Identification of PLX4032‐resistance mechanisms and implications for novel RAF inhibitors

BRAF inhibitors improve melanoma patient survival, but resistance invariably develops. Here we report the discovery of a novel BRAF mutation that confers resistance to PLX4032 employing whole‐exome sequencing of drug‐resistant BRAF^V600K melanoma cells. We further describe a new screening approach, a genome‐wide piggyBac mutagenesis screen that revealed clinically relevant aberrations (N‐terminal BRAF truncations and CRAF overexpression). The novel BRAF mutation, a Leu505 to His substitution (BRAF^L505H), is the first resistance‐conferring second‐site mutation identified in BRAF mutant cells. The mutation replaces a small nonpolar amino acid at the BRAF‐PLX4032 interface with a larger polar residue. Moreover, we show that BRAF^L505H, found in human prostate cancer, is itself a MAPK‐activating, PLX4032‐resistant oncogenic mutation. Lastly, we demonstrate that the PLX4032‐resistant melanoma cells are sensitive to novel, next‐generation BRAF inhibitors, especially the ‘paradox‐blocker’ PLX8394, supporting its use in clinical trials for treatment of melanoma patients with BRAF‐mutations.     

Deep inspiration breath hold in post-operative radiotherapy for right breast cancer: a retrospective analysis

BackgroundThe aim of our study is to determine whether deep inspiration breath hold (DIBH) is effective for reducing exposure of the heart, left coronary artery (LAD) and both lungs in right breast radiotherapy.Materials and methodsWe have analyzed 10 consecutive patients with right-sided breast cancer (BC), simulated during free breathing (FB) and in DIBH modality. For all patients we contoured breast PTV and organs at risk (right and left lungs, heart, LAD) on both CT scans (FB and DIBH). Finally, 5 patients were treated with IMRT and 5 with VMAT techniques.ResultsAll patients were able to end the treatments in DIBH modalities regardless of the longer treatment time in comparison to FB. The maximum and mean dose to the heart are lower in the DIBH modality. The mean values of the heart mean dose were 1.76 Gy in DIBH and 2.19 Gy in FB. The mean heart maximum dose in DIBH and FB were, respectively, 9.3 Gy and 11 Gy. Likewise, the maximum dose to the LAD is lower in DIBH; 2.57 Gy versus 3.56 Gy in FB. Noteworthy, 3 patients with hepatomegaly treated with the DIBH technique showed a higher ipsilateral lung dose than FB, but a decrease of liver dose.ConclusionWe report that the use of DIBH for right-sided BC allows the dose to the heart, LAD and to the liver to be reduced in case of hepatomegaly. This technique is well tolerated by patients, when adequately trained, and could be considered effective even in right sided BC.     

Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis,Immaturity and Oxidative Stress

Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2′-deoxyguanosine [8-OHdG] and malondialdehyde [MDA]), apoptosis (caspase activity and cleaved poly[ADP-ribose] polymerase [cPARP]) and sperm immaturity (creatine phosphokinase [CK] and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies.     

Hospital Admission of Cancer Patients: Avoidable Practice or Necessary Care?

Background

Cancer patients are frequently admitted to hospital due to acute conditions or refractory symptoms. This occurs through the emergency departments and requires medical oncologists to take an active role. The use of acute-care hospital increases in the last months of life.

Patients and methods

We aimed to describe the admissions to a medical oncology inpatient service within a 16-month period with respect to patients and tumor characteristics, and the outcome of the hospital stay.

Results

672 admissions of 454 patients were analysed. The majority of admissions were urgent (74.1%), and were due to uncontrolled symptoms (79.6%). Among the chief complaints, dyspnoea occurred in 15.7%, pain in 15.2%, and neurological symptoms in 14.5%. The majority of the hospitalizations resulted in discharge to home (60.6%); in 26.5% the patient died and in 11.0% was transferred to a hospice. Admissions due to symptoms correlated with a longer hospital stay and a higher incidence of in-hospital death.

Conclusion

We suggest that hospital use is not necessarily a sign of inappropriately aggressive care: inpatient care is probably an unavoidable step in the cancer trajectory. Optimization of inpatient supportive procedures should be a specific task of modern medical oncology.     

Lipase immobilization on differently functionalized vinyl-based amphiphilic polymers: influence of phase segregation on the enzyme hydrolytic activity

Microbial lipase from Candida rugosa was immobilized by physical adsorption onto an ethylene-vinyl alcohol polymer (EVAL) functionalized with acyl chlorides. To evaluate the influence of the reagent chain-length on the amount and activity of immobilized lipase, three differently long aliphatic fatty acids were employed (C8, C12, C18), obtaining EVAL functionalization degrees ranging from 5% to 65%. The enzyme-polymer affinity increased with both the length of the alkyl chain and the matrix hydrophobicity. In particular, the esterified polymers showed a tendency to give segregated hydrophilic and hydrophobic domains. It was observed the formation of an enzyme multilayer at both low and high protein concentrations. Desorption experiments showed that Candida rugosa lipase may be adsorbed in a closed form on the polymer hydrophilic domains and in an open, active structure on the hydrophobic ones. The best results were found for the EVAL-C18 13% matrix that showed hyperactivation with both the soluble and unsoluble substrate after enzyme desorption. In addition, this supported biocatalyst retained its activity for repetitive cycles.     

Protein kinase Akt2/PKBβ is involved in blastomere proliferation of preimplantation mouse embryos

Activation of Akt/Protein Kinase B (PKB) by phosphatidylinositol-3-kinase (PI3K) controls several cellular functions largely studied in mammalian cells, including preimplantation embryos. We previously showed that early mouse embryos inherit active Akt from oocytes and that the intracellular localization of this enzyme at the two-cell stage depends on the T-cell leukemia/lymphoma 1 oncogenic protein, Tcl1. We have now investigated whether Akt isoforms, namely Akt1, Akt2 and Akt3, exert a specific role in blastomere proliferation during preimplantation embryo development. We show that, in contrast to other Akt family members, Akt2 enters male and female pronuclei of mouse preimplantation embryos at the late one-cell stage and thereafter maintains a nuclear localization during later embryo cleavage stages. Depleting one-cell embryos of single Akt family members by microinjecting Akt isoform-specific antibodies into wild-type zygotes, we observed that: (a) Akt2 is necessary for normal embryo progression through cleavage stages; and (b) the specific nuclear targeting of Akt2 in two-cell embryos depends on Tcl1. Our results indicate that preimplantation mouse embryos have a peculiar regulation of blastomere proliferation based on the activity of the Akt/PKB family member Akt2, which is mediated by the oncogenic protein Tcl1. Both Akt2 and Tcl1 are essential for early blastomere proliferation and embryo development.     

Relaxation induced by N-terminal fragments of chromogranin A in mouse gastric preparations

A definitive role for chromogranin A (CGA)-derived fragments in the control of the gastrointestinal smooth muscle contractility has not been yet established. The purpose of the present study was to evaluate, in vitro, the effects of the recombinant vasostatin 1-78 (VS-1), CGA 7-57 and CGA 47-66 on the mouse gastric mechanical activity, recording the changes of intraluminal pressure. VS-1, CGA 7-57 and CGA 47-66 produced concentration-dependent relaxations. Mouse anti-vasostatin-1 monoclonal antibody 5A8, recognising the region 53-57, abolished the relaxation induced by VS-1, indicating the specificity of the effect. The relaxation was significantly reduced by tetrodotoxin (TTX), blocker of neuronal voltage-dependent Na(+) channels, l-NAME, inhibitor of nitric oxide (NO) synthase, or apamin, blocker of small conductance Ca(2+)-dependent K(+) channels. The joint application of TTX and l-NAME did not show any additive effects, whereas TTX plus apamin abolished the VS-1 response. The results suggest that the N-terminal CGA-derived peptides are able to relax mouse gastric muscle and, therefore, they point out an inhibitory role of vasostatin I in the gastrointestinal tract. The relaxation is mediated in part by neural mechanisms through NO production and in part by non-neural mechanisms involving the opening of small conductance Ca(2+)-dependent K(+) channels.