ALBERT

Description: In vitro studies have suggested that AML cells are sensitive to treatment with the mammalian target of rapamycin (mTOR) inhibitor, rapamycin. In particular, mTOR inhibition is known to enhance the sensitivity of primary AML cells and AML stem cells to etoposide based chemotherapy leading to inhibition of leukemic SRC activity in NOD/SCID mice. To determine the feasibility of applying this approach in vivo, we performed a Phase I dose escalation study of the mTOR inhibitor sirolimus (rapamycin) with a combination chemotherapy induction regimen in adults with relapsed or refractory non-M3 AML. The purpose of this trial was to determine the safety and dose limiting toxicities of sirolimus and chemotherapy in this patient population. Patients received a loading dose of oral sirolimus on day 1 followed 24 hours later by daily doses of oral sirolimus on days 2–7 plus MEC (mitoxantrone 8 mg/m2/day IV, etoposide 100 mg/m2/day IV, and cytarabine 1000 mg/m2/day IV) on days 1–5. Five sirolimus dose levels were explored by a standard 3+3 design. Sirolimus was studied at loading doses from 3–15 mg and daily doses from 1–5 mg/d. Clinical response was assessed by bone marrow biopsy upon hematologic recovery or day 42, whichever occurred first. 23 adults (14 women, 9 men) of median age 58 (range 22 to 65) with relapsed, refractory, or secondary AML were treated with sirolimus and MEC. Five subjects had antecedent hematologic disorders or prior leukemogenic chemotherapy, 18 had relapsed or refractory disease. Sirolimus was well tolerated and did not increase non-hematologic toxicity of MEC chemotherapy. Asymptomatic, reversible liver transaminase or bilirubin elevations occurred in 4 patients, two of which were 〉 grade 2. One patient with a history of prior cytarabine cerebellar toxicity (unknown at the time of study entry) developed reversible cerebellar ataxia. Three patients died of complications related to bacterial infections during chemotherapy-induced aplasia. Dose limiting toxicity was prolonged myelosuppression at the highest planned dose level and was responsible for one treatment-related death due to infectious complications from unresolved aplasia on study day 119. For the first four dose levels the median time to ANC recovery 〉500/uL among evaluable patients was 27 days (range16–38). Pharmacokinetic data showed that doses of 3 mg and higher consistently achieved rapamycin levels considered therapeutic in solid organ transplantation (4–9.2 ug/L). Bone marrow studies in 2/2 evaluable patients on dose level 4 (12 mg loading dose and 4 mg per day sirolimus) showed inhibition of p70S6 kinase phosphorylation consistent with effective inhibition of mTOR at this dose level. Complete remissions occurred in four patients, all treated for first relapse. Two patients subsequently proceeded to allogeneic transplantation. These results indicate that the combination of mTOR inhibition and chemotherapy is feasible in human AML and establish an appropriate dose for phase II studies to be 12mg loading dose followed by 4 mg daily. Patient recruitment at this dose is ongoing. Confirmation of the efficacy of this regimen, which targets signal transduction in leukemic 〈 stem cells, is planned in a randomized phase II trial at the cooperative group level.

Description: Introduction: Iron chelation therapy (ICT) is essential in removing excess iron deposited in body organs, ultimately preventing organ failure and extending the lives of patients (pts) with transfusion-dependent hematological disorders such as β-thalassemia and myelodysplastic syndromes (MDS). As a life-long treatment, traditional ICT (deferoxamine, Desferal®, DFO) is based on a burdensome regimen (subcutaneous delivery 5–7 times a week) that has been shown to negatively impact on pts’ health-related quality of life (HRQoL). The oral chelator deferasirox (Exjade®) is less burdensome to pts offering 24-hour ICT, 7 days a week. Methods: This substudy was part of a single arm, multicenter, 1-year open-label trial (the EPIC study) to investigate the efficacy/safety of deferasirox. The first 558 pts with a variety of hematological disorders were recruited. These pts came from sites in seven countries: Australia, Belgium, France, Germany, UK, Greece, and Italy. Treatment-naïve pts and those having previously received ICT (DFO or deferiprone [Ferriprox®] exclusively, or combined) participated (n=558). Pts were asked at baseline, week 4 and week 52 (end of study [EOS]) to complete the 36-item Short Form health survey (SF-36). The SF-36 is a self-administered questionnaire and measures eight HRQoL domains: physical functioning; role-physical; bodily pain; general health; vitality; social functioning; role-emotional; and mental health. Mean change in SF-36 domain scores were calculated for all pts who had completed data at baseline and week 4, as well all those with completed data at baseline and EOS. All domains are scored so that higher scores indicate a better QoL. Results: Overall, the mean age of the 558 pts (274 β-thalassemia, 168 MDS, 50 sickle cell disease and 66 other anemias) recruited to take part in this substudy was 40.8 years (SD=22.58); 51.5% of patients (n=289) were male and 48.5% (n=272) were female. Within this sample, 337 pts aged ≥16 years completed the SF-36 at baseline, 322 at week 4 and 277 at EOS. Mean domain scores for pts at baseline, week 4 and EOS are presented in Table 1. With the exception of role-emotional (mean=0.78, SD=40.56), mean change in SF-36 domain scores significantly improved (P

Description: BACKGROUND: Conventional treatment options for patients with relapsed hematologic malignancies are both limited and highly toxic driving the pursuit of more tumor specific and less toxic therapies. RNA targeted oligonucleotides are potentially powerful drugs with the ability to silence genes required for malignant hematopoietic cell growth at the post transcriptional level. Studies from our laboratory have validated the c-myb proto-oncogene, which regulates important hematopoietic cell functions and is overexpressed in many hematologic malignancies, as a target for this technology. A prior Phase I trial using a 24 nucleotide phosphorothioated antisense oligodeoxynucleotide targeted to c-Myb mRNA (C-MYB AS ODN) did not identify a maximum tolerated dose (MTD) of the drug. Here we report initial results of a follow up Phase I dose escalation trial using C-MYB AS ODN at higher dose levels than previously studied in subjects with refractory hematologic malignancies. METHODS: C-MYB AS ODN is administered as a 7 day continuous infusion. 5 dose levels ranging from 3mg/kg/day to 12mg/kg/day are planned. Subjects are enrolled using an accelerated dose escalation scheme in which one subject is enrolled on each dose level (DL) with plans to revert to the standard 3+3 design in the event of significant attributable toxcity. C-MYB AS ODN concentrations are measured in peripheral blood (PB) and in mononuclear cells (MNC) by slot blotting at baseline, days 3 and 7 of infusion and two weeks after cessation of infusion. C-myb expression is assessed at these timepoints though QRT-PCR for c-myb RNA. Disease specific assessments of response are measured at predefined timepoints after therapy. RESULTS: 6 subjects, all with refractory acute myelogenous leukemia (AML) have enrolled to date. Escalation through the first 3 DLs occurred without any toxicities. At DL4 (10mg/kg/day) abnormalities have been noted in coagulation assays. The first subject enrolled on DL 4 developed a grade 3 prolongation of the activated partial thromboplastin time (PTT) attributable to drug which returned to normal within 48 hours of drug cessation. Factor levels, DIC parameters and reptilase time were normal. The PTT abnormality was consistent with a “lupus like” inhibitor effect (DRVVT was abnormal and the PTT corrected with the addition of phospholipid in two independent tests.) The 2nd subject treated at DL 4 developed a milder but similar PTT prolongation. The 3rd subject enrolled on DL 4 had a normal PTT throughout therapy. No subjects developed bleeding complications. Plasma and intracellular drug concentrations were dose related (320–640pg/l and 2–80 ng/5×10e6 cells respectively). Peak drug concentrations were found on Days 3–7. By 14 days after infusion, most ODN was cleared from plasma, but remained measurable in MNC at concentrations 30–50% of the maximum value detected. QRT-PCR for c-myb mRNA was performed in 3 subjects. Subject 1’s (DL 1) c-myb mRNA levels gradually decreased from baseline during infusion and nadired two weeks after cessation of infusion. Subjects 3 (DL 3) & 5 (DL 4) had a decrease in c-myb mRNA levels midway through infusion but c-myb RNA levels increased back to baseline by cessation of infusion. To date no subject has had a clinically important response to therapy. Accrual continues for DL 5. CONCLUSIONS: C-MYB AS ODN is detectable in plasma and MNCs of subjects during continuous drug infusion with a steady state reached by day 3 of infusion. Plasma drug levels were markedly reduced 14 days after cessation of infusion but MNC drug levels remained elevated. C-MYB AS ODN at DL 4 (10mg/kg/day) is associated with a PTT prolongation consistent with a “lupus inhibitor” like effect. While encouraging biological activity was identified optimal dose and delivery remain to be established before clinically significant effects can be reasonably expected.

Description: Iron overload has been shown to induce DNA damage and may be implicated in leukocyte apoptosis. Previously we have shown that indirect markers of apoptosis such as caspase activity and Bax levels were higher in leukocytes of thalassemia patients. We have now assessed whether nucleosomal DNA fragmentation, a hallmark of apoptosis, is higher in leukocytes of iron-overloaded thalassemia patients compared with control subjects. Methods: Thalassemia Clinical Research Network patients participating in the Novartis CICL670A0107 trial (a randomized comparison of deferasirox, an oral iron chelator, vs. DFO) were eligible and 44 (25 male, mean age: 21.8 ± 11.1 yrs) were enrolled in the study. Fasting blood samples were obtained after a 5-day washout of DFO prior to commencing treatment with study drug, and 24 hours post-chelator at 1, 6, and 12 months on study. Thirty healthy controls matched for age, sex, race and antioxidant usage (15 male, mean age 24.5 ± 9 yrs) also supplied a blood sample. Plasma, serum and cells were separated by centrifugation. The classic marker for apoptosis, nucleosomal DNA fragmentation was determined by a high throughput ELISA assay that used monoclonal antibodies directed against single- and double-stranded DNA bound to histones (H1, H2A, H2B, H3, and H4) and thus specifically detected mono- and oligonucleosomes (measured in ng/μg leukocyte protein). Detection of free mono- or oligonucleosomes is a direct measurement of apoptotic DNA fragmentation. DNA fragmentation results were log-transformed prior to analysis and means are reported. Results: At baseline, thalassemia patients had elevated nucelosomes compared to the controls (351.5 vs. 74.9 ng/μg leukocyte protein, p = 0.001). This high level of leukocyte DNA fragmentation was unchanged throughout the study. Furthermore, nucleosomal DNA fragmentation was positively correlated with CRP (r = 0.3, p = 0.01), WBC (r = 0.2, p = 0.05) and ANC (r = 0.2, p = 0.04), suggesting apoptosis is present in increased inflammatory states. Conclusions: This finding demonstrates that nucleosomal DNA fragmentation is higher in thalassemia compared to control patients and thus provides further evidence that there is increased apoptosis in circulating leukocytes of thalassemia patients.

Description: Introduction: Iron chelation therapy (ICT) is essential for the treatment of iron overload and is routinely used life-long to help extend patients’ (pts) lives. Conventional ICT (deferoxamine, Desferal®, DFO) is burdensome to pts in terms of method (subcutaneous infusions) and duration (typically 8–12 hours, 5–7 days per week). Deferasirox (Exjade®) is an oral chelator that offers 24-hour ICT, 7 days a week and consequently is less burdensome to pts. Pt-reported outcomes can augment what is known about ICT from clinical and physiological assessments, and they are important since they represent how a pt feels. Methods: As part of a large, single arm, multicenter, 1-year open-label trial (the EPIC study) to assess the efficacy and safety of deferasirox in pts with transfusion-dependent iron overload, β-thalassemia (n=270) and MDS pts (n=87), previously receiving ICT (DFO, deferiprone [Ferriprox®] or combined), were recruited from sites in Australia, Belgium, France, Germany, UK, Greece and Italy. All pts aged ≥16 years were asked to complete the 19-item Satisfaction with ICT questionnaire (SICT) at baseline and week 52 (end of study [EOS]). The SICT is a validated questionnaire assessing pt satisfaction over 4 domains of ICT: perceived effectiveness; side effects; acceptance; burden. Higher SICT scores represent greater levels of satisfaction with each domain. Mean change domain scores were calculated for all pts who had completed data at both time points. Adherence to deferasirox was assessed by asking pts how often they thought about stopping their ICT, and how often they took their ICT exactly as directed by their doctor. Pts responded from 1 ‘Never’ to 5 ‘Always’, and the frequency of pts responding ‘Never’ and ‘Always’ for each item at baseline and EOS was calculated. Results: Mean age of β-thalassemia pts was 26.2 years (SD=11.36); 45.6% (n=123) were male and 54.4% (n=147) were female. MDS pts had a mean age of 66.4 years (SD=10.75); 57.5% (n=50) males and 42.5% (n=37) females. There were 181 β-thalassemia and 57 MDS pts aged ≥16 years who completed the SICT at study baseline. Baseline and EOS mean SICT scores are presented in Table 1. Significant improvements in mean change domain scores were reported in β-thalassemia (P

Description: In vitro, bexarotene inhibits the proliferation of non-M3 AML cell lines and induces differentiation of leukemic blasts. Our previous phase I study in non-M3 AML showed evidence of leukemic response as manifested by reduction in bone marrow blast counts (15% response rate), improved platelet counts (41%) and improved neutrophil counts (26%). Based on these results, a phase II trial in non-M3 AML was initiated at the phase I MTD. In the current phase II trial, bexarotene (300mg/m2) was administered daily as monotherapy until disease progression or unacceptable side effects occurred. Fourteen patients have been enrolled: 8M/6F, median age 74 (range 20–83), 9 secondary AML (MDS or prior chemotherapy), 9 primary refractory or relapsed 〈 1 year after induction, 5 no prior induction chemotherapy, 5 requiring hydroxyurea at the time of enrollment for leukemic blast control, 4 prior allogeneic stem cell transplant, 12 blood transfusion dependent, 11 platelet transfusion dependent, and 8 neutropenic. Overall, no significant adverse events were noted. All patients received prophylactic antihyperlipidemic agents and achieved good lipid control. Two patients developed mild hypothyroidism related to bexarotene. Five patients were evaluable with bone marrow biopsy at 2 months: 1 50% reduction in absolute blasts, 1 SD and 3 PD. Similar to data from our prior phase I study, evidence of clinical activity was manifested as platelet count response in 1 patient and neutrophil increases attributable to bexarotene in 2 patients. When combining the results of our phase I experience (27 patients) with our phase II data (14 patients), there is a suggestion of increased activity in patients with 5q minus abnormalities with 4/7 (57%) benefiting (2 BM response, 4 neutrophil improvements and 1 platelet response). Conversely rates of clinical benefit were lower in patients with multiple (〉3) cytogenetic abnormalities (2/13), relapse after stem cell transplant (1/9) or requiring hydroxyurea for peripheral blast control at the time of study enrollment (0/10). Bexarotene is very well tolerated at the dose level studied. Early evidence for clinical activity has been seen as exemplified by improvement in platelet count, increased neutrophil counts and decreased bone marrow blasts. In summary, we conclude that bexarotene is an active agent in a subgroup of patients with AML. Study enrollment continues with amended inclusion criteria to focus on patients more likely to benefit from treatment.

Description: Purpose: Despite therapeutic advancements, biological markers that predict the natural history of primary central nervous system lymphoma (PCNSL) are lacking and age and performance status are the only consistently identified independent prognostic variables. BCL6 rearrangements and deletion of the tumor suppressor gene R-PTP-κ (PTPRK) at 6q22 are thought to be common genetic abnormalities in PCNSL but their prognostic significance is unknown. The aim of this study is to determine the prevalence and survival impact of del(6)(q22), BCL6, immunoglobulin heavy chain (IGH), and MYC gene rearrangements and prevalence of Epstein-Barr virus (EBV) infection in PCNSL affecting immunocompetent patients. Patients and methods: Seventy-six specimens from 76 HIV-negative, immunocompetent patients with PCNSL newly diagnosed and treated at Mayo Clinic between 1985 and 2006 were studied. Interphase fluorescence in-situ hybridization (FISH) was performed using two-color break apart probes (BAP) for BCL6 and MYC, a two-color dual-fusion probe for IGH-BCL6, and a two-color probe for del(6)(q22) on thin sections of paraffin-embedded tumor samples. Two-color IGH BAP FISH probes were also used to confirm IGH rearrangements in cases showing extra IGH signals without fusion using the IGH-BCL6 probe. In situ hybridization was performed using probes that recognize EBV-encoded RNA (EBER) on paraffin-embedded tumor samples. Survival data were analyzed for patients diagnosed after 1997 (n=53), corresponding to the change to high dose methotrexate as the standard of care. Survival was calculated from the date of tissue diagnosis to date of death or last contact. Survival curves were estimated using the Kaplan-Meier method. The log-rank test was used to compare survival across groups. Two-tailed p-values

Description: Benzene, a common industrial solvent and ubiquitous environmental contaminant, is hematotoxic and chronic exposure increases the risk of leukemia and lymphoma. Benzene is also immunotoxic and has been shown to increase susceptibility to infectious agents. Benzene depresses B and T lymphocyte numbers as well as their mitogenic responses and benzene metabolites mediate the impairment of macrophage activity. The immunotoxic effects of benzene have been demonstrated mainly in animal and in vitro models exposed to high levels of benzene or its metabolites. In order to elucidate the molecular mechanisms underlying the hematotoxic and immunotoxic effects of benzene, we previously performed a global gene expression study of a small number of workers (N = 6) exposed to benzene (〉10ppm) compared with unexposed controls (N = 6), and identified several genes associated with benzene exposure. Here, we greatly expanded the study to include 125 individuals exposed to a range of benzene concentrations, including 59 individuals exposed to

Description: Background: Under conditions of iron overload, ascorbic acid is oxidised at an increased rate leading to a risk of vitamin C deficiency. With deferoxamine (DFO) standard therapy, vitamin C is usually given at a dose of 2–3mg/kg on the days of DFO infusion as this increases iron excretion by up to 30%. With deferarisox (DFX) chelation treatment, although supplementation is permitted, there is currently no information about the effects of vitamin C supplementation on iron excretion and it is often left to patients or their clinician’s discretion as to whether supplementation is given. With long-term treatment, in the absence of supplementation there is a potential risk that vitamin C deficiency will develop and this could influence response to treatment. Patients and Methods: We have measured fasting plasma vitamin C in 41 patients who have been on long term deferasirox treatment for transfusional iron overload for between 1.5 and 5 years. 32 of these patients had received no supplementation and 9 patients had received 2–3 mg/kg/ day of supplementation. We have examined whether trends in serum ferritin, myocardial T2* and liver iron, during the final year of observation, relate to plasma levels of vitamin C. Results: Fasting plasma Vitamin C was significantly lower in the 41 patients (mean=30.3μmol/l, SD=20.8) than healthy control patients (mean=60.29μmol/l SD=12.6) (P

Description: Background: Steroid-resistant patients with Diamond-Blackfan anemia (DBA) require life-long transfusion therapy, which ultimately results in toxic iron overload. Efficacy and safety of a once-daily, oral iron chelator, deferasirox (Exjade®), in patients with DBA have been demonstrated in one trial in which patients were dosed based on liver iron concentration (LIC) at enrollment. More recently, patients with various transfusion dependant anemias have been enrolled as part of a large, prospective, multicenter trial, the EPIC study, to evaluate whether fixed starting doses of deferasirox, based on transfusion history and subsequent dose titration, can provide clinically acceptable chelation, as measured by serum ferritin (SF). Data for the DBA sub-group are presented. Methods: Fourteen patients with transfusion-dependent DBA and SF levels of ≥1000 ng/mL, or 20 transfusions or 100 mL/kg of blood) and a R2 MRI confirmed LIC 〉2 mg Fe/g dry weight, were enrolled. Deferasirox was administered at an initial dose of 10–30 mg/kg/day depending on transfusion requirements, with dose adjustments in steps of 5–10 mg/kg/day (in the range 0–40 mg/kg/day) based on assessment of 3-month SF trends and safety markers. SF was assessed every 4 weeks; the primary efficacy endpoint was change in SF from baseline at 52 weeks. Safety assessments included adverse event (AE) monitoring and assessment of laboratory parameters. Results: Male (n=7) and female (n=7) patients with DBA (mean age 17.3 years), had a median baseline SF of 2288.8 ng/mL, and received a mean of 185.0 mL/kg of blood in the year prior to enrollment. Most patients had received previous chelation therapy with deferoxamine (DFO; 71.4%); two patients (14.3%) had received combination DFO/deferiprone and two (14.3%) had received no prior chelation therapy. All patients completed 12 months of treatment with no discontinuation. At 12 months, a significant reduction in SF from baseline was observed in the total population at an average actual dose of 21.0±4.8 mg/kg/day (−789.5 ng/mL; 2288.8 ng/mL [baseline]; P=0.0121). A similar reduction was seen in patients receiving an average actual dose of deferasirox ≥20–10xULN (upper limit of normal) on two consecutive visits; all had elevated ALT levels at baseline. Conclusions: Over a 1-year treatment period, deferasirox significantly reduced iron burden in transfusion-dependent DBA patients with iron overload. Efficacy was demonstrated in patients receiving doses above and below 20 mg/kg/day, suggesting the need to individually titrate the dose according to the rate of iron intake from ongoing blood transfusions, as well as current iron burden and target SF levels. Deferasirox was well tolerated, with an AE profile similar to that observed in other patient groups.