Temsirolimus, an mTOR inhibitor, in combination with lower-dose clofarabine as salvage therapy for older patients with acute myeloid leukaemia: results of a phase II GIMEMA study (AML-1107)

Authors


  • Results presented, in part, at the 52nd Annual Meeting of the American Society of Hematology, December 2010, Orlando, FL, USA.

Professor Sergio Amadori, Department of Haematology, Tor Vergata University Hospital, Viale Oxford 81, Roma, 00133, Italy.
E-mail: sergio.amadori@ptvonline.it

Summary

The mammalian target of rapamycin (mTOR) signalling pathway has emerged as an important therapeutic target for acute myeloid leukaemia (AML). This study assessed the combination of temsirolimus, an mTOR inhibitor, and lower-dose clofarabine as salvage therapy in older patients with AML. Induction consisted of clofarabine 20 mg/m2 on days 1–5 and temsirolimus 25 mg (flat dose) on days 1, 8 and 15. Patients achieving complete remission with (CR) or without (CRi) full haematological recovery could receive monthly temsirolimus maintenance. In 53 evaluable patients, the overall remission rate (ORR) was 21% (8% CR, 13% CRi). Median disease-free survival was 3·5 months, and median overall survival was 4 months (9·1 months for responders). The most common non-haematological severe adverse events included infection (48%), febrile neutropenia (34%) and transaminitis (11%). The 30-d all-cause induction mortality was 13%. Laboratory data from 25 patients demonstrated that a >50%in vivo inhibition of S6 ribosomal protein phosphorylation was highly correlated with response rate (75% with inhibition versus 0% without inhibition; = 0·0001), suggesting that targeting the mTOR pathway is clinically relevant. The acceptable safety profile and the predictive value of target inhibition encourage further investigation of this novel regimen.

Considerable progress has been made in the treatment of younger patients with acute myeloid leukaemia (AML), but current therapeutic results in adults aged 60 years or older remain unsatisfactory. Older patients respond less well to standard chemotherapy than younger individuals, as reflected by the higher incidence of treatment-related mortality, lower complete remission rates, and shorter survival in major clinical trials (Appelbaum et al, 2006; Burnett et al, 2009; Estey, 2009). Moreover, induced remissions are typically short-lived, and therapeutic options are limited at the time of relapse. Thus, combined with older patients refractory to upfront chemotherapy, there exist a substantial number of individuals where novel therapeutic agents and treatment paradigms are sorely needed.

The major thrust of novel therapeutics in AML is the development of agents targeting critical cellular and molecular events that ultimately lead to leukaemic transformation. One promising target for molecular therapy in AML is the PI3K/Akt signalling pathway, which is constitutively activated in 90% of AML samples and has been shown to be central to the proliferation and survival of the leukaemic blasts (Chapuis et al, 2010a). The serine-threonine kinase mammalian target of rapamycin (mTOR) is downstream of PI3K/Akt and can be inhibited by selective inhibitors including rapamycin (sirolimus) and its second-generation analogues (rapalogs). mTOR inhibitors have been shown to induce apoptosis of AML cells in vitro, to have in vivo activity in experimental models of AML, and to enhance the antileukaemic activity of a variety of cytotoxic agents, including etoposide, anthracyclines and cytarabine (Recher et al, 2005a). Early clinical studies of mTOR inhibitors as monotherapy in patients with advanced AML demonstrated tolerability, inhibition of mTOR signalling in patient samples and modest clinical activity (Recher et al, 2005b; Yee et al, 2006). The feasibility of combining mTOR inhibition with intensive chemotherapy has been assessed in a recent phase I dose escalation study of sirolimus in patients with relapsed and refractory AML, with promising results (Perl et al, 2009).

Temsirolimus, currently approved by the US Food and Drug Administration (FDA) for use in renal-cell carcinoma and mantle-cell lymphoma, is a water-soluble ester analogue of sirolimus that is rapidly converted to the parent compound after intravenous administration. Temsirolimus infused weekly at a flat dose of 25 mg has shown encouraging clinical activity in a variety of solid tumours as well as in mantle cell lymphoma (Atkins et al, 2004; Hess et al, 2009), with an improved safety profile as compared to higher doses. This dosing schedule results in high peak plasma concentrations, is associated with effective biological activity, and is recommended as the optimum regimen for use in trials combining temsirolimus with other myelosuppressive agents (Atkins et al, 2004; Raymond et al, 2004; Hess et al, 2009; Teachey et al, 2009). Clofarabine, a rationally designed deoxyadenosine analogue, approved by the US FDA for the treatment of children with relapsed/refractory acute lymphoblastic leukaemia, has been shown to have significant activity in both advanced and previously untreated AML, when used as a single agent and in combinations at daily doses ranging from 30 to 40 mg/m2 administered intravenously for 5 consecutive days (Kantarjian et al, 2007; Burnett et al, 2010).

On these grounds, we hypothesized that mTOR inhibition induced by temsirolimus may enhance the clinical activity of lower-dose clofarabine (20 mg/m2/d for 5 d) in older patients with advanced AML and conducted a phase II study to determine the efficacy and safety of this combination. As an adjunct to the trial, a correlative laboratory study was performed to assess the relationship between clinical activity and in vivo suppression of phosphorylated S6 ribosomal protein (p-S6RP), a downstream biomarker of mTOR activity (Martelli et al, 2011).

Patients and methods

Study design and eligibility

AML1107 was a multicentre, open-label phase II trial performed by the Gruppo Italiano Malattie Ematologiche dell’Adulto (GIMEMA) cooperative group. The study was approved by the Local Ethics Committee at each participating institution and conducted in accordance with the Declaration of Helsinki. Written, informed consent was obtained from all patients before study enrolment.

Patients with AML in first untreated relapse or refractory to no more than one initial induction attempt with standard chemotherapy were considered eligible for the study if they were aged more than 60 years and had a World Health Organization (WHO) performance status (PS) grade 0–2. Other requirements to be met on study entry included adequate hepatic (serum total bilirubin ≤1·5 upper limit of normal (ULN), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤2·5 ULN), renal (serum creatinine ≤88·4 μmol/l) and cardiac function, no uncontrolled infection, and a life expectancy of at least 4 weeks. Exclusion criteria included a diagnosis of acute promyelocytic leukaemia or AML developing after a myeloproliferative disorder, active central nervous system involvement, and prior therapy with clofarabine or mTOR inhibitors.

Treatment regimen

Clofarabine (Genzyme Europe, Naarden, The Netherlands) and temsirolimus (Pfizer Inc., New York, NY, USA) were supplied by the manufacturers. Induction therapy consisted of a single course of clofarabine 20 mg/m2 intravenously (iv) over 1 h daily on days 1–5, and temsirolimus 25 mg (flat dose) iv over 30 min on days 1, 8 and 15. Patients with a partial remission could receive a second identical course if the treating physician considered this to be in the patients’ best interest. Patients achieving a morphological complete remission, with (CR) or without (CRi) full blood count recovery, could receive up to twelve courses of maintenance therapy with single agent temsirolimus at 25 mg (flat dose) on days 1 and 8 of each 28-d course. Prophylactic antibiotics and standard supportive care were provided according to institutional guidelines, but the use of azole antifungals was permitted only after completion of all scheduled temsirolimus doses. Growth factors were not administered routinely to patients during the study.

Assessment of toxicity and response

Patients were monitored with physical examination, complete blood count and chemistry profile at least twice weekly during induction, and then at least every 2 weeks as long as they received maintenance temsirolimus. Bone marrow aspiration was done on day 28 of induction therapy, and then every week as indicated to document response. Subsequently, responding patients were to have a bone marrow aspiration every 3 months until disease progression. Response to treatment was defined according to the revised recommendations of the International Working Group for AML (Cheson et al, 2003). Toxicities were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE), version 3 (http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf).

Correlative laboratory study

In vivo inhibition of mTOR signalling was assessed by comparison of S6RP phosphorylation in bone marrow blasts collected from consenting patients pre-therapy (day 0) with that seen in samples obtained at one later time point, between 24 and 48 h after the first or second temsirolimus infusion. Given that pharmacodynamic data in cancer patients treated with weekly doses of 25 mg of temsirolimus have shown that maximum in vivo inhibition of p-S6RP is reached within 24 to 48 h post-infusion, and that such inhibitory effect is no longer visible 7 d after dosing when temsirolimus and its major metabolite, sirolimus, are already expected to be eliminated (Peralba et al, 2003), it is plausible to assume that sampling the bone marrow within 24 to 48 h of the first or second temsirolimus infusion would be equally informative of the in vivo biological effects induced by the mTOR inhibitor. Only bone marrow aspirates of sufficient cellularity were considered evaluable for serial assessment of in vivo inhibition of mTOR signalling. Quantitative flow cytometry to measure levels of p-S6RP was done as follows: bone marrow mononuclear cells were separated by Ficoll/Hypaque density centrifugation and stained with a phycoerythrin (PE)-conjugated anti-CD33 and a phycoerythrin-cyanin 7 (PC7)-conjugated anti-CD45 antibody (both from Beckman Coulter, Miami, FL, USA). They were then washed with phosphate-buffered saline (PBS) and processed for intracytoplasmic staining of p-S6RP, using an AlexaFluor488-conjugated antibody (Cell Signaling Technology, Danvers, MA, USA), as reported elsewhere (Chiarini et al, 2010). Blast cells were identified by CD45/CD33/side scatter gating (Borowitz et al, 1993), and p-S6RP expression was quantified by a test-to-control fluorescence intensity ratio (RFI) between the mean fluorescence intensity (MFI) of the stained cells and the MFI of the isotypic control (rabbit IgG conjugated to AlexaFluor488). Cells were analysed on a FC500 flow cytometer (Beckman Coulter), and at least 5000 events per sample were acquired.

Statistical analysis

The primary endpoint was morphological complete remission rate, defined as CR plus CRi; all other responses were considered treatment failures. Secondary endpoints included the safety profile of the combination, overall survival (OS), disease-free survival (DFS), and the relationship of clinical response to in vivo inhibition of mTOR signalling. The sample size was based on an α level of 0·05 and a power of 0·90. Given a target CR+CRi rate of at least 40% and an acceptable CR+CRi rate not <20%, 54 patients were required. Patients were recruited using a Simon two-stage design, allowing early closure if <5 of the first 19 patients achieved CR or CRi (Simon, 1989). OS was defined as the time interval from the date of start of therapy to death from any cause or the date last known alive. DFS was calculated from achievement of CR or CRi until disease recurrence or death in remission. Results are presented for all treated patients. Kaplan–Meier methodology is used to describe time-to-event outcomes. Data cut-off was December 31, 2010.

Results

Patient characteristics

Between April 2009 and June 2010, a total of 54 patients meeting the eligibility criteria were enrolled at 14 GIMEMA centres, but only 53 received any amount of chemotherapy and were included in the analysis (one patient died of disease progression before beginning therapy). Table I lists the baseline characteristics of the 53 evaluable patients. The median age was 69 years (range, 60–78 years); 85% had a WHO performance score of 0 or 1; and the majority (72%) of patients had intermediate cytogenetics at study entry (Grimwade et al, 2001). Eighteen patients (34%) were refractory to the initial induction therapy, and 35 (66%) were in first untreated relapse. Among the latter, 12 had experienced a first remission duration of <6 months, and 23 patients were in remission for at least 6 months or longer.

Table I.   Patient characteristics.
CharacteristicNo. of patients (%)
  1. WHO, World Health Organization; CR1, first complete remission.

  2. *Based on the criteria of Grimwade et al (2001), patients with t(8;21) or inv(16)/t(16;16) were assigned to the favourable risk group; those with monosomy 5 or 7, del(5q), 3q abnormalities, or complex changes (≥5 unrelated abnormalities) were assigned to the adverse risk group; those with normal karyotype or other noncomplex abnormalities were assigned to the intermediate risk group.

No. of patients53 (100)
Age (years)
 Median69
 Range60–78
Sex
 Male31 (58)
 Female22 (42)
WHO performance score
 0–145 (85)
 28 (15)
Status of disease
 Primary refractory18 (34)
 First relapse35 (66)
 CR1 duration <6 months12 (23)
 R1 duration ≥6 months23 (43)
Cytogenetic risk group*
 Favourable4 (8)
 Intermediate38 (72)
 Adverse6 (11)
 Not available5 (9)

Response and outcome

Clinical responses are summarized in Table II. Among all 53 treated patients, 4 (8%) achieved CR, and 7 (13%) achieved CRi, for an overall response rate (ORR) of 21% [95% confidence interval (CI), 11–34%]. One patient with relapsed AML required two courses to achieve remission; all others achieved a CR or CRi following one course. The median time to remission was 36 d (range, 27–87 d). Of the remaining patients, 34 (64%) had resistant disease; 7 (13%) died within 30 d of induction, including three patients from infection (two pneumonia, on days 10 and 24; one sepsis on day 16), one patient from cerebrovascular accident (on day 19), and three patients from rapidly progressive disease (on days 9, 15 and 19); one patient had a partial remission (PR) but declined further therapy and was lost to follow-up. As shown in Table III, the ORR was higher in patients whose first remission lasted for at least 6 months (30%), whereas only two of 12 (17%) patients with a shorter first remission duration and two of 18 (11%) with primary induction failure achieved CR or CRi. Responses were observed in five of 21 (24%) patients who were aged ≥70 years and in six of 32 (19%) patients younger than age 70 years. The impact of cytogenetics on induction outcome can hardly be assessed, as the majority of patients were in the intermediate risk group. However, none of the six patients with adverse cytogenetics achieved a response.

Table II.   Induction outcomes (53 patients).
ResponseNo. of patients (%)
  1. ORR, overall response rate; CR, complete remission; CRi, complete remission with incomplete blood count recovery; PR, partial remission.

ORR (CR+CRi)11 (21)
CR4 (8)
CRi7 (13)
PR1 (2)
Resistance34 (64)
Induction death (within 30 d)7 (13)
Table III.   Analysis of response by baseline characteristics.
CharacteristicPatients (n)CRCRiORR (%)
  1. ORR, overall response rate; CR, complete remission; CRi, complete remission with incomplete blood count recovery; CR1, first complete remission.

  2. *Based on the criteria of Grimwade et al (2001), patients with t(8;21) or inv(16)/t(16;16) were assigned to the favourable risk group; those with monosomy 5 or 7, del(5q), 3q abnormalities, or complex changes (≥5 unrelated abnormalities) were assigned to the adverse risk group; those with normal karyotype or other noncomplex abnormalities were assigned to the intermediate risk group.

Overall534721
Age (years)
 <70322419
 ≥70212324
Status of disease
 Primary refractory181111
 First relapse353626
 CR1 < 6 months121117
 CR1 ≥ 6 months232530
Cytogenetic risk group*
 Favourable40250
 Intermediate384421
 Adverse6000
 Not available50120

With a median follow-up of 10·9 months (range 0·3–20·2), median overall survival for all 53 treated patients was 4·0 months (range, 0·3–20·2), and 9·1 months (range, 2·3–20·2) for responders (Fig 1). Of the 11 patients achieving CR or CRi, one patient suffered from an early relapse and 10 were started on monthly temsirolimus maintenance, two of whom completed the scheduled 12-month courses. The median remission duration was 3·5 months. Nine patients developed recurrent disease after a median of 3·2 months (range, 1–14·7); the duration of response was censored at 2 months for one patient who withdrew from the study whilst still in remission, and at 16 months for one patient who remained in remission at data cut-off.

Figure 1.

 Overall survival. All treated patients (solid line) versus responding patients (dashed line).

Toxicity

All patients developed grade 4 neutropenia and thrombocytopenia, with complete responders (CR or CRi) requiring a median of 28 d (range, 22–44 d) from the start of treatment to recover a neutrophil count >0·5 × 109/l and a median of 32 d (range, 19–47 d) to recover a platelet count >50 × 109/l. The frequency of additional treatment-related toxicities that occurred during induction in at least 5% of patients is summarized in Table IV. The most commonly encountered severe adverse events included infections (48%) and febrile neutropenia (34%). Of note, mucositis was usually mild, whereas diarrhoea was unusual. Hepatic grade 3–4 toxicity was 11%, but increased transaminases were largely transient. During induction, temsirolimus dose delays/reductions occurred occasionally and were mostly due to neutropenic fever or infection. Grade 3 or higher cytopenias as well as non-haematological toxicities during maintenance temsirolimus were uncommon.

Table IV.   Treatment-associated non-haematological adverse events occurring during induction in at least 5% of patients.
EventGrade, patients (n, %)
Any gradeGrade 3Grade 4
Infection34 (64)20 (38)5 (10)
Febrile neutropenia30 (57)14 (26)4 (8)
Nausea/vomiting29 (55)4 (8)0
Elevated transaminases25 (52)5 (9)1 (2)
Fatigue23 (43)3 (6)1 (2)
Mucositis17 (32)1 (2)1 (2)
Hyperglycaemia16 (30)1 (2)1 (2)
Diarrhoea12 (23)2 (4)0
Haemorrhage12 (23)1 (2)0
Metabolic (other)4 (7)4 (7)0

Correlative laboratory study

Paired day 0 and one later time point pharmacodynamic assessments of mTOR signalling were performed in bone marrow samples of 25 patients for whom sufficient cell numbers were available. All baseline samples demonstrated S6RP phosphorylation, consistent with activation of the mTOR pathway (data not shown). Flow-cytometric examination of paired samples revealed two distinct patterns of pharmacodynamic response to temsirolimus-based treatment (two representative cases are illustrated in Fig 2). Overall, as shown in Fig 3, S6RP phosphorylation was strongly inhibited (median residual p-S6RP 39%, range 19–47%) in 12 (48%) patients (pharmacodynamic responders), while weak to no detectable target inhibition (median residual p-S6RP 101%, range 59–161%) was detected in 13 cases (pharmacodynamic non-responders). Of the 25 assayed patients, eight had levels of p-S6RP measured on trial day 2 or 3, and 17 on trial day 9 or 10: the proportion of patients with evidence of pharmacodynamic response was comparable in the two cohorts (4/8 vs. 8/17, respectively), supporting equivalence of the two sampling time points. Direct correlation of in vivo p-S6RP inhibition and induction outcome provided important information in the 25 patients tested. Clinical responses only occurred in the subset of 12 patients where maximum residual p-S6RP level was reduced to below 50% of baseline: 9 (75%) responded (3 CR, 5 CRi, 1 PR), including three of four with primary induction failure and six of eight in first relapse. In contrast, none the 13 patients showing >50% residual p-S6RP activity in AML blasts responded clinically to therapy (= 0·0001).

Figure 2.

 Flow cytometric analysis of the effects of treatment on S6RP phosphorylation. Shown are the representative histograms of a pharmacodynamic (PD) responder (A) and a non-responder (B), analysed for the level of p-S6RP in marrow blasts collected at baseline (day 0) and day +2 of treatment. Blasts were selected using a CD45/CD33/side scatter gating strategy. White histograms, negative control (irrelevant antibody); black histograms, antibody to p-S6RP.

Figure 3.

 Treatment-induced changes in S6RP phosphorylation relative to baseline in 25 patients for whom paired bone marrow samples were available. Two distinct patterns are evident: a >50% inhibition was detected in 12 patients (pharmacodynamic responders, PD-R), whereas 13 patients had weaker or no target inhibition (pharmacodynamic non-responders, PD-NR). The horizontal lines inside the boxes indicate median values. The lower and upper borders of the boxes correspond to the 25th and 75th percentile respectively. The whiskers extend to the 100% range.

Discussion

Defects in signalling pathways are increasingly recognized as an essential element of leukaemogenesis. One commonly dysregulated pathway in AML is the PI3K/Akt/mTOR signalling network, suggesting that components of this pathway could serve as rational therapeutic targets. In this study, the mTOR inhibitor temsirolimus was administered in combination with clofarabine as salvage therapy in older patients with relapsed or refractory AML. The choice of clofarabine was based on the encouraging clinical activity shown by the drug when administered to patients with relapsed/refractory AML at the recommended dosing schedule of 40 mg/m2 daily for 5 d (Kantarjian et al, 2007). However, at this dose profound myelosuppression was ubiquitous and severe liver dysfunction was not negligible, especially in the older age population. Given that, as recently reported (Burnett et al, 2010), reduced doses of clofarabine as upfront monotherapy maintain efficacy with improved tolerability in elderly AML, a dosing schedule of 20 mg/m2 daily for 5 d was selected for combination with temsirolimus.

The results of this multicentre trial indicate that the combination of lower-dose clofarabine and temsirolimus has activity and acceptable toxicity in older adults with AML refractory to or relapsing after initial intensive chemotherapy. The overall response rate (CR + CRi) was 21%, with a median duration of response of 3·5 months, a median survival for all patients of 4·0 months, and a median survival for responders of 9·1 months. All responses occurred following one induction course, with the exception of one patient who required two courses to achieve remission. Responses were seen in all patient subgroups with the exception of the six patients presenting with an adverse karyotype, but tended to be less frequent among those with shorter first CR duration or primary induction failure.

The combination regimen was reasonably well tolerated, and both agents could be given at full doses in more than 90% of patients. Consistent with prior studies of single-agent clofarabine (Kantarjian et al, 2007; Burnett et al, 2010), severe toxicities were primarily cytopenias and infections, as would be expected in a population of older patients with advanced disease. The non-haematological safety profile was favourable; mucosal and gastrointestinal toxicities occurred infrequently and at low grades, and transient grade 3 or higher liver function abnormalities were seen in only a minority of patients. The all-cause 30-d mortality of 13% compares favourably with that in studies with similar patient populations treated with higher doses clofarabine or intensive chemotherapy (Kantarjian et al, 2007; Mato et al, 2008), especially in light of the fact that only four of the seven induction deaths were attributable to treatment-related adverse events.

Although these results may appear relatively modest, they are indeed quite significant for the association with in vivo mTOR inhibition. In fact, the key finding emerging from our correlative study is that effective dephosphorylation of S6RP, an established biomarker of mTOR activity, in response to temsirolimus is necessary for clinical response to the combination regimen. A very encouraging ORR of 75% was documented in patients showing inhibition of p-S6RP to below 50% of baseline level, whereas none of the patients who failed to achieve that level of target inhibition responded to therapy. While we cannot exclude a role for clofarabine in mediating p-S6RP inhibition, these findings strongly support the concept that inhibition of mTOR activity in response to rapalogs could enhance chemotherapy efficacy in AML.

Why did temsirolimus fail to induce an effective mTOR inhibition in roughly 50% of our assayed patients? At present, we do not have a precise explanation for this but several factors may have contributed. Given the fact that a pharmacokinetic (PK) study was not built into this trial when it was designed, possible correlations between individual temsirolimus PK parameters and extent of mTOR inhibition or clinical activity cannot be ruled out, although data from previous studies in cancer patients do not support such an association when single-agent temsirolimus was administered weekly at a wide range of doses (Raymond et al, 2004). Alternatively, cell-intrinsic mechanisms could plausibly be responsible for the insufficient p-S6RP dephosphorylation observed in these patients. It is known that rapamycin, a major metabolite of temsirolinus, interacts with the mTORC1 subunit of mTOR in a manner that is competitive with phosphatidic acid (PA), the metabolic product of phospholipase D (PLD) (Chen et al, 2003). As PLD activity has been reported to be upregulated in most tumours, elevated levels of PA are likely to reduce the sensitivity of cancer cells to mTOR-targeted therapy (Foster, 2009). This data, if confirmed in AML, could provide a rationale for developing combination strategies aimed at overcoming rapamycin resistance by targeting PLD activity. Additionally, the Ser 235/236 residues of S6RP, besides being a target of mTORC1 signalling, are phosphorylated by other kinases including p90 ribosomal S6 (p90RSK), a downstream mediator of the MEK/ERK signalling pathway which is frequently dysregulated in AML (Wullschleger et al, 2006; Steelman et al, 2008). Consistent with these findings, activation of the MEK/ERK effector network downstream of RAS was recently found to be predictive of poor response to mTOR inhibitors in patients with solid tumours (Di Nicolantonio et al, 2010). From these data, one can speculate that a strategy targeting RAS/MEK/ERK signalling might be useful for enhancing the efficacy of rapalog-based therapeutic approaches in AML.

Although the ORR in this difficult-to-treat patient population is encouraging, particularly when an effective target inhibition is reached, many patients did not respond and most responders eventually progressed on temsirolimus maintenance. Clearly, this may be a reflection of the limited biological activity of rapamycin/rapalogs as these agents are known to inhibit mTORC1 but not, as a general rule, the rapamycin-insensitive mTORC2 functional complex. This differential inhibitory effect can lead to a paradoxical activation of pro-survival signalling pathways upstream of mTOR including those mediated by PI3K and Akt, ultimately resulting in rapamycin resistance (Liu et al, 2009). Whether these preclinical findings are of clinical relevance is still unknown, but much effort is being made to develop a new generation of agents targeting the PI3K/Akt/mTOR network at multiple sites (Meric-Bernstam & Gonzalez-Angulo, 2009). Inhibitors targeting both mTORC1 and mTORC2 (ATP-competitive catalytic site inhibitors), as well as dual catalytic PI3K/mTOR inhibitors, are currently undergoing early phase clinical testing, and it is hoped that they will lead to more optimal targeting of this pathway for AML therapy (Chapuis et al, 2010b; Markman et al, 2010).

In summary, the results from this study show that temsirolimus can be safely combined with lower-dose clofarabine as salvage therapy for older patients with AML, and that the combination has encouraging clinical activity, particularly when associated with a strong on-target effect on mTOR signalling. Further investigation of this novel regimen as upfront therapy in older patients with AML who are considered unsuitable for intensive chemotherapy is planned at GIMEMA.

Acknowledgements

This work was supported in part by research grants from PRIN 2008 to S.A., A.M.M., D.C., and G. Martinelli. We would like to thank Dr Domenico Magro (Pugliese Hospital, Catanzaro) and Dr Giuseppe Fioritoni (Santo Spirito Hospital, Pescara) for contributing patients to the trial. We also thank Pfizer Inc. and Genzyme Europe for providing free investigational drugs (Temsirolimus and Clofarabine, respectively) for the study.

Authorship contributions

S.A. designed the research and coordinated the clinical trial; A.M.M., F.C. and F.R. performed the flow cytometric assays, analysed and interpreted the laboratory data; S.A., R.S., E.A., P.F., and M.V. collected, analysed and interpreted the clinical data; E.L.S. performed statistical analysis; S.A., R.S., and A.M.M. wrote the manuscript; A.V., G. Meloni, F.P., G. Martinelli, M.L., L.P., D.C., E.R., F.D.R., C.F., and L.A. contributed patients to the trial; and all authors reviewed and approved the manuscript.

Competing Interests

The authors declare no competing interests.

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