Regulation of MEK inhibitor selumetinib sensitivity by AKT phosphorylation in the novel BRAF L525R mutant

Background

Oncogenic mutations in BRAF genes are found in approximately 5–10% of colorectal cancers. The majority of BRAF mutations are located within exons 11–15 of the catalytic kinase domains, with BRAF V600E accounting for more than 80% of the observed BRAF mutations. Sensitivity to BRAF- and mitogen-activated protein kinase (MEK) inhibitors varies depending on BRAF mutations and tumor cell types. Previously, we newly identified, BRAF L525R-mutation, in the activation segment of the kinase in colorectal cancer patient. Here, we characterized the function of the BRAF L525R mutation.

Methods

HEK293 cells harboring a BRAF mutation (V600E or L525R) were first characterized and then treated with cetuximab, dabrafenib, and selumetinib. Cell viability was measured using WST-1 assay and the expression of proteins involved in the extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways was evaluated using western blot analysis.

Results

The MEK inhibitor selumetinib effectively inhibited cell proliferation and ERK phosphorylation in BRAF L525R cells but not in BRAF V600E cells. Further studies revealed that AKT phosphorylation was reduced by selumetinib in BRAF L525R cells but not in BRAF V600E cells or selumetinib-resistant BRAF L525R cells. Moreover, the AKT inhibitor overcame the selumetinib resistance.

Conclusions

We established a model system harboring BRAF L525R using HEK293 cells. BRAF L525R constitutively activated ERK. AKT phosphorylation caused sensitivity and resistance to selumetinib. Our results suggest that a comprehensive network analysis may provide insights to identify effective therapies.

     

Leukocyte-Mimetic Liposomes Penetrate Into Tumor Spheroids and Suppress Spheroid Growth by Encapsulated Doxorubicin

As leukocytes can penetrate into deep regions of a tumor mass, leukocyte-mimetic liposomes (LM-Lipo) containing leukocyte membrane proteins are also expected to penetrate into tumors by exerting properties of those membrane proteins. The aim of the present study was to examine whether LM-Lipo, which were recently demonstrated to actively pass through inflamed endothelial layers, can penetrate into tumor spheroids, and to investigate the potential of LM-Lipo for use as an anticancer drug carrier. We prepared LM-Lipo via intermembrane protein transfer from human leukemia cells; transfer of leukocyte membrane proteins onto the liposomes was determined by Western blotting. LM-Lipo demonstrated a significantly high association with human lung cancer A549 cells compared with plain liposomes, which contributed to effective anti-proliferative action by encapsulated doxorubicin hydrochloride (DOX). Confocal microscopic images showed that LM-Lipo, but not plain liposomes, could efficiently penetrate into A549 tumor spheroids. Moreover, DOX-encapsulated LM-Lipo significantly suppressed tumor spheroid growth. Thus, leukocyte membrane proteins transferred onto LM-Lipo retained their unique function, which allowed for efficient penetration of the liposomes into tumor spheroids, similar to leukocytes. In conclusion, these results suggest that LM-Lipo could be a useful tumor-penetrating drug delivery system for cancer treatment.     

Is routine abdominal drainage necessary after liver resection?

Purpose

Prophylactic abdominal drainage is performed routinely after liver resection in many centers. The aim of this study was to examine the safety and validity of liver resection without abdominal drainage and to clarify whether routine abdominal drainage after liver resection is necessary.

Methods

Patients who underwent elective liver resection without bilio-enteric anastomosis between July, 2006 and June, 2012 were divided into two groups, based on whether surgery was performed before or after, we adopted the no-drain strategy. The “former group” comprised 256 patients operated on between July, 2006 and June, 2009 and the “latter group” comprised 218 patients operated between July, 2009 and June, 2012. We compared the postoperative complications, percutaneous drainage, and postoperative hospital stay between the groups, retrospectively.

Results

There were no significant differences in the rates of postoperative bleeding, intraabdominal infection, or bile leakage between the groups. Drain insertion after liver resection did not reduce the rate of percutaneous drainage. Postoperative hospital stay was significantly shorter in the latter group.

Conclusion

Routine abdominal drainage is unnecessary after liver resection without bilio-enteric anastomosis.

     

Antiarrhythmic effect of bisoprolol, a highly selective beta1-blocker, in patients with paroxysmal atrial fibrillation

In the treatment of arrhythmia, beta-blockers are mainly used to regulate the heart rate. However, beta-blockers are also known as drugs with an antiarrhythmic effect due to the suppression of sympathetic activity. We evaluated the antiarrhythmic effects of a highly selective beta(1)-blocker, bisoprolol, in patients with diurnal paroxysmal atrial fibrillation (P-AF). A total of 136 patients with symptomatic diurnal P-AF were enrolled. Patients were divided into a diurnal-specific P-AF group and a diurnal & nocturnal P-AF group, as well as into a bisoprolol single use group and a combined use group with an antiarrhythmic drug. The effects of bisoprolol were evaluated in 3 categories: subjective symptom improvement, quality of life (QOL) improvement, and elimination of P-AF episode in Holter electrocardiograms (ECGs). For patients with effective treatment, a long-term effect up to 24 months was evaluated. Five patients (3.7%) discontinued bisoprolol due to side effects. Following administration of bisoprolol, 109 patients (80%) experienced subjective symptom improvement, 103 patients (76%) experienced QOL improvement, and elimination of P-AF episodes in ECGs was observed in 84 patients (62%). The elimination rate of P-AF episodes in ECGs was higher in the diurnal P-AF group than in the diurnal & nocturnal P-AF group (P=0.042). There was no significant difference between the bisoprolol single use group and the combined use group. A long-term suppressive effect by bisoprolol was observed in 70 of 83 patients (84%). The results demonstrate that bisoprolol has an antiarrhythmic effect against sympathetic diurnal P-AF, improving subjective symptoms and QOL and eliminating P-AF episodes in ECGs.     

Monitoring EGFR T790M with plasma DNA from lung cancer patients in a prospective observational study

Use of plasma DNA to detect mutations has spread widely as a form of liquid biopsy. EGFR T790M has been observed in half of lung cancer patients who have acquired resistance to EGFR tyrosine kinase inhibitors (EGFR‐TKI). Effectiveness of monitoring T790M via plasma DNA during treatment with EGFR‐TKI has not been established as an alternative to re‐biopsy. This was a prospective multicenter observational study involving non‐small cell lung cancer patients carrying EGFR L858R or exon 19 deletions, treated with EGFR‐TKI. The primary objective was to determine whether T790M could be detected using plasma DNA in patients with progressive disease (PD). T790M was examined using the mutation‐biased PCR and quenching probe (MBP‐QP) method, a sensitive, fully‐automated system developed in our laboratory. Eighty‐nine non‐small cell lung cancer patients were enrolled from seven hospitals in Japan. Sequential examinations revealed T790M in plasma DNA among 40% of patients who developed PD. Activating mutations, such as L858R and exon 19 deletions, were detected in 40% of patients using plasma DNA, and either T790M or activating mutations were observed in 62%. Dividing into four periods (before PD, at PD, at discontinuation of EGFR‐TKI and subsequently), T790M was detected in 10, 19, 24 and 27% of patients, respectively. Smokers, males, patients having exon 19 deletions and patients who developed new lesions evidenced significantly frequent presence of T790M in plasma DNA. Monitoring T790M with plasma DNA using MBP‐QP reflects the clinical course of lung cancer patients treated with EGFR‐TKI. Detection of T790M with plasma DNA was correlated with EGFR mutation type, exon 19 deletions and tumor progression. Re‐biopsy could be performed only in 14% of PD cases, suggesting difficulty in obtaining re‐biopsy specimens in practice. Monitoring T790M with plasma DNA reflects the clinical course, and is potentially useful in designing strategies for subsequent treatment.     

Antitumor effects of minodronate,a third-generation nitrogen-containing bisphosphonate,in synergy with γδT cells in human glioblastoma in vitro and in vivo

Nitrogen-containing bisphosphonates (N-BPs), which prevent bone resorption, exert direct and γδT cell (GDT)-mediated antitumor effects against several tumor cell types, including glioblastoma (GBM). However, limited information is available regarding the antitumor effects of N-BPs in GBM. Specifically, the antitumor effects of minodronate (MDA), a third-generation N-BP, in GBM are yet unclear. This study aimed to investigate the antitumor effects of MDA in GBM in vitro and in vivo. We performed growth inhibition and apoptosis detection assays using the GBM cell lines U87MG and U138MG. Apoptosis inhibition assays were also conducted. In vivo xenograft assays were performed in highly immunodeficient NOD.Cg-Prkdc^scid Il2rg^tm1Sug/Jic mice subcutaneously implanted with U87MG and U138MG cells. Growth inhibition and apoptosis detection assays demonstrated that MDA inhibited GBM cell growth via apoptosis, which was markedly enhanced by ex vivo expanded GDT. A pan-caspase inhibitor, z-VAD-fmk, inhibited MDA-induced U138MG apoptosis and MDA/GDT-induced U87MG and U138MG apoptosis. But z-VAD-fmk increased MDA-induced U87MG apoptosis. MDA/GDT-mediated apoptosis was blocked by the anti-T cell receptor (TCR) Vγ9, mevalonate pathway inhibitor, granzyme B inhibitor, and antitumor necrosis factor (TNF)-α. In vivo xenograft assays showed that combined intraperitoneal administration of MDA/GDT induced antitumor effects on unestablished U87MG-derived subcutaneous tumors. MDA exerted direct and GDT-mediated anti-GBM apoptotic effects in a caspase-dependent manner. GDT recognized MDA-exposed GBM cells via TCRVγ9 and induced apoptosis via granzyme B and TNF-α release. Because MDA elicited anti-GBM effects in synergy with GDT in vivo, a combination of MDA and ex vivo-generated GDT could be an effective treatment in patients with GBM.     

Initial experience of transnasal endoscopic biliary drainage without conscious sedation for the treatment of acute cholangitis (with video)

BACKGROUND: Endoscopic biliary drainage has been established as providing effective treatment for acute obstructive jaundice and cholangitis. A recently developed ultrathin transnasal videoendoscope is less invasive, even for patients who were critically ill, and can be performed without conscious sedation. OBJECTIVE: To evaluate the clinical efficacy and safety of biliary drainage (BD) by using transnasal videoendoscopy (TNE) without conscious sedation. DESIGN: Case series. SETTING: This procedure was performed at Tokyo Medical University Hospital. PATIENTS: Three patients with bile-duct stones and acute cholangitis and with a previous biliary endoscopic sphincterotomy (ES) were included in this study. INTERVENTION: All patients underwent BD by using a front-viewing TNE. Two 5F stents were placed into the bile duct across the major papilla. MAIN OUTCOME MEASUREMENT: The efficacy and safety of the TNE technique. RESULTS: Transnasal insertion of a TNE endoscope was feasible in all patients, without epistaxis. TNE BD was achieved in all patients. Abdominal pain, fever, and jaundice improved 24 hours after the procedure in all patients. Despite the absence of intravenous conscious sedation, all patients would agree to undergo the procedure again, if necessary. LIMITATIONS: Maneuverability of the TNE endoscope; limited to patients with a previous ES. CONCLUSIONS: In this small series, unsedated TNE can be used to successfully drain the biliary system in patients with a previous ES. Additional studies to validate this hypothesis are needed.     

Transnasal ultrathin endoscopy for placement of a long intestinal tube in patients with intestinal obstruction

BACKGROUND: The technical difficulties related to the insertion of a long intestinal tube into the jejunum under fluoroscopy present a considerable problem in patients with an intestinal obstruction. OBJECTIVE: To evaluate the usefulness of endoscopic long intestinal-tube placement with the ultrathin esophagogastroduodenoscope (UT-EGD). DESIGN: A prospective randomized clinical trial was conducted. PATIENTS: Twenty-eight consecutive patients who presented with an intestinal obstruction were included in the study. INTERVENTION: The UT-EGD was inserted nasally into at least the second portion of the duodenum or beyond. After a guidewire was introduced through the working channel, with fluoroscopic guidance, the UT-EGD itself was carefully removed with the guidewire left in place. Next, a hydrophilic intestinal tube was advanced over the guidewire into the jejunum, and then the guidewire was removed. MAIN OUTCOME MEASUREMENTS: Primary end points are the total procedure time, the radiation exposure time, and the rate of complications, all compared with the conventional method. RESULTS: The mean (+/-SD) total procedure time was 18.7 +/- 8.4 minutes for the UT-EGD method and 39.5 +/- 15.0 minutes for the conventional method, with a significant time difference between the 2 methods (P < .0005). The mean (+/-SD) radiation exposure time was also shorter with the UT-EGD method (11.1 +/- 6.0 minutes) than with the conventional method (30.3 +/- 13.7 minutes) (P < .0005). There were no complications, except for mild nasal bleeding with each method. CONCLUSIONS: The UT-EGD method has definite advantages in the placement of a long intestinal tube for patients with an intestinal obstruction in comparison with the conventional method.