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Clinical Cancer Research Vol. 11, 6615-6624, September 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Clinical

Phase 1 Study of ABT-751, a Novel Microtubule Inhibitor, in Patients with Refractory Hematologic Malignancies

Karen W.L. Yee1, Anne Hagey2, Srdan Verstovsek1, Jorge Cortes1, Guillermo Garcia-Manero1, Susan M. O'Brien1, Stefan Faderl1, Deborah Thomas1, William Wierda1, Steven Kornblau1, Alessandra Ferrajoli1, Maher Albitar1, Evelyn McKeegan2, David R. Grimm2, Toby Mueller2, Rhonda R. Holley-Shanks2, Leonardo Sahelijo2, Gary B. Gordon2, Hagop M. Kantarjian1 and Francis J. Giles1

Authors' Affiliations: 1 Department of Leukemia, University of Texas M.D. Anderson Cancer Center, Houston, Texas and 2 Abbott Laboratories, Abbott Park, Illinois

Requests for reprints: Francis J. Giles, Department of Leukemia, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 428, Houston, TX 77030. Phone: 713-792-8217; Fax: 713-794-4297; E-mail: frankgiles{at}aol.com.


   Abstract
Top
 Abstract
Patients and Methods
 Results
 Discussion
 References
 
Purpose: ABT-751 is an oral antimitotic agent that binds to the colchicine site on ß-tubulin. A phase 1 study was conducted to determine the maximum tolerated dose and toxicities of ABT-751 in patients with advanced myelodysplastic syndrome and relapsed or refractory acute leukemias.

Study Design: Thirty-two patients were treated: nine with 100 (n = 3), 125 (n = 3), or 150 mg/m2 (n = 3) of ABT-751 given orally once daily for 7 days every 3 weeks and 23 with 75 (n = 3), 100 (n = 3), 125 (n = 5), 150 (n = 5), 175 (n = 3), or 200 mg/m2 (n = 4) of ABT-751 given orally once daily for 21 days every 4 weeks. Consenting patients had pharmacogenetic sampling and enumeration of circulating endothelial cells (CEC).

Results: Dose-limiting toxicity consisted of ileus in one patient at 200 mg/m2, with a subsequent patient developing grade 2 constipation at the same dose level. One patient with relapsed acute myelogenous leukemia achieved a complete remission that was sustained for 2 months. Four other patients had transient hematologic improvements, consisting of a decrease in peripheral blood blasts and improvements in platelet counts. CEC number was reduced in three patients with a concomitant reduction in peripheral blasts. A previously undescribed nonsynonymous single nucleotide polymorphism, encoding Ala185Thr, was identified in exon 4 of the ß-tubulin gene, TUBB, in three other patients. The recommended phase 2 dose in hematologic malignancies is 175 mg/m2 daily orally for 21 days every 4 weeks.

Conclusion: Further assessment of ABT-751, especially in combination with other agents, in patients with acute leukemias is warranted.

Microtubules are key components of the cytoskeleton and are important for a variety of cellular processes, including maintenance of cell shape, intracellular transport, and segregation of chromosomes during cell division by mitotic spindle formation (1). The mitotic spindle is composed of microtubules and therefore is sensitive to alterations in microtubule dynamics. Microtubules are dynamic polymers, composed of {alpha}–tubulin and ß-tubulin heterodimers, which constantly undergo growth (polymerization) or shortening (destabilization). Agents that bind to tubulin and disrupt microtubule dynamics block cell cycle progression at the G2-M phase with resultant apoptosis. These antimitotic agents can be classified into three distinct groups based on their tubulin binding domains: the Vinca domain (e.g., vincristine, vinblastine, vinorelbine, and cryptophycin 52), the taxane domain (e.g., paclitaxel, docetaxel, and epothilones), and the colchicine site (e.g., ABT-751, CI-980, and combretastatins; refs. 2–7).

Vinca alkaloids have shown efficacy in hematologic malignancies, whereas taxanes are more effective in solid tumors. The use of either class of agents is frequently limited by the development of resistance, which may be mediated by overexpression of membrane transporter proteins, such as P-glycoprotein encoded by the MDR1 gene. ABT-751 (previously known as E7010) is an orally available antimitotic methoxybenzene sulfonamide agent (Fig. 1.) that binds preferentially and competitively to the colchicine site of the ß3 isoform of tubulin with a Ki of 3.3 µmol/L and inhibits microtubule polymerization with an IC50 value of 3.1 µmol/L (8–11). ABT-751 is not a multidrug resistance substrate (12). In preclinical studies, ABT-751 inhibited cellular proliferation of a broad range of human tumor cell lines and xenograft models (9, 13), including those that are paclitaxel and doxorubicin resistant due to the multidrug-resistant phenotype.



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  		Fig. 1. Structure of ABT-751 (N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide).

 
Similar to other tubulin-destabilizing agents, such as colchicine, the Vinca alkaloids, and combretastatin, high doses of ABT-751 selectively reduce tumor blood flow and induce tumor necrosis, with negligible effects on normal vascular function (14–19). This vascular effect may be independent of a direct cytotoxic effect on tumor cells (19). It is unclear whether this is mediated by antiangiogenesis, as has been reported for several microtubule inhibitors (20–23).

Increased angiogenesis, determined by greater microvessel density and elevated surrogate angiogenic markers such as vascular endothelial growth factor, is observed in acute leukemias and myelodysplastic syndromes (24–28) and may predict outcome (27–29). Microvessel density in bone marrow biopsies and levels of angiogenic factors, such as vascular endothelial growth factor, do not reliably reflect total tumor angiogenic activity (30). Conditioned media from primary acute myelogenous leukemia (AML) cultures can enhance endothelial cell proliferation (31). Increased plasma vascular endothelial growth factor levels have been correlated with endothelial precursor/progenitor cell (EPC) mobilization (32, 33). Circulating endothelial cells (CEC) and circulating EPCs that originate from the bone marrow contribute to tumor angiogenesis by incorporating into newly forming blood vessels to support tumor growth and metastasis (34–38). Unlike terminally differentiated CECs that detach from the vessel walls, enter the circulation after vascular injury, and have a limited proliferation capacity, EPCs are able to form highly proliferative endothelial colonies when exposed to angiogenic factors (36, 39–41). CECs can be detected at elevated concentrations in patients with cancer (32, 33, 35) and with a variety of nonmalignant disorders (36–38) compared with healthy controls (32). In some patients with cancer, achievement of complete remission correlated with reductions in the number of CECs to levels comparable with those observed in healthy controls (32). These findings provide a strong basis for investigating CECs and circulating EPCs as pharmacodynamic markers of efficacy for cancer drugs and for cancer surveillance.

ABT-751 is currently being evaluated in patients with advanced solid tumors (42, 43). In phase 1 studies of ABT-751 given to adults with solid tumors for either seven consecutive days during a 21-day cycle or 21 consecutive days during a 28-day cycle, the maximum plasma concentration (Cmax) and area under the plasma concentration-time curve over a dosing interval (AUC{tau}) increased proportionally with increasing doses, indicating linear pharmacokinetics (12). ABT-751 was rapidly absorbed following oral dosing with the time to Cmax (Tmax) of ~2 hours. ABT-751 had a half-life of ~5 hours and showed minimal accumulation after daily or twice-daily dosing (12). Efficacious concentrations (0.5-1.5 µg/mL) in preclinical models were achieved in all dose groups (12).

A phase 1 study was conducted to determine the safety and tolerability of ABT-751 in patients with refractory or relapsed acute leukemias or advanced myelodysplastic syndrome. In addition, in consenting patients, additional blood samples were collected to assess ß-tubulin gene mutations and levels of CECs and circulating EPCs at baseline and at various treatment time points.


   Patients and Methods
Top
 Abstract
 Patients and Methods
Results
 Discussion
 References
 
Patients. Adults with a histologically confirmed diagnosis of AML, acute lymphocytic leukemia (ALL), or advanced myelodysplastic syndrome (MDS; refractory anemia with excess blasts or refractory anemia with excess blasts in transformation) refractory to standard therapy, or for whom no standard therapy was available, were eligible for enrollment. Other inclusion criteria were adequate performance status (Eastern Cooperative Oncology Group, 0-2), age >16 years, and adequate hepatic and renal function. All patients had to practice effective birth control and give written informed consent indicating that they were aware of the investigational nature of the study, in keeping with the policies of the M.D. Anderson Cancer Center.

Study design. This was a phase 1 trial designed to determine the safety and tolerability of oral ABT-751 in patients with hematologic malignancies. Based on preliminary experience from phase 1 trials conducted in patients with solid tumors, the initial dose level was 100 mg/m2 orally once daily for 7 days every 3 weeks (i.e., 7 of 21 days; refs. 19–21). After the first three cohorts were accrued, preliminary clinical experience from a phase 1 trial conducted in solid tumors indicated that a 21-day regimen followed by 7 days off drug was well tolerated (43). Preclinical models had shown enhanced tumor activity with chronic dosing on a 21- to 28-day schedule (12). Therefore, after approval by the institutional review board, the treatment schema was modified to an initial dose level of 75 mg/m2 daily orally for 21 days every 4 weeks (i.e., 21 of 28 days). Patients were assigned successively to one of six dose cohorts, which ranged from 75 to 200 mg/m2/d. Doses were escalated by 25 mg/m2/d per dose level.

Complete blood counts were obtained at least weekly. Bone marrow assessments were done once every 2 to 3 weeks during the first cycle of treatment and then once every 2 to 4 weeks thereafter, as clinically indicated until remission, at which time they were done after every cycle. Patients without evidence of disease progression or unacceptable drug-related toxicity were eligible to receive additional cycles of treatment.

For dose escalation to occur, three assessable patients had to complete their first cycle without a dose-limiting toxicity (DLT). With each DLT, three additional assessable patients had to be accrued, and further escalation could occur only if no more DLTs were observed. Hematologic DLT was defined using the National Cancer Institute Common Toxicity Criteria version 2.0 (44) as myelosuppression with bone marrow hypoplasia (cellularity <5%) without evidence of leukemia for ≥42 days. Nonhematologic DLT was defined as any grade ≥3 adverse event, except for the following: nausea and vomiting ≥ grade 3, drug-related fever of any grade, alopecia, or clinically insignificant biochemical abnormalities. Maximum tolerated dose was defined as the highest dose for which the incidence of DLT was ≤33%.

Assessment of toxicity and response. All patients were evaluable for toxicity if they received at least one dose of ABT-751. Evaluation of response was done at the end of each cycle of treatment. A complete response (CR) required disappearance of all signs and symptoms related to disease, normalization of the peripheral counts (absolute neutrophil count of ≥1 x 109/L and platelet count of ≥100 x 109/L), and a normal bone marrow morphology with no evidence of dysplasia and 5% or fewer blasts. A partial response was defined as fulfilling the criteria for CR in the peripheral blood but with 6% to 25% abnormal cells in the marrow or ≥50% decrease in bone marrow blasts compared with pretreatment values. CRp was defined as per CR but with a platelet count of <100 x 109/L.

Sample collection. Whole blood samples were collected after obtaining written informed consent. Patient blood was collected into an acid citrate dextrose tube (Becton Dickinson, Sparks, MD) and shipped with cold packs to arrive at and be processed at Abbott within 24 hours of collection. Samples for ß-tubulin gene, CEC, and circulating EPC analyses were collected on day 1 (baseline) and day 21 of cycle 1, at the time of second and third bone marrow aspirates and/or biopsies, and at the end of treatment or off-study.

Analysis of ß-tubulin gene (TUBB) mutations. Blood was then transferred to a cell preparation tube (Becton Dickinson) for peripheral blood mononuclear cell isolation. Leukemic cells were enriched from the peripheral blood mononuclear cell fraction by positive immunoselection (Miltenyi Biotec, Auburn, CA) with a cocktail of antibodies defined by the patient's bone marrow flow cytometry report. Once an antibody cocktail was assigned to a patient, all subsequent samples from that patient were enriched similarly. Three different antibody cocktails were used: (a) CD13 + CD33 + CD34; (b) CD10 + CD19 + CD34; and (c) CD13 + CD33 (BD PharMingen, San Diego, CA). Enriched cancer cells were washed and centrifuged. The resulting negative fraction from this enrichment/immunoselection procedure was used as the control. Cell pellets were stored at –80°C until DNA extraction.

Genomic DNA was isolated from sorted peripheral WBC using the Gentra PUREGENE System according to manufacturer's instructions (Gentra Systems, Minneapolis, MN). The primer sets used to amplify exons 1 and 4 from the most common isoform of the ß-tubulin gene family, TUBB (class I ß-tubulin subtype M40; ß5), and their resulting fragment sizes are listed in Table 1. Exons 1 and 4 were evaluated as the colchicines-binding site on ß-tubulin and have been localized to the NH2 terminus domain (amino acids 1-36 corresponding to exon 1 and part of exon 2) and the COOH terminus domain (residues 214-241 corresponding to exon 4; refs. 45–48). The exon 1 PCR primer sets were designed to amplify all of exon 1, whereas the exon 4 primer sets were used to amplify the majority of the exon 4 coding region, –102 bp in the middle of the exon. PCR was done using the Platinum Taq DNA Polymerase Hi Fi kit (Invitrogen Life Technologies, Carlsbad, CA) under the conditions suggested by the manufacturer's protocol. The reactions contained 0.03 unit/µL of Platinum Taq DNA Polymerase Hi Fi, 1x Platinum Taq Hi Fi PCR Buffer, 2 mmol/L MgSO4, 0.2 mmol/L deoxynucleotide triphosphates (Roche, Indianapolis, IN), and 300 nmol/L of each primer (Integrated DNA Technologies, Inc., Coralville, IA). Briefly, 50 ng of genomic DNA were amplified in 50 µL reaction volumes in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). For each PCR primer set, the following cycling conditions were used: one cycle of 94°C for 90 seconds; 55 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds; and one final elongation cycle of 72°C for 3 minutes. Each PCR fragment was amplified in a separate PCR reaction.


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  		Table 1. ß-Tubulin gene primers

 
The PCR products amplified from TUBB exons 1 and 4 were subjected to dideoxy sequencing using the primer sets listed in Table 1. Both DNA strands of each amplified fragment were sequenced on the Applied Biosystems 3130 Genetic Analyzer using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's protocol. Briefly, 2 µg of each PCR fragment were sequenced in a 20-µL reaction with 4 µL BigDye Terminator Ready Reaction Mix, 4 µL Cycle Sequencing buffer, and 1.6 µmol/L primer. The reactions were amplified on a GeneAmp PCR System 9700 (Applied Biosystems) using the following protocol: 25 cycles of 94°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 minutes. Sequencing fragments were purified using Sephacryl S-300 High Resolution (Amersham Biosciences, Piscataway, NJ) columns before analyzing on the 3130 Genetic Analyzer.

Circulating endothelial cell and circulating endothelial precursor/progenitor cell analyses. Whole blood was incubated with a panel of fluorescent monoclonal antibodies, reacting with the endothelial markers P1H12-FITC (CD146; Calbiochem, San Diego, CA) and CD31-PE (BD Biosciences, San Jose, CA), and CD45-APC (BD Biosciences; to exclude hematopoietic cells), along with a nuclear stain (LDS -751; Molecular Probes, Eugene, OR). By switching the CD31-PE antibody to either CD133-PE (Miltenyi Biotec) or Annexin V-PE (BD Biosciences), characterization of EPCs and apoptotic endothelial cells were done, respectively. Stained whole blood then underwent red cell lysis and fixation using FACSLyse (DAKO, Carpinteria, CA) and was read using four-color flow cytometry on either a BD LSR-II (BD Biosciences) or a BD FACSCalibur (BD Biosciences). A panel of isotype control antibodies (BD Biosciences) was used to establish the appropriate negative controls. At least 50,000 events were acquired in duplicate for each measurement.3

CECs were defined as being nucleated (LDS-751 positive), positive for CD146 and CD31, and negative or dimly positive for CD45. EPCs were defined as coexpressing CD133 and CD146, being CD45 dim or negative and nucleated.3 All phenotypic analyses were reported as a percentage of total CECs measured in that run.

Statistical analysis. Statistical comparisons of CEC and EPC levels between controls and leukemic patients were done using paired Student's t test. Statistical differences with P ≤ 0.05 were considered significant. Associations between variables were evaluated by Pearson's correlation coefficient R.


   Results
Top
 Abstract
 Patients and Methods
 Results
Discussion
 References
 
Study group. A total of 32 patients were entered into the study between January 2003 and June 2004. Patient characteristics are listed in Table 2. Twenty-nine patients (91%) had a diagnosis of AML or MDS, most of whom had poor-risk cytogenetics (83%) and/or had a previous history of solid tumor or other hematologic disorder requiring chemotherapy or immunotherapy (55%). One patient with a t(8;21) had additional clonal abnormalities, including trisomy 6 and +der(21)t(8;21). Patients were heavily pretreated with 87% having received two or more prior chemotherapy regimens and 19% having received an allogeneic stem cell transplant. A 77-year-old man had not received any prior therapy for AML but had received treatment with a farnesyltransferase protein inhibitor for MDS.


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  		Table 2. Patient characteristics

 
Dose escalation. A total of 46 cycles of therapy were given on either a 7 of 21-day or 21 of 28-day regimen, with a median of one cycle (range, 0-8 cycles). The first nine patients were treated on a 7 of 21-day schedule. The first cohort received 100 mg/m2/dose, the second 125 mg/m2/dose, and the third 150 mg/m2/dose. No DLTs were observed in the initial three patient cohorts (Table 3). Patients received a median of two cycles of therapy (range, 1-8) on the 7 of 21-day schedule.


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  		Table 3. Drug-related adverse effects at all dose levels for the 7-day every 3 weeks schedule (n = 9)

 
After the first three cohorts were accrued, the treatment schema was modified to an initial dose level of 75 mg/m2 daily orally for 21 days every 4 weeks. No DLTs were observed in the initial five cohorts of patients. One of four patients experienced small bowel obstruction/ileus as a DLT at the 200 mg/m2 dose level. The next patient enrolled at this dose level experienced grade 2 constipation with absent bowel sounds not responsive to laxatives, but which resolved with the discontinuation of ABT-751. Further expansion at this dose level was not attempted because of concern that ileus/small bowel obstruction could recur. Patients received a median of one cycle of therapy (range, 0-2) on the 21 of 28-day schedule.

Medication and protocol adherence. Patient adherence to medication was excellent with only five patients missing one or two doses of ABT-751. One patient on the 7 of 21-day regimen required a transient dose interruption with subsequent reduction from 100 to 75 mg/m2/dose for grade 1 confusion, dizziness, and somnolence, which were subsequently determined to have been caused by leukemia-related leptomeningeal involvement. Another patient on the 7 of 21-day regimen, who required a transient dose interruption while undergoing a splenectomy for symptomatic splenomegaly, had a subsequent dose escalation from 100 to 125 mg/m2/dose for the third cycle of therapy. Six patients received concomitant therapy with hydroxyurea for leukocytosis, all of whom were subsequently taken off study for disease progression. One patient received intrathecal instillations of chemotherapy for leptomeningeal disease and was also later taken off study for disease progression.

Side effects. All 32 patients were evaluable for toxicity. The nonhematologic toxicities for nine patients treated with the 7 of 21-day dosing regimen are summarized in Table 3. The most common grade 1 or 2 adverse effects were nausea and/or vomiting (33%), diarrhea (33%), constipation (33%), increased alkaline phosphatase and/or bilirubin (33%), peripheral paresthesias (22%), somnolence (22%), and anorexia (22%). One patient receiving ABT-751 at a dose of 150 mg/m2 developed grade 1 hyperbilirubinemia during the fifth cycle of therapy. This progressed to grade 3 (peak total bilirubin, 3.2 mg/d) on cycle 8 with the concomitant development of nonneutropenic sepsis, pneumonia, and worsening ischemic cardiomyopathy.

Grade 1 or 2 adverse events were documented at all dose levels for patients receiving the 21 of 28-day regimen (Table 4). No consistent or significant increment in the frequency of grade 2 toxicities was observed as the dose of ABT-751 was escalated. The most frequent toxicities were gastrointestinal [i.e., nausea and/or vomiting (56%), constipation (43%), diarrhea (39%), anorexia (26%), abdominal pain (13%), mucositis (13%), and elevations of transaminases, bilirubin, and/or alkaline phosphatase (13%)]. Neurologic symptoms included peripheral neuropathy (17%), dizziness (4%), and somnolence (4%). Fatigue was observed in 17% of patients, bone pain in 13%, and weight loss in 13%. One patient experienced a grade 3 ileus/small bowel obstruction during the first cycle of treatment at the 200 mg/m2/dose, which resolved with conservative management and discontinuation of ABT-751. The subsequent patient enrolled at this dose experienced grade 2 constipation unresponsive to laxatives, abdominal pain, and nausea and vomiting. ABT-751 was held and the patient continued treatment with laxatives, with resolution of these symptoms. The patient subsequently withdrew from the study. Further expansion at this dose level was not attempted because of the concern of recurrent ileus/small bowel obstruction. Therefore, the recommended phase 2 dose for hematologic malignancies is 175 mg/m2 daily orally for 21 days every 4 weeks.


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  		Table 4. Drug-related adverse effects at all dose levels for the 21-day every 4 weeks schedule (n = 23)

 
Twenty-four infectious episodes were noted in 19 patients (59%). These included fever of unknown origin in four patients (12%) and documented infections in 15 patients (47%): five episodes of bacteremia, eight pneumonias, two cellulitis, one perirectal abscess, one pseudomembranous colitis, and three urinary tract infections. Three patients with AML died while on study from neutropenic sepsis.

Response to treatment. Twenty-nine patients were evaluable for response. Two patients withdrew from study without completing one cycle of therapy; a third was taken off study after developing a DLT before completion of at least one cycle of treatment.

One patient with relapsed AML achieved a CRp after two cycles of therapy on the 7 of 21-day regimen at a dose of 150 mg/m2/dose that was sustained for 2 months, at which time a repeat bone marrow assessment revealed the recurrence of dysplastic features consisting of dyerythropoiesis and 5% blasts. Further treatment with ABT-751 was discontinued after the eighth cycle of therapy because of nonneutropenic sepsis with pneumonia, worsening ischemic cardiomyopathy with pulmonary edema, and grade 3 hyperbilirubinemia. Four patients had hematologic improvement. Two patients, with diagnoses of AML and ALL, treated with ABT-751 at a dose of 175 mg/m2/dose for 21 days every 28 days had a reduction in the percentage of peripheral blood blasts from 43% to 5% and from 94% to 0%, respectively. In the first patient, the blasts increased to 33% with the discontinuation of ABT-751 during the 7-day drug-free period but disappeared completely on reinitiation of the drug. This patient subsequently decided to withdraw from further therapy. The second patient was taken off-study because of disease progression in the bone marrow. One patient with AML receiving the 125 mg/m2/dose showed a transient elevation in the platelet count from 23 x 109/L to 75 x 109/L. Another patient with AML treated with a dose of 150 mg/m2/dose had an elevation in the platelet count from 32 x 109/L to 81 x 109/L. Both patients were later taken off study for disease progression.

ß-Tubulin gene (TUBB) mutations. Genomic DNA was isolated from the immunomagnetic bead sorted peripheral blood leukemic cells and nonleukemic control cells of 24 patients (82 cell fractions from 20 patients with AML, three with ALL, and one with MDS) and sequenced for variants in exons 1 and 4. One patient had genomic DNA isolated only during therapy and not at baseline.

Two single nucleotide polymorphisms (SNP) were discovered in the TUBB promoter region (i.e., G -> A and C -> T at nucleotides –49 and –38, respectively, in 75% and 79% of patients, respectively; data not shown). Six SNPs were detected in genomic DNA in exon 4 (i.e., 567 C -> T, 616 G -> A, 627 T -> G, 665 C -> G, 627 T -> C, and 806 C -> T in 4%, 12.5%, 75%, 46%, 12.5%, and 12.5% of patients, respectively; data not shown). Three of these SNPs encoded for nonsynonymous amino acid changes. Of these three nonsynonymous SNPs, two have been previously reported (665 C -> G for Ser201Cys and 806 C -> T for Ala248Val; ref. 49). The Ala248Val change seems close to the colchicines-binding site (amino acids 214-241; refs. 45, 46). The third novel nonsynonymous SNP, 616 G -> A (Ala185Thr), was detected in the control fraction and leukemia samples of three patients with AML not responding to therapy. Loss of heterozygosity was detected at baseline in two patients with AML, involving both SNPs in the promoter region in one patient and nucleotide 627 in exon 4 in one patient. No changes in the TUBB gene sequence were detected during the course of therapy. There was no discernible association between individual polymorphisms and response to treatment.

Circulating endothelial cell and circulating endothelial precursor/progenitor cell enumeration. Phenotypic analyses of CECs, EPCs, and apoptotic endothelial cells were done on patients with a WBC count of >1 x 109/L. Twenty-four of 26 patients had blood analyzed for CECs. These patients with refractory leukemia and advanced MDS (20 patients with AML, three with ALL, and one with MDS) exhibited wide ranges in CEC and phenotypic numbers compared with normal subjects (Table 5; Fig. 2). The baseline mean CEC concentrations and the percentage of EPCs were significantly different from normal controls (P = 0.037 and P < 0.0001, respectively). Similarly, the proportion of apoptotic CECs was significantly decreased in patients with hematologic malignancies compared with healthy controls (P < 0.0001).


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  		Table 5. CEC phenotypic analysis

 


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  		Fig. 2. CECs and EPCs in normal volunteers and in patients with hematologic malignancies during the course of ABT-751 therapy (i.e., cycle 1, day 21). The levels of CECs (A), percentage of EPCs (B), and percentage of apoptotic CECs (C) in normal volunteers (diamonds) and in patients with hematologic malignancies at baseline (squares) and after treatment (triangles) with ABT-751. The CEC enumeration graph is in logarithmic scale because two patients had very high CEC concentrations. Horizontal bars, means for each group. Points, means of at least two fluorescence-activated cell sorting determinations.

 
There was no difference in the CEC levels and proportion of EPCs or apoptotic CECs observed in patients with AML, ALL, or MDS, albeit the small number of patients with diagnoses of ALL or MDS. There were no differences observed with age (>60 versus <60 years) or de novo versus secondary or therapy-related AML.

A strong correlation was observed between changes in the CEC levels and WBC counts during treatment (R = 0.89; Fig. 3); there was no correlation with EPCs. Decreases in CECs were not detected until patients were dosed at ≥175 mg/m2, with four of five patients showing decreased CEC levels and the fifth stable levels. There were no differences in the proportion of EPCs and apoptotic CECs in patients receiving ABT-751 at doses ≥175 mg/m2 compared with those receiving lower doses. Two patients with leukemia who received ABT-751 at a dose of 175 mg/m2/d had decreases in CECs from 230.9/µL and 18.8/µL, respectively, on day 1, to 6.1/µL and 3.6/µL, respectively, on day 21. These changes correlated with a reduction in absolute peripheral blasts from 9.87 x 109/L and 5.5 x 109/L, respectively, to 0 x 109/L and 0.17 x 109/L, respectively. One patient with AML who received ABT-751 at a dose of 200 mg/m2/d showed a decrease in CEC concentration from 32.8/µL to 1.8/µL, correlating with a reduction in peripheral blasts from 1.54 x 109/L to 0.75 x 109/L.



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  		Fig. 3. Correlation of WBC count and CECs. The percentage change in CEC values was positively correlated with the patients' change in WBC count (from baseline at all measured time points). CEC levels were determined by fluorescence-activated cell sorting analysis. The association between WBC count and CEC was evaluated by Pearson's R correlation coefficient.

 
Baseline evaluation for the subject who experienced a CRp while receiving ABT-751 at doses of 150 mg/m2 was not done; however, all subsequent analyses showed low concentrations of CECs ranging from 0.1/µL to 1.7/µL, with a value of 1.3/µL at study completion. The CEC levels did not decrease in the two patients with hematologic improvements in platelet counts, both of whom received doses of <175 mg/m2.


   Discussion
Top
 Abstract
 Patients and Methods
 Results
 Discussion
References
 
ABT-751 exposure results in inhibition of microtubule assembly with resultant G2-M arrest and apoptosis. ABT-751 is a candidate novel drug for development, as approved antineoplastic agents do not target the colchicine-binding domain of tubulin. It has a broad spectrum of antitumor activity that is not affected by P-glycoprotein overexpression and has good absorption after oral administration. (11, 12, 42).

The current study indicates that the recommended phase 2 dose for patients with hematologic malignancies is 175 mg/m2 daily orally for 21 days every 28 days. None of the three patients treated at this dose experienced any grade 3 or 4 toxicities. Although only one of the patients treated with 200 mg/m2 daily (i.e., 450 mg/d) orally for 21 days every 28 days developed a DLT consisting of ileus/small bowel obstruction, a subsequent patient (also receiving an actual dose of 450 mg/d) developed grade 2 constipation. Therefore, further expansion at this dose was not pursued, as this level of toxicity was felt to be dose limiting.

This recommended phase 2 dose is higher than the maximum tolerated dose (i.e., 200 or ~100 mg/m2) reported for ABT-751 given in patients with advanced solid tumors in phase 1 dose-escalating trials using this schedule (43). The patients in the 175 mg/m2/dose cohort received actual doses of either 350 mg/d (n = 1) or 375 mg/d (n = 2). Eighteen of 23 patients (78%) in the current study treated with ABT-751 on the 21 of 28-day schedule received ABT-751 at doses of ≥200 mg/d up to a maximum of 450 mg/d; only four of them received a dose of 200 mg/d. Most patients completed at least one cycle of therapy, except for one patient each receiving 200, 225, 325, and 300 mg/d, and two patients receiving 450 mg/d. DLT was observed only in one patient receiving a dose of 450 mg/d. The DLTs reported in patients with solid tumors treated with ABT-751 were ileus, small bowel obstruction, and peripheral neuropathy (42, 43). The majority had colon cancer (n = 25 of 69, 36%), some with prior abdominal surgery and prior oxaliplatin therapy. In the current study, only three patients (9%) with ALL had received prior vincristine therapy, two of whom had grade 1 or 2 peripheral paresthesias at baseline without any worsening of these symptoms on study. Eleven patients (34%) had prior intra-abdominal surgery, including the patient who developed the ileus.

Antileukemic activity of ABT-751 was observed in five patients, four of whom had been heavily pretreated, with one patient achieving a CRp. The effect was modest and transient in the four other patients, consisting of a decrease in peripheral blood blasts and elevations in platelet counts. Although ABT-751 pharmacokinetics were not determined in this study, the daily doses given to these patients were within the range of those given to patients with solid tumors in ongoing phase 1 studies (11, 12, 42). In patients with solid tumors, ABT-751 accumulation measured by trough plasma concentrations was minimal after doses once (25-300 mg) or twice (25-175 mg) daily during multiple dosing for 7 or 21 days. Although ABT-751 plasma concentrations at steady state were about ≥0.5 µg/mL at dose levels similar to those in the current study. Based on the pharmacokinetics of ABT-751 in patients with solid tumors, it was expected that efficacious concentrations in preclinical models (0.5-1.5 g/mL) would be achieved in all patients in this study.

The modest activity of single-agent ABT-751 in this group of patients may be due to acquired or inherent resistance mediated in part by mutations in genes encoding the ß-tubulin subunits that impair binding of drugs (47, 48). Therefore, we investigated whether mutations occurred in the colchicines-binding site of ß-tubulin. Like other investigators (49, 50), we did not detect the ß-tubulin mutations previously described by Monzó et al. in patients with paclitaxel resistant non–small cell lung cancer (48). This discrepancy may be due to amplification of homologous nonfunctional ß-tubulin pseudogenes by Monzó et al. (49–51). We did however detect several polymorphisms. Two of the nonsynonymous SNPs in exon 4, detected in this study, have been described previously (52). However, a novel SNP on exon 4, resulting in the Ala185Thr polymorphism, lying outside of the stated colchicine-binding sites (45, 46), was observed. The significance of the ß-tubulin polymorphisms is currently unknown. Further evaluation of the Ala185Thr polymorphism, along with the two promoter SNPs (–49G>A and –38C>T), and complete sequencing of the ß-tubulin gene (TUBB), in particular exon 2 that contains sequences important for colchicine binding, are under way.

Increased CECs and circulating EPCs have been described previously in patients with solid tumors and lymphomas. This is the first study to evaluate CECs and EPCs in patients with acute leukemias and advanced MDS. This patient population has increased angiogenesis, as shown by elevated microvessel density or vascular endothelial growth factor levels, which may correlate with prognosis. Correlation between vascular endothelial growth factor levels and CECs have been shown (32, 33). A correlation between CECs and WBC counts existed at baseline, and changes in WBC on therapy were generally reflected in changes in CEC levels (Fig. 3). Significantly higher and more variable CEC and circulating EPCs concentrations and lower proportions of apoptotic CECs were observed in patients with relapsed or refractory leukemia or advanced MDS compared with healthy normal subjects (Table 5; Fig. 2). In addition, patients with reductions in peripheral blood blasts had reductions in WBC counts and CEC levels. These results suggest a close relationship between CEC levels and bone marrow activity in patients with leukemia. Therefore, CECs, both in number and phenotype, may play a role as a leukemic biomarker. Measurements of CECs may provide information on the activity of specific cancer drugs (e.g., increased apoptosis of CECs being observed in effective antiangiogenic treatments). There was no increase in the proportion of apoptotic CECs, although only a small number of patients were treated at doses ≥175 mg/m2. This may indicate that the mechanism of action of ABT-751 is not mediated by antiangiogenesis (53). An important issue for future studies to examine is the dynamics of the relationship between changes in CEC and clinical responses, the currently reported study was not large enough to address this issue.

One patient with relapsed AML-M2 was noted to have an extremely high number of CECs at baseline (i.e., 599.7/µL, of which 93.8% were EPCs). This patient had received a prior matched related donor stem cell transplant, while in relapse, achieved a CRp of short duration and developed chronic graft-versus-host disease. She commenced ABT-751 therapy 5 months after transplantation and at the time and was taken off study for disease progression after completing 21 days of therapy. The WBC count had increased from 19.5 x 109/L to 53.1 x 109/L and CECs to 846.4/µL (of which 96% were EPCs). The percentage of peripheral and bone marrow blasts increased from 81% to 91% and from 45% to 88%, respectively. This differs significantly from mean baseline CEC, EPC, and apoptotic CEC levels of 2.4/µL ± 0.9/µL (median, 1.9/µL), 25.6 ± 10.0% (median, 16.4%), and 36.4 ± 9.9% (median, 30.7%), respectively, in five other patients with AML who had received a nonmyeloablative allogeneic stem cell transplant after a similar fludarabine-based conditioning regimen. Median interval between allogeneic stem cell transplant and therapy with ABT-751 was 4.3 months (range, 2.7-18). Median number of cycles completed was 1 (range, 0-1). No patient achieved a CR nor had hematologic improvements. No patient had active graft-versus-host disease while receiving therapy with ABT-751; graft-versus-host disease has been documented to increase CEC levels (54).

Thus, the high variability in CECs observed in patients may be to due to the presence of comorbidities (e.g., cardiovascular disease; ref. 55), medications (e.g., statins and cyclosporin; refs. 56–58), prior therapy (e.g., stem cell transplant; refs. 59, 60), or sepsis (61). Therefore, individual medical history is critical when CECs are evaluated in a given patient.

ABT-751 is a well-tolerated oral microtubule inhibitor with some evidence of clinical activity in heavily pretreated patients with acute leukemia. The recommended phase 2 dose is 175 mg/m2 daily orally for 21 days every 28 days in this patient population. Further investigation of this agent in this population is warranted.


   Footnotes
 
Grant support: Cancer Care Ontario fellowship (K.W.L. Yee).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Presented in part at the 45th Annual Meeting of the American Society of Hematology, San Diego, California, November 16, 2003.

3 E. McKeegan, et al. A method to characterize human and canine circulating endothelial cells in healthy and cancer populations. Submitted for publication. Back

Received 3/22/05; revised 5/11/05; accepted 5/25/05.


   References
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 Abstract
 Patients and Methods
 Results
 Discussion
 References
 

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