A randomised comparison of CPX-351 and FLAG-Ida in adverse karyotype AML and high-risk MDS: the UK NCRI AML19 trial

Liposomal daunorubicin and cytarabine (CPX-351) improves overall survival (OS) compared to 7+3 chemotherapy in older patients with secondary acute myeloid leukaemia (AML); to date there have been no randomized studies in younger patients. The high-risk cohort of the UK NCRI AML19 trial (ISRCTN78449203) compared CPX-351 with FLAG-Ida in younger adults with newly-diagnosed adverse cytogenetic AML or high-risk myelodysplastic syndromes (MDS). 189 patients were randomized (median age 56y). By clinical criteria 49% had de novo AML, 20% secondary AML and 30% high risk MDS. MDS-related cytogenetics were present in 73% of patients, with complex karyotype in 49%. TP53 was the most commonly mutated gene, in 43%. Myelodysplasia-related gene mutations were present in 75 patients (44%). The overall response rate (CR + CRi) after course two was 64% and 76% for CPX-351 and FLAG-Ida (OR:0.54, 95%CI 0.28-1.04, p=0.06). There was no difference in OS (13.3 months vs 11.4 months, HR:0.78, 95%CI 0.55-1.12, p=0.17) or event-free survival (HR:0.90, 95%CI 0.64-1.27, p=0.55) in multivariable analyses. However, relapse-free survival was significantly longer with CPX-351 (median 22.1 vs 8.35 months, HR:0.58, 95% CI 0.36-0.95, p=0.03). There was no difference between the treatment arms in patients with clinically defined secondary AML (HR:1.1, 95%CI 0.52-2.30) or those with MDS-related cytogenetic abnormalities (HR:0.94, 95%CI 0.63-1.40), however an exploratory sub-group of patients with MDS-related gene mutations had significantly longer OS with CPX-351 (median 38.4 vs 16.3 months, HR:0.42, 95%CI 0.21-0.84, heterogeneity p=0.05). In conclusion, OS in younger patients with adverse risk AML/MDS was not significantly different between CPX-351 and FLAG-Ida

The biochemical genetics of sulphate assimilation in barley (Hordeum vulgare)

The sulphate assimilation pathway was investigated in barley (Hordeum vulgare). The enzymes ATP sulphurylase, thiosulphate reductase, sulphite reductase, serine acetyltransferase and cysteine synthase proposed as steps in the pathway of higher plants, have been detected in barley seedling leaves. Cysteine synthase was purified ca 400 fold from extract of 7 day old barley seedling leaves to give an activity of 52 μmols cysteine produced/min/mg protein. Differential centrifugation of mechanically ruptured protoplasts showed cysteine synthase was likely to be predominantly or exclusively located within the chloroplast. Specific staining for cysteine synthase activity of non-denaturing polyacrylamide gels suggest the presence of a single form of the enzyme in barley. Development of cysteine synthase activity in barley seedlings was shown not to be dependent on light but was influenced by nutritional status. Selenate, which was shown to be toxic to barley seedlings, was used as the positive selection agent in a screen designed to isolate whole plant mutants defective in sulphate assimilation. Over 70,000 M₂ seedlings (mutagenised in the Mi with 2mM sodium azide) were screened for resistance to selenate. Nine M₂ seedlings were selected as most likely to be defective in sulphate assimilation. M₃ progeny were obtained from four of the M₂ selections. The M₃ progeny showed no resistance to selenate and specific activity staining of non-denaturing polyacrylamide gels showed the presence of functional ATP sulphurylase, thiosulphate reductase, sulphite reductase and cysteine synthase. These results suggest it is unlikely that these M₂ selections carry a heritable mutation in sulphate assimilation

Molecular, clinical and therapeutic determinants of outcome in NPM1 mutated AML

Although NPM1-mutated acute myeloid leukemia (AML) carries a generally favorable prognosis, many patients still relapse and die. Previous studies identified several molecular and clinical features associated with poor outcome, however only FLT3-ITD mutation and adverse karyotype are currently used for risk stratification due to inconsistent results and uncertainty around how other factors should influence treatment, particularly given the strong prognostic impact of post-induction measurable residual disease (MRD). Here we analyzed a large group of patients with NPM1mut AML enrolled in prospective trials (NCRI AML17 and AML19, n=1357) to delineate the impact of baseline molecular and clinical features, post induction MRD status and treatment intensity on outcome. FLT3-ITD (HR 1.28, 95%CI 1.01-1.63), DNMT3A (HR 1.65, 95%CI 1.32-2.05), WT1 (HR 1.74, 95%CI 1272-2.38) and non-ABD NPM1 mutations (HR 1.64, 95%CI 1.22-2.21) were independently associated with poorer overall survival (OS). These factors were also strongly associated with MRD positivity. For patients achieving MRD negativity, these mutations (except FLT3-ITD) were associated with an increased cumulative incidence of relapse (CIR) and poorer OS. However, apart from the few patients with adverse cytogenetics, we could not identify any group of MRD negative patients with a CIR >40% or with benefit from allograft in first remission. Intensified chemotherapy with the FLAG-Ida regimen was associated with improved outcomes in all subgroups, with greater benefits observed in the highest risk molecular subgroups

Overlapping features of therapy-related and de novo NPM1-mutated AML

NPM1-mutated AML represents a WHO leukemia entity with unique pathological and clinical features. Little is known about the characteristics of "therapy-related" NPM1-mutated AML. We compared the genetics, transcriptional profile and clinical outcome of therapy-related NPM1-mutated AML (t-NPM1 AML), de-novo NPM1-mutated AML (dn-NPM1 AML) and therapy-related AML with wild-type NPM1 (t-AML). A normal karyotype was more frequent in t-NPM1 AML (n=78/96 cases, 88%) and dn-NPM1 (n=1986/2394,88%) than in t-AML (n=103/390,28%; p 0.1), but more frequently than t-AML (n=162; 14% and 10%; p-values <0.001). TP53 and PPM1D, typically mutated in t-AML, were consistently wild-type in t-NPM1 AML (97% and 96%). t-NPM1 and dn-NPM1 AML were transcriptionally similar, displaying upregulation of HOX genes and down-regulation of CD133 and CD34. With a median follow-up of 62 months, 3-year overall survival (OS) for t-NPM1 AML (n=96), dn-NPM1 AML (n=2394) and t-AML (n=390) was 54%, 60% and 31%. In multivariable analysis OS was similar for the two NPM1-mutated groups (HR 0.9, 95%CI 0.65-1.25, p=0.45) but better in t-NPM1 AML than t-AML (HR 1.86, 95%CI 1.30-2.68, p<0.001). Relapse-free survival did not differ between t-NPM1 and dn-NPM1 AML (HR 1.02, 95%CI 0.72-1.467, p=0.90) but was significantly higher in t-NPM1 AML than t-AML (HR 1.77, 95%CI 1.19-2.64, p=0.0045).t-NPM1 and dn-NPM1 AML have similar clinical, genomic and transcriptomic features, suggesting that they should be classified as a single disease entit

Overlapping features of therapy-related and de novoNPM1-mutated AML

: NPM1-mutated AML represents a WHO leukemia entity with unique pathological and clinical features. Little is known about the characteristics of "therapy-related" NPM1-mutated AML. We compared the genetics, transcriptional profile and clinical outcome of therapy-related NPM1-mutated AML (t-NPM1 AML), de-novo NPM1-mutated AML (dn-NPM1 AML) and therapy-related AML with wild-type NPM1 (t-AML). A normal karyotype was more frequent in t-NPM1 AML (n=78/96 cases, 88%) and dn-NPM1 (n=1986/2394,88%) than in t-AML (n=103/390,28%; p 0.1), but more frequently than t-AML (n=162; 14% and 10%; p-values <0.001). TP53 and PPM1D, typically mutated in t-AML, were consistently wild-type in t-NPM1 AML (97% and 96%). t-NPM1 and dn-NPM1 AML were transcriptionally similar, displaying upregulation of HOX genes and down-regulation of CD133 and CD34. With a median follow-up of 62 months, 3-year overall survival (OS) for t-NPM1 AML (n=96), dn-NPM1 AML (n=2394) and t-AML (n=390) was 54%, 60% and 31%. In multivariable analysis OS was similar for the two NPM1-mutated groups (HR 0.9, 95%CI 0.65-1.25, p=0.45) but better in t-NPM1 AML than t-AML (HR 1.86, 95%CI 1.30-2.68, p<0.001). Relapse-free survival did not differ between t-NPM1 and dn-NPM1 AML (HR 1.02, 95%CI 0.72-1.467, p=0.90) but was significantly higher in t-NPM1 AML than t-AML (HR 1.77, 95%CI 1.19-2.64, p=0.0045).t-NPM1 and dn-NPM1 AML have similar clinical, genomic and transcriptomic features, suggesting that they should be classified as a single disease entity

FLT3 inhibitors as MRD-guided salvage treatment for molecular failure in FLT3 mutated AML

Patients with FLT3-mutated AML have a high relapse rate and suboptimal outcomes. Many have co-mutations suitable for measurable residual disease (MRD) monitoring by RT-qPCR and those destined to relapse can be identified by high or rising levels of MRD, called molecular failure. This provides a window for pre-emptive intervention, but there is little evidence to guide treatment. The use of FLT3 inhibitors (FLT3i) appears attractive but their use has not yet been evaluated. We identified 56 patients treated with FLT3i at molecular failure. The FLT3 mutation was an ITD in 52, TKD in 7 and both in 3. Over half of patients had previously received midostaurin. Molecular failure occurred at a median 9.2 months from diagnosis and was treated with gilteritinib (n = 38), quizartinib (n = 7) or sorafenib (n = 11). 60% achieved a molecular response, with 45% reaching MRD negativity. Haematological toxicity was low, and 22 patients were bridged directly to allogeneic transplant with another 6 to donor lymphocyte infusion. 2-year overall survival was 80% (95%CI 69-93) and molecular event-free survival 56% (95%CI 44-72). High-sensitivity next-generation sequencing for FLT3-ITD at molecular failure identified patients more likely to benefit. FLT3i monotherapy for molecular failure is a promising strategy which merits evaluation in prospective studies.unknownNot hel

Molecular MRD status and outcome after transplantation in NPM1 mutated AML: results from the UK NCRI AML17 study.

Relapse remains the most common cause of treatment failure for patients with acute myeloid leukaemia (AML) who undergo allogeneic stem cell transplantation (alloSCT) and carries a grave prognosis. Multiple studies have identified the presence of measurable residual disease (MRD) assessed by flow cytometry (FCM) prior to alloSCT as a strong predictor of relapse, but it is not clear how these findings apply to patients who test positive in molecular MRD assays which have far greater sensitivity. We analysed pre-transplant blood and bone marrow samples by reverse-transcription polymerase chain reaction (RT-qPCR) in 107 patients with NPM1 mutant AML enrolled in the UK National Cancer Research Institute (NCRI) AML17 study. After a median follow-up of 4.9 years, patients with negative, low (<200 copies / 105 ABL in the PB and <1000 copies in the BM) and high levels of MRD had an estimated 2y overall survival (OS) of 83%, 63% and 13% respectively (p<0.0001). Focussing on patients with low level MRD prior to alloSCT, those with FLT3 ITD had significantly poorer outcome (hazard ratio, HR, 6.14, p=0.01). Combining these variables was highly prognostic, dividing patients into two groups with 2y OS of 17% and 82% (HR 13.2, p<0.0001). T-depletion was associated with significantly reduced survival both in the entire cohort (2y OS 56% vs 96%, HR 3.24, p=0.0005) and in MRD positive patients (2y OS 34% vs 100%, HR 3.78, p=0.003) but there was no significant effect of either conditioning regimen or donor source on outcome. Registered at ISRCTN (http://www.isrctn.com/ISRCTN55675535)