Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma

Background

There is much discussion in the cancer drug development community about how to incorporate molecular tools into early-stage clinical trials to assess target modulation, measure anti-tumor activity, and enrich the clinical trial population for patients who are more likely to benefit. Small, molecularly focused clinical studies offer the promise of the early definition of optimal biologic dose and patient population.

Methods and Findings

Based on preclinical evidence that phosphatase and tensin homolog deleted on Chromosome 10 (PTEN) loss sensitizes tumors to the inhibition of mammalian target of rapamycin (mTOR), we conducted a proof-of-concept Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. We aimed to assess the safety profile of daily rapamycin in patients with glioma, define the dose of rapamycin required for mTOR inhibition in tumor tissue, and evaluate the antiproliferative activity of rapamycin in PTEN-deficient glioblastoma. Although intratumoral rapamycin concentrations that were sufficient to inhibit mTOR in vitro were achieved in all patients, the magnitude of mTOR inhibition in tumor cells (measured by reduced ribosomal S6 protein phosphorylation) varied substantially. Tumor cell proliferation (measured by Ki-67 staining) was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test) but not the intratumoral rapamycin concentration. Tumor cells harvested from the Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, indicating that clinical resistance to biochemical mTOR inhibition was not cell-intrinsic. Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test).

Conclusions

Rapamycin has anticancer activity in PTEN-deficient glioblastoma and warrants further clinical study alone or in combination with PI3K pathway inhibitors. The short-term treatment endpoints used in this neoadjuvant trial design identified the importance of monitoring target inhibition and negative feedback to guide future clinical development.Trial registration: http://www.ClinicalTrials.gov (#{"type":"clinical-trial","attrs":{"text":"NCT00047073","term_id":"NCT00047073"}}NCT00047073).     

Differential selection for B-raf and Ha-ras mutated liver tumors in mice with high and low susceptibility to hepatocarcinogenesis

Activation of the Ras/Raf/MEK/ERK pathway is frequently observed in animal and human tumors. In our study, we analyzed B-raf codon 637 (formerly 624) and Ha-ras codon 61 mutations in liver tumors from C3H, B6C3F1 and C56BL mice which differ considerably with regard to their susceptibility to hepatocarcinogenesis. In total, 73% (102/140) of tumors induced by a single application of N-nitrosodiethylamine or 7,12-dimethylbenz[a]anthracene contained either B-raf or Ha-ras mutations and only <3% (4/140) were mutated in both genes. In addition, B-raf mutations were present in 76% (19/25) of early precancerous liver lesions. The prevalence of Ha-ras mutated tumors was significantly higher in the susceptible C3H and B6C3F1 mouse strains (39-50%) than in the comparatively resistant C57BL mouse (7%). B-raf mutated tumors, by contrast, were more frequent in C57BL mice (68%) than in the other two strains (17-45%). Taken together, our findings indicate that alterations affecting the Ras/Raf/MEK/ERK signalling pathway are a hallmark of carcinogen-induced liver tumors in mice. Moreover, our results show that mutational activation of B-raf in liver tumors of different mouse strains is, by contrast to Ha-ras, inversely related to their susceptibility to hepatocarcinogenesis. Although activated Ras and Raf proteins are assumed to have similar biological effects because they feed into the same signalling pathway, there seem to be subtle strain-specific differences in selection processes favouring the preferential outgrowth of either B-raf or Ha-ras mutated tumor populations in mouse liver.     

Anticoagulation intensity and outcomes among patients prescribed oral anticoagulant therapy: a systematic review and meta-analysis

Background

Patients taking oral anticoagulant therapy balance the risks of hemorrhage and thromboembolism. We sought to determine the association between anticoagulation intensity and the risk of hemorrhagic and thromboembolic events. We also sought to determine how under-or overanticoagulation would influence patient outcomes.

Methods

We reviewed the MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials and CINAHL databases to identify studies involving patients taking anticoagulants that reported person-years of observation and the number of hemorrhages or thromboemboli in 3 or more discrete ranges of international normalized ratios. We estimated the overall relative and absolute risks of events specific to anticoagulation intensity.

Results

We included 19 studies. The risk of hemorrhage increased significantly at high international normalized ratios. Compared with the therapeutic ratio of 2–3, the relative risk (RR) of hemorrhage (and 95% confidence intervals [CIs]) were 2.7 (1.8–3.9; p < 0.01) at a ratio of 3–5 and 21.8 (12.1–39.4; p < 0.01) at a ratio greater than 5. The risk of thromboemboli increased significantly at ratios less than 2, with a relative risk of 3.5 (95% CI 2.8–4.4; p < 0.01). The risk of hemorrhagic or thromboembolic events was lower at ratios of 3–5 (RR 1.8, 95% CI 1.2–2.6) than at ratios of less than 2 (RR 2.4, 95% CI 1.9–3.1; p = 0.10). We found that a ratio of 2–3 had the lowest absolute risk (AR) of events (AR 4.3%/yr, 95% CI 3.0%–6.3%).

Conclusions

The risks of hemorrhage and thromboemboli are minimized at international normalized ratios of 2–3. Ratios that are moderately higher than this therapeutic range appear safe and more effective than subtherapeutic ratios.Oral anticoagulant therapy is essential for the treatment and prevention of many thromboembolic disorders. Since anticoagulants can cause serious adverse events,^1–3 physicians monitor the international normalized ratios of patients taking these drugs to ensure that their ratios fall within a target range.An international normalized ratio of 2–3 is the most common target range. Results of previous studies revealed an increased risk of bleeding among patients whose ratios exceeded 4, an increased risk of stroke among patients whose ratios were 1.5–2 and a decreased risk of stroke at a ratio of 2.4.^4,5 However, the evidence supporting the range of 2–3 has some deficiencies. We sought to determine whether the risk of hemorrhagic and thromboembolic events is minimized at an international normalized ratio of 2–3 among patients taking anticoagulants. In addition, it has been observed that patients spend more time with a ratio below 2 than above 3.^6,7 The impact of such systematic underanticoagulation on patient outcomes is unknown. We sought to determine the effect of under-or overanticoagulation on the risk of thromboemboli and hemorrhage.     

The essential role of centrosomal NDE1 in human cerebral cortex neurogenesis

We investigated three families whose offspring had extreme microcephaly at birth and profound mental retardation. Brain scans and postmortem data showed that affected individuals had brains less than 10% of expected size (≤10 standard deviation) and that in addition to a massive reduction in neuron production they displayed partially deficient cortical lamination (microlissencephaly). Other body systems were apparently unaffected and overall growth was normal. We found two distinct homozygous mutations of NDE1, c.83+1G>T (p.Ala29GlnfsX114) in a Turkish family and c.684_685del (p.Pro229TrpfsX85) in two families of Pakistani origin. Using patient cells, we found that c.83+1G>T led to the use of a novel splice site and to a frameshift after NDE1 exon 2. Transfection of tagged NDE1 constructs showed that the c.684_685del mutation resulted in a NDE1 that was unable to localize to the centrosome. By staining a patient-derived cell line that carried the c.83+1G>T mutation, we found that this endogeneously expressed mutated protein equally failed to localize to the centrosome. By examining human and mouse embryonic brains, we determined that NDE1 is highly expressed in neuroepithelial cells of the developing cerebral cortex, particularly at the centrosome. We show that NDE1 accumulates on the mitotic spindle of apical neural precursors in early neurogenesis. Thus, NDE1 deficiency causes both a severe failure of neurogenesis and a deficiency in cortical lamination. Our data further highlight the importance of the centrosome in multiple aspects of neurodevelopment.     

Crystal structures of a cysteine-modified mutant in loop D of acetylcholine-binding protein

Covalent modification of α7 W55C nicotinic acetylcholine receptors (nAChR) with the cysteine-modifying reagent [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET(+)) produces receptors that are unresponsive to acetylcholine, whereas methyl methanethiolsulfonate (MMTS) produces enhanced acetylcholine-gated currents. Here, we investigate structural changes that underlie the opposite effects of MTSET(+) and MMTS using acetylcholine-binding protein (AChBP), a homolog of the extracellular domain of the nAChR. Crystal structures of Y53C AChBP show that MTSET(+)-modification stabilizes loop C in an extended conformation that resembles the antagonist-bound state, which parallels our observation that MTSET(+) produces unresponsive W55C nAChRs. The MMTS-modified mutant in complex with acetylcholine is characterized by a contracted C-loop, similar to other agonist-bound complexes. Surprisingly, we find two acetylcholine molecules bound in the ligand-binding site, which might explain the potentiating effect of MMTS modification in W55C nAChRs. Unexpectedly, we observed in the MMTS-Y53C structure that ten phosphate ions arranged in two rings at adjacent sites are bound in the vestibule of AChBP. We mutated homologous residues in the vestibule of α1 GlyR and observed a reduction in the single channel conductance, suggesting a role of this site in ion permeation. Taken together, our results demonstrate that targeted modification of a conserved aromatic residue in loop D is sufficient for a conformational switch of AChBP and that a defined region in the vestibule of the extracellular domain contributes to ion conduction in anion-selective Cys-loop receptors.     

Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats

Calorie restriction [CR; ~65% of ad libitum (AL) intake] improves insulin-stimulated glucose uptake (GU) and Akt phosphorylation in skeletal muscle. We aimed to elucidate the effects of CR on 1) processes that regulate Akt phosphorylation [insulin receptor (IR) tyrosine phosphorylation, IR substrate 1-phosphatidylinositol 3-kinase (IRS-PI3K) activity, and Akt binding to regulatory proteins (heat shock protein 90, Appl1, protein phosphatase 2A)]; 2) Akt substrate of 160-kDa (AS160) phosphorylation on key phosphorylation sites; and 3) atypical PKC (aPKC) activity. Isolated epitrochlearis (fast-twitch) and soleus (slow-twitch) muscles from AL or CR (6 mo duration) 9-mo-old male F344BN rats were incubated with 0, 1.2, or 30 nM insulin and 2-deoxy-[(3)H]glucose. Some CR effects were independent of insulin dose or muscle type: CR caused activation of Akt (Thr(308) and Ser(473)) and GU in both muscles at both insulin doses without CR effects on IRS1-PI3K, Akt-PP2A, or Akt-Appl1. Several muscle- and insulin dose-specific CR effects were revealed. Akt-HSP90 binding was increased in the epitrochlearis; AS160 phosphorylation (Ser(588) and Thr(642)) was greater for CR epitrochlearis at 1.2 nM insulin; and IR phosphorylation and aPKC activity were greater for CR in both muscles with 30 nM insulin. On the basis of these data, our working hypothesis for improved insulin-stimulated GU with CR is as follows: 1) elevated Akt phosphorylation is fundamental, regardless of muscle or insulin dose; 2) altered Akt binding to regulatory proteins (HSP90 and unidentified Akt partners) is involved in the effects of CR on Akt phosphorylation; 3) Akt effects on GU depend on muscle- and insulin dose-specific elevation in phosphorylation of Akt substrates, including, but not limited to, AS160; and 4) greater IR phosphorylation and aPKC activity may contribute at higher insulin doses.     

Evaluation of an electrostatic toxicity model for predicting Ni(2+) toxicity to barley root elongation in hydroponic cultures and in soils

Assessing environmental risks of metal contamination in soils is a complex task because the biologically effective concentrations of metals in soils vary widely with soil properties. The factors influencing the toxic effect of nickel (Ni) on root growth of barley (Hordeum vulgare) were re-evaluated using published data from both soil and hydroponic cultures. The electrical potential (ψ(0) (o) ) and ion activities ({I(z) }(0) (o) ) at the outer surfaces of root-cell plasma membranes (PMs) were computed as the basis of the re-evaluation. The reanalyses demonstrated that root growth was related to: the Ni(2+) activity at the PM surface, ({Ni(2+) }(0) (o) ); calcium (Ca) deficiency (related to {Ca(2+) }(0) (o) ); osmotic effects; and modification of intrinsic Ni(2+) toxicity by magnesium (Mg(2+) ; this appeared to exert an intrinsic (specific) ameliorating effect on intrinsic Ni(2+) toxicity). Electrostatic toxicity models (ETM) were developed to relate root growth to these factors (R(2) > 0.751). Based on the ETM developed in soil culture and a Ni(2+) solid-solution partitioning model, critical metal concentrations in soils linked to a biological effect were well predicted for 16 European soils with a wide range of properties, indicating the potential utility of ETM in risk assessment of metals in terrestrial ecosystems.     

Air-pollutant chemicals and oxidized lipids exhibit genome-wide synergistic effects on endothelial cells

Background  

Ambient air pollution is associated with increased cardiovascular morbidity and mortality. We have found that exposure to ambient ultrafine particulate matter, highly enriched in redox cycling organic chemicals, promotes atherosclerosis in mice. We hypothesize that these pro-oxidative chemicals could synergize with oxidized lipid components generated in low-density lipoprotein particles to enhance vascular inflammation and atherosclerosis.     

"Pseudosarcoma" in a pregnant woman

Background

As a result of improvements in diagnostic accuracy, the primary source of the tumour is identified in more than 99% of cases presenting with a malignancy. Whilst the axial skeleton is a common site of metastases, the sternum is rarely affected, especially by isolated metastases.

Case presentation

We report a case of a 68 year old male who was referred to the surgical outpatient clinic with a six month history of sternal pain. The patient was known to have essential thrombocythaemia, which had recently transformed into acute myeloid leukaemia but a sternal biospy showed mucinous adenocarcinoma. He had not localising symptoms and full evaluation failed to localise the primary tumour.

Conclusion

Solitary sternal metastases are rare and when found an underlying neoplasm is usually identified allowing targeted treatment. If however, there is no symptomatic tumour, the metastasis should simply be treated symptomatically.