A chemogenomic approach to identify personalized therapy for patients with relapse or refractory acute myeloid leukemia: results of a prospective feasibility study.

Targeted next-generation sequencing (tNGS) and ex vivo drug sensitivity/resistance profiling (DSRP) have laid foundations defining the functional genomic landscape of acute myeloid leukemia (AML) and premises of personalized medicine to guide treatment options for patients with aggressive and/or chemorefractory hematological malignancies. Here, we have assessed the feasibility of a tailored treatment strategy (TTS) guided by systematic parallel ex vivo DSRP and tNGS for patients with relapsed/refractory AML (number NCT02619071). A TTS issued by an institutional personalized committee could be achieved for 47/55 included patients (85%), 5 based on tNGS only, 6 on DSRP only, while 36 could be proposed on the basis of both, yielding more options and a better rationale. The TSS was available in <21 days for 28 patients (58.3%). On average, 3 to 4 potentially active drugs were selected per patient with only five patient samples being resistant to the entire drug panel. Seventeen patients received a TTS-guided treatment, resulting in four complete remissions, one partial remission, and five decreased peripheral blast counts. Our results show that chemogenomic combining tNGS with DSRP to determine a TTS is a promising approach to propose patient-specific treatment options within 21 days.

Nectin-4: a new prognostic biomarker for efficient therapeutic targeting of primary and metastatic triple-negative breast cancer.

Background: Triple-negative breast cancers (TNBCs) are associated with a poor prognosis. In contrast to other molecular subtypes, they have no identified specific target and chemotherapy remains the only available systemic treatment. The adhesion molecule nectin-4 represents a new potential therapeutic target in different cancer models. Here, we have tested the prognostic value of nectin-4 expression and assessed the therapeutic efficiency of an anti-nectin 4 antibody drug conjugate (ADC) on localised and metastatic TNBC in vitro and in vivo. Materials and methods: We analysed nectin-4/PVRL4 mRNA expression in 5673 invasive breast cancers and searched for correlations with clinicopathological features including metastasis-free survival (MFS). Immunohistochemistry was carried out in 61 TNBCs and in samples of primary TNBC Patient-Derived Xenografts (PDXs). An anti-nectin-4 antibody eligible for ADC was produced and tested in vitro and in vivo in localised and metastatic TNBC PDXs. Results: High nectin-4/PVRL4 mRNA expression was associated with poor-prognosis features including the TN and basal subtypes. High PVRL4 mRNA expression showed independent negative prognostic value for MFS in multivariate analysis in TNBCs. Nectin-4 protein expression was not detected in adult healthy tissues including mammary tissue. Membranous protein expression was found in 62% of TNBCs, with strong correlation with mRNA expression. We developed an ADC (N41mab-vcMMAE) comprising a human anti-nectin-4 monoclonal antibody conjugated to monomethyl auristatin-E (MMAE). In vitro, this ADC bound to nectin-4 with high affinity and specificity and induced its internalisation as well as dose-dependent cytotoxicity on nectin-4-expressing breast cancer cell lines. In vivo, this ADC induced rapid, complete and durable responses on nectin-4-positive xenograft TNBC samples including primary tumours, metastatic lesions, and local relapses; efficiency was dependent on both the dose and the nectin-4 tumour expression level. Conclusion: Nectin-4 is both a new promising prognostic biomarker and specific therapeutic target for ADC in the very limited armamentarium against TNBC.

A robust and rapid xenograft model to assess efficacy of chemotherapeutic agents for human acute myeloid leukemia.

Relevant preclinical mouse models are crucial to screen new therapeutic agents for acute myeloid leukemia (AML). Current in vivo models based on the use of patient samples are not easy to establish and manipulate in the laboratory. Our objective was to develop robust xenograft models of human AML using well-characterized cell lines as a more accessible and faster alternative to those incorporating the use of patient-derived AML cells. Five widely used AML cell lines representing various AML subtypes were transplanted and expanded into highly immunodeficient non-obese diabetic/LtSz-severe combined immunodeficiency IL2Rγc(null) mice (for example, cell line-derived xenografts). We show here that bone marrow sublethal conditioning with busulfan or irradiation has equal efficiency for the xenotransplantation of AML cell lines. Although higher number of injected AML cells did not change tumor engraftment in bone marrow and spleen, it significantly reduced the overall survival in mice for all tested AML cell lines. On the basis of AML cell characteristics, these models also exhibited a broad range of overall mouse survival, engraftment, tissue infiltration and aggressiveness. Thus, we have established a robust, rapid and straightforward in vivo model based on engraftment behavior of AML cell lines, all vital prerequisites for testing new therapeutic agents in preclinical studies.

Effects of intermittent suckling on body composition of Iberian piglets weaned at 35 days of age.

Piglet body composition at weaning could be a determinant for pig's viability and may be influenced by factors such as the nutritional management followed during suckling. An experiment was conducted to study whether intermittent suckling (IS) affects body composition at weaning and nutrient and energy retention during a 34-day lactation period in Iberian piglets. Litters were subjected to conventional suckling (CS) or IS (n=10 litters of six piglets per treatment) in two trials. All piglets had ad libitum access to creep feed from day 15 onwards. In IS, piglets were progressively separated from the sow for 6, 8 and 10 h daily during the last week of lactation, whereas in CS piglets had continuous access to their dams. Creep feed intake in litters and BW development of individual piglets were measured throughout the 34-day lactation. Within each litter, both at birth and at weaning (day 35), one piglet was used to assess nutrient retention and body composition by the comparative slaughter approach. During days 29 to 35 of the experiment, daily creep feed intake was greater in IS piglets (IS 124, CS 67 g/piglet, P=0.040), and average daily gain differed significantly between groups (IS 190, CS 150 g/day, P=0.010). BW at weaning was higher in the IS than in the CS piglets (IS 8.19, CS 7.48 kg, P=0.011). Empty-body fat and energy content at weaning were higher in the IS compared with CS litters, as well as fat content in the carcass (P=0.04). The IS treatment did not affect empty-body protein deposition, but significantly increased daily retention of fat, energy, ash and calcium, compared with CS litters (P<0.05). Thus, IS in Iberian piglets seems to enhance feed intake, growth rate and retention of some body components, which may contribute to a higher body fat content at weaning and facilitate the weaning process.

Anti-leukemia activity of chaetocin via death receptor-dependent apoptosis and dual modulation of the histone methyl-transferase SUV39H1.

Epigenetic deregulation is involved in acute myeloid leukemia (AML) pathogenesis and epigenetic targeting drugs are in clinical trial. Since the first results with histone-deacetylase inhibitors in AML are controversial, novel single and combined treatments need to be explored. It is tempting to combine chromatin-targeting drugs. SUV39H1, the main methyl-transferase for lysine 9 tri-methylation on histone H3, interacts with oncogenes involved in AML and acts as a transcriptional repressor for hematopoietic differentiation and immortalization. We report here that pharmacological inhibition of SUV39H1 by chaetocin induces apoptosis in leukemia cell lines in vitro and primary AML cells ex vivo, and that it interferes with leukemia growth in vivo. Chaetocin treatment upregulates reactive oxygen species (ROS) production as well as the transcription of death-receptor-related genes, in a ROS-dependent manner, leading to death receptor-dependent apoptosis. In addition to its direct inhibition by chaetocin, SUV39H1 is indirectly modulated by chaetocin-induced ROS. Accordingly, chaetocin potentiates other anti-AML drugs, in a ROS-dependent manner. The decryption of a dual mechanism of action against AML involving both direct and indirect SUV39H1 modulation represents an innovative read-out for the anticancer activity of chaetocin and for its synergy with other anti-AML drugs, suggesting new therapeutic combination strategies in AML.

Loss of AF6/afadin, a marker of poor outcome in breast cancer, induces cell migration, invasiveness and tumor growth.

Afadin/AF6, an F-actin-binding protein, is ubiquitously expressed in epithelia and has a key role during development, through its regulatory role in cell-cell junction organization. Afadin loss of expression in 15% of breast carcinoma is associated with adverse prognosis and increased risk of metastatic relapse. To determine the role of afadin in breast cancer, we studied the functional consequences of afadin protein extinction using in vitro and in vivo models. Three different breast cancer cell lines representative of the major molecular subtypes were stably repressed for afadin expression (knockdown of afadin (afadin KD)) using RNA interference. Collective and individual migrations as well as Matrigel invasion were markedly increased in afadin KD cells. Heregulin-ß1 (HRG-ß1)-induced migration and invasion were increased by twofold in afadin KD cells. Conversely, ectopic expression of afadin in the afadin-negative T47D cell line inhibited spontaneous and HRG-ß1-induced migrations. RAS/MAPK and SRC kinase pathways were activated in afadin KD cells. Activation levels positively correlated with migration and invasion strength. Use of MEK1/2 (U0126) and SRC kinases (SU6656) inhibitors reduced afadin-dependent migration and invasion. Afadin extinction in the SK-BR-3 cell line markedly accelerated tumor growth development in mouse mammary gland and lung metastasis formation. These results may explain why the loss of afadin expression in tumors correlates with high tumor size and poor metastasis-free survival in patients.

Carotid Endarterectomy: experience in 8743 cases.

INTRODUCTION: Recently published case series of patients undergoing carotid endarterectomy suggested a reduction in the rate of perioperative neurologic events when compared to those reported in the large randomized trials performed in the 1990s, without great differences between high and low risk patients. METHODS: As a major center of Vascular Surgery we prospectively collected data on 8743 carotid endarterectomy procedures (eversion technique 75%, patch closure 17.5%) performed in the period 1992-2009. RESULTS: Perioperative mortality was 0.32% (27/8743) with myocardial infarction being the most frequent cause (9 patients). Perioperative neurological morbidity was 1.04% (91/8743) with 51 major and 40 minor strokes. In 201 cases (2.3%) a cervical hematoma (suture-line bleeding in 41 cases and or diffuse oozing in 160 cases) in the early postoperative period necessitated urgent wound revision. In 262 (3.0%) cases we observed permanent or transient lesions of cranial nerves in the postoperative period. There was no significant difference in the combined ipsilateral stroke and perioperative death rate in octogenarian patients (2.1% in octogenarians and 1.2% in younger patients, p>0.05), even though an increasing trend was evident. CONCLUSIONS: Carotid endarterectomy has a reduced rate of perioperative complications when compared to those previously reported in literature. The low complication rate is related to improved preoperative patients evaluation, surgeons' increasing experience and to surgical and anesthesiological techniques. Carotid angioplasty and stenting should have their results compared to these real world results of carotid endarterectomy in order to asses their reliability when treating extracranial cerebrovascular disease.

Mouse vanin-1 is cytoprotective for islet beta cells and regulates the development of type 1 diabetes.

AIMS/HYPOTHESIS: Islet cell death is a key initiating and perpetuating event in type 1 diabetes and involves both immune-mediated and endogenous mechanisms. The epithelial pantetheinase vanin-1 is proinflammatory and cytoprotective via cysteamine release in some tissues. We investigated the impact of a vanin-1 deficiency on islet death and type 1 diabetes incidence. METHODS: Vanin-1-deficient mice were produced and tested in drug-induced and autoimmune diabetes models. The contribution of vanin-1 to islet survival versus immune responses was evaluated using lymphocyte transfer and islet culture experiments. RESULTS: The vanin-1/cysteamine pathway contributes to the protection of islet beta cells from streptozotocin-induced death in vitro and in vivo. Furthermore, vanin-1-deficient NOD mice showed a significant aggravation of diabetes, which depended upon loss of vanin-1 expression by host tissues. This increased islet fragility was accompanied by greater CD4+ insulitis without impairment of regulatory cells. Addition of cystamine, the product of pantetheinase activity, protected islets in vitro and compensated for vanin-1 deficiency in vivo. CONCLUSIONS/INTERPRETATION: This study unravels a major cytoprotective role of cysteamine for islet cells and suggests that modulation of pantetheinase activity may offer alternative strategies to maintain islet cell homeostasis.

Visceral artery aneurysms.

Visceral artery aneurysms (VAA) frequently present as life-threatening emergencies. The purpose of this study was to review our experience with VAA treatment. Between 1988 and April 2002, 31 VAA were treated in 28 patients (14 males, 14 females) with average age of 55 +/- 15 years. The most common locations were the splenic artery (16) and the hepatic artery (7). Three patients underwent emergency surgery, 22 patients had elective open surgery, and 7 patients underwent endovascular treatment. In the surgical group the perioperative mortality rate was 3.6%. The perioperative morbidity rate was 7.1% (one case of respiratory distress manifested in the immediate postoperative period and one urgent case of bilious fistula). In the endovascular group none of the patients died; the perioperative morbidity rate was of 14.3% (one case of hepatic artery thrombosis after failure of gastroduodenal artery aneurysm embolization). Failure of the procedure was 42.9% (3 cases of aneurysm recanalization). In conclusion, we believe that an aggressive surgical approach is justified, even in the case of asymptomatic VAA, because of the low morbidity and mortality rates. Endovascular treatment should be reserved for selected cases.

Vanin-1-/- mice exhibit a glutathione-mediated tissue resistance to oxidative stress.

Vanin-1 is an epithelial ectoenzyme with pantetheinase activity and generating the amino-thiol cysteamine through the metabolism of pantothenic acid (vitamin B(5)). Here we show that Vanin-1(-/-) mice, which lack cysteamine in tissues, exhibit resistance to oxidative injury induced by whole-body gamma-irradiation or paraquat. This protection is correlated with reduced apoptosis and inflammation and is reversed by treating mutant animals with cystamine. The better tolerance of the Vanin-1(-/-) mice is associated with an enhanced gamma-glutamylcysteine synthetase activity in liver, probably due to the absence of cysteamine and leading to elevated stores of glutathione (GSH), the most potent cellular antioxidant. Consequently, Vanin-1(-/-) mice maintain a more reducing environment in tissue after exposure to irradiation. In normal mice, we found a stress-induced biphasic expression of Vanin-1 regulated via antioxidant response elements in its promoter region. This process should finely tune the redox environment and thus change an early inflammatory process into a late tissue repair process. We propose Vanin-1 as a key molecule to regulate the GSH-dependent response to oxidative injury in tissue at the epithelial level. Therefore, Vanin/pantetheinase inhibitors could be useful for treatment of damage due to irradiation and pro-oxidant inducers.

Traumatic rupture of the thoracic aorta.

Traumatic rupture of the thoracic aorta is a life threatening situation, and may be secondary to several mechanisms; mainly penetrating or iatrogenic lesions and blunt trauma. Although penetrating mechanisms predominate, the number of patients with aortic disruption due to blunt trauma has continued to increase. This paper shows an overview focusing on the pathogenesis, diagnosis, timing and type of treatment regarding traumatic injuries of the thoracic aorta; it also reports the experience of one single center that deals with these lesions. The major difficulty in the evaluation data on blunt aortic injury is that retrospective reviews often group together patients with all types of aortic lesions, comparing outcomes for injuries in different locations, with diverse methods of repair and different surgeons and/or institutions.

Carotid endarterectomy in the octogenarian: outcomes of 345 procedures performed from 1995-2000.

The purpose of this study is to evaluate the efficacy and the safety of carotid endarterectomy (CEA) in the octogenarian patient. From January 1995 to December 2000, we have performed 3430 CEAs in 2743 patients: 345 CEAs in 269 octogenarian patients (Group 1) and 3085 CEAs in 2474 younger patients (Group 2). Age was the only selection criteria for including patients in Group 1. Octogenarians' perioperative mortality (1.4%) was greater than that in Group 2 (0.3%) (p<0.05). No differences can be found between the groups' perioperative ipsilateral stroke rates (1.7% in Group 1 vs 1.2% in Group 2) and combined ipsilateral stroke and death rates (2.3% in Group 1 vs 1.3% in Group 2) (p>0.05). The octogenarians' Kaplan-Meier 6-year overall and free-stroke survival rates were 86 and 76% respectively. CEA can be performed in selected octogenarian patients with low early and late mortality and neurologic morbidity rates.

Vascular prosthetic graft infection: epidemiology, bacteriology, pathogenesis and treatment.

UNLABELLED: Vascular prosthetic graft infection remains a major surgical challenge. Prevention of risk factors and antibiotic therapy can reduced but not eradicate it. Management of infected vascular grafts depends on several factors, including the location of the infected prosthesis, the extent of infection, and the underlying micro-organism. Classic treatment consists of extra-anatomic bypass grafting. The disappointing results due to the high mortality and amputation rate have kindled interest in alternative approaches, such as in situ reconstruction with antibiotic-bonded prostheses, autogenous veins or arterial allografts. PURPOSE: We focused on the treatment of aortic graft infection by means of both fresh and cryopreserved arterial allograft. Here, the experience of the Italian Collaborative Vascular Homograft Group is reported. METHODS: Between March 1994 and December 2000 seventy-nine patients with aortic graft infection were treated. The results of 68 patients are analysed. Eleven patients were treated with fresh, and 57 with cryopreserved homograft. Emergency surgical procedures were performed in 12 patients (17%). Aortoenteric fistula was diagnosed in 22 patients. The mean interval between the first procedure and the insertion of a homograft for patients with infected aortic graft was 3 years (range 1-15). The mean duration of follow-up was 30 months (range 1-68). Clinical and duplex scanning evaluation were routinely performed. Computer tomography (CT), magnetic resonance (MR), or arteriography were performed on the basis of duplex scanning results. RESULTS: The analysis was performed on 68 cases for which there were sufficient reliable data. Eleven deaths occurred during the early postoperative period (30 days), a mortality rate of 16%. There were also seventeen late deaths, a mortality rate of 25%. Eleven patients had graft occlusion; six cases were successfully treated with thrombectomy. In three cases leg amputation was necessary. The results of fresh and cryopreserved homografts were compared. No significant differences of early postoperative mortality, late mortality, homograft-related mortality, graft failure were observed. The presence of aortoenteric fistula is a negative predicting factor of perioperative early mortality, which causes a rapid decline in the survival curve. Thirty-six months after the surgery the actuarial survival of the patients was 57% and the actuarial patency of the allograft was 41%. CONCLUSION: No significant difference in terms of clinical outcome was observed when using fresh, rather than cryopreserved homografts. The only factor that significantly influenced the survival rate appeared to be the aorto-enteric fistula.

Results of carotid endarterectomy performed with preoperative duplex ultrasound assessment alone.

Contrast injection cerebral angiography has been considered for several decades the "gold-standard" technique for diagnosis and operative planning of carotid disease. More recently, however, an increasing number of institutions are using duplex ultrasound as the single independent preoperative test. The objective of this investigation was to evaluate the impact of the utilization of duplex ultrasonography as the only preoperative test on the outcome of the procedure. Between 1993 and 1996, the authors performed 1,149 carotid procedures. Duplex ultrasound as the only preoperative test was employed with increasing frequency in a total of 728 cases. In 1995 and 1996, a cerebral arteriogram was performed only if duplex ultrasound was technically inadequate or questionable or showed an atypical pattern of disease. During the 4 years analyzed in this study, the number of the procedures increased from 165 in 1993 to 412 in 1996. The thirty-day mortality rate was 0.43%, and neurologic morbidity was 1.65%. According to the year in which the procedure was performed, the mortality/morbidity rates were 1.2/2.4 in 1993, 0.52/2.08 in 1994, 0.26/1.57 in 1995, and 0.24/1.21 in 1996. Indication to perform an arteriogram became very selective in 1995. Regardless of these changes in the diagnostic work-up, some degree of reduction in both 30-day mortality and neurologic morbidity was recorded. Considering a cost of 724 European Currency Units (ECU) per arteriogram, 527,072 ECU were saved in this period. In the last 4 years, duplex ultrasound has replaced arteriography as the first-choice technique for preoperative assessment of carotid disease at the authors' institution. There was definitely no detrimental effect on the clinical results that, on the contrary, improved during the same period. This policy has allowed a significant reduction in the cost of the procedure and has most likely prevented several arteriography-related complications. The authors recommend this policy to all institutions in which accurate duplex ultrasound is available.

Remarkable spectator ligand effect on the rate constant of ligand substitution of (aqua)ruthenium(II) complexes.

The influence of two different di(1-pyrazolyl)alkane ligands on the rate constant of aqua ligand substitution of ruthenium(II) complexes with the formula [Ru(H2O)(L2)(tpmm)]2+ (L2 = di(1-pyrazolyl)methane (DPMet) or 2,2-di(1-pyrazolyl)propane (DPPro)) was investigated. A 9.4 x 10(5)-fold increase in the rate constant of ligand substitution at pH = 6.86 was observed when DPMet was replaced with DPPro. This remarkable increase was unexpected, considering that these bidentate ligands appear quite similar. To help lend insight into this dramatic spectator ligand effect, the activation parameters for the ligand substitution reactions were determined, and single-crystal X-ray data were collected on the structurally analogous (chloro)ruthenium(II) complexes, [Ru(Cl)(L2)(tpmm)]+. These results are discussed in the context of a heteroscorpionate effect exerted by the DPPro ligand.

Emergent mechanical properties of self-assembled polymeric capsules.

Synthetic self-assembled systems combine responsiveness and reversibility with the ability to perform chemical tasks such as molecular recognition and catalysis. An unmet challenge is the construction of polymeric materials that, like nature's tubulin, are simultaneously reversible and capable of useful physical tasks. We report here a class of reversibly formed polymers that show covalent-polymer mechanical integrity in solution and in the solid state. Non-Newtonian, polymeric behavior is observed despite the low molecular weight of the individual subunits and the seemingly weak forces holding the assemblies together. These polymers assemble through self-complementary hydrogen bonding and by physical encapsulation of small molecules; accordingly, the emergent macroscopic structure and function can be controlled by appropriate chemical signals.