Addition of the mammalian target of rapamycin inhibitor, everolimus, to consolidation therapy in acute myeloid leukemia: experience from the UK NCRI AML17 trial.

As part of the UK NCRI AML17 trial, adult patients with acute myeloid leukemia in remission could be randomized to receive the mammalian target of rapamycin inhibitor everolimus, sequentially with post-induction chemotherapy. Three hundred and thirty-nine patients were randomised (2:1) to receive everolimus or not for a maximum of 84 days between chemotherapy courses. The primary endpoint was relapse-free survival. At 5 years there was no difference in relapse-free survival [29% versus 40%; odds ratio 1.19 (0.9-1.59) P=0.2], cumulative incidence of relapse [60% versus 54%: odds ratio 1.12 (0.82-1.52): P=0.5] or overall survival [45% versus 58%: odds ratio 1.3 (0.94-1.81): P=0.11]. The independent Data Monitoring Committee advised study termination after randomization of 339 of the intended 600 patients because of excess mortality in the everolimus arm without any evidence of beneficial disease control. The delivery of the everolimus dose was variable, but there was no evidence of clinical benefit in patients with adequate dose delivery compared with no treatment. This study suggests that the addition of mammalian target of rapamycin inhibition to chemotherapy provides no benefit.

X-linked sideroblastic anemia due to ALAS2 intron 1 enhancer element GATA-binding site mutations.

X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia. In affected males, it is uniformly associated with partial loss-of-function missense mutations in the erythroid-specific heme biosynthesis protein 5-aminolevulinate synthase 2 (ALAS2). Here, we report five families with XLSA owing to mutations in a GATA transcription factor binding site located in a transcriptional enhancer element in intron 1 of the ALAS2 gene. As such, this study defines a new class of mutations that should be evaluated in patients undergoing genetic testing for a suspected diagnosis of XLSA.

Missense SLC25A38 variations play an important role in autosomal recessive inherited sideroblastic anemia.

BACKGROUND: Congenital sideroblastic anemias are rare disorders with several genetic causes; they are characterized by erythroblast mitochondrial iron overload, differ greatly in severity and some occur within a syndrome. The most common cause of non-syndromic, microcytic sideroblastic anemia is a defect in the X-linked 5-aminolevulinate synthase 2 gene but this is not always present. Recently, variations in the gene for the mitochondrial carrier SLC25A38 were reported to cause a non-syndromic, severe type of autosomal-recessive sideroblastic anemia. Further evaluation of the importance of this gene was required to estimate the proportion of patients affected and to gain further insight into the range and types of variations involved. DESIGN AND METHODS: In three European diagnostic laboratories sequence analysis of SLC25A38 was performed on DNA from patients affected by congenital sideroblastic anemia of a non-syndromic nature not caused by variations in the 5-aminolevulinate synthase 2 gene. RESULTS: Eleven patients whose ancestral origins spread across several continents were homozygous or compound heterozygous for ten different SLC25A38 variations causing premature termination of translation (p.Arg117X, p.Tyr109LeufsX43), predicted splicing alteration (c.625G>C; p.Asp209His) or missense substitution (p.Gln56Lys, p.Arg134Cys, p.Ile147Asn, p.Arg187Gln, p.Pro190Arg, p.Gly228Val, p.Arg278Gly). Only three of these variations have been described previously (p.Arg117X, p.Tyr109LeufsX43 and p.Asp209His). All new variants reported here are missense and affect conserved amino acids. Structure modeling suggests that these variants may influence different aspects of transport as described for mutations in other mitochondrial carrier disorders. CONCLUSIONS: Mutations in the SLC25A38 gene cause severe, non-syndromic, microcytic/hypochromic sideroblastic anemia in many populations. Missense mutations are shown to be of importance as are mutations that affect protein production. Further investigation of these mutations should shed light on structure-function relationships in this protein.

Heteroduplex analysis for the three common HFE variants: methodology, reliability and analysis of over 5000 requests for testing.

OBJECTIVE: To describe the analysis of over 5300 patient samples for the HFE genotype. METHODS: Blood samples received from hospitals in England, Wales and Ireland were analysed with a single, multiplex PCR using heteroduplex generators for the C282Y, H63D and S65C variants of the HFE gene. PCR products labelled with fluorescent dyes were analysed by capillary electrophoresis. Genotype frequencies were analysed according to the reasons given for testing. RESULTS: Analysis of 400 samples sent in duplicate revealed one error that was associated with reporting rather than the methodology. Of 5327 samples received, 1122 were for family testing, 2470 for diagnostic testing and in 1735 cases no reason was given. Overall, homozygosity for C282Y was found in 14% of samples received for family testing and in 16% of the remaining samples. Clinical indications such as "liver disease" were of little predictive value for homozygosity for C282Y, but this increased if a raised serum ferritin concentration or transferrin saturation was indicated. When the diagnosis was iron overload, 39% of subjects tested were homozygous for C282Y. Compound heterozygosity (C282Y/H63D) was more frequent than in the general population but the frequency was not further increased in subjects for whom there was a diagnosis of iron overload. The frequencies of heterozygosity for H63D or S65C and homozygosity for H63D were not significantly increased in any group compared with the general population frequency. CONCLUSION: These results demonstrate the reliability of the methodology and confirm the difficulty of identifying genetic haemochromatosis purely on the basis of clinical suspicion that haemochromatosis may be responsible for liver disease, diabetes or arthritis.

Rapid and sensitive detection of internal tandem duplication and activating loop mutations of FLT3.

Mutations of the FLT3 gene, a receptor tyrosine kinase, are the most frequent genetic alteration reported in acute myeloid leukaemia, with internal tandem duplications (ITD) or mutations within the activating loop (AL) reported at a frequency of around 24% and 6%, respectively. ITD mutations have associated with a poor prognosis. In this study we have used polymerase chain reaction (PCR), combined with restriction enzyme digestion for the detection of AL mutations, with the DNA products separated on the Agilent 2100 Bioanalyser using a DNA-500 kit. This analysis enabled the rapid identification of mutations in FLT3, approximate sizing of the ITD, an estimate of the proportion of mutant RNA and in some cases, specific heteroduplex patterns associated with triplet deletions. Our data shows that approximately 16% of the patients examined had an ITD mutation and over 13% had a mutation in the AL including triplet deletions involving codons 835/836 and point mutations in codon D839. Based on the sensitivity and speed of the bioanalyser, we suggest that this method is invaluable and provides an improvement to the current use of agarose gels for the analysis of FLT3 PCR products.

Protein kinase C mediates mutant N-Ras-induced developmental abnormalities in normal human erythroid cells.

RAS mutations are one of the most frequent molecular abnormalities associated with myeloid leukemia and preleukemia, yet there is a poor understanding of how they contribute to the pathogenesis of these conditions. Here, we describe the consequences of ectopic mutant N-Ras (N-Ras*) expression on normal human erythropoiesis. We show that during early (erythropoietin [EPO]-independent) erythropoiesis, N-Ras* promoted the amplification of a phenotypically primitive but functionally defective subpopulation of CD34(+) erythroblasts. N-Ras* also up-regulated the expression of megakaryocyte antigens on human erythroblasts. Although early erythroblasts expressing N-Ras* were able to respond to erythropoietin and generate mature progeny, this occurred with greatly reduced efficiency, probably explaining the poor colony growth characteristics of these cells. We further report that this oncogene promoted the expression and activation of protein kinase C (PKC) and that the effects of N-Ras* on erythropoiesis could be abrogated or attenuated by inhibition of PKC. Similarly, the effects of this oncogene could be partially mimicked by treatment with PKC agonist. Together, these data suggest that expression of N-Ras* is able to subvert the normal developmental cues that regulate erythropoiesis by activating PKC. This gives rise to phenotypic and functional abnormalities commonly observed in preleukemia, suggesting a direct link between RAS mutations and the pathogenesis of preleukemia.