TP53 mutation status divides myelodysplastic syndromes with complex karyotypes into distinct prognostic subgroups.

Risk stratification is critical in the care of patients with myelodysplastic syndromes (MDS). Approximately 10% have a complex karyotype (CK), defined as more than two cytogenetic abnormalities, which is a highly adverse prognostic marker. However, CK-MDS can carry a wide range of chromosomal abnormalities and somatic mutations. To refine risk stratification of CK-MDS patients, we examined data from 359 CK-MDS patients shared by the International Working Group for MDS. Mutations were underrepresented with the exception of TP53 mutations, identified in 55% of patients. TP53 mutated patients had even fewer co-mutated genes but were enriched for the del(5q) chromosomal abnormality (p < 0.005), monosomal karyotype (p < 0.001), and high complexity, defined as more than 4 cytogenetic abnormalities (p < 0.001). Monosomal karyotype, high complexity, and TP53 mutation were individually associated with shorter overall survival, but monosomal status was not significant in a multivariable model. Multivariable survival modeling identified severe anemia (hemoglobin < 8.0 g/dL), NRAS mutation, SF3B1 mutation, TP53 mutation, elevated blast percentage (>10%), abnormal 3q, abnormal 9, and monosomy 7 as having the greatest survival risk. The poor risk associated with CK-MDS is driven by its association with prognostically adverse TP53 mutations and can be refined by considering clinical and karyotype features.

Impact of socioeconomic status on disease phenotype, genomic landscape and outcomes in myelodysplastic syndromes.

Genetic and epigenetic alterations contribute to the biological and clinical characteristics of myelodysplastic syndromes (MDS), but a role for socioeconomic environment remains unclear. Here, socioeconomic status (SES) for 283 MDS patients was estimated using the Scottish Index of Multiple Deprivation tool. Indices were assigned to quintile categorical indicators ranked from SES1 (lowest) to SES5 (highest). Clinicopathological features and outcomes between SES quintiles containing 15%, 20%, 19%, 30% and 16% of patients were compared. Prognostic scores identified lower-risk MDS in 82% of patients, with higher-risk disease in 18%. SES quintiles did not associate with age, gender, cytogenetics, International Prognostic scores or, in sub-analysis (n = 95), driver mutations. The odds ratio of a diagnosis of refractory anaemia was greater than other MDS sub-types in SES5 (OR 1·9, P = 0·024). Most patients (91%) exclusively received supportive care. SES did not associate with leukaemic transformation or cause of death. Cox regression models confirmed male gender (P < 0·05), disease-risk (P < 0·0001) and age (P < 0·01) as independent predictors of leukaemia-free survival, with leukaemic transformation an additional determinant of overall survival (P = 0·07). Thus, if access to healthcare is equitable, SES does not determine disease biology or survival in MDS patients receiving supportive treatment; additional studies are required to determine whether outcomes following disease-modifying therapies are influenced by SES.

Dysregulation of autophagy in chronic lymphocytic leukemia with the small-molecule Sirtuin inhibitor Tenovin-6.

Tenovin-6 (Tnv-6) is a bioactive small molecule with anti-neoplastic activity. Inhibition of the Sirtuin class of protein deacetylases with activation of p53 function is associated with the pro-apoptotic effects of Tnv-6 in many tumors. Here, we demonstrate that in chronic lymphocytic leukemia (CLL) cells, Tnv-6 causes non-genotoxic cytotoxicity, without adversely affecting human clonogenic hematopoietic progenitors in vitro, or murine hematopoiesis. Mechanistically, exposure of CLL cells to Tnv-6 did not induce cellular apoptosis or p53-pathway activity. Transcriptomic profiling identified a gene program influenced by Tnv-6 that included autophagy-lysosomal pathway genes. The dysregulation of autophagy was confirmed by changes in cellular ultrastructure and increases in the autophagy-regulatory proteins LC3 (LC3-II) and p62/Sequestosome. Adding bafilomycin-A1, an autophagy inhibitor to Tnv-6 containing cultures did not cause synergistic accumulation of LC3-II, suggesting inhibition of late-stage autophagy by Tnv-6. Thus, in CLL, the cytotoxic effects of Tnv-6 result from dysregulation of protective autophagy pathways.

Heterogeneity of p53-pathway Protein Expression in Chemosensitive Chronic Lymphocytic Leukemia: A Pilot Study.

The presence of p53-pathway dysfunction in chronic lymphocytic leukemia (CLL) can be used to identify patients with chemotherapy-refractory disease. Therapeutic responses are known to vary between patients with chemosensitive CLL and may relate to differences in p53-pathway activity. We hypothesized that the magnitude or type of p53-pathway protein expression is heterogeneous in patients with chemosensitive disease and could associate with white cell responses. In this pilot study, changes in p53 and its transcriptional targets, p21/waf1 and MDM2 were analyzed by immunoblotting and densitometry in CLL cells from 10 patients immediately prior to the start of chemotherapy, and after culture for 24 hours (h) with fludarabine (n=7) or chlorambucil (n=3). The in vitro response was also compared to that in vivo in circulating cells pre-treatment, and at 24h and 96h of chemotherapy. Disease responses were evident in all patients after the first treatment-cycle. Significant p53 induction was observed in CLL cells treated in vitro and in vivo. Greater heterogeneity in the expression-intensity was observed in vivo (σ2=45.15) than in vitro (σ2=1.33) and the results failed to correlate (r(2)=0.18, p=0.22). p21/waf1 and MDM2 expression-profiles were also dissimilar in vitro and in vivo. Higher in vivo (but not in vitro) responses associated with changes in white cell count (p=0.026). Thus, heterogeneity of p53-pathway activity exists in chemosensitive CLL; in unselected patients, in vivo changes do not correlate with those in vitro, but may associate with post-treatment white cell responses.

Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.

In a previous study, we identified somatic mutations of SF3B1, a gene encoding a core component of RNA splicing machinery, in patients with myelodysplastic syndrome (MDS). Here, we define the clinical significance of these mutations in MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). The coding exons of SF3B1 were screened using massively parallel pyrosequencing in patients with MDS, MDS/MPN, or acute myeloid leukemia (AML) evolving from MDS. Somatic mutations of SF3B1 were found in 150 of 533 (28.1%) patients with MDS, 16 of 83 (19.3%) with MDS/MPN, and 2 of 38 (5.3%) with AML. There was a significant association of SF3B1 mutations with the presence of ring sideroblasts (P < .001) and of mutant allele burden with their proportion (P = .002). The mutant gene had a positive predictive value for ring sideroblasts of 97.7% (95% confidence interval, 93.5%-99.5%). In multivariate analysis including established risk factors, SF3B1 mutations were found to be independently associated with better overall survival (hazard ratio = 0.15, P = .025) and lower risk of evolution into AML (hazard ratio = 0.33, P = .049). The close association between SF3B1 mutations and disease phenotype with ring sideroblasts across MDS and MDS/MPN is consistent with a causal relationship. Furthermore, SF3B1 mutations are independent predictors of favorable clinical outcome, and their incorporation into stratification systems might improve risk assessment in MDS.

Absolute SILAC-compatible expression strain allows Sumo-2 copy number determination in clinical samples.

Quantitative mass spectrometry-based proteomics is a vital tool in modern life science research. In contrast to the popularity of approaches for relative protein quantitation, the widespread use of absolute quantitation has been hampered by inefficient and expensive production of labeled protein standards. To optimize production of isotopically labeled standards, we genetically modified a commonly employed protein expression Escherichia coli strain, BL21 (DE3), to construct an auxotroph for arginine and lysine. This bacterial strain allows low-cost, high-level expression of fully labeled proteins with no conversion of labeled arginine to proline. In combination with a fluorescence-based quantitation of standards and nontargeted LC-MS/MS analysis of unfractionated total cell lysates, this strain was used to determine the copy number of a post-translational modifier, small ubiquitin-like modifier (SUMO-2), in HeLa, human sperm, and chronic lymphocytic leukemia cells. By streamlining and improving the generation of labeled standards, this production system increases the breadth of absolute quantitation by mass spectrometry and will facilitate a far wider uptake of this important technique than previously possible.

Predicted costs of iron-chelators in myelodysplastic syndromes: a 10-year analysis based on actual prevalence and red cell transfusion rates.

Consideration of iron-chelation (IC) in transfusion-dependent patients is recommended in most clinical-practice guidelines on myelodysplastic syndromes (MDS). The financial impact of IC on health-care systems is predicted through economic modeling, but an analysis based on actual prevalence is lacking. Here, we have investigated the potential drug-costs and need for IC in a cohort of 189 United Kingdom-based MDS patients diagnosed from 2000 to 2010. Patients with low or intermediate-1 IPSS scores were identified as eligible for IC if ≥24 red cell units (RCU) had been transfused over 12 consecutive months or the transfusion-intensity averaged ≥2 RCU per month. Drug-costs were calculated from the time patients qualified for IC until death or last follow-up. In 159 patients with low/intermediate-1 MDS, survival was superior with a low IPSS score (P = 0.014), age <70 years (P = 0.043), transfusion-independence at diagnosis (P = 0.0056) and transfusion-intensity of <2 RCU per month (P = 0.009). Reflecting the time elapsed since diagnosis, longer survival was observed with a cumulative red cell load of ≥75 U (P = 0.046). By logistic-regression analysis, transfusion-intensity independently predicted survival (P = 0.0035) in low and intermediate-1 risk MDS patients. Forty-one patients fulfilled criteria for consideration of IC. Of these, 6 patients died within 1 month; 35 patients survived for a median of 16 months (range 1-61). Had patients commenced IC, the anticipated drug-costs alone would have been ~$526,880-$2,064,800 over 10 years. The lack of association between cumulative transfusion-load and survival calls for a prospective evaluation of the cost-utility of IC in patients surviving long-term, to enable evidence-based recommendations in MDS management.

Factors influencing a second myeloid malignancy in patients with Philadelphia-negative -7 or del(7q) clones during tyrosine kinase inhibitor therapy for chronic myeloid leukemia.

The detection of Philadelphia-negative (Ph(neg)) cells with non-random karyotypic abnormalities after tyrosine kinase inhibitor (TKI) therapy of chronic myeloid leukaemia (CML) can be associated with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). To our knowledge, however, there have been no studies on variables influencing the risk of MDS/AML in patients with specific Ph(neg) karyotypes. We systematically examined studies reporting -7 or del(7q) within Ph(neg) cells in TKI-treated CML patients, and abstracted clinical and cytogenetic data from individual reports into a standardized format for further analysis. Of 53 patients, 43 had Ph(neg) -7 clones [as the sole abnormality (-7(sole)) in 29, or with other clones (-7(dual)) in 14], and del(7q) was present in 10. A total of 16/51 evaluable patients, all with -7, transformed to MDS/AML. Transformation was more frequent (15/16 patients) within 6 months of Ph(neg) -7 detection rather than subsequently (P < 0.0001). At first detection after TKI therapy, Ph(neg) abnormal clones comprised ≥50% of Ph(neg) cells in a greater proportion of patients with -7 than del(7q) (P = 0.035). Upon comparing -7(sole) and -7(dual), the latter was likely to be transient (P = 0.004), and AML was frequently observed with persistent -7 clones (P = 0.03). By logistic regression analysis (n = 36), clone size (P = 0.017), time-to-detection longer than 15 months (P = 0.02), and CML response (P = 0.085) were associated with MDS/AML. Validation of these novel associations in registry-based studies will help develop predictive criteria that define the MDS/AML risk in individual patients.

Gene expression profile in rat dorsal root ganglion following sciatic nerve injury and systemic neurotrophin-3 administration.

Following sciatic nerve transection in adult rats, a proportion of injured dorsal root ganglion (DRG) neurons die, through apoptosis, over the following 6 months. Previous studies showed that axotomy and neurotrophin-3 administration may have effects on expression of neurotrophins and their receptors in DRG. In the current study, the fourth and fifth lumbar DRGs of rats were examined 2 weeks after right sciatic nerve transection and ligation. The effects of axotomy and systemic NT-3 treatment on neuronal genes were investigated by microarray. The results demonstrated that bone morphogenetic protein (BMP) and Janus protein tyrosine kinase signaling pathways are induced in axotomized DRG, and PI-3 kinase and BMP pathways and genes controlling various cellular functions were induced after axotomy and NT-3 administration.

Δ160p53 is a novel N-terminal p53 isoform encoded by Δ133p53 transcript.

p53 gene expresses several protein isoforms modulating p53-mediated responses through regulation of gene expression. Here, we identify a novel p53 isoform, Δ160p53, lacking the first 159 residues. By knockdown experiments and site-directed mutagenesis, we show that Δ160p53 is encoded by Δ133p53 transcript using ATG160 as translational initiation site. This hypothesis is supported by endogenous expression of Δ160p53 in U2OS, T47D and K562 cells, the latter ones carrying a premature stop codon that impairs p53 and Δ133p53 protein expression but not the one of Δ160p53. Overall, these results show that the Δ133p53 transcript generates two different p53 isoforms, Δ133p53 and Δ160p53.

RAS mutation in acute myeloid leukemia is associated with distinct cytogenetic subgroups but does not influence outcome in patients younger than 60 years.

The pathogenesis of acute myeloid leukemia (AML) involves the cooperation of mutations promoting proliferation/survival and those impairing differentiation. The RAS pathway has been implicated as a key component of the proliferative drive in AML. We have screened AML patients, predominantly younger than 60 years and treated within 2 clinical trials, for NRAS (n = 1106), KRAS (n = 739), and HRAS (n = 200) hot-spot mutations using denaturing high-performance liquid chromatography or restriction fragment length polymorphism (RFLP) analysis. NRAS mutations were confirmed in 11% of patients (126/1106) and KRAS mutations in 5% (39/739). No HRAS mutations were detected in 200 randomly selected samples. Codons most frequently mutated were N12 (43%), N13 (21%), and K12 (21%). KRAS mutations were relatively overrepresented in French-American-British (FAB) type M4 (P < .001). NRAS mutation was over-represented in the t(3;5)(q21 approximately 25;q31 approximately q35) subgroup (P < .001) and underrepresented in t(15;17)(q22;q21) (P < .001). KRAS mutation was overrepresented in inv(16)(p13q22) (P = .004). Twenty-three percent of KRAS mutations were within the inv(16) subgroup. RAS mutation and FLT3 ITD were rarely coexistent (14/768; P < .001). Median percentage of RAS mutant allele assayed by quantitative RFLP analysis was 28% (N12), 19% (N13), 25% (N61), and 21% (K12). RAS mutation did not influence clinical outcome (overall/disease-free survival, complete remission, relapse rate) either for the entire cohort or within cytogenetic risk groups.

Stabilization of somatropin by heparin.

Somatropin, human growth hormone (hGH), is an unstable protein, posing challenging problems for its formulation and long-term stability. Since hGH formed insoluble adducts with heparin our aim was to evaluate heparin as a stabilizing agent for the drug. These adducts were characterized by particle diameter, tertiary structure variations and release studies. Studies were also carried out to determine the stability of hGH in the presence and absence of heparin by an interfacial denaturation method and real-time stability studies by measuring hGH activity and particle diameter. Moreover, biological activity of hGH and hGH/UH (unfractionated heparin) adducts was identified by daily subcutaneous injections to hypophysectomized rats. There was a decrease in mean hydrodynamic particle diameter of hGH/UH adducts with increased pH (54.4 to 12.2 nm from pH 3 to pH 7) indicating that the adducts were either dissociating or dissolving at high pH. Furthermore, second-derivative spectroscopy indicated that complexation of hGH with heparin did not cause a major disruption in the tertiary structure of hGH but decreased the hydrophilic environment around the tyrosine residues. Release of hGH from hGH/UH adducts was pH and ionic strength dependent with the highest release at pH 8 (93%) and lowest release at pH 3 (0%) over the first hour. Interfacial denaturation methods indicated that vortex agitation over 120 s resulted in no change in the optical density of hGH/UH adducts compared with a substantial increase for hGH alone at pH 6.8. Real-time stability studies over 93 days demonstrated that hGH/UH adducts at both pH 3 and 7 with an excess of heparin produced the highest percent of active hGH remaining in the solution at 4 degrees C and 37 degrees C. The higher stability of hGH/UH adducts with excess heparin compared with the stoichiometric ratio was also confirmed by particle size measurements during storage. The biological activity of these adducts was comparable with hGH alone by weight-gain studies in hypophysectomized rats. The findings suggest the value of using hGH/heparin adducts to stabilize the protein.

Effects of systemically administered NT-3 on sensory neuron loss and nestin expression following axotomy.

Previous work has shown that administration of the neurotrophin NT-3 intrathecally or to the proximal stump can prevent axotomy-induced sensory neuron loss and that NT-3 can stimulate sensory neuron differentiation in vitro. We have examined the effect of axotomy and systemic NT-3 administration on neuronal loss, apoptosis (defined by morphology and activated caspase-3 immunoreactivity), and nestin expression (a protein expressed by neuronal precursor cells) in dorsal root ganglia (DRG) following axotomy of the adult rat sciatic nerve. Systemic administration of 1.25 or 5 mg of NT-3 over 1 month had no effect on the incidence of apoptotic neurons but prevented the overall loss of neurons seen at 4 weeks in vehicle-treated animals. Nestin-immunoreactive neurons began to appear 2 weeks after sciatic transection in untreated animals and steadily increased in incidence over the next 6 weeks. NT-3 administration increased the number of nestin-immunoreactive neurons at 1 month by two- to threefold. Nestin-IR neurons had a mean diameter of 20.78 +/- 2.5 microm and expressed the neuronal markers neurofilament 200, betaIII-tubulin, protein gene product 9.5, growth associated protein 43, trkA, and calcitonin gene-related peptide. Our results suggest that the presence of nestin in DRG neurons after nerve injury is due to recent differentiation and that exogenous NT-3 may prevent neuron loss by stimulating this process, rather than preventing neuron death.

Hereditary sensory neuropathy is caused by a mutation in the delta subunit of the cytosolic chaperonin-containing t-complex peptide-1 (Cct4 ) gene.

A spontaneous autosomal recessive mutation was identified in the Sprague-Dawley rat strain with an early onset sensory neuropathy. The main clinical features of the mutation (mutilated foot, mf ), detectable shortly after birth, include ataxia, insensitivity to pain and foot ulceration. The pathological features include a severe reduction in the number of sensory ganglia and fibres. This mutant is therefore an excellent model for human hereditary sensory neuropathies. Here, we demonstrate that the mf locus maps to the distal end of rat chromosome 14, a region syntenic to human 2p13-p16 and proximal mouse 11. Sequence analysis of four candidate genes in this interval revealed a 1349G>A mutation in the chaperonin (delta) subunit 4 (Cct4) gene associated with the mf mutant. This change resulted in the substitution of a highly conserved cysteine for tyrosine at amino acid 450. Although we did not identify a mutation in the human CCT4 gene in a set of HSN patients, this result clearly demonstrates the pathological consequences of a defect in Cct4, a subunit of CCT (cytosolic chaperonin-containing t-complex peptide-1), involved in folding tubulin, actin and other cytosolic proteins. This is the first report of a mutation in a molecular chaperonin causing a hereditary neuropathy and raises the possibility that mis-folding proteins may be a cause of this group of neuropathies.