Genomic and pathological heterogeneity in clinically diagnosed small cell lung cancer in never/light smokers identifies therapeutically targetable alterations

Small‐cell lung cancer (SCLC) occurs infrequently in never/former light smokers. We sought to study this rare clinical subset through next‐generation sequencing (NGS) and by characterizing a representative patient‐derived model. We performed targeted NGS, as well as comprehensive pathological evaluation, in 11 never/former light smokers with clinically diagnosed SCLC. We established a patient‐derived model from one such patient (DFCI168) harboring an NRAS ^Q61K mutation and characterized the sensitivity of this model to MEK and TORC1/2 inhibitors. Despite the clinical diagnosis of SCLC, the majority (8/11) of cases were either of nonpulmonary origin or of mixed histology and included atypical carcinoid (n = 1), mixed non‐small‐cell lung carcinoma and SCLC (n = 4), unspecified poorly differentiated carcinoma (n = 1), or small‐cell carcinoma from different origins (n = 2). RB1 and TP53 mutations were found in four and five cases, respectively. Predicted driver mutations were detected in EGFR (n = 2), NRAS (n = 1), KRAS (n = 1), BRCA1 (n = 1), and ATM (n = 1), and one case harbored a TMPRSS2‐ERG fusion. DFCI168 (NRAS ^Q61K) exhibited marked sensitivity to MEK inhibitors in vitro and in vivo. The combination of MEK and mTORC1/2 inhibitors synergized to prevent compensatory mTOR activation, resulting in prolonged growth inhibition in this model and in three other NRAS mutant lung cancer cell lines. SCLC in never/former light smokers is rare and is potentially a distinct disease entity comprised of oncogenic driver mutation‐harboring carcinomas morphologically and/or clinically mimicking SCLC. Comprehensive pathologic review integrated with genomic profiling is critical in refining the diagnosis and in identifying potential therapeutic options.     

Spinal gene transfer using ultrasound and microbubbles.

Spinal gene therapy is a promising option for treating various spinal-related disorders. Several previous studies using viral vectors reported successful transfer of therapeutic genes into the spinal nerve system. However, because of the considerable immunogenicity related to the use of viruses, non-viral gene transfer still needs to be developed. One possible approach is the combined use of ultrasound and echo-contrast microbubbles. The present study shows that this method can be applied for targeted intrathecal gene delivery. We intrathecally injected a mixture of plasmid-DNA encoded with luciferase and commercially available albumin microbubbles by needle puncture at the lower lumbar intervertebral space in mice. Subsequent percutaneous ultrasonication on the lumbar vertebrae significantly enhanced the luciferase expression, analyzed by imaging luciferin bioluminescence, in the dorsal meningeal cells at the insonated region. No apparent neurological damages were induced by the present spinal interventions. In addition to the general benefits of the combined use of ultrasound and microbubbles, our approach can offer some advantages specific to spinal gene transfection including minimal invasiveness of simple percutaneous dural puncture, targetability due to the limited access of ultrasound waves through anatomical apertures of the vertebrae, and possible paracrine delivery of therapeutic molecules to the spinal nerve system.     

Cognitive- and motor-related regions in Parkinson's disease: FDOPA and FDG PET studies

OBJECTIVE: Using 6-[(18)F]fluoro-L-dopa (FDOPA) and [(18)F]fluorodeoxyglucoce (FDG) positron emission tomography (PET), multiple regression analyses were performed to determine the specific brain regions that are related to cognitive and motor symptoms in nondemented patients with Parkinson's disease. METHODS: Spatially normalized images of FDOPA influx rate constant (Ki) values and relative regional cerebral metabolic rates for glucose (rrCMRglc) were created. Raven's Coloured Progressive Matrices (RCPM) scores and the Unified Parkinson's Disease Rating Scale (UPDRS) motor scores were used to determine the patients' cognitive and motor functions, respectively. Multiple correlation analyses between the FDOPA and FDG images and the cognitive and motor scores were performed for each voxel. RESULTS: RCPM score was significantly positively correlated with the FDOPA Ki in the left hippocampus and with the rrCMRglc in the left middle frontal gyrus and right retrosplenial cortex. Motor function was significantly positively correlated with the FDOPA Ki in the bilateral striatum and with the rrCMRglc in association areas and primary visual cortex. The level of motor function was significantly inversely correlated with the FDOPA Ki in the anterior cingulate gyrus and with the rrCMRglc in bilateral primary motor cortex and right putamen. CONCLUSIONS: Changes of striatal FDOPA uptake and rrCMRglc in the primary motor cortex likely represent dysfunction in the motor system involving the corticobasal ganglia-thalamocortical loop. Change of FDOPA uptake in the anterior cingulate gyrus may be related to up-regulation of dopamine synthesis in surviving dopamine neurons. The regions where correlation with cognitive function was observed belong to a cognitive frontoparietal-hippocampal network.     

Wash-out of DMSO does not improve the speed of engraftment of cord blood transplantation: follow-up of 46 adult patients with units shipped from a single cord blood bank

BACKGROUND: Prolonged periods of marrow hypoplasia have been a problem in cord blood transplantation. DMSO is thought to produce osmotic shock to the progenitors when the thawed cells are infused into the patients. To solve this problem, a 2x dilution method originally developed in the New York Blood Center showed earlier myeloid engraftment,1 although follow-up clinical studies have not performed. STUDY DESIGN AND METHODS: To clarify the influence of the removal of DMSO by this method on the speed of engraftment in unrelated cord blood transplantation, 46 adult patients with cord blood units processed by the Tokyo Cord Blood Bank from September 1998 to March 31, 2002 were studied. Twenty-four patients received 2.6 +/- 0.71 x 10(7) nucleated cells per kg without washing (nonwashed group), while 22 patients were received 2.7 +/- 0.52 x 10(7) nucleated cells per kg after 2x dilution washing (washed group). RESULTS: The cumulative incidence of engraftment was not significantly different between the two groups. Median neutrophil recovery (>/=5 x 10(9)/L) in the nonwashed and washed groups was 26 and 25 days, respectively, and the median platelet recovery (>/=20 x 10(9)/L) in patients with myeloid engraftment was 44 and 40 days, respectively (NS). On the other hand, the doses of CFCs and CD34+ cells showed the influence on myeloid and platelet recovery. CONCLUSION: A 2x dilution after thawing cord blood did not result in the improvement of myeloid engraftment speed.     

Activity of cofilin can be regulated by a mechanism other than phosphorylation/dephosphorylation in muscle cells in culture

Cofilin plays a critical role in actin filament dynamics in a variety of eukaryotic cells. Its activity is regulated by phosphorylation/dephosphorylation of a Ser3 residue on the N-terminal side and/or its binding to a phosphoinositide, PIP₂. To clarify how cofilin activity is regulated in muscle cells, we generated analogues of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser3) of cofilin to Ala and Asp, respectively. These mutated proteins, as well as the cofilin having Ser3 residue (S3-cofilin), were produced in an E. coli expression system and conjugated with fluorescent dyes. In an in vitro functional assay, A3-cofilin retained the ability to bind to F-actin. Upon injection into cultured muscle cells, A3-cofilin and S3-cofilin promptly disrupted actin filaments in the cytoplasm, and many cytoplasmic rods containing both the exogenous cofilin and actin were generated, while D3-cofilin was simply diffused in the cytoplasm without affecting actin filaments. Several hours after the injection, however, the activity of A3-cofilin and S3-cofilin was suppressed: the actin-A3-cofilin (or S3-cofilin) rods disappeared, the cofilin diffused in the cytoplasm like D3-cofilin, and actin filaments reformed. Both GFP-fused A3-cofilin and S3-cofilin that were produced by cDNA transfection were also suppressed in the cytoplasm of muscle cells in culture. Thus, some mechanism(s) other than phosphorylation can suppress A3-cofilin activity. We observed that PIP₂ can bind to A3-cofilin just as to S3-cofilin and inhibits the interaction of A3-cofilin with actin. Our results suggest that the activity of A3-cofilin and also S3-cofilin can be regulated by PIP₂ in the cytoplasm of muscle cells.     

Epstein-Barr virus-associated B-cell lymphoma in a patient with DNA ligase IV (LIG4) syndrome

A 14-year-old Japanese girl with a progressing combined immunodeficiency had developed non-Hodgkin's diffuse large B cell lymphoma. Her molecular analysis showed a compound heterozygote of novel mutations in the LIG4 gene, M249V substitution and a five nucleotides deletion from nucleotide position 1,270-1,274. She had also a set of characteristic clinical features of LIG4 syndrome. Mutations in the LIG4 gene, which plays a critical role in the repair of DNA double-strand breaks, imply a correlation with malignancies and several cases with leukemia or lymphoma have already been reported. We report here on a case of LIG4 syndrome complicated with distinct EBV-associated B-cell lymphoma.     

Physiological role of L-type Ca2+ channels in marginal cells in the stria vascularis of guinea pigs

Using immunohistochemical and electrophysiological methods, we investigated the role of L-type Ca(2+) channels in the regulation of the endocochlear potential (EP) of the endolymphatic surface cells (ESC) of the guinea pig stria vascularis. The following findings were made: (1) Administration of 30 microg/ml nifedipine via a vertebral artery significantly suppressed the transient asphyxia-induced decrease in the EP (TAID) and the transient asphyxia-induced increase in the Ca(2+), referred to as TAIICa, concentration in the endolymph ([Ca](e)). (2) The endolymphatic administration of 1 microg/ml nifedipine significantly inhibited the TAID as well as the TAIICa. The endolymphatic administration of nifedipine (0.001-10 microg/ml) inhibited the TAID in a dose-dependent manner. (3) The endolymphatic administration of (+)-Bay K8644, an L-type Ca(2+) channel closer, significantly inhibited the TAID, whereas (-)-Bay K8644, an L-type Ca(2+) channel opener, caused a large decrease in the EP from approximately +75 mV to approximately +20 mV at 10 min after the endolymphatic administration. (4) By means of immunohistochemistry, a positive staining reaction with L-type Ca(2+) channels was detected in the marginal cells of the stria vascularis. (5) Under the high [Ca](e) condition, we examined the mechanism of the TAIICa and hypothesized that the TAIICa might have been caused by the decrease in the EP through a shunt pathway in the ESC. (6) The administration of nifedipine to the endolymph significantly inhibited the Ba(2+)-induced decrease in the EP. These findings support the view that L-type Ca(2+) channels in the marginal cells regulate the EP, but not directly the TAIICa.     

Prenatal exposure to bisphenol A and phthalates and behavioral problems in children at preschool age: the Hokkaido Study on Environment and Children’s Health

Background

Studies reported adverse behavioral development including internalizing and externalizing problems in association with prenatal exposure to bisphenol A (BPA) and phthalates; however, findings were not sufficient due to using different assessment tools and child ages among studies. This study aimed to examine associations between maternal serum levels of BPA and phthalate metabolites and behavioral problems at preschool age.

Methods

The Strengths and Difficulties Questionnaire (SDQ) was used to assess behavioral problems at 5 years of age. BPA and phthalate metabolite levels in the first trimester maternal serum was determined by LC-MS/MS for 458 children. Variables used for adjustment were parental ages, maternal cotinine levels, family income during pregnancy, child sex, birth order, and age at SDQ completed.

Results

The median concentrations of BPA, MnBP, MiBP, MEHP, and MECPP, primary and secondary metabolites of phthalates, were 0.062, 26.0, 7.0, 1.40, and 0.20 ng/ml, respectively. MECPP level was associated with increase conduct problem risk (OR?=?2.78, 95% CI 1.36–5.68) overall and the association remained after child sex stratification, and odds ratios were increased with wider confidence interval (OR?=?2.85, 95% CI 1.07–7.57 for boys, OR?=?4.04, 95% CI 1.31–12.5 for girls, respectively). BPA, ∑DBP_m (MnBP + MiBP), and ∑DEHP_m (MEHP+MECPP) levels were not associated with any of the child behavioral problems.

Conclusions

Our analyses found no significant association between BPA or summation of phthalate metabolite levels and any of the behavioral problems at 5 years of age but suggested possible association between MECPP levels and increased risk of conduct problems.