To determine the association of RUNX1 mutations with therapeutic outcome in younger and older patients with primary cytogenetically normal acute myeloid leukemia (CN-AML) and with gene/microRNA expression signatures.
Patients and Methods
Younger (< 60 years; n = 175) and older (≥ 60 years; n = 225) patients with CN-AML treated with intensive cytarabine/anthracycline-based first-line therapy on Cancer and Leukemia Group B protocols were centrally analyzed for RUNX1 mutations by polymerase chain reaction and direct sequencing and for established prognostic gene mutations. Gene/microRNA expression profiles were derived using microarrays.
RUNX1 mutations were found in 8% and 16% of younger and older patients, respectively (P = .02). They were associated with ASXL1 mutations (P < .001) and inversely associated with NPM1 (P < .001) and CEBPA (P = .06) mutations. RUNX1-mutated patients had lower complete remission rates (P = .005 in younger; P = .006 in older) and shorter disease-free survival (P = .058 in younger; P < .001 in older), overall survival (P = .003 in younger; P < .001 in older), and event-free survival (P < .001 for younger and older) than RUNX1 wild-type patients. Because RUNX1 mutations were more common in older patients and almost never coexisted with NPM1 mutations, RUNX1 mutation–associated expression signatures were derived in older, NPM1 wild-type patients and featured upregulation of genes normally expressed in primitive hematopoietic cells and B-cell progenitors, including DNTT, BAALC, BLNK, CD109, RBPMS, and FLT3, and downregulation of promoters of myelopoiesis, including CEBPA and miR-223.
RUNX1 mutations are twice as common in older than younger patients with CN-AML and negatively impact outcome in both age groups. RUNX1-mutated blasts have molecular features of primitive hematopoietic and lymphoid progenitors, potentially leading to novel therapeutic approaches.
To determine the frequency of DNMT3A mutations, their associations with clinical and molecular characteristics and outcome, and the associated gene- and microRNA-expression signatures in primary cytogenetically normal acute myeloid leukemia (CN-AML).
Patients and Methods
Four hundred fifteen previously untreated adults were analyzed for DNMT3A mutations and established prognostic gene mutations and expression markers. Gene- and microRNA-expression profiles were derived using microarrays.
Younger (< 60 years; n = 181) and older (≥ 60 years; n = 234) patients had similar frequencies of DNMT3A mutations (35.3% v 33.3%). Missense mutations affecting arginine codon 882 (R882-DNMT3A) were more common (n = 92; 62%) than those affecting other codons (non–R882-DNMT3A). DNMT3A-mutated patients did not differ regarding complete remission rate, but had shorter disease-free survival (DFS; P = .03) and, by trend, overall survival (OS; P = .07) than DNMT3A–wild-type patients. In multivariable analyses, DNMT3A mutations remained associated with shorter DFS (P = .01), but not with shorter OS. When analyzed separately, the two DNMT3A mutation types had different significance by age group. Younger patients with non–R882-DNMT3A mutations had shorter DFS (P = .002) and OS (P = .02), whereas older patients with R882-DNMT3A mutations had shorter DFS (P = .005) and OS (P = .002) after adjustment for other clinical and molecular prognosticators. Gene- and microRNA-expression signatures did not accurately predict DNMT3A mutational status.
DNMT3A mutations are frequent in CN-AML, and their clinical significance seems to be age dependent. DNMT3A-R882 mutations are associated with adverse prognosis in older patients, and non–R882-DNMT3A mutations are associated with adverse prognosis in younger patients. Low accuracy of gene- and microRNA-expression signatures in predicting DNMT3A mutation status suggested that the role of these mutations in AML remains to be elucidated.
Resynthesized (Resyn) Brassica napus L. can be used to broaden the genetic diversity and to develop a heterotic genepool for rapeseed hybrid breeding. Domesticated vegetable types are usually employed as B. oleracea parents. We sought to evaluate the potential of wild species as parents for Resyn lines. Fifteen Resyn lines were derived by crossing wild B. oleracea ssp. oleracea and oilseed B. rapa, and 29 Resyn lines were generated from 10 wild Brassica species (B. bourgaei, B. cretica, B. incana, B. insularis, B. hilarionis, B. macrocarpa, B. montana, B. rupestris, B. taurica, B. villosa). Genetic distances were analyzed with AFLP markers for 71 Resyn lines from wild and domesticated B. oleracea, and compared with 55 winter, spring, vegetable, and Asian B. napus genotypes. The genetic distances clearly showed that Resyn lines with wild species provide a genetic diversity absent from the breeding material or Resyn lines from domesticated species. Forty-two Resyn lines were crossed with one or two winter oilseed rape testers, resulting in 64 hybrids that were grown in one year and four locations in Germany and France. The correlation between hybrid yield and genetic distance was slightly negative (r = −0.29). Most of the hybrids with Resyn lines from wild B. oleracea were lower in yield than hybrids with Resyn lines from domesticated B. oleracea. It is promising that Resyn lines descending from unselected wild B. oleracea accessions produced high-yielding hybrids when crossed with adapted genotypes: these Resyn lines would be suited to develop heterotic pools in hybrid breeding.
Electronic supplementary material
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Acute myeloid leukemia (AML) is the result of a multistep transforming process of hematopoietic precursor cells (HPCs) which enables them to proceed through limitless numbers of cell cycles and to become resistant to cell death. Increased proliferation renders these cells vulnerable to acquiring mutations and may favor leukemic transformation. Here, we review how deregulated cell cycle control contributes to increased proliferation in AML and favors genomic instability, a prerequisite to confer selective advantages to particular clones in order to adapt and independently proliferate in the presence of a changing microenvironment. We discuss the connection between differentiation and proliferation with regard to leukemogenesis and outline the impact of specific alterations on response to therapy. Finally, we present examples, how a better understanding of cell cycle regulation and deregulation has already led to new promising therapeutic strategies.
Acute myeloid leukemia (AML); cell cycle; genetic instability; proliferation; differentiation
To determine the frequency of TET2 mutations, their associations with clinical and molecular characteristics and outcome, and the associated gene- and microRNA-expression signatures in patients with primary cytogenetically normal acute myeloid leukemia (CN-AML).
Patients and Methods
Four-hundred twenty-seven patients with CN-AML were analyzed for TET2 mutations by polymerase chain reaction and direct sequencing and for established prognostic gene mutations. Gene- and microRNA-expression profiles were derived using microarrays.
TET2 mutations, found in 23% of patients, were associated with older age (P < .001) and higher pretreatment WBC (P = .04) compared with wild-type TET2 (TET2-wt). In the European LeukemiaNet (ELN) favorable-risk group (patients with CN-AML who have mutated CEBPA and/or mutated NPM1 without FLT3 internal tandem duplication [FLT3-ITD]), TET2-mutated patients had shorter event-free survival (EFS; P < .001) because of a lower complete remission (CR) rate (P = .007), and shorter disease-free survival (DFS; P = .003), and also had shorter overall survival (P = .001) compared with TET2-wt patients. TET2 mutations were not associated with outcomes in the ELN intermediate-I–risk group (CN-AML with wild-type CEBPA and wild-type NPM1 and/or FLT3-ITD). In multivariable models, TET2 mutations were associated with shorter EFS (P = .004), lower CR rate (P = .03), and shorter DFS (P = .05) only among favorable-risk CN-AML patients. We identified a TET2 mutation-associated gene-expression signature in favorable-risk but not in intermediate-I–risk patients and found distinct mutation-associated microRNA signatures in both ELN groups.
TET2 mutations improve the ELN molecular-risk classification in primary CN-AML because of their adverse prognostic impact in an otherwise favorable-risk patient subset. Our data suggest that these patients may be candidates for alternative therapies.
To evaluate the prognostic significance of expression levels of a single microRNA, miR-181a, in the context of established molecular markers in cytogenetically normal acute myeloid leukemia (CN-AML), and to gain insight into the leukemogenic role of miR-181a.
Patients and Methods
miR-181a expression was measured in pretreatment marrow using Ohio State University Comprehensive Cancer Center version 3.0 arrays in 187 younger (< 60 years) adults with CN-AML. Presence of other molecular prognosticators was assessed centrally. A gene-expression profile associated with miR-181a expression was derived using microarrays and evaluated by Gene-Ontology analysis.
Higher miR-181a expression associated with a higher complete remission (CR) rate (P = .04), longer overall survival (OS; P = .01) and a trend for longer disease-free survival (DFS; P = .09). The impact of miR-181a was most striking in poor molecular risk patients with FLT3-internal tandem duplication (FLT3-ITD) and/or NPM1 wild-type, where higher miR-181a expression associated with a higher CR rate (P = .009), and longer DFS (P < .001) and OS (P < .001). In multivariable analyses, higher miR-181a expression was significantly associated with better outcome, both in the whole patient cohort and in patients with FLT3-ITD and/or NPM1 wild-type. These results were also validated in an independent set of older (≥ 60 years) patients with CN-AML. A miR-181a-associated gene-expression profile was characterized by enrichment of genes usually involved in innate immunity.
To our knowledge, we provide the first evidence that the expression of a single microRNA, miR-181a, is associated with clinical outcome of patients with CN-AML and may refine their molecular risk classification. Targeted treatments that increase endogenous levels of miR-181a might represent novel therapeutic strategies.
Hybrid breeding relies on the combination of parents from two differing heterotic groups. However, the genetic diversity in adapted oilseed rape breeding material is rather limited. Therefore, the use of resynthesized Brassica napus as a distant gene pool was investigated. Hybrids were derived from crosses between 44 resynthesized lines with a diverse genetic background and two male sterile winter oilseed rape tester lines. The hybrids were evaluated together with their parents and check cultivars in 2 years and five locations in Germany. Yield, plant height, seed oil, and protein content were monitored, and genetic distances were estimated with molecular markers (127 polymorphic RFLP fragments). Resynthesized lines varied in yield between 40.9 dt/ha and 21.5 dt/ha, or between 85.1 and 44.6% of check cultivar yields. Relative to check cultivars, hybrids varied from 91.6 to 116.6% in yield and from 94.5 to 103.3% in seed oil content. Mid-parent heterosis varied from −3.5 to 47.2% for yield. The genetic distance of parental lines was not significantly correlated with heterosis or hybrid yield. Although resynthesized lines do not meet the elite rapeseed standards, they are a valuable source for hybrid breeding due to their large distance from present breeding material and their high heterosis when combined with European winter oilseed rape.
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Lenalidomide is effective in myeloma and low-risk myelodysplastic syndromes with deletion 5q. We report results of a phase I dose-escalation trial of lenalidomide in relapsed or refractory acute leukemia.
Patients and Methods
Thirty-one adults with acute myeloid leukemia (AML) and four adults with acute lymphoblastic leukemia (ALL) were enrolled. Lenalidomide was given orally at escalating doses of 25 to 75 mg daily on days 1 through 21 of 28-day cycles to determine the dose-limiting toxicity (DLT) and maximum-tolerated dose (MTD), as well as to provide pharmacokinetic and preliminary efficacy data.
Patients had a median age of 63 years (range, 22 to 79 years) and a median of two prior therapies (range, one to four therapies). The DLT was fatigue; 50 mg/d was the MTD. Infectious complications were frequent. Plasma lenalidomide concentration increased proportionally with dose. In AML, five (16%) of 31 patients achieved complete remission (CR); three of three patients with cytogenetic abnormalities achieved cytogenetic CR (none with deletion 5q). Response duration ranged from 5.6 to 14 months. All responses occurred in AML with low presenting WBC count. No patient with ALL responded. Two of four patients who received lenalidomide as initial therapy for AML relapse after allogeneic transplantation achieved durable CR after development of cutaneous graft-versus-host disease, without donor leukocyte infusion.
Lenalidomide was safely escalated to 50 mg daily for 21 days, every 4 weeks, and was active with relatively low toxicity in patients with relapsed/refractory AML. Remissions achieved after transplantation suggest a possible immunomodulatory effect of lenalidomide, and results provide enthusiasm for further studies in AML, either alone or in combination with conventional agents or other immunotherapies.
Because of its high growth rate at low temperatures in early spring, there is renewed interest in Brassica rapa as a winter crop for biomass production in Europe. The available cultivars are not developed for this purpose however. An approach for breeding bioenergy cultivars of B. rapa could be to establish populations from two or more different cultivars with high combining ability. The objective of this study was to evaluate the heterosis for biomass yield in the European winter B. rapa genepool. The genetic variation and heterosis of the biomass parameters: dry matter content, fresh and dry biomass yields were investigated in three cultivars representing different eras of breeding by comparing full-sibs-within and full-sibs-between the cultivars. Field trials were performed at two locations in Germany in 2005–2006. Mean mid-parent heterosis was low with 2.5% in fresh and 3.0% in dry biomass yield in full-sibs-between cultivars. Mean values of individual crosses revealed a higher variation in mid-parent heterosis ranging from 14.6% to −7.5% in fresh biomass yield and from 19.7% to −12.7% in dry biomass yield. The low heterosis observed in hybrids between European winter cultivars can be explained by the low genetic variation between these cultivars as shown earlier with molecular markers. In conclusion, a B. rapa breeding program for biomass production in Europe should not only use European genetic resources, but should also utilize the much wider worldwide variation in this species.
Genetic variation; Heterosis
Near-infrared reflectance spectroscopy (NIRS) of intact seeds allows the non-destructive estimation of seed quality parameters which is highly desirable in plant breeding. Together with yield, oil content and quality, a main aim in safflower (Carthamus tinctorius L.) breeding is the selection of genotypes with a low percentage of empty seeds even under cooler climates. We developed NIRS calibrations for seed oil content, oleic and linoleic acid content, the seed hull fraction and the percentage of empty seeds using seed meal and intact seeds. For the different calibrations 108–534 samples from a safflower breeding program with lines adapted to German conditions, were analyzed with reference analyses (Soxhlet, gas chromatography), and scanned by NIRS as intact seeds and seed meal. Calibration equations were developed and tested through cross validation. The coefficient of determination of the calibration (R2) for intact seeds ranged from 0.91(oil content), 0.90 (seed hull fraction), 0.84 (empty seeds), 0.73 (linoleic acid) to 0.68 (oleic acid). The coefficient of determination of the cross validation was higher for seed meal than for intact seeds except for the parameter seed hull fraction. The results show that NIRS calibrations are applicable in safflower breeding programs for a fast screening.
Safflower breeding; Carthamus tinctorius; Near-infrared reflectance spectroscopy (NIRS); Seed oil; Oleic acid; Linoleic acid; Seed hull
To analyze the frequency and associations with prognostic markers and outcome of mutations in IDH genes encoding isocitrate dehydrogenases in adult de novo cytogenetically normal acute myeloid leukemia (CN-AML).
Patients and Methods
Diagnostic bone marrow or blood samples from 358 patients were analyzed for IDH1 and IDH2 mutations by DNA polymerase chain reaction amplification/sequencing. FLT3, NPM1, CEBPA, WT1, and MLL mutational analyses and gene- and microRNA-expression profiling were performed centrally.
IDH mutations were found in 33% of the patients. IDH1 mutations were detected in 49 patients (14%; 47 with R132). IDH2 mutations, previously unreported in AML, were detected in 69 patients (19%; 13 with R172 and 56 with R140). R172 IDH2 mutations were mutually exclusive with all other prognostic mutations analyzed. Younger age (< 60 years), molecular low-risk (NPM1-mutated/FLT3-internal tandem duplication–negative) IDH1-mutated patients had shorter disease-free survival than molecular low-risk IDH1/IDH2-wild-type (wt) patients (P = .046). R172 IDH2-mutated patients had lower complete remission rates than IDH1/IDH2wt patients (P = .007). Distinctive microarray gene- and microRNA-expression profiles accurately predicted R172 IDH2 mutations. The highest expressed gene and microRNAs in R172 IDH2-mutated patients compared with the IDH1/IDH2wt patients were APP (previously associated with complex karyotype AML) and miR-1 and miR-133 (involved in embryonal stem-cell differentiation), respectively.
IDH1 and IDH2 mutations are recurrent in CN-AML and have an unfavorable impact on outcome. The R172 IDH2 mutations, previously unreported in AML, characterize a novel subset of CN-AML patients lacking other prognostic mutations and associate with unique gene- and microRNA-expression profiles that may lead to the discovery of novel, therapeutically targetable leukemogenic mechanisms.
The biologic and clinical significance of KIT overexpression that associates with KIT gain-of- function mutations occurring in subsets of acute myeloid leukemia (AML) (i.e., core binding factor AML) is unknown. Here, we show that KIT mutations lead to MYC-dependent miR-29b repression and increased levels of the miR-29b target Sp1 in KIT-driven leukemia. Sp1 enhances its own expression by participating in a NFκB/HDAC complex that further represses miR-29b transcription. Upregulated Sp1 then binds NFκB and transactivates KIT. Therefore, activated KIT ultimately induces its own transcription. Our results provide evidence that the mechanisms of Sp1/NFκB/HDAC/miR-29b-dependent KIT overexpression contribute to leukemia growth and can be successfully targeted by pharmacological disruption of the Sp1/NFκB/HDAC complex or synthetic miR-29b treatment in KIT-driven AML.
To analyze the prognostic significance of NPM1 mutations, and the associated gene- and microRNA-expression signatures in older patients with de novo, cytogenetically normal acute myeloid leukemia (CN-AML) treated with intensive chemotherapy.
Patients and Methods
One hundred forty-eight adults age ≥ 60 years with de novo CN-AML, enrolled onto Cancer and Leukemia Group B protocols 9720 and 10201, were studied at diagnosis for NPM1, FLT3, CEBPA, and WT1 mutations, and gene- and microRNA-expression profiles.
Patients with NPM1 mutations (56%) had higher complete remission (CR) rates (84% v 48%; P < .001) and longer disease-free survival (DFS; P = .047; 3-year rates, 23% v 10%) and overall survival (OS; P < .001; 3-year rates, 35% v 8%) than NPM1 wild-type patients. In multivariable analyses, NPM1 mutations remained independent predictors for higher CR rates (P < .001) and longer DFS (P = .004) and OS (P < .001), after adjustment for other prognostic clinical and molecular variables. Unexpectedly, the prognostic impact of NPM1 mutations was mainly observed in patients ≥ 70 years. Gene- and microRNA-expression profiles associated with NPM1 mutations were similar across older patient age groups and similar to those in younger (< 60 years) patients with CN-AML. These profiles were characterized by upregulation of HOX genes and their embedded microRNAs and downregulation of the prognostically adverse MN1, BAALC, and ERG genes.
NPM1 mutations have favorable prognostic impact in older patients with CN-AML, especially those age ≥ 70 years. The gene- and microRNA-expression profiles suggest that NPM1 mutations constitute a marker defining a biologically homogeneous entity in CN-AML that might be treated with specific and/or targeted therapies across age groups.
MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA’s seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins.
Functional loss of the tumor suppressor Smad4 is involved in pancreatic and colorectal carcinogenesis and has been associated with the acquisition of invasiveness. We have previously demonstrated that the heterotrimeric basement membrane protein laminin-332 is a Smad4 target. Namely, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-332; its loss is thus implicated in the reduced or discontinuous deposition of the heterotrimeric basement membrane molecule as evident in carcinomas. Uncoupled expression of laminin genes, on the other hand, namely overexpression of the laminin-γ2 chain is an impressive marker at invasive edges of carcinomas where tumor cells are maximally exposed to signals from stromal cell types like macrophages. As Smad4 is characterized as an integrator of multiple extracellular stimuli in a strongly contextual manner, we asked if loss of Smad4 may also be involved in uncoupled expression of laminin genes in response to altered environmental stimuli. Here, we address Smad4 dependent effects of the prominent inflammatory cytokine TNFα on tumor cells.
Smad4-reconstituted colon carcinoma cells like adenoma cells respond to TNFα with an increased expression of all three chains encoding laminin-332; coincubation with TGFβ and TNFα leads to synergistic induction and to the secretion of large amounts of the heterotrimer. In contrast, in Smad4-deficient cells TNFα can induce expression of the γ2 and β3 but not the α3 chain. Surprisingly, this uncoupled induction of laminin-332 chains in Smad4-negative cells rather than causing intracellular accumulation is followed by the release of γ2 into the medium, either in a monomeric form or in complexes with as yet unknown proteins. Soluble γ2 is associated with increased cell migration.
Loss of Smad4 may lead to uncoupled induction of laminin-γ2 in response to TNFα and may therefore represent one of the mechanisms which underlie accumulation of laminin-γ2 at the invasive margin of a tumor. The finding, that γ2 is secreted from tumor cells in significant amounts and is associated with increased cell migration may pave the way for further investigation to better understand its functional relevance for tumor progression.
Linkage disequilibrium was investigated in canola quality winter rapeseed to analyze (1) the prospects for whole-genome association analyses and (2) the impact of the recent breeding history of rapeseed on linkage disequilibrium. A total of 845 mapped AFLP markers with allele frequencies ≥0.1 were used for the analysis of linkage disequilibrium in a population of 85 canola quality winter rapeseed genotypes. A low overall level of linkage disequilibrium was found with a mean r2 of only 0.027 over all 356,590 possible marker pairs. At a significance threshold of P = 2.8 × 10−7, which was derived by a Bonferroni correction from a global α-level of 0.1, only 0.78% of the marker pairs were in significant linkage disequilibrium. Among physically linked marker pairs, the level of linkage disequilibrium was about five times higher with more than 10% of marker pairs in significant linkage disequilibrium. Linkage disequilibrium decayed rapidly with distance between linked markers with high levels of linkage disequilibrium extending only for about 2 cM. Owing to the rapid decay of linkage disequilibrium with distance association analyses in canola quality rapeseed will have a significantly higher resolution than QTL analyses in segregating populations by interval mapping, but much larger number of markers will be necessary to cover the whole genome. A major impact of the recent breeding history of rapeseed on linkage disequilibrium could not be observed.
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Improving oil and protein quality for food and feed purposes is an important goal in rapeseed (Brassica napus L.) breeding programs. Rapeseed contains phytosterols, used to enrich food products, and sinapate esters, which are limiting the utilization of rapeseed proteins in the feed industry. Increasing the phytosterol content of oil and lowering sinapate ester content of meal could increase the value of the oilseed rape crop. The objective of the present study was to identify quantitative trait loci (QTL) for phytosterol and sinapate ester content in a winter rapeseed population of 148 doubled haploid lines, previously found to have a large variation for these two traits. This population also segregated for the two erucic acid genes. A close negative correlation was found between erucic acid and phytosterol content (Spearman’s rank correlation, rs = −0.80**). For total phytosterol content, three QTL were detected, explaining 60% of the genetic variance. The two QTL with the strongest additive effects were mapped on linkage groups N8 and N13 within the confidence intervals of the two erucic acid genes. For sinapate ester content four QTL were detected, explaining 53% of the genetic variance. Again, a close negative correlation was found between erucic acid and sinapate ester content (rs = −0.66**) and the QTL with the strongest additive effects mapped on linkage groups N8 and N13 within the confidence intervals of the two erucic acid genes. The results suggests, that there is a pleiotropic effect of the two erucic acid genes on phytosterol and sinapate ester content; the effect of the alleles for low erucic acid content is to increase phytosterol and sinapate ester content. Possible reasons for this are discussed based on known biosynthetic pathways.
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