Monica Pallis

The impact of dose escalation and resistance modulation in older patients with acute myeloid leukaemia and high risk myelodysplastic syndrome : the results of the LRF AML14 trial

The acute myeloid leukaemia (AML)14 trial addressed four therapeutic questions in patients predominantly aged over 60 years with AML and High Risk Myelodysplastic Syndrome: (i) Daunorubicin 50 mg/m2 vs. 35 mg/m2; (ii) Cytarabine 200 mg/m2 vs. 400 mg/m2 in two courses of DA induction; (iii) for part of the trial, patients allocated Daunorubicin 35 mg/m2 were also randomized to receive, or not, the multidrug resistance modulator PSC‐833 in a 1:1:1 randomization; and (iv) a total of three versus four courses of treatment. A total of 1273 patients were recruited. The response rate was 62% (complete remission 54%, complete remission without platelet/neutrophil recovery 8%); 5‐year survival was 12%. No benefits were observed in either dose escalation randomization, or from a fourth course of treatment. There was a trend for inferior response in the PSC‐833 arm due to deaths in induction. Multivariable analysis identified cytogenetics, presenting white blood count, age and secondary disease as the main predictors of outcome. Although patients with high Pgp expression and function had worse response and survival, this was not an independent prognostic factor, and was not modified by PSC‐833. In conclusion, these four interventions have not improved outcomes in older patients. New agents need to be explored and novel trial designs are required to maximise prospects of achieving timely progress.

P-glycoprotein in acute myeloid leukaemia: Therapeutic implications of its association with both a multidrug-resistant and an apoptosis-resistant phenotype

P-glycoprotein (Pgp) expression is an independent prognostic factor for response to remission-induction chemotherapy in acute myeloblastic leukaemia, particularly in the elderly. There are several potential agents for modulating Pgp-mediated multi-drug resistance, such as cyclosporin A and PSC833, which are currently being evaluated in clinical trials. An alternative therapeutic strategy is to increase the use of drugs which are unaffected by Pgp. However, in this review, we explain why this may be more difficult than it appears. Evidence from in vitro studies of primary AML blasts supports the commonly held supposition that chemoresistance may be linked to apoptosis-resistance. We have found that Pgp has a drug-independent role in the inhibition of in vitro apoptosis in AML blasts. Modulation of cytokine efflux, signalling lipids and intracellular pH has all been suggested as ways by which Pgp may affect cellular resistance to apoptosis include modulation of cytokine efflux, signalling lipids and intracellular pH; these are discussed in this review. For a chemosensitising agent to be successful, it may be more important for it to enhance apoptosis than to increase drug uptake.

DNA repair contributes to the drug-resistant phenotype of primary acute myeloid leukaemia cells with FLT3 internal tandem duplications and is reversed by the FLT3 inhibitor PKC412

The presence of internal tandem duplications (ITD) mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor influences the risk of relapse in acute myeloid leukaemia (AML). We have investigated DNA repair in FLT3-ITD and wild-type (WT) cells. Using the comet assay, we have demonstrated that the FLT3 inhibitor PKC412 significantly inhibits repair of DNA damage in the MV4-11-FLT3-ITD cell line and FLT3-ITD patient samples but not in the HL-60-FLT3-WT cell line or FLT3-WT patient samples. Following the discovery that transcript levels of the DNA repair gene RAD51 are significantly correlated with FLT3 transcript levels in FLT3-ITD patients, we further investigated the role of RAD51 in FLT3-ITD-AML. The reduction in DNA repair in PKC412-treated FLT3-ITD cells was shown to be associated with downregulation of RAD51 mRNA and protein expression and correlates with the maintenance of phosphorylated H2AX levels, implying that PKC412 inhibits the homologous recombination double-strand break repair pathway in FLT3-ITD cells. Using FLT3-short interfering RNA (siRNA), we also demonstrated that genetic silencing of FLT3 results in RAD51 downregulation in FLT3-ITD cells but not in FLT3-WT cells. This work suggests that the use of FLT3 inhibitors such as PKC412 may reverse the drug-resistant phenotype of FLT3-ITD-AML cells by inhibiting repair of chemotherapy-induced genotoxic damage and thereby reduce the risk of disease relapse.

Analysis of factors that affect in vitro chemosensitivity of leukaemic stem and progenitor cells to gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukaemia.

Relapse in acute myeloid leukaemia (AML) is considered to result from the persistence of drug-resistant leukaemic stem and progenitor cells (LSPC) within a bone marrow ‘niche’ microenvironment. Identifying novel agents that have the potential to target these LSPC in their niche microenvironment will aid in the characterization of candidate agents for post-remission chemotherapy. Using an in vitro model, we found that 48-h culture with gemtuzumab ozogamicin (Mylotarg) resulted in a 34% reduction in CD34+CD38−CD123+ LSPC number, whereas normal CD34+CD38− haemapoietic stem cells were insensitive to this agent. As there was considerable heterogeneity in LSPC response to Mylotarg treatment, various factors potentially underpinning the differential response were assessed. LSPC that overexpressed CD33 (P=0.01), which were P-glycoprotein-negative (P=0.008) and with internal tandem duplication (ITD) of the FLT3 gene (FLT3/ITD) status (P=0.006) responded better to Mylotarg treatment. LSPC from patient samples that have these combined characteristics as well as low LSPC burden showed significantly more chemosensitivity to Mylotarg compared with all other cases (P=0.002). In multivariate analysis, LSPC burden and FLT3 status were found to be predictors of LSPC chemosensitivity to Mylotarg treatment (P<0.0001). In conclusion, we have shown heterogeneity in the LSPC compartment of AML patients underpinning differential in vitro sensitivity to Mylotarg.

Flow cytometric measurement of phosphorylated STAT5 in AML: lack of specific association with FLT3 internal tandem duplications

STAT5 phosphorylation has been noted in 69-95% of AML cases by Western blotting. We used flow cytometry to measure phosphorylated STAT5 on a semi-quantitative scale. The method was validated on K562 cells, which constitutively express phosphorylated STAT5, but lose this when BCR-abl tyrosine kinase activity is blocked by STI571. Phosphorylated STAT5 was found to measure 2.22+/-0.09 relative fluorescence units (RFU) falling to 0.925+/-0.005RFU in the presence of STI571. Phosphorylated STAT5 expression was 0.99 to 2.09RFU in 28 primary AML samples. There was no logical cut-off point between positive and negative fluorescence. FLT3 internal tandem duplications, found in 11/28 samples, were not significantly associated with the level of phosphorylated STAT5 expression. We conclude that STAT5 phosphorylation can be measured sensitively by flow cytometry in AML and that its expression should not be dichotomised as present or absent.

Sequential Influences of Leukemia-Specific and Genetic Factors on P-Glycoprotein Expression in Blasts from 817 Patients Entered into the National Cancer Research Network Acute Myeloid Leukemia 14 and 15 Trials

Purpose: P-glycoprotein (Pgp) is a major prognostic factor for chemotherapy failure in acute myeloid leukemia (AML). This study compared the influence of genetic and leukemia-specific factors on Pgp. Experimental Design: Eight hundred and seventeen samples were studied prospectively for Pgp protein expression and function and G1199A, G2677T, and C3435T polymorphisms in the encoding gene ABCB1. Results: Age, low WBC count, high bcl-2, secondary AML and myelodysplastic syndrome, and adverse cytogenetics all correlated strongly with high Pgp (MRK16) protein expression. However, ABCB1 3435TT homozygosity was negatively correlated with Pgp. Pgp protein is only expressed in 41% of samples such that the negative effect of the polymorphism was not seen at baseline Pgp levels but was marked in the upper 41% of samples (MRK16 Δmean fluorescence intensity of 75th centile sample = 9 units for TT variant samples and 26 units for CC/CT; P = 0.003). However, no association was found between genetic factors and Pgp function using rhodamine 123 accumulation. Conclusions: The genetic polymorphism 3435TT (which results in unstable mRNA) has a significant effect on Pgp expression, but this is only seen in ∼40% of cases in which mRNA and protein are detectable. Moreover, leukemia-specific factors, such as low WBC count and poor risk cytogenetics, have a much greater effect than genetic polymorphisms on Pgp expression in AML blasts.

Use of standardized flow cytometric determinants of multidrug resistance to analyse response to remission induction chemotherapy in patients with acute myeloblastic leukaemia

We have used a combination of flow cytometric assays to define multidrug resistance (MDR) positive and negative blasts in cryopreserved samples from 47 MRC trial patients with acute myeloblastic leukaemia (AML). Our primary test is a standardized assay for daunorubicin accumulation. Confirmatory assays for MDR comprised the cyclosporin modulation assay for rhodamine‐123 uptake as a measure of functional P‐glycoprotein and the measurement of lung resistance protein and multidrug resistance associated protein (with LRP‐56 and MRPr1 respectively).

Clonal haemopoiesis may occur after conventional chemotherapy and is associated with accelerated telomere shortening and defects in the NQO1 pathway; possible mechanisms leading to an increased risk of t-AML/MDS.

The molecular pathogenesis of therapy‐related acute myeloid leukaemia/myelodysplastic syndrome (t‐AML/MDS) remains uncertain. However, clonal haemopoiesis may develop following stem cell transplantation and precede the development of t‐AML/MDS. Moreover, accelerated telomere shortening may be induced by replicative stress or oxidative damage, leading to genomic instability, and inactivating polymorphisms of the gene encoding NADPH‐quinone oxidoreductase (NQO1) are more frequently observed in patients with t‐AML. We studied clonal haemopoiesis, telomere length and NQO1 status in 146 patients receiving conventional chemotherapy for non‐myeloid malignancies. Clonal haemopoiesis was demonstrated in eight of 98 (8%) patients. Telomere length was reduced in patients following chemotherapy (n = 52) compared with controls (n = 42) (P < 0·001), particularly in those with clonal haemopoiesis (P < 0·002). Whilst there was a trend towards telomere shortening in control subjects polymorphic for NQO1‐187Ser (n = 12), chemotherapy‐exposed patients polymorphic for the NQO1‐187Ser allele (n = 29) had significantly shorter telomeres (P < 0·001). Furthermore, chemotherapy‐treated patients with the NQO1‐187Ser, polymorphism were more likely to develop clonal haemopoiesis than patients with wild type NQO1 (odds ratio = 7; 1·16–42·6). We conclude that a switch to clonal haemopoiesis may occur after conventional chemotherapy and lead to accelerated telomere shortening. Patients with the NQO1‐187Ser polymorphism have an increased risk of developing both clonal haemopoiesis and telomere shortening, which may partly explain the predisposition to t‐AML in NQO1‐187Ser null individuals.

Resistance to FLT3 inhibition in an in vitro model of primary AML cells with a stem cell phenotype in a defined microenvironment

Relapse in acute myeloid leukaemia (AML) is mediated by survival of leukaemic stem cells following remission-induction chemotherapy. It would therefore be useful to identify therapeutic agents that target leukaemic stem cells. We devised a flow cytometric chemosensitivity assay allowing 48 h culture of leukaemic blasts in a defined microenvironment followed by enumeration of viable CD34+CD38−CD123+ leukaemic stem and progenitor cells (LSPC). The assay was used to investigate the LSPC response to cytosine arabinoside (Ara-C) and to the FLT3 inhibitor AG1296. There was a 3.6-fold increase in Ara-C-treated LSPC survival under defined ‘niche-like’ conditions compared to culture without microenvironmental support. Nine AML samples with internal tandem duplications of FLT3 (FLT3/ITDs) were treated with AG1296. Three samples were very sensitive (>50% kill) and 4 were moderately sensitive (10–50% kill) in bulk suspension culture without microenvironmental support. However, under defined ‘niche-like’ conditions, the survival of LSPC was enhanced rather than inhibited by AG1296 treatment. We conclude that an interaction between LSPC and a defined in vitro microenvironment models a chemoresistant niche. Our data point to a need to investigate more novel chemotherapeutic agents under these stringent conditions to identify agents that may be suitable to target minimal residual disease in AML.

The expression of P‐glycoprotein in AML cells with FLT3 internal tandem duplications is associated with reduced apoptosis in response to FLT3 inhibitors

P‐glycoprotein (pgp), a membrane efflux pump, is recognized to have an anti‐apoptotic function. Internal tandem duplications (ITDs) of the Fms‐like tyrosine kinase 3 (FLT3) receptor are the most common mutations in acute myeloid leukaemia (AML). Both ITDs and pgp positivity confer an adverse clinical prognosis. FLT3 inhibitors induce variable apoptosis in cell lines transfected with FLT3 ITDs. We studied the effect of herbimycin A, AG1296 and PKC412 on primary AML blasts. All compounds showed significantly higher cell kill after 48‐h incubation in samples with an ITD compared with wild type (Herbimicin P < 0·001; AG1296 P = 0·001, PKC412, P = 0·002). Pgp‐positive samples were significantly less sensitive to herbimycin and AG1296 than pgp‐negative samples, although neither molecule inhibited the efflux function of pgp. The concurrent incubation with the pgp inhibitor PSC833 resulted in an enhanced cell kill in 4/5 ITD pgp‐positive samples versus two of nine ITD pgp‐negative samples. PKC412 inhibited pgp function and induced cell death in FLT3 ITD/pgp‐positive samples. We conclude that AML samples with a FLT3 ITD are more susceptible to these inhibitors than wild‐type samples. However, the expression of pgp in cells with FLT3 ITDs can reduce their sensitivity to FLT3 inhibitors and therefore pgp expression should be assessed in clinical trials of FLT3 inhibitors.