. We now report TLK199 treatment of myeloid progenitor cells isolated from normal human blood resulted in the increased formation of CFU-GM (46%) CFU-MK (47%) and BFU-E (142%) lineages over baseline. A corresponding increase in the percentage of cells expressing CD11b, a granulocyte and monocyte marker, was observed in the CFU-GM cells. Since TLK199 is currently being evaluated in a Phase 2a trial in patients with refractory MDS, we examined the effect of treatment on formation of BFU-E, CFU-GM and CFU-GEMM before and after TLK199 treatment at doses of 50 to 400 mg/m2. A significant increase in the number of hematopoietic progenitor cell colonies measured from patient peripheral blood and bone marrow was observed as early as Day 4 of Cycle 1 as compared to pretreatment baseline. Ten of 12 patients showed an increase in at least one colony forming type (BFU-E, CFU-GM and CFU-GEMM) and 7 of 12 had an increase in all three colony forming types following TLK199 administration. These results correlate with clinical improvement in hematological parameters in peripheral blood and bone marrow observed in MDS patients treated with TLK199. Studies are underway to define the mechanism of bone marrow and peripheral blood count recovery observed following treatment of MDS patients with TLK199 and the role of GST P1-1 as a regulatory element in myeloid proliferation and differentiation.

STI571: targeting BCR-ABL as therapy for CML.

Therapeutic agent STI571 (signal transduction inhibitor number 571) is a rationally developed, potent, and selective inhibitor for abl tyrosine kinases, including bcr-abl, as well c-kit and the platelet-derived growth factor receptor tyrosine kinases. Results of clinical trials to date have demonstrated the crucial role of the bcr-abl tyrosine kinase in chronic myelogenous leukemia (CML) pathogenesis and the potential of anticancer agents designed to target specific molecular abnormalities in human cancer. An initial phase I study of STI571 included 83 Ph(+) CML patients who had failed interferon-based therapy. Patients were required to be in chronic phase, defined liberally as less than 15% blasts in blood or bone marrow. Patients were treated with once-daily oral doses of STI571 in 14 successive dose cohorts ranging from 25-1,000 mg. In this phase I study, no dose-limiting toxicity was encountered and toxicity at all dose levels was minimal. The threshold for a maximally effective dose was found at 300 mg; for patients treated at or above this level, complete hematologic response was seen in 98% of patients, with complete cytogenetic responses in 13% and major cytogenetic responses in 31%. With a median duration of follow-up of 310 days, ongoing responses are evident in 96% of patients. In the phase II study of the accelerated phase of CML, 233 patients were treated with either 400 or 600 mg of STI571. With similar follow-up to the chronic phase trial, 91% of patients showed a hematological response; 63% of patients achieved a complete hematological response but not all patients had recovery of peripheral blood counts. In addition to the phase II clinical trials with STI571, a phase III trial randomizing newly diagnosed patients to either interferon with low-dose s.c. cytosine arabinoside versus STI571 is ongoing; this trial accrued rapidly and data collection is ongoing. Integration of STI571 into CML treatment algorithms will require long-term follow-up data from the ongoing phase II and III clinical studies.

Tempo of hematologic recovery correlates with peripheral blood CD34+ cell level in patients undergoing stem cell mobilization

This study was undertaken to evaluate the relationship between the time to recovery of peripheral blood counts and CD34+ cells in the peripheral blood (PB) and apheresis collections of patients undergoing intensive chemotherapy followed by rhG-CSF. Twenty-three patients with a median age of 42 years (range 17–64) with malignancies underwent peripheral blood stem cell (PBSC) collection after cyclophosphamide (CY) 4 g/m2 and etoposide (600 mg/m2) followed by rhG-CSF (10 μg/kg/day). The WBC, platelet counts, CD34+ cell counts per ml of PB, and CD34+ cells in apheresis products were followed in all patients. The relationship of the time to recovery of WBC >1,000/μl, >3,000/μl, >10,000/μl and platelets >20,000/μl and 50,000/μl was compared to the average daily CD34+ cells/ml in each patient using the Spearman Correlation test. The tempo of recovery of WBC and platelets were highly correlated with the average CD34+ cell count in blood. In order to derive some useful guidelines for the timing of apheresis, the patients were divided into two groups, early recover (ER) and late recover (LR) based on the median time (day 10) to reach WBC count greater than 1,000/μl. ER patients had an average daily PB CD34+ cell count of 9.04 × 104/ml (range 0.44–17.5) and a median yield of CD34+ cells of 10.43 × 106/kg (range 0.60–25.95) compared to LR patients, who had 1.87 × 104/ml (range 0.32–5.44) in the PB (P = .001) and a yield 3.20 × 106/kg CD34+ cells (range 0.037–9.39) (P = .001). Patients recovering their WBC to 1,000/ml within 10 days of completing this regimen may undergo PBSC collection and achieve minimum-target cell doses of >2.5 × 106 CD34+ cells/kg—100% of the time. J. Clin. Apheresis 13:1–6, 1998. © 1998 Wiley-Liss, Inc.

7E3 F(ab′)2 , a Monoclonal Antibody to the Platelet GPIIb/IIIa Receptor, Protects Against Microangiopathic Hemolytic Anemia and Microvascular Thrombotic Renal Failure in Baboons Treated With C4b Binding Protein and a Sublethal Infusion of Escherichia coli

AbstractWe have used our previously described baboon model of infusion of both a sublethal dose of Escherichia coli and C4b binding protein to assess the impact of inhibiting platelet function with the F(ab′ )2 fragment of the monoclonal antibody 7E3, directed against the platelet glycoprotein (GP)IIb/IIIa receptor, on the characteristic microvascular changes. At a dose of 0.25 to 0.35 mg/kg bolus plus an infusion of 0.25 to 0.35 mg/kg over 6 hours, c7E3 F(ab′ )2 had only a minimal impact on fibrinogen consumption and delayed but did not prevent, the development of thrombocytopenia. Treatment with 7E3 F(ab′ )2 , however, produced significant protection from the development of microangiopathic hemolysis and renal insufficiency. Histologic examination supported these observations, with treated animals having fewer schistocytes on blood smear and less evidence of ischemic renal changes. Treated animals also had more rapid recovery of peripheral white blood counts, suggesting a possible protective effect of treatment on ischemic damage to the bone marrow. These data indicate that potent inhibition of platelet function via GPIIb/IIIa receptor blockade can decrease ischemic organ damage in this animal model that has features similar to those found in diffuse intravascular coagulation, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura.

Effects of Recombinant Human Granulocyte Colony-Stimulating Factor on the Hematologic Recovery and Survival of Irradiated Mice

We studied the effects of intraperitoneal injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF) according to various administration schedules on the recovery of spleen colony-forming units (CFU-S) and peripheral blood counts, and on the survival of irradiated mice. The sooner and more frequently the mice were injected with rhG-CSF after irradiation, the more enhanced the recovery of CFU-S in bone marrow was obtained on day 7. Twice-daily injections of rhG-CSF from day 0 to day 2 significantly enhanced the recovery of platelets and hematocrit, but two injections of rhG-CSF on only day 0 did not. Twice-daily injections of rhG-CSF from day 0 to day 6 enhanced the recovery of platelets more effectively than twice-daily injections of rhG-CSF from day 1 to day 7, and increased the survival of irradiated mice more effectively than any other examined administration schedules. Twice-daily injections of rhG-CSF from day 0 to day 6 were significantly effective in enhancing the survival of mice irradiated with 8.5-, 9.0-, and 9.5-Gy x-rays, although not effective after irradiation of 10.5-Gy x-rays.