Treatment plan

Sorafenib was supplied as 200 mg tablets for oral administration. Two different schedules of administration were investigated; Schedule “A”: once or twice daily, five days per week, every week for a 21 day cycle, and Schedule “B”: once or twice daily, for 14 days every 21 days. The starting dose for both schedules was 200 mg twice daily (BID) (i.e. dose level 0) (Table 1). One cycle of therapy was defined as 21 days for both schedules. Patients with persistent grade 2 or with any grade 3-4 drug-related toxicity had treatment interrupted until toxicity resolved to grade 1 or less, to be restarted at the next lower dose level without making up for missed doses. For patients who achieved a remission or normalized their counts, therapy could be interrupted for cytopenias (granulocytes < 1×10⁹/L or platelets <50×10⁹/L) with dose reduction upon resumption of therapy if recovery took over two weeks. Intrapatient dose escalation was permitted once the next dose level was considered safe. Treatment could continue for six months from the time of the first dose or until disease progression or unacceptable adverse event.

Table 1.

Dose levels of sorafenib.

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Response evaluation

Response was evaluated according to the modified International Working Group (IWG) criteria.¹⁶ A complete response (CR) required disappearance of all signs and symptoms related to disease, peripheral blood counts with absolute neutrophil count 1×10⁹/L or over and platelet count 100×10⁹/L or over, and normal bone marrow morphology with no evidence of dysplasia and 5% blasts or under. Complete response with incomplete platelet recovery (CRp) was defined as a CR but with a platelet count of less than 100×10⁹/L without platelet transfusion requirements. Partial response (PR) was defined as fulfilling the criteria for CR in the peripheral blood but with 6% to 25% blasts in the marrow or at least a 50% decrease in bone marrow blasts compared with pre-treatment values.

Translational studies

Peripheral blood mononuclear cell isolation: heparinized whole-blood (10 ml) collected at baseline (day 0, before sorafenib administration), day +1 and day +4, was subjected to RBC lysis in hypotonic buffer (0.15 M NH4Cl, 0.02 M Tris–HCl), and mononuclear cells were resuspended and washed once with PBS.

For evaluation of apoptosis in leukemic blasts, the mononuclear cells from patient blood samples were stained with APC-conjugated anti-CD34 or anti-CD33 antibodies and annexin-V-FLUOS, and the changes in cellular mitochondrial inner transmembrane potential were determined by staining with chloromethyl-X-rosamine (CMXRos, Invitrogen Co). The samples were analyzed by three-color flow cytometry as previously described.^{17, 18}

For immunoblot analyses, rabbit polyclonal phospho-FLT3 (Tyr591) (Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal FLT3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal phospho-ERK (Thr202/Tyr204), horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG secondary antibodies (Santa Cruz Biotechnology) were used. Antibody binding was visualized with the use of an enhanced chemiluminescence detection system (ECL-plus; Amersham Pharmacia Biotech). The semiquantitative immunoblotting data were generated by Scion Imaging software (Beta 4.03; Scion Corporation, Frederick, MD, USA).

Toxicity

Treatment overall was well tolerated. The most common adverse events (regardless of causality) were fatigue in 29 (58%), nausea/vomiting in 22 (44%), diarrhea in 18 (36%) and dyspnea in 15 (30%) patients (Table 3). Grade 3-4 non-DLT defining toxicities included fatigue in one patient, nausea/vomiting in 2 patients, dyspnea in one, creatinine elevation in one patient, transient electrolyte abnormalities in 14 patients and bone pain in one patient. Grade 1-2 skin rash was encountered in 12 (24%) patients and 5 (10%) patients developed grade 1-2 hand-foot syndrome. There was no clear difference in the incidence or type of adverse events between the two treatment schedules. On Schedule A, two DLTs (grade 3 hyperbilirubinemia and hypertension, respectively) were encountered at dose level 3 (i.e. 600 mg twice daily for five days per week). A total of 15 patients (one received less than 21 days of therapy and was replaced) were treated at dose level 2 (400 mg twice daily) without any DLT. On Schedule B one DLT (grade 3 hyperbilirubinemia) was encountered at dose level 2 (400 mg twice daily) with no additional DLT after expansion of the cohort to 6 patients. Two DLTs (congestive heart failure/atrial fibrillation and asymptomatic elevation of amylase/lipase, respectively) were encountered in the next dose cohort. Thus, for both schedules, dose level 2 (400mg twice daily) was deemed to be the MTD. Toxicities regardless of attribution and according to schedule and dose level are summarized in Table 3.

Table 3.

Toxicities irrespective of attribution.

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Responses

A total of 5 (10%) patients responded, 3 with CR and 2 with CRp (Schedule A=3, Schedule B=2) (Online Supplementary Figure S1). The age range of the responders was 21–75 years and median number of prior therapies was 3 (range 1–4). All but one of the responders had reduction of bone marrow blasts to below 5% by C1D21. Two of the responders proceeded with stem cell transplant and in the other 3 patients responses lasted for four weeks, four weeks, and more than six months, respectively. The patient with the longest response (Patient 41), with AML evolving from CMML, had received 2 prior therapies (with a brief response to decitabine) and had FLT3-ITD. On Schedule B at dose of 600 mg twice daily, he developed elevation of amylase and lipase after five days of administration of sorafenib requiring interruption of therapy. Still, by day 15 his bone marrow blast had decreased from 40% to 2%. He had also cleared his circulating blasts by day 5 and his platelets improved from 25×10⁹/L to 103×10⁹/L by day 8. Upon resolution of toxicity, he resumed therapy at 400 mg twice daily and is currently on cycle 12 of treatment.

In addition, 3 patients (all FLT3-ITD) had clearance of marrow blasts (morphological leukemia free status) from pre-treatment blast count of 85%, 83%, and 55%, respectively without recovery of blood counts; one of these patients proceeded to stem cell transplant (SCT). Twelve (24%) additional patients had reduction in bone marrow blasts (≥50% in 9 and 25–49% in 3 patients) (Figure 1). The improvement in blast count lasted four weeks or longer in 11 of these patients. Two additional patients had a reduction of over 50% in circulating blasts that lasted for four weeks. In total, 7 patients (one achieving CR, one CRp and 5 with significant reduction in marrow blasts) were able to proceed to SCT after responding to sorafenib. The changes in peripheral blood blasts are shown in Figure 2 stratified by FLT3 ITD status and the marrow blast changes are summarized in the Online Supplementary Table S1 and S2.

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Figure 1.

Percentage changes in bone marrow blasts from baseline according to FLT3 mutation status. Patients with both internal tandem duplication (ITD) and D835 mutation are counted as ITD mutation. Only information from patients with at least one follow-up bone marrow examination in addition to the baseline is included. *= no follow-up marrow done due to disease progression (ITD=4, WT=3, D835=3). 0=no change in marrow blast percentage.

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Figure 2.

Changes in median number of peripheral blood absolute blast count (total white blood cell count X % blasts) with time according to FLT3 mutation status. Patients with both internal tandem duplication (ITD) and D835 mutation are counted as ITD mutation. (N, ITD=34, Wild-type/D835=16) in a compassionate use

Responses were seen at all dose levels but confined to patients with FLT3 mutation. No response was seen in patients without FLT3 mutation or among the 3 patients with FLT3-ITD mutation who had received prior therapy with other FLT3 inhibitors. These last 3 patients had transient reductions in marrow blasts without achieving remission on their prior therapy with other FLT3 inhibitors.