Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis.

Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that >60% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.

Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia.

To diagnose juvenile myelomonocytic leukemia (JMML) is sometimes challenging, because around 10% of patients lack molecular abnormalities affecting Ras-MAPK (mitogen-activated protein kinase) pathway and other diseases such as cytomegalovirus infection can mimic clinical signs of JMML. In order to validate a phospho-specific flow cytometry assay assessing phospho-signal transducer and activator of transcription factor 5 (p-STAT5) as a new diagnostic tool for JMML, we examined 22 samples from children with JMML and 47 controls. CD33+/CD34+ cells from 22 patients with JMML showed hyperphosphorylation of STAT5 induced by sub-saturating doses of granulocyte-macrophage colony-stimulating factor (GM-CSF). Using a training set of samples (11 JMML and 23 controls), we identified a threshold for p-STAT5-positive after stimulation with 0.1 ng/ml GM-CSF (17.17%) that discriminates JMML from controls. This threshold was validated in an independent series (11 JMML, 24 controls and 7 cases with diseases other than JMML) where we demonstrated that patients with JMML could be distinguished from other subjects with a sensitivity of 91% (confidence interval (CI) 59-100%) and a specificity of 87% (CI 70-96%). Positive and negative predictive values were 71% (CI 42-92%) and 96% (CI 82-100%), respectively. In conclusion, flow cytometric p-STAT5 profiling is a reliable diagnostic tool for identifying patients with JMML and can contribute to consistency of current diagnostic criteria.

Identification of germline susceptibility loci in ETV6-RUNX1-rearranged childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a malignant disease of the white blood cells. The etiology of ALL is believed to be multifactorial and likely to involve an interplay of environmental and genetic variables. We performed a genome-wide association study of 355 750 single-nucleotide polymorphisms (SNPs) in 474 controls and 419 childhood ALL cases characterized by a t(12;21)(p13;q22) - the most common chromosomal translocation observed in childhood ALL - which leads to an ETV6-RUNX1 gene fusion. The eight most strongly associated SNPs were followed-up in 951 ETV6-RUNX1-positive cases and 3061 controls from Germany/Austria and Italy, respectively. We identified a novel, genome-wide significant risk locus at 3q28 (TP63, rs17505102, P(CMH)=8.94 × 10(-9), OR=0.65). The separate analysis of the combined German/Austrian sample only, revealed additional genome-wide significant associations at 11q11 (OR8U8, rs1945213, P=9.14 × 10(-11), OR=0.69) and 8p21.3 (near INTS10, rs920590, P=6.12 × 10(-9), OR=1.36). These associations and another association at 11p11.2 (PTPRJ, rs3942852, P=4.95 × 10(-7), OR=0.72) remained significant in the German/Austrian replication panel after correction for multiple testing. Our findings demonstrate that germline genetic variation can specifically contribute to the risk of ETV6-RUNX1-positive childhood ALL. The identification of TP63 and PTPRJ as susceptibility genes emphasize the role of the TP53 gene family and the importance of proteins regulating cellular processes in connection with tumorigenesis.

In high risk hypertensive subjects with incidental and unilateral renal artery stenosis percutaneous revascularization with stent improves blood pressure control but not glomerular filtration rate.

AIM: In high-risk hypertensive subjects (HTs) with incidental unilateral renal artery stenosis (RAS), the effectiveness of percutaneous revascularization with stent (PR-STENT) on blood pressure (BP) and glomerular filtration rate (GFR) is not established. METHODS: Eighteen HTs aged 65.7 ± 9.2 years with angiographically diagnosed unilateral RAS (≥ 60%) were randomized to receive PR-STENT (N=9) or to NO-STENT (N=9). BP (mercury sphygmomanometer) and GFR (99mTc-DTPA clearances during renal scintigraphy) were evaluated yearly for three years. Echo-Doppler of renal arteries was performed to verify the anatomic patency and flow velocities of the reperfused artery. Analysis of variance compared BP and GFR values changes from baseline to the follow-up; differences for continuous variables were evaluated between groups with the Tukey's post hoc test after adjustment for age, change of BP between baseline and at the follow-up, GFR and body mass index (BMI). RESULTS: Baseline systolic BP and GFR values were not different between groups. The significantly greater GFR increase observed in PR-STENT than in NO-STENT at univariate analysis at the end of follow-up (62.5 ± 19.2 vs. 42.24 ± 17.6, P<0.02) disappeared after adjustment for confounding factors. However, systolic BP remained significantly lower in PR-STENT than in NO-STENT (140.1 ± 4.6 vs. 170.0 ± 8.3, P<0.0001) also after adjustment for age, GFR and BMI. CONCLUSION: PR-STENT reduces systolic BP without improving GFR. Due to the strong association between high BP and renal damage, this study raises the question on whether PR-STENT should be performed in all HTs with unilateral and incidental RAS.

DNA copy-number abnormalities do not occur in infant ALL with t(4;11)/MLL-AF4.

The pathogenesis of infant acute lymphoblastic leukemia (ALL) is still not well defined. Short latency to leukemia and very high concordance rate for ALL in Mixed-Lineage Leukemia (MLL)-positive infant twins suggest that the MLL rearrangement itself could be sufficient for overt leukemia. Attempts to generate a suitable mouse model for MLL-AF4-positive ALL did not thoroughly resolve the issue of whether cooperating mutations are required to reduce latency and to generate overt leukemia in vivo. In this study, we applied single-nucleotide polymorphism array technology to perform genomic profiling of 28 infant ALL cases carrying t(4;11) to detect MLL-cooperating aberrations hidden to conventional techniques and to gain new insights into infant ALL pathogenesis. In contrast to pediatric, adolescent and adult ALL cases, the MLL rearrangement in infant ALL is associated with an exceptionally low frequency of copy-number abnormalities, thus confirming the unique nature of this disease. By contrast, additional genetic aberrations are acquired at disease relapse. Small-segmental uniparental disomy traits were frequently detected, mostly constitutional, and widely distributed throughout the genome. It can be argued that the MLL rearrangement as a first hit, rather than inducing the acquisition of additional genetic lesions, has a major role to drive and hasten the onset of leukemia.

Safety and long-term results of patent foramen ovale transcatheter closure in patients with thrombophilia.

AIM: Trials on transcatheter closure of patent foramen ovale (PFO) in different settings attempted to exclude patients with thrombophilia for the risk of device thrombosis. Authors sought to retrospectively evaluate safety and results of transcatheter PFO closure in patients with confirmed coagulation abnormalities. METHODS: Between December 2006 and December 2008, 30 out of 98 consecutive patients (mean age 40+/-10.9 years, 23 females) referred to Rovigo General Hospital for transcatheter closure had coagulation abnormalities including mutations of factor V Leiden, factors X, VIII, protein C, S, MHFTR factors, and antiphospholipid and anticardiolipin antibodies, hyperhomocisteinimia. All patients underwent preoperative transesophageal echo and brain magnetic resonance imaging, and intra-cardiac echo-guided transcatheter PFO closure. RESULTS: Success rate was 100%; there was no difference in occlusion and complications rates between patients with and without thrombophilia: in particular no device thrombosis or recurrent cerebral ischemia or stroke were observed during the follow-up. Patients with thrombophilia had a higher incidence of atrial septal aneurysm, migraine with aura and deep venous thrombosis in the previous medical history compared to patients without. CONCLUSIONS: Despite its small sample, this study suggests that patients with coagulation abnormalities should not be excluded from the trial; they have potentially a higher risk of stroke through a PFO compared to other patients, and transcatheter closure is as safe and effective as in general population with almost no additional therapy rather than aspirin.

Early post-procedural migraine attack predicts migraine resolution after patent foramen ovale transcatheter closure.

AIM: In patients with patent foramen ovale-related migraine, the procedure of transcatheter closure itself is likely to cause a migraine attack. Our study is aimed to evaluate the incidence of migraine attacks immediately after closure procedure and their clinical and potential prognostic significance. METHODS: We reviewed our database from January 2005 to April 2007 searching for patients with severe disabling migraine despite anti-headache therapy who were submitted to transcatheter closure of patent foramen ovale (PFO). Medical records of these patients were carefully reviewed in order to record migraine episodes immediately (0 to 6 h) after closure procedure. RESULTS: Twenty-one patients with previous stroke and migraine underwent PFO closure: the procedure was successful in all of the patients with no perioperative and in-hospital complications. Ten patients (47.6%) experienced a migraine attack of mean duration 3.5+/-2.4 h immediately after the closure procedure. Those patients had the same procedure time compared with other patients, but had larger PFO: patients with migraine attack immediately after closure had higher rate of complete abolition of migraine in the follow-up. CONCLUSION: Although more larger studies are needed to evaluate the exact relationships between migraine and PFO, in patients with a tight correlation between migraine and PFO, a prolonged opening of the PFO, as during closure procedure, may cause a migraine attack immediately after the closure. This fact can be considered a positive prognostic factor for migraine abolishment in the follow-up.

Intracardiac echocardiography-guided atrial septal defect transcatheter closure of a very old surgical residual shunt.

Although some studies have suggested excellent long-term outcome, arrhythmias, pulmonary hypertension, and paradoxical cerebral embolism are mentioned as results of residual shunts in the long-term follow-up after surgical atrial septal defect (ASD) closure at a young age. In cases of previous patch closure, transcatheter repair of residual shunts can be problematic due both to clinical decision-making in the presence of elevated pulmonary pressure and to a very old patch. A 70-year-old woman operated for an ASD with synthetic patch closure when she was 35 years old was referred to our center because of recurrent paroxysmal atrial fibrillation, initially decompensated right heart failure with rest and exercise-induced dyspnea as results of a residual shunt and moderate pulmonary hypertension. Complete right heart catheterization confirmed a mean pulmonary pressure of about 55 mm Hg and a Qp:Qs ratio of 1.78. A mechanical intracardiac echocardiography study with a 9F 9 MHz UltraICE catheter (Boston Scientific Corp.) showed a highly echogenous interatrial patch with a very stiff appearance and a very high residual defect of 8.7 and 11.2 mm on the aortic valve plane and on the four-chamber views, respectively. An occlusion test with a compliant AGA medical balloon demonstrated a decrease in mean pulmonary pressure to 36 mm Hg. A 10 mm Amplatzer's ASD occluder was implanted after a first unsuccessful attempt due to patch stiffness. Three-month echocardiography follow-up demonstrated almost normal pulmonary pressure and only slight dilation of the right chambers. At six-month follow-up, the patient no longer experienced dyspnea. This case demonstrates that transcatheter closure of a residual shunt following surgical ASD repair can be successfully accomplished also in elderly patients with a very old patch and decompensated right heart failure: the balloon occlusion test and intracardiac echocardiography appear to be effective in the operative decision-making process.

Vascular remodeling after carotid artery stenting.

Carotid stenting is an alternative to endarterectomy for the treatment of carotid stenosis. To determine the role of vascular remodeling after stent placement, we studied 19 high surgical risk patients undergoing carotid stenting for severe stenosis. Using high-resolution ultrasound, we evaluated the intima-media thickness (IMT), the intima-intima diameter, and the adventitia-adventitia diameter at prespecified sites of the carotid artery tree during 3 years of follow-up. The IMT of internal carotid artery, at the site of maximum stenosis, increased significantly from 0 mm after 24 hours, to 0.41 mm at 3 months, to 0.48 mm at 6 months, and to 0.51 mm at 3 years of follow-up. In the same site, diameters and residual stenosis (range 29-24%) did not change over time. Our study showed that stent is self-expanding against the atherosclerotic plaque within the 3-year follow-up period. Despite neointima formation, the intima-intima diameter does not change without worsening of the residual stenosis.

Endovascular treatment of femoro-popliteal obstructive disease.

The systemic nature of vascular atherosclerosis is beginning to involve not only the angiologists and the vascular surgeons, but also the clinical and the invasive cardiologists. Femoral occlusive disease is one of the most challenging field due to the particular anatomical morphology of the femoral arterial wall that is prone to obstructive disease and high restenosis rate after percutaneous revascularization. Acute and chronic arterial diseases are the main clinical scenario involving femoral vessels. Percutaneous techniques include endoluminal recanalization, subintimal recanalization, stent implantation, mechanical and rheolytic thrombectomy, laser angioplasty, and cryoplasty. In this review the authors propose an overview and an update of the most recent advances in techniques and results in the field of endovascular treatment of femoral artery occlusive disease.

Percutaneous revascularization of coexisting severe carotid and coronary artery disease: a case report.

Following the NASCET and ACAS trials, the use of carotid endarterectomy for the treatment of carotid artery stenosis has become widespread. However, in high-risk patients, the perioperative morbidity and mortality have reached 18%. In such populations, a percutaneous approach including coronary angioplasty and stenting of the carotid lesion could be an option worth exploring. In this report we discuss a case that is representative of our experience with the simultaneous treatment of critical carotid and coronary stenosis. A 74-year-old patient with advanced coronary artery disease and severe bilateral carotid pathology was submitted to coronary angioplasty and stenting of the carotid lesions.